Hydrophobically modified biopolymers with benzenediol functional groups and their oxidized forms

Abstract

The present disclosure provides hydrophobically modified biopolymers, including hydrophobically modified chitosan, that contain benzenediol groups, optionally oxidized to the corresponding quinones. The biopolymers demonstrate a surprising ability to tune the mucoadhesion, cohesion, and hemostatic properties of materials, resulting in next-generation hemostatic agents, among other uses. Thus, in another aspect, the present disclosure provides compositions comprising the modified biopolymers of the present disclosure, and methods for treating bleeding and wounds.

Description

[Background technology]

[0001] Biopolymer materials have been used in wound care and hemostasis, among other health and personal care applications. For hemostasis (i.e., bleeding control), biopolymers must be engineered to balance several critical properties. For example, chitosan exhibits good tissue adhesion, but native chitosan itself exhibits minimal hemostatic activity. Modified chitosans with enhanced hemostatic properties have been described, including hydrophobically modified chitosans. See, for example, U.S. Pat. No. 8,932,560. However, hydrophobically modified chitosans may lose tissue adhesion properties and material cohesion, such as when presented in a flowable form. That is, while modified chitosans can provide enhanced hemostatic activity, their ability to adhere to wound tissue and maintain cohesion may be impaired, especially in the presence of significant blood flow.

[0002] Therefore, modified biopolymers such as modified chitosan that balance tissue adhesion with hemostatic activity and material cohesion are desirable for use in bleeding control, such as in surgical bleeding. Other objects of the present disclosure will become apparent from the description that follows. Summary of the Invention

[0003] The present disclosure provides hydrophobically modified biopolymers comprising covalently attached hydrophobic grafts and benzenediol groups, and optionally oxidized forms thereof, along the polymer backbone. Hydrophobically modified biopolymers include hydrophobically modified chitosan (hm-chitosan), which comprises benzenediol groups, optionally with some benzenediol groups oxidized to the corresponding quinone. The biopolymers demonstrate a surprising ability to tune and balance the mucoadhesion, cohesion, and hemostatic properties of materials, resulting in next-generation hemostatic agents. Thus, in another aspect, the present disclosure provides compositions comprising the modified biopolymers of the present disclosure, as well as methods and uses for treating bleeding and wounds.

[0004] In one aspect, the present disclosure provides hydrophobically modified biopolymers comprising covalently attached hydrophobic grafts and benzenediol groups along the polymer backbone, optionally with some of the benzenediol groups oxidized to the corresponding quinones.

[0005] In various embodiments, the hydrophobically modified biopolymer is a modified polysaccharide, such as chitosan, alginate, or cellulose. In some embodiments, the hydrophobically modified biopolymer is hydrophobically modified chitosan. Hydrophobic substituents that find use in accordance with the present disclosure include (but are not limited to) saturated and unsaturated hydrocarbons. In some embodiments, the hydrophobic substituent is aliphatic, including straight-chain or branched-chain hydrocarbons. In various embodiments, the hydrophobically modified biopolymer (e.g., chitosan) contains a hydrophobic graft (i.e., hydrophobic substituent) that is a linear or branched hydrocarbon of 6 to 18 carbon atoms. In one exemplary embodiment, the hydrophobic graft is a linear hydrocarbon and can be a uniform size or a combination of sizes.

[0006] In some embodiments, in the case of hm-chitosan, the hydrophobic graft may be present at about 0.01% to about 15%, or about 0.01% to about 10% of the chitosan monomers. In some embodiments, the hm-chitosan has about 0.1% to about 5%, or about 0.5% to about 3% of the chitosan monomers independently modified with hydrocarbon chains selected from the range of C6 to C18. In some embodiments, the hm-chitosan includes a hydrophobic graft selected from one or a combination of C8, C12, C14, C16, and C18. In some embodiments, the hydrophobic graft is selected from C8, C10, and / or C12, and the graft is present at about 0.1% to about 5%, or about 1% to about 3%, or about 1% to about 2% of the chitosan monomers. In some embodiments, the selection of the hydrophobic graft can provide another layer of control over hemostatic activity and material cohesion.

[0007] In various embodiments, the hydrophobically modified biopolymer (e.g., hm-chitosan) can further include smaller hydrocarbon substituents (C2, i.e., including acetyl substituents such as those found in chitin) to adjust the density of positive charges and the rate of biodegradation of the material. In some embodiments, the smaller hydrocarbon substituents are selected from the C1-C4 hydrocarbon range, which allows chitosan to be more predictably degraded by lysozyme activity in the body.

[0008] According to the present disclosure, the hydrophobically modified biopolymer further comprises benzenediol groups substituted along the polymer backbone, optionally with some oxidized to the corresponding quinone. For example, the benzenediol groups may comprise catechol groups. The catechol moieties can be grafted to the biopolymer using, for example, hydrocaffeic acid or L-DOPA reagents. The addition of benzenediol groups and their oxidized forms to the hydrophobically modified biopolymer increases tissue adhesion (e.g., mucoadhesion). Furthermore, such benzenediol groups added to the hydrophobically modified biopolymer help the biopolymer remain in solution and form a usable gel.

[0009] In various embodiments, the benzenediol groups and their oxidized forms are grafted onto the biopolymer at a density of 0.1% to about 15% of the polymer's monomers. For example, the hydrophobically modified biopolymer can be hm-chitosan, and the benzenediol groups and their oxidized forms are grafted onto the hm-chitosan at a density of about 0.1% to about 10% of the chitosan monomers, or about 0.1% to about 5% of the chitosan monomers, or about 0.1% to about 2% (e.g., about 0.8%) of the chitosan monomers. In various embodiments, about 10% to about 90% of the benzenediol groups, or about 25% to about 75% of the benzenediol groups, or about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinone. In various embodiments, the ratio of non-oxidized benzenediol (e.g., catechol) to oxidized benzenediol (e.g., quinone) is about 1:2, or about 1:1, or in another embodiment, the ratio may be about 2:1, about 3:1, about 4:1, or about 5:1. As the level of oxidation increases, the cohesive strength of the material also increases. Similarly, as the level of non-oxidized molecules increases, the adhesive strength of the modified biopolymer also increases.

[0010] Partial oxidation of the benzenediol groups allows for great tuning of both the adhesive and cohesive properties (eg, in aqueous hydrogel format) of the hydrophobically modified biopolymer.

[0011] In another aspect, the present disclosure provides compositions comprising the modified biopolymers of the present disclosure. Generally, the compositions will further comprise a solvent or carrier, and in some embodiments, are in the form of a hydrogel. In some embodiments, the solvent comprises water. Alternatively, the biopolymer compositions may be formulated as a solid, powder, liquid, foam, or putty.

[0012] In various embodiments, the hydrophobically modified biopolymer is present in the composition at a concentration of about 1 wt% to about 5 wt%, e.g., about 1 wt% to about 3 wt%, based on the total weight of the composition. In various embodiments, the composition has a pH of about 3.0 to about 6.0, or about 3.5 to about 5.5, or about 4.0 to about 5.5, or about 4.5 to about 5.5. These pH levels make the gel biocompatible, and adjusting the pH within this range allows for further adjustment of the gel's adhesiveness and cohesiveness. In various embodiments, the solvent further comprises an organic or inorganic acid to adjust the pH. In some embodiments, the organic or inorganic acid may be selected from acetic acid, lactic acid, glycolic acid, glutamic acid, carbonic acid, citric acid, ascorbic acid, maleic acid, and combinations thereof.

[0013] In various embodiments, the composition further comprises one or more second polymers. In some embodiments, the second polymer is selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, polyvinyl alcohol, and combinations thereof. In some embodiments, the second polymer is in particulate or granular form (e.g., gelatin granules). In one embodiment, the composition may further comprise one or more additional components, such as an anti-agglomerating agent, an anti-foaming agent, and an antibacterial agent.

[0014] In another aspect, the present disclosure provides a method for treating bleeding or a wound, the method comprising applying a hydrophobic modified biopolymer described herein or a composition described herein to the bleeding or wound. In various embodiments, the bleeding or wound can be surgical bleeding or cavity bleeding. In some embodiments, the bleeding is in an area at risk for pressure- or compression-related injury. Because the gel compositions disclosed herein do not swell after application, the compositions have desirable safety characteristics for application to areas at risk for pressure- or compression-related injury. In some embodiments, the surgical bleeding is a bleeding scale of 1 to 3 (out of 5). Due to the cohesive and adhesive properties of the material and its hemostatic effect, even moderate to severe bleeding can be controlled using the modified biopolymers and compositions of the present disclosure.

[0015] In various embodiments, the composition can be applied in a flowable form (e.g., a hydrogel). Such materials are useful for treating surgical bleeding, including arterial and organ bleeding (e.g., liver bleeding), and are easy to handle. In some embodiments, the composition is applied to a skin laceration. In another embodiment, the modified biopolymer or composition thereof is coated onto or incorporated into other materials, such as bandages and wound dressings, to treat trauma and bleeding, among other things.

[0016] Other aspects and embodiments of the present disclosure will become apparent from the following detailed description.

[0017] These and other features, aspects, and advantages of the present invention will be discussed in more detail below in conjunction with the description of the embodiments thereof as illustrated in the accompanying drawings. [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1 compares hydrophobically modified chitosans with various catechol substitutions. The vial on the right shows that a 2 wt% composition of hydrophobically modified chitosan with 8% chitosan monomer units containing catechol grafts fails to produce a stable gel. On the other hand, the vial on the left, with 0.8% monomer units containing catechol grafts, yields a stable gel. [Figure 2] 1 shows a UV spectrum analysis of a composition according to the present invention, where two shoulders are observed at 280 nm and 330 nm, indicating partial oxidation of the benzenediol group. The peak at 280 nm corresponds to the unoxidized catechol. The peak at 330 nm corresponds to the quinone group, which is an oxidized catechol group. [Figure 3] 1 shows an example of a composition of the present disclosure maintained at pH 5.4 (e.g., with partial oxidation of the catechol groups) and titrated to pH 12.0. The pH 5.4 composition provides strong hemostatic properties reflected by complete binding of the gel to the blood sample. On the other hand, the pH 12.0 composition is unable to interact with blood. In the pH 12 gel, the catechol groups are fully oxidized, and the combination of high pH and complete catechol oxidation results in a structure that cannot react with the blood sample. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present disclosure provides hydrophobically modified biopolymers comprising covalently attached hydrophobic grafts and benzenediol groups, and optionally oxidized forms thereof, along the biopolymer backbone. The hydrophobically modified biopolymers, including hydrophobically modified chitosan (hm-chitosan), comprise benzenediol groups, some of which are optionally oxidized to the corresponding quinones. As described in detail below, the present biopolymers have demonstrated a surprising ability to adjust or balance the mucoadhesion, cohesion, and hemostatic properties of materials, resulting in next-generation hemostatic agents. Thus, in another aspect, the present disclosure provides compositions comprising the modified biopolymers of the present disclosure, as well as methods and uses for treating bleeding and wounds.

[0020] In one aspect, the present disclosure provides hydrophobically modified biopolymers comprising covalently attached hydrophobic grafts and benzenediol groups along the biopolymer backbone, optionally wherein at least a portion of the benzenediol groups are oxidized to the corresponding quinones.

[0021] In various embodiments, the hydrophobically modified biopolymer is a modified polysaccharide, such as chitosan, alginate, or cellulose, all of which are abundant natural biopolymers. These polysaccharides have diverse natural origins; cellulosic compounds are found in plants, while chitosan and alginate are found in the exoskeletons or outer membranes of various organisms. Hydrophobically modified biopolymers, including hydrophobically modified chitosan and alginate, are described in U.S. Pat. Nos. 8,932,560, 8,668,899, and 10,179,145, each of which is incorporated herein by reference in its entirety. In some embodiments, the biopolymer is chitosan. Chitosan is a stable, robust, and durable biopolymer that can retain its functionality during extremely long storage periods at room temperature.

[0022] Chitosan is the generic name for a linear, random copolymer of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. Chitosan's molecular structure is a linear backbone connected by glycosidic bonds. Chitosan can be derived by deacetylation of chitin obtained from one or more of crab, shrimp, krill, and crayfish. Commercial chitosan preparations are typically prepared by chemical deacetylation of chitin under alkaline conditions. Depending on the natural source of chitin (e.g., extracted from crustacean shells) and its manufacturing process, chitosan can vary in size (average molecular weight Mw) and degree of N-acetylation (%DA). Chitosan's poor solubility in water and common organic solvents limits its applications, but the presence of reactive amino groups in the chitosan backbone allows chitosan to be chemically conjugated to various molecules to tailor its properties for various applications.

[0023] The degree of deacetylation of chitin (to provide native chitosan for modification) generally ranges from about 40% to 100%, or in some embodiments, from 50% to 100%, which determines the charge density and makes the biopolymer more reactive and amenable to modification. The charge density of chitosan is an important parameter for its tissue adhesive properties. The amount of acetylation can be adjusted by adding acetyl groups back to chitosan in accordance with the present disclosure. Thus, according to one embodiment of the present disclosure, modified chitosan will have free amines on about 40% or more of its monomers, about 50% or more of its monomers, or about 60% or more of its monomers. In various embodiments, modified chitosan according to the present disclosure contains free amines on about 40% to about 75% of its monomers, about 50% to about 75%, or about 55% to about 75% of its monomers. The structure of chitosan (deacetylated structure) is shown in Formula 1.

[0024] [ka]

[0025] In various embodiments, the biopolymer is hydrophobically modified chitosan (hm-chitosan). The chitosan can be high molecular weight chitosan, medium molecular weight chitosan, or low molecular weight chitosan. Generally, the molecular weight of the biopolymer (e.g., chitosan) will be in the range of about 25,000 to about 1,500,000 grams per mole. In various embodiments, the molecular weight of the biopolymer (e.g., chitosan) will be in the range of about 40,000 to about 500,000 grams per mole, about 50,000 to about 250,000 grams per mole, or about 50,000 to about 100,000 grams per mole. As used herein, the term "molecular weight" refers to the weight-average molecular weight. Methods for determining the average molecular weight of a biopolymer include low-angle laser light scattering (LLS) and size-exclusion chromatography (SEC).

[0026] In some embodiments, the biopolymer is chitosan having a low molecular weight (before modification) of less than 150,000 daltons. In another embodiment, the biopolymer is chitosan having a medium molecular weight of about 150,000 to about 350,000 daltons (before modification). In yet another embodiment, the biopolymer is chitosan having a high molecular weight of about 400,000 daltons or greater (before modification).

[0027] The natural biopolymers used may take various forms. For example, hm-cellulose compounds may be formed from, but are not limited to, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, and / or hydroethyl methyl cellulose. hm-chitosan may be prepared from, but is not limited to, the following chitosan salts: chitosan lactate, chitosan salicylate, chitosan pyrrolidone carboxylate, chitosan itaconate, chitosan nicotinate, chitosan formate, chitosan acetate, chitosan gallate, chitosan glutamate, chitosan maleate, chitosan aspartate, chitosan glycolate, and quaternary amine-substituted chitosans and their salts. hm-alginates may be prepared from, but are not limited to, sodium alginate, potassium alginate, magnesium alginate, calcium alginate, and / or aluminum alginate.

[0028] The hydrophobic modification of the chitosan backbone is achieved by associating an amphiphilic compound with the amino groups of chitosan, thereby binding the hydrophobic tail of the amphiphilic compound to the hydrophilic backbone structure. The process of adding hydrophobic modifications to the chitosan backbone has previously been described in U.S. Patent Nos. 8,664,199, 8,668,899, 8,932,560, 9,066,885, 9,616,088, 10,179,145, 10,493,094, 11,274,194, and 11,298,517 (each of which is incorporated herein by reference in its entirety).

[0029] The hydrophobic substituents found to be useful in accordance with the present disclosure can be classified as saturated or unsaturated hydrocarbons. In some embodiments, the hydrophobic substituents are aliphatic, including straight-chain or branched-chain hydrocarbons, or cyclic hydrocarbons. Generally, hydrophobic substituents are not substituted with hydrophilic groups (such as substituents containing O, S, N, or halogen atoms). For example, according to the present disclosure, benzenediols (e.g., catechol) and their oxidized forms are not considered hydrophobic substituents.

[0030] The hydrophobic substituent, in various embodiments, may be a linear or branched hydrocarbon, saturated (e.g., entirely single-bonded, an "alkane"), or may contain one, two, or three double bonds (an "alkene").

[0031] In some embodiments, the hydrophobic substituent is a linear or branched hydrocarbon chain. In some embodiments, the hydrophobic modification of the chitosan backbone is achieved by associating a fatty aldehyde with the amino groups of chitosan, such that the hydrophobic tail of the fatty aldehyde is attached to the hydrophilic backbone structure via an amine bond. For example, chitosan can be reacted with an alkyl aldehyde in acetic acid and aqueous ethanol, and the resulting Schiff base can be reduced to a stable secondary amine by adding a reducing agent such as sodium cyanoborohydride. See U.S. Pat. No. 8,932,560, which is incorporated herein by reference in its entirety. Alternatively, hydrophobically modified biopolymers can be prepared using fatty acid anhydride chemistry, resulting in amide bonds between the chitosan biopolymer and the hydrocarbon chain. The amide bonds formed between chitosan and fatty acid anhydrides are storage-stable, even in the presence of dilute acid, which is necessary to maintain the solubility of the hydrophobically modified chitosan. In this way, modified biopolymers can be prepared using one-pot synthesis without the need for very strong reagents, including reducing agents. The materials can be precipitated and dried after the reaction for processing and incorporation into products, including solutions, gels, and foams, among others. See U.S. Patent No. 11,274,194, which is incorporated herein by reference in its entirety.

[0032] In various embodiments, the hydrophobically modified biopolymer (e.g., chitosan) contains a hydrophobic graft (i.e., hydrophobic substituent) that is a linear or branched hydrocarbon of 6 to 18 carbon atoms. In one exemplary embodiment, the hydrophobic graft is a linear hydrocarbon and can be of a uniform size or a combination of sizes. In some embodiments, the hydrophobic graft is present in about 0.01% to about 15%, or about 0.01% to about 10% of the chitosan monomers. In some embodiments, hm-chitosan has about 0.1% to about 5%, or about 0.5% to about 3% of the chitosan monomers modified with hydrocarbon chains independently selected from the range of C6 to C18. In one exemplary embodiment, hm-chitosan has about 1% to about 3%, or about 1% to about 2% of the chitosan monomers modified with hydrocarbon chains independently selected from the range of C6 to C18. In some embodiments, the hm-chitosan comprises a hydrophobic graft selected from one or a combination of C8, C12, C14, C16, and C18. In some embodiments, the hydrophobic graft is selected from C8, C10, and / or C12, and the graft is present at about 0.1% to about 5%, or about 1% to about 3%, or about 1% to about 2% of the chitosan monomers.

[0033] In some embodiments, the choice of hydrophobic graft can provide another layer of tailoring for hemostasis and material cohesion. For example, C8, C10, and / or C12 acyl groups can be used to provide effective hemostasis, while C14, C16, and / or C18 acyl groups can provide cohesiveness under exudate flow. Smaller hydrophobic grafts, such as C8, are fluid at room and body temperature, allowing the biopolymer to spread effectively over tissue surfaces, while larger grafts, such as C18, can hold the chains strong and together even in the presence of high exudate or blood flow. See U.S. Patent Application Publication No. 2020 / 0121825, which is incorporated herein by reference in its entirety.

[0034] In various embodiments, the hydrophobically modified biopolymer (e.g., hm-chitosan) can further include smaller hydrocarbon substituents (C2, i.e., including acetyl substituents such as those found in chitin) to adjust the density of positive charges and the rate of biodegradation. In some embodiments, the smaller hydrocarbon substituents are selected from the C1-C4 hydrocarbon range, allowing the chitosan to degrade more predictably from lysozyme activity in the body. Such embodiments result in a material that can be left in the body after treatment of a wound or bleeding. See US 2020 / 0121825, which is incorporated by reference in its entirety. In various embodiments, the density of C1-C4 hydrocarbon substituents (e.g., C2) along the biopolymer backbone (e.g., chitosan backbone) can be in the range of about 5% to about 50% of the monomers in the polymer, or in some embodiments, in the range of about 20% to about 45% of the monomers in the polymer. In some embodiments, the hydrophobically modified polymer is a hydrophobically modified chitosan, wherein about 5% to about 50% of the hm-chitosan monomers contain acetyl groups, or about 10% to about 40% of the hm-chitosan monomers contain acetyl groups.

[0035] According to the present disclosure, the hydrophobically modified biopolymer further comprises benzenediol groups substituted along the biopolymer backbone, and optionally, some of the benzenediol groups are oxidized. For example, the benzenediol groups may comprise catechol groups. The catechol moieties can be grafted onto the biopolymer using, for example, hydrocaffeic acid or L-DOPA reagents.

[0036] The addition of benzenediol groups to hydrophobically modified biopolymer compositions increases the tissue adhesion (e.g., mucoadhesion) of the hydrophobically modified composition. Furthermore, such benzenediol groups added to the hydrophobically modified biopolymer help the biopolymer remain in solution and form a usable gel. The chemical structure of native chitosan with benzenediol groups added by linkage to available amines is shown below.

[0037] [ka]

[0038] Below is a diagram of each monomer unit separated by square brackets (m monomers contain a catechol moiety, n monomers contain a free amine, and p monomers contain an acetyl).

[0039] [ka]

[0040] Shown below is the formula for a chitosan molecule in which the benzenediol substituents have been fully oxidized to their corresponding quinones.

[0041] [ka]

[0042] Below is the formula for the hydrophobically modified chitosan of the present disclosure, which has benzenediol substituents, some of which are oxidized.

[0043] [ka]

[0044] According to this formula, hydrophobic grafts (C8 shown above) are present in various ratios and densities along with benzenediol and its oxidized forms, as described herein. The modified chitosan may also contain C1-C4 groups (e.g., acetyl), as described elsewhere herein.

[0045] In various embodiments, the hydrophobically modified chitosan can be described according to the following formula, where the amount of chitosan monomers having substituents containing catechol is represented by the integer m, the amount of chitosan monomers having substituents with free amines is represented by the integer n, and the amount of chitosan monomers having hydrophobic substituents is represented by the integer q:

[0046] [ka]

[0047] In various embodiments, benzenediol groups and their oxidized forms (m and p) are grafted onto the biopolymer at a density of 0.1% to about 15% of the biopolymer's monomers. For example, the hydrophobically modified biopolymer can be hm-chitosan, and benzenediol groups and their oxidized forms (m and p above) are grafted onto the hm-chitosan at a density of about 0.1% to about 10% of the chitosan monomers, or about 0.1% to about 5% of the chitosan monomers, or about 0.1% to about 2% (e.g., about 0.8%) of the chitosan monomers. In various embodiments, about 10% to about 90% of the benzenediol groups, or about 25% to about 75% of the benzenediol groups, or about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinone (monomer p in the above formula). In various embodiments, the ratio of non-oxidized benzenediol (e.g., catechol) to oxidized benzenediol (e.g., quinone) is about 1:2 or about 1:1 (e.g., in the range of about 1:2 to about 1:1), or in another embodiment, the ratio can be about 2:1, about 3:1, about 4:1, or 5:1 (e.g., in the range of about 1:2 to about 5:1, or about 1:1 to about 5:1, or about 2:1 to about 5:1). As the level of oxidation increases, the cohesiveness of the material increases. Similarly, as the level of non-oxidized molecules increases, the adhesiveness of the modified biopolymer increases. The biopolymer of the above formula may further include a monomer having an acetyl group, as described above.

[0048] Partial oxidation of the benzenediol groups allows for significant tuning of both the adhesive and cohesive properties of the aqueous-modified biopolymer, for example, in the form of an aqueous hydrogel. There are two measures of hemostatic gel functionality: cohesive strength and adhesive strength. The cohesive properties of a hemostatic gel are exemplified by the gel's ability to maintain its integrity under pressure. For example, in a burst pressure test, in which a stream of water is directed at the gel, the higher the cohesive strength of the gel, the greater the pressure required for water to penetrate the gel. Meanwhile, the adhesive properties of the gel can be measured in a similar manner. The higher the level of adhesive strength, the greater the pressure required to detach the gel from the tissue or surface to which it is attached. The most functional hemostatic gels possess both high cohesive and adhesive values.

[0049] While the hydrocarbon chains as hydrophobic grafts also provide a framework for tuning or balancing the adhesiveness and cohesiveness of the resulting hydrophobically modified biopolymer (as described above), the partial oxidation of benzenediol groups along the backbone provides an additional layer of tunability independent of the hydrophobic interactions imparted by the hydrophobic groups. For example, non-oxidized benzenediol groups enhance the adhesiveness (e.g., mucoadhesion) of the biopolymer, while oxidized benzenediol groups enhance the cohesiveness of the biopolymer. Overall, combining the hydrophobic modification with the addition of both non-oxidized and oxidized benzenediol groups onto the biopolymer backbone results in a highly tunable hydrogel system with optimized adhesiveness and cohesiveness, as well as hemostatic properties, to effectively treat bleeding from damaged tissue (including moderate to severe surgical bleeding) and successfully manage wound exudate. Gel cohesiveness can be measured based on the elastic modulus. For example, in various embodiments, the elastic modulus of the hydrogels of the present disclosure ranges from about 50 to about 5,000 Pascals. In some embodiments, the elastic modulus is less than about 4000 Pascals, or less than about 2000 Pascals, or less than about 1000 Pascals, or less than about 500 Pascals.

[0050] Figure 1 shows a comparison of hydrophobically modified chitosans further containing 8% or 0.8% catechol substitution (relative to the number of monomers in the biopolymer). As shown in Figure 1, the 8% catechol (right) fails to form a gel matrix and forms a precipitate. The 0.8% vial (left) shows a homogeneous, stable gel. While it has been described that catechol attachment to chitosan results in good tissue adhesion, it was surprising that these properties were exhibited at extremely low levels of catechol attachment when added to hydrophobically modified chitosan. Consequently, in one embodiment, the hydrophobically modified biopolymer (e.g., chitosan) has benzenediol grafts (including their oxidized forms) at a concentration of at least about 0.1% of the monomer units, but less than about 10%, or less than about 8%, or less than about 5%, or less than about 3%, or less than about 2%. In various embodiments, the benzenediol graft (including its oxidized form) is present in a range of 0.2% to about 2%, or in a range of about 0.5% to about 1.5% of the monomers of the polymer.

[0051] The properties of partial oxidation of catechol are unexpected and provide significant benefits to the material. Specifically, it allows the adhesive and cohesive properties of HMC-C to be tuned based on the level of oxidation of the benzenediol groups present in the molecule. The partially oxidized catechol groups on the biopolymer backbone are quinone functional groups (i.e., the hydroxyl in the benzenediol is oxidized to a quinone). Unoxidized hm-chitosan is colorless; the gel is translucent. With increasing oxidation, the gel color ranges from clear to dark orange, and fluidity and hemostatic properties decrease, as shown in Figure 3. The level of oxidation can be monitored using UV spectroscopy, as shown in Figure 2, for example.

[0052] A typical process for attaching benzenediol groups to hydrophobically modified chitosan (HMC) involves the following steps: Dissolve and / or suspend the hydrophobically modified chitosan in distilled water. Add an acid, such as HCl, to the HMC to bring the solution to the desired pH and further solubilize the HMC. Add a catalyst, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride and ethanol to the mixture. Add a source of benzenediol, such as 3,4-dihydroxyhydrocinnamic acid (hydrocaffeic acid) (HCA), to the mixture, which is then allowed to react for approximately 1 hour. While the reaction is taking place, add an oxidizing agent, such as NaOH, to bring the pH to about 7.0 to about 12.5 for several minutes. For example, the reaction can be allowed to proceed for up to 10 minutes, at which point the reaction is shocked with ethanol to quench the oxidation. The higher the pH the solution can achieve, the greater the level of oxidation. After the reaction is quenched, the resulting solution can be recovered and processed, for example, by drying and / or grinding to a powder. Other processes for preparing materials (including powders) containing HMC containing partially oxidized benzenediol groups can be used in accordance with the present disclosure.

[0053] In another aspect, the present disclosure provides a composition comprising a hydrophobically modified biopolymer of the present disclosure (e.g., one having benzenediol groups as described, some of which are oxidized). Generally, the composition will further comprise a solvent or carrier, and in some embodiments, is in the form of a hydrogel. In some embodiments, the solvent comprises water. Alternatively, the biopolymer composition may be formulated as a solid, powder, liquid, foam, or putty. For example, the biopolymer may be a solid, lyophilized, or dehydrated solution, or a dehydrated foam or powder. Thus, the biopolymer may form a solid matrix. Usable foam formulations of hydrophobically modified chitosan are described in US 2021 / 0353501 or US 2022 / 0226625 (each of which is incorporated herein by reference in its entirety). In various embodiments, the foam can be prepared and applied using a propellant or CO2 generated by a double-barrel syringe system. In yet another embodiment, the formulation is a putty, e.g., as described in US2014 / 0314706, which is incorporated herein by reference in its entirety. A typical "putty" composition can include a biopolymer, such as polyvinyl alcohol, and an ionic crosslinker, such as sodium borate.

[0054] In various embodiments, the hydrophobically modified biopolymer (for example, but not limited to, ground into a powder) is present in the composition at a concentration of about 1 wt% to about 5 wt%, e.g., about 1 wt% to about 3 wt%, based on the total weight of the composition.

[0055] In various embodiments, the composition has a pH of about 3.0 to about 6.0, or about 3.5 to about 5.5, or about 4.0 to about 5.5, or about 4.5 to about 5.5. These pH levels make the gel biocompatible, and adjusting the pH within this range allows for further adjustment of the gel's adhesiveness and cohesiveness. In various embodiments, the solvent further comprises an organic or inorganic acid to adjust the pH. In some embodiments, the organic or inorganic acid may be selected from acetic acid, lactic acid, glycolic acid, glutamic acid, carbonic acid, citric acid, ascorbic acid, maleic acid, and combinations thereof. In some embodiments, the acid comprises an organic acid selected from acetic acid, L-lactic acid, and combinations thereof. For example, the solvent may comprise an organic acid present at a concentration of about 0.03 M to about 0.1 M, or about 0.04 to about 0.8 M, and optionally about 0.05 M.

[0056] In various embodiments, the composition further comprises one or more second polymers. In some embodiments, the second polymer is selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, polyvinyl alcohol, and combinations thereof. In some embodiments, the second polymer is in particulate or "granular" form. In some embodiments, the composition comprises gelatin granules. In various embodiments, the particles or granules have an approximate average diameter of about 10 microns to about 2000 microns, or about 50 to about 1000 microns, or about 100 microns to about 750 microns, or about 250 microns to about 750 microns. In some embodiments, the second polymer (e.g., gelatin granules) is present in a concentration of about 0.01 wt% to about 5 wt%, or about 0.05 wt% to about 2 wt%, or about 0.1 wt% to about 1 wt% of the composition.

[0057] In one embodiment, the composition may further comprise one or more additional ingredients, such as an anti-flocculant, an anti-foaming agent, and an antibacterial agent. For example, the composition may comprise EDTA as an anti-flocculant. EDTA may be present at a concentration of 1 ppm to about 1000 ppm, or about 10 ppm to about 500 ppm, or about 10 ppm to about 100 ppm. A representative anti-foaming agent is ethanol, which in some embodiments is present at a concentration of about 0.1 wt% to about 5.0 wt%, or about 0.1 wt% to about 2.0 wt%, or about 0.1 wt% to about 1.0 wt% of the composition.

[0058] In one exemplary embodiment, the composition comprises hydrophobically modified chitosan-catechol (HMC-C) (in the described embodiment) at a concentration of about 1.0 to about 4.0 wt %, and about 0.2 to about 1.0 wt % gelatin granules, and 0.01 to 0.10 M acetic acid, about 30 to about 50 ppm EDTA, about 0.1 to about 1.0 wt % ethanol, and about 85 wt % to about 98 wt % water.

[0059] In one exemplary embodiment, the composition contains about 1.8 wt% HMC-C (a medium- or high-molecular-weight chitosan having about 35% C2 and about 1% C8 relative to the total chitosan monomers, grafted with catechol at a density of about 0.5 to about 1.5% of the chitosan monomers), about 0.5 wt% gelatin granules, about 0.05 M acetic acid, about 40 ppm EDTA, about 0.5 wt% ethanol, and about 97% water. The exemplary composition yields a gel, as shown on the left side of Figure 1, which is homogeneous and transparent. In various embodiments, the HMC-C is partially oxidized as described.

[0060] In another aspect, the present disclosure provides a method for treating bleeding or a wound, comprising applying a hydrophobic modified biopolymer described herein or a composition described herein to the bleeding or wound. In various embodiments, the bleeding or wound is surgical bleeding and may be bleeding into a body cavity. For example, the bleeding may be arterial or venous bleeding, or in some embodiments, organ bleeding. In some embodiments, the bleeding is liver bleeding or large or small intestinal bleeding. In some embodiments, the bleeding is in a location at risk for pressure- or compression-related injury. Because the gel compositions disclosed herein do not expand after application, the compositions have desirable safety for application to areas at risk for pressure- or compression-related injury. Such injury may occur, for example, during surgery selected from neurosurgery, orthopedic surgery, brain surgery, ophthalmic surgery, ear, nose, and throat surgery, thoracic surgery, prostate surgery, thyroid surgery, cardiac surgery, vascular surgery, spinal surgery, and gynecological surgery. For example, such injury may occur in the context of spinal surgery, such as a discectomy, laminectomy, laminotomy, lumbar decompression, arthrodesis, or anterior cervical discectomy. In various embodiments, such methods and uses of the compositions avoid injury to one or more neural structures, such as thecal sac compression, spinal cord compression, and nerve compression.

[0061] In one embodiment, the composition is applied to a site selected from one or more of the following: nerve structure, spinal cord, optic chiasm, spine, thecal sac, peritoneal sac, blood vessel, nerve, pulmonary artery, superior vena cava, inferior vena cava, brain tissue, bladder, cavernous nerve, muscle, bone, and joint.The surgical site may include bleeding selected from one or more of subcutaneous and muscle bleeding, bone bleeding, epidural bleeding, and large vessel bleeding.Other types of surgery that can use this method include prostatectomy, and bleeding may involve the cavernous nerve.

[0062] In some embodiments, the surgical bleeding is a scale of 1 to 3 (out of a scale of 5). In some embodiments, the bleeding is at least a scale of 3. In some embodiments, the bleeding is a scale of 4. In general, the bleeding scale can be defined as follows: 0 (no bleeding, hemostasis), 1 (minimal bleeding), 2 (mild bleeding), 3 (moderate bleeding), 4 (severe bleeding), and 5 (extreme bleeding). In some embodiments, the patient has one or more factors that affect the amount of blood loss during surgery selected from older age, higher body mass index, presence of osteoporotic bone, neuromuscular scoliosis, bone metastases, and anticoagulant therapy.

[0063] Due to the cohesive and adhesive properties of the material, as well as its hemostatic properties, the modified biopolymers and compositions of the present disclosure can be used to control even moderate to severe bleeding.

[0064] In this context, bleeding scales (e.g., those associated with skin lacerations) can be modeled as described in U.S. Patent No. 10,283,015, which is incorporated herein by reference in its entirety. The device can be used to simulate various scales of bleeding ex vivo by pumping natural or synthetic blood through a porous surface (e.g., determined by an exchangeable plate) under various flow rates.

[0065] In various embodiments, the composition can be applied in a flowable form (e.g., a hydrogel). Such materials are useful for treating bleeding due to surgery and are easy to handle. In another embodiment, the modified biopolymer or composition thereof is coated onto or incorporated into other materials, such as bandages and wound dressings, to treat trauma and bleeding, among other things.

[0066] The term "about" is used herein to mean ±10% of the associated numerical value, unless the context dictates otherwise. [Example]

[0067] The following examples were conducted to investigate hydrophobically modified chitosan materials bearing catechol functional groups and their oxidized forms.

[0068] [Example 1] In a representative process, hydrophobically modified chitosan (HMC) with catechol functional groups was prepared essentially according to the following method: 10.0 g of HMC (C2 / 35, C8 / 1) was dissolved in distilled water. This HMC contained 35% amide-containing chitosan monomers (i.e., not deacetylated) and 1% C8 hydrocarbon-grafted chitosan monomers. Thus, 64% of the chitosan monomers had available amines. The molecular weight of the chitosan was approximately 400 kDa. After dissolution, 18.4 mL of 1 M HCl was added, increasing the viscosity and solubilizing the HMC. In a separate container, 1.375 g of EDC was dissolved in 500 mL of ethanol and 500 mL of water. After dissolving the EDC, 0.650 g of hydrocaffeic acid was added to the EDC solution.

[0069] In one example, a predetermined amount of EDC-hydrocaffeic acid solution was added to an HMC solution to prepare HMC-catechol having about 8% chitosan monomer with catechol grafts. In another example, a predetermined amount of EDC-hydrocaffeic acid solution was added to an HMC solution, 10 times lower than in the previous example, to prepare HMC-catechol having about 0.8% chitosan monomer with catechol grafts.

[0070] Figure 1 shows a comparison of the physical properties of an aqueous solution containing 2 wt.% HMC-catechol (8%) and an aqueous solution containing 2 wt.% HMC-catechol (0.8%), both at pH 4.5. The composition with 8% catechol failed to form a stable gel due to lack of solubility (right), while the vial with 0.8% catechol (left) produced a stable gel with interesting mechanical properties.

[0071] Because catechol-modified chitosan is thought to have good mucoadhesive properties, based on the results shown here with HMC-catechol, we investigated whether HMC-catechol could be tailored to prepare a next-generation hemostatic agent that exhibits ideal properties of tissue adhesion, material coagulation, and hemostatic activity.

[0072] [Example 2] Hydrophobically modified chitosan (HMC) with partially oxidized catechol functional groups was prepared by dissolving 10.0 g of HMC (C2 / 35, C8 / 1) in distilled water. This HMC contained 35% amide-containing chitosan monomers (i.e., not deacetylated) and 1% C8 hydrocarbon-grafted chitosan monomers. Therefore, approximately 64% of the chitosan monomers have available amines. After dissolution, 18.4 mL of 1 M HCl was added, increasing the viscosity and solubilizing the HMC. In a separate container, 1.375 g of EDC was dissolved in 500 mL of ethanol and 500 mL of water. After dissolving the EDC, 0.650 g of hydrocaffeic acid was added to the EDC solution. The EDC-hydrocaffeic acid solution was then added to the HMC solution. The resulting solution contained HMC-catechol with approximately 5 mol % catechol (i.e., approximately 5% of the monomers had catechol substituents). The HMC-catechol was then partially oxidized by adding 40 ml of 2 M NaOH to bring the reaction to a pH of 12.27 for approximately 10 minutes. In a comparative example, the reaction pH was brought to 6.93 (Example 4). The composition brought to pH 12.27 had more oxidized catechol groups than the composition brought to pH 6.93. The reaction was stopped by shocking the solution with 1 L of ethanol.

[0073] The composition was dried to a powder. The powder was reconstituted to form a gel with a pH in the range of 4.5-5.5 (i.e., approximately 5.0). The addition of partially oxidized catechol groups is believed to balance gel cohesiveness and mucoadhesiveness while maintaining the hemostatic properties of HMC. As oxidation increases, the gel becomes more cohesive. If oxidation becomes too severe (presumably occurring above approximately pH 12.0 for more than 10 minutes prior to ethanol shock), the resulting powder no longer dissolves.

[0074] [Example 3] Hydrophobically modified chitosan with partially oxidized catechol was prepared by dissolving 10.0 g of HMC (C2 / 35, C8 / 1) in distilled water. After dissolution, 21.4 mL of 1 M HCl was added, which increased the viscosity and solubilized the HMC. In a separate container, 1.376 g of EDC was dissolved in 330 mL of ethanol. After dissolving the EDC, 0.650 g of hydrocaffeic acid was added to the EDC solution. The EDC-hydrocaffeic acid solution was added to the HMC solution. The resulting solution contained HMC-catechol. The HMC-catechol was then partially oxidized by adding 8 mL of 2 M NaOH to bring the reaction to pH 6.93. The reaction was stopped by shocking the solution with 1.5 L of ethanol.

[0075] Materials prepared essentially according to this example (formulated as hydrogels) were tested for tissue adhesion and material cohesion by testing burst pressure. Additionally, materials prepared essentially according to this example (formulated as hydrogels) were tested for in vivo hemostatic activity using a liver bleeding test. See, e.g., U.S. Pat. No. 1,274,194, which is incorporated herein by reference in its entirety. The resulting material is more adherent than HMC. This material was determined to exhibit favorable properties for tissue adhesion, material cohesion, and hemostatic activity.

[0076] The analysis of partially oxidized HMC-catechol (pH 12.3) can be seen in Figure 2. This is a UV spectrum analysis, where two shoulders at 280 nm and 330 nm can be seen, representing the partially oxidized state of catechol in the composition. The peak at 280 nm corresponds to the non-oxidized catechol group. The peak at 330 nm corresponds to the quinone group, which is the oxidized catechol molecule.

[0077] [Example 4] A 1% (w / v) partially oxidized HMC-catechol solution (essentially as prepared in Example 2) was prepared in 0.05 M acetic acid in deionized water. The solution was viscous but flowable in nature. The pH of the solution was measured to be 5.4. When mixed 50 / 50% (v / v) with citrated bovine whole blood and vortexed for 1 second, the resulting mixture formed a gel and held its own weight when the vial was inverted. See Figure 3 (top). The same stock solution of 1% (w / v) partially oxidized HMC-catechol solution was titrated to pH 12 by dropwise addition of 1.0 M NaOH. The color of the solution (now containing more oxidized catechol) darkened to a dark orange and became non-flowing in nature. This color change from a relatively clear, slightly red-orange hue to a deep, dark orange indicates the complete oxidation of the catechol groups attached to the hydrophobically modified chitosan backbone. When mixed with citrate-supplemented bovine whole blood at a 50 / 50% (v / v) ratio and vortexed for 1 second, the resulting mixture did not form a gel. The blood still flowed freely and separated from the orange gel. The orange gel retained its bulk and showed little interaction with the blood, except for a small layer of blood diffused within the gel surface. As shown in Figure 3 (bottom), gels with fully oxidized catechols were unable to interact with blood or form gel complexes. On the other hand, partially oxidized HMC-catechol gels showed good interaction with blood, forming gel complexes, while also exhibiting good tissue adhesion and material cohesion.

[0078] [Example 5] A preliminary preclinical study was conducted using an in vivo sheep model to investigate the safety and efficacy of HMC-catechol gel after epidural application. This example uses HMC-catechol gel substantially as described in Example 1. The purpose of this study was to evaluate the local histological response to HMC-catechol gel after lumbar laminectomy at 30, 60, and 90 days postoperatively, as well as to perform a histopathological evaluation of tissues from the spinal cord, exiting nerve roots, and vertebral elements of the surgery.

[0079] Animals underwent lumbar laminectomy at the L3 and L5 levels on day 0 of the study, and the surgical sites were treated with HMC-catechol. In each animal, one site was treated as a "clinical case," where excess HMC-catechol not involved in the clot was removed according to the manufacturer's instructions (TFU), and the second site was treated as a "worst case," where excess HMC-catechol was not removed. Animals were allowed to survive for 30, 60, or 90 days postoperatively and then euthanized. At necropsy, a postmortem laminectomy was performed at the midpoint (non-operated, L4) level to allow perfusion, and this level served as a control. Tissues intended for histopathology and immunohistochemistry (IHC) analysis were collected and immersion-fixed in 10% neutral buffered formalin (NBF).

[0080] Spinal cord tissue was trimmed, decalcified, processed, and embedded in paraffin blocks. The resulting blocks were sectioned using a microtome and mounted on glass slides. From each block, one slide was stained with hematoxylin and eosin (H&E), one slide was stained with Masson's trichrome (MT), and one slide was immunohistochemically labeled for the detection of ionized calcium-binding adaptor molecule 1 (IBA-1).

[0081] Under the conditions of this study, microscopic evaluation of lumbar vertebrae with spinal cords from three ovine animals treated with HMC-catechol after laminectomy at the L3 and L5 levels and euthanized at 30, 60, or 90 days demonstrated the following notable findings: All tissue reactions at the surgical site of all laminectomies observed at 30, 60, and 90 days were similar, as expected for this surgical model, indicating normal healing progression across all time points. At the surgical access site (dorsal to all tissue in the epidural space), healing was characterized by fibrosis tending to be replaced by bridging new bone, minimal residual inflammatory cells, and minimal or mild amounts of residual HMC-catechol associated with minimal numbers of macrophages, multinucleated giant cells, and rare lymphocytes. The level of fibrosis was as expected for this surgical model, and no adverse effects were observed from HMC-catechol treatment.

[0082] Importantly, the amount of residual HMC-catechol tended to decrease between each time interval, indicating progressive degradation. The pattern of HMC-catechol-associated resorption was relatively benign and associated with minimal or mild numbers of macrophages, multinucleated giant cells, and rare lymphocytes. There was no difference in healing between "clinical" and "worst-case" laminectomy sites. HMC-catechol-associated changes were limited to low-grade lymphocyte, macrophage, and multinucleated giant cell infiltration that did not affect healing, suggesting excellent biocompatibility in the sheep model.

[0083] The tissue response in the epidural space was limited to occasional foci of fibrosis, minimal residual inflammation, and residual HMC-catechol. Fibrosis in the epidural space was scored as minimal to mild, with no evidence of excessive fibrosis in response to HMC-catechol. Epidural HMC-catechol was detected at the 30- and 60-day time intervals, but importantly, there was no evidence of residual HMC-catechol in the epidural space at 90 days.

[0084] Spinal cord changes consisted of nerve fiber degeneration and microglial reactivity, both of which were interpreted as secondary to surgery and unrelated to HMC-catechol. Another important finding was the lack of evidence of antemortem compression-related injury in the spinal cord. HMC-catechol was not detected in the spinal cord or subdural space. Rarely, insignificant inflammatory cell infiltration or adhesions were noted in the dura-pia mater in the subdural space. These changes were not associated with the presence of HMC-catechol.

[0085] Under the conditions of this study, treatment of sheep lumbar spine with HMC-catechol demonstrated excellent biocompatibility, high levels of successful healing after laminectomy and 30-, 60-, or 90-day survival, without adverse findings or safety concerns.

Claims

1. 1. A hydrophobically modified biopolymer comprising a biopolymer, said biopolymer comprising covalently attached hydrophobic grafts and benzenediol groups and optionally oxidized forms thereof along said biopolymer backbone.

2. The hydrophobically modified biopolymer of claim 1 , wherein the biopolymer is chitosan.

3. The hydrophobically modified biopolymer of claim 2 , wherein the chitosan is a medium or high molecular weight chitosan.

4. 4. The hydrophobically modified biopolymer of any one of claims 1 to 3, wherein the hydrophobic graft comprises a linear hydrocarbon of 6 to 18 carbon atoms.

5. 5. The hydrophobically modified biopolymer of any one of claims 2 to 4, wherein the hydrophobically modified biopolymer is a hydrophobically modified chitosan (hm-chitosan) having from about 0.01% to about 10% chitosan monomers modified with hydrocarbon chains independently selected from the range of C6 to C18.

6. 6. The hydrophobically modified biopolymer of claim 5, wherein the hm-chitosan has from about 0.1% to about 5%, or from about 0.5% to about 3%, of chitosan monomers independently modified with hydrocarbon chains selected from the range of C6 to C18.

7. 7. The hydrophobically modified biopolymer of claim 5 or 6, wherein the hm-chitosan comprises one or more hydrophobic grafts selected from C8, C12, C14, C16, and C18.

8. 8. The hydrophobically modified biopolymer of any one of claims 5 to 7, wherein from 5% to about 50% of the chitosan monomers contain acetyl groups, optionally from about 10% to about 40% of the hm-chitosan monomers contain acetyl groups, and optionally the modified chitosan contains free amines in from about 40% to about 75% of its monomers.

9. 9. The hydrophobically modified biopolymer of any one of claims 1 to 8, wherein the benzenediol group comprises a catechol moiety and optionally an oxidized form thereof.

10. 10. The hydrophobically modified biopolymer of claim 9, wherein the catechol moiety is grafted onto the hydrophobically modified biopolymer using a hydrocaffeic acid or L-DOPA reagent.

11. 11. The hydrophobically modified biopolymer of any one of claims 1 to 10, wherein the benzenediol groups and optionally oxidized forms thereof are grafted onto the biopolymer at a density of 0.1% to about 15% of the monomers of the biopolymer.

12. 12. The hydrophobically modified biopolymer of claim 11, wherein the hydrophobically modified biopolymer is hm-chitosan, and benzenediol groups, and optionally oxidized forms thereof, are grafted to the hm-chitosan at a density of about 0.1% to about 10% of the chitosan monomers, or about 0.1% to about 8% of the chitosan monomers, or about 0.1% to about 5% of the chitosan monomers, or about 0.1% to about 2% of the chitosan monomers.

13. 13. The hydrophobically modified biopolymer of any one of claims 1 to 12, wherein at least some of the benzenediol groups are oxidized to the corresponding quinone.

14. 14. The hydrophobically modified biopolymer of claim 13, wherein about 10% to about 90% of the benzenediol groups, or about 25% to about 75% of the benzenediol groups, or about 30% to about 60% of the benzenediol groups are oxidized to the corresponding quinones.

15. The hydrophobically modified biopolymer of claim 14 in powder form.

16. A composition comprising the hydrophobically modified biopolymer of any one of claims 1 to 15 and a solvent.

17. 17. The composition of claim 16, wherein the hydrophobically modified biopolymer is present at a concentration of about 1 wt % to about 5 wt %, based on the total weight of the composition.

18. 18. The composition of claim 17, wherein the hydrophobically modified biopolymer is present at a concentration of about 1 wt % to about 3 wt %, based on the total weight of the composition.

19. The composition of any one of claims 16 to 18, wherein the solvent comprises water.

20. 20. The composition of claim 19, wherein the composition has a pH of about 3.0 to about 6.0, or about 3.5 to about 5.5, or about 4.0 to about 5.5, or about 4.5 to about 5.

5.

21. 21. The composition of claim 20, which is a hydrogel.

22. 22. The composition of claim 20 or 21, wherein the solvent optionally further comprises an organic or inorganic acid, which may be selected from acetic acid, lactic acid, glycolic acid, glutamic acid, carbonic acid, citric acid, ascorbic acid, maleic acid, and combinations thereof.

23. 23. The composition of claim 22, wherein the acid comprises an organic acid selected from acetic acid, L-lactic acid, and combinations thereof.

24. 24. The composition of claim 22 or 23, wherein the solvent comprises an organic acid present at a concentration of about 0.03 M to about 0.1 M, or about 0.04 to about 0.8 M, and optionally about 0.05 M.

25. The composition of any one of claims 16 to 24, further comprising one or more second polymers.

26. 26. The composition of claim 25, wherein the second polymer is selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, and combinations thereof.

27. 27. The composition of claim 26, wherein the second polymer is gelatin.

28. The composition of any one of claims 25 to 27, wherein the second polymer is in particulate or granular form.

29. 29. The composition of claim 28, wherein the particles or granules have an average diameter of from about 10 microns to about 2000 microns, or from about 50 to about 1000 microns, or from about 100 microns to about 750 microns, or from about 250 microns to about 750 microns.

30. 30. The composition of any one of claims 25 to 29, wherein the second polymer is present at a concentration of from about 0.01 wt % to about 5 wt %, or from about 0.05 wt % to about 2 wt %, or from about 0.1 wt % to about 1 wt % of the composition.

31. The composition of any one of claims 16 to 30, further comprising one or more of an anti-agglomerating agent, an anti-foaming agent, and an anti-microbial agent.

32. 32. The composition of claim 31, wherein the composition comprises EDTA as an anti-aggregating agent.

33. 33. The composition of claim 32, wherein the EDTA is present at a concentration of from 1 ppm to about 1000 ppm, or from about 10 ppm to about 500 ppm, or from about 10 ppm to about 100 ppm.

34. The composition of any one of claims 31 to 33, wherein the antifoaming agent is ethanol.

35. 35. The composition of claim 34, wherein ethanol is present at a concentration of from about 0.1 wt % to about 5.0 wt %, or from about 0.1 wt % to about 2.0 wt %, or from about 0.1 wt % to about 1.0 wt % of the composition.

36. 36. A method for treating bleeding or wounds, comprising applying to said bleeding or wound a hydrophobic modified biopolymer according to any one of claims 1 to 15, or a composition according to any one of claims 16 to 35.

37. 37. The method of claim 36, wherein the bleeding or wound is surgical bleeding.

38. 38. The method of claim 37, wherein the surgical bleeding is bleeding into a body cavity or bleeding at an area at risk of pressure or compression related injury.

39. 39. The method of claim 38, wherein the surgical bleeding is an injury resulting from spinal surgery, neurosurgery, or vascular surgery.

40. 40. The method of any one of claims 37 to 39, wherein the bleeding is a bleeding of scale 1 to 3.

41. 40. The method of any one of claims 37 to 39, wherein the bleeding is at least a 3 scale bleeding.

42. 40. The method of any one of claims 37 to 39, wherein the bleeding is a scale 4 bleeding.

Images