Hydrophobically modified biopolymers with benzenediol functionality and oxidized forms thereof

Abstract

The present invention provides hydrophobically modified biopolymers comprising a hydrophobically modified chitosan comprising benzenediol groups, optionally a portion of which is oxidized to the corresponding quinones. The biopolymers exhibit a surprising ability to modulate mucosal adhesion, material cohesiveness, and material hemostatic effects to provide next generation hemostatic agents and the like. Accordingly, in other aspects, the invention provides compositions comprising the modified biopolymers of the invention and methods for the treatment of bleeding and wounds.

Description

BACKGROUND OF THE INVENTION

[0001] Biopolymeric materials have been used for wound care and hemostasis, as well as other uses in health and personal care. Particularly for hemostasis (i.e., controlling bleeding), biopolymers must be engineered to balance several key properties. For example, while chitosan shows good tissue adhesion properties, native chitosan itself exhibits little hemostatic effect. Modified chitosans have been documented to have better hemostatic properties, including hydrophobically modified chitosans. See, for example, U.S. Patent No. 8,932,560. However, hydrophobically modified chitosans may lose tissue adhesion and material cohesion, including when in a flowable form. That is, while modified chitosans can provide better hemostatic effects, the ability of modified chitosan to adhere to injured tissue and maintain cohesion, particularly in the presence of substantial blood flow, may be impaired.

[0002] Accordingly, modified biopolymers, such as modified chitosan, that achieve a balance between tissue adhesion properties and hemostatic effects and material cohesion are suitable for bleeding control, including surgical bleeding. Other objects of the invention will be apparent from the following disclosure. SUMMARY OF THE INVENTION

[0003] The present invention provides hydrophobically modified biopolymers that include covalently linked hydrophobic grafts and hydroquinone groups along the polymer backbone, and optionally their oxidized forms. The hydrophobically modified biopolymers include hydrophobically modified chitosan (hm-chitosan), which optionally includes hydroquinone groups, where a portion of the hydroquinone groups are oxidized to the corresponding quinones. The biopolymers exhibit a surprising ability to modulate and balance mucoadhesion, material cohesion, and material hemostatic effects to provide a next-generation hemostatic agent. Accordingly, in other aspects, the present invention provides compositions that include the modified biopolymers of the invention and methods and uses for bleeding and wound treatment.

[0004] Accordingly, in one aspect, the present invention provides a hydrophobically modified biopolymer that includes covalently linked hydrophobic grafts and hydroquinone groups along the polymer backbone, optionally where a portion of the hydroquinone groups are 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 used according to the present invention include (but are not limited to) saturated and unsaturated hydrocarbons. In some embodiments, the hydrophobic substituent is aliphatic and includes straight-chain or branched-chain hydrocarbons. In various embodiments, the hydrophobically modified biopolymer (e.g., chitosan) contains a hydrophobic graft (i.e., a hydrophobic substituent) that is a straight-chain or branched-chain hydrocarbon having 6 to 18 carbon atoms. In an exemplary embodiment, the hydrophobic graft is a straight-chain hydrocarbon and can be of uniform size or a combination of sizes.

[0006] In some embodiments, in the case of hm-chitosan, the hydrophobic graft can account for 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 a hydrocarbon chain independently selected from C6 to C18. In some embodiments, hm-chitosan contains 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 on 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 an additional layer to modulate the hemostatic effect and material cohesion.

[0007] In various embodiments, the hydrophobically modified biopolymer (e.g., hm-chitosan) can also contain smaller hydrocarbon substituents (including C2, i.e., the acetyl substituent in chitin) to modulate the density of positive charges and to modulate the biodegradation rate of the material. In some embodiments, the smaller hydrocarbon substituents are selected from C1 to C4 hydrocarbons, which enables chitosan to degrade more predictably in vivo from lysozyme activity.

[0008] According to the present invention, the hydrophobically modified biopolymer further includes hydroquinone groups substituted along the polymer backbone, and optionally a portion of the hydroquinone groups is oxidized to the corresponding quinone. For example, the hydroquinone groups can include catechol groups. The catechol moiety can be grafted onto the biopolymer using, for example, hydrogenated caffeic acid or L-DOPA reagents. Adding the hydroquinone groups and their oxidized forms to the hydrophobically modified biopolymer increases the tissue adhesion properties (e.g., mucoadhesive properties). In addition, such hydroquinone groups added to the hydrophobically modified biopolymer help the biopolymer to remain in solution and form a gel that is easy to use.

[0009] In various embodiments, the hydroquinone groups and their oxidized forms are grafted onto the biopolymer at a density of from 0.1% to about 15% of the polymer monomers. For example, the hydrophobically modified biopolymer can be hm-chitosan, and the hydroquinone groups and their oxidized forms are grafted onto 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% of the chitosan (e.g., about 0.8%). In various embodiments, about 10% to about 90% of the hydroquinone groups are oxidized, or about 25% to about 75% of the hydroquinone groups are oxidized, or about 30% to about 60% of the hydroquinone groups are oxidized to the corresponding quinones. In various embodiments, the ratio of unoxidized hydroquinone (e.g., catechol) to oxidized hydroquinone (e.g., quinone) is about 1:2 or about 1:1; or in other embodiments, the ratio can be about 2:1, about 3:1, about 4:1 or about 5:1. As the degree of oxidation increases, the cohesiveness of the material also increases. Similarly, the higher the content of unoxidized molecules, the greater the adhesiveness of the modified biopolymer.

[0010] The partial oxidation of the hydroquinone groups allows for great tunability of the adhesion and cohesion properties of the hydrophobically modified biopolymer, for example, in the form of an aqueous hydrogel.

[0011] In other aspects, the present invention provides a composition comprising the modified biopolymer of the present invention. 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 can be formulated as a solid, powder, liquid, foam or putty.

[0012] In various embodiments, based on the total weight of the composition, the concentration of the hydrophobically modified biopolymer in the composition is from about 1 wt.% to about 5 wt.%, such as from about 1 wt.% to about 3 wt.%. In various embodiments, the pH of the composition is from about 3.0 to about 6.0, or from about 3.5 to about 5.5, or from about 4.0 to about 5.5, or from about 4.5 to about 5.5. These pH values provide biocompatible gels and further adjust the adhesiveness and cohesiveness of the gels by adjusting the pH value within this range. In various embodiments, the solvent also comprises an organic or inorganic acid for adjusting the pH. The organic or organic acids in some embodiments can 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 secondary polymers. In some embodiments, the secondary polymer is selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, polyvinyl alcohol, and combinations thereof. In some embodiments, the secondary polymer is particulate or granular (e.g., gelatin particles). In embodiments, the composition may further comprise one or more additional components such as an anti - flocculant, an anti - foaming agent, and an antibacterial agent.

[0014] In other aspects, the present invention provides a method for treating bleeding or a wound, comprising applying the hydrophobically modified biopolymer described herein or the composition described herein to the bleeding or wound. In various embodiments, the bleeding or wound is surgical bleeding or may be cavity bleeding. In some embodiments, the bleeding is located at a site at risk of pressure or compression - related injury. Since the gel compositions disclosed herein do not swell after administration, the compositions have desirable safety properties and are suitable for use at sites at risk of pressure or compression - related injury. In some embodiments, the surgical bleeding is grade 1 to 3 bleeding (out of a total of 5 grades). Due to the cohesiveness and adhesiveness of the material and its hemostatic effect, the modified biopolymers and compositions of the present invention can even control moderate to severe bleeding.

[0015] In various embodiments, the composition can be administered in a flowable form (e.g., a hydrogel). Such materials can be used to treat surgical bleeding, including arterial bleeding and organ bleeding (such as liver bleeding), and are easy to handle. In some embodiments, the composition is applied to skin lacerations. In other embodiments, the modified biopolymer or its composition is coated or incorporated into other materials such as bandages and wound dressings, particularly for treating external wounds and bleeding.

[0016] Other aspects and embodiments of the present invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other features, aspects, and advantages of the present invention will be considered in more detail in conjunction with the following description of embodiments of the present invention shown in the accompanying drawings, in which:

[0018] Figure 1 Hydrophobically modified chitosans with different catechol substitutions were compared. The vial on the right shows that a 2 wt.% composition of hydrophobically modified chitosan containing 8% chitosan monomer units does not form a stable gel. On the other hand, the vial on the left contains 0.8% of the monomer units containing catechol grafts, thereby forming a stable gel.

[0019] Figure 2The ultraviolet spectral analysis of the composition of the present invention is shown, where two shoulders can be seen at 280 nm and 330 nm, indicating partial oxidation of the hydroquinone group. The peak at 280 corresponds to unoxidized catechol. The peak at 330 corresponds to the quinone group, i.e., the oxidized catechol group.

[0020] Figure 3 An example of a composition according to the present invention that remains stable at pH 5.4 (e.g., with partial oxidation of the catechol group) is shown, as well as the composition titrated to pH 12.0. The composition with a pH of 5.4 has strong hemostatic properties, which are reflected in the complete binding of the gel to the sample blood. On the other hand, the composition at pH 12.0 cannot interact with the blood. In the gel at pH 12, the catechol group is completely oxidized, and the combination of the high pH value and the complete oxidation of the catechol results in a structure that cannot react with the blood in the sample. Detailed Description

[0021] The present invention provides a hydrophobically modified biopolymer that includes covalently linked hydrophobic grafts and hydroquinone groups along the biopolymer backbone, as well as optionally its oxidized form. The hydrophobically modified biopolymer, including hydrophobically modified chitosan (hm-chitosan), optionally contains hydroquinone groups, a portion of which is oxidized to the corresponding quinone. As described in detail below, the biopolymer shows a surprising ability to regulate or balance mucoadhesion, material cohesion, and material hemostasis to provide the next generation of hemostatic agents. Thus, in other aspects, the present invention provides compositions comprising the modified biopolymer of the present invention and methods and uses for bleeding and wound treatment.

[0022] Thus, in one aspect, the present invention provides a hydrophobically modified biopolymer that includes covalently linked hydrophobic grafts and hydroquinone groups along the biopolymer backbone, optionally, wherein at least a portion of the hydroquinone groups is oxidized to the corresponding quinone.

[0023] In various embodiments, the hydrophobically modified biopolymer is a modified polysaccharide, such as chitosan, alginate, or cellulose, all of which are abundant natural biopolymers. The natural sources of these polysaccharides vary: cellulose is present in plants, while chitosan and alginate are present in the exoskeletons or outer membranes of various organisms. Hydrophobically modified biopolymers, including hydrophobically modified chitosan and alginate, are described in U.S. Patent No. 8,932,560, U.S. Patent No. 8,668,899, and U.S. Patent No. 10,179,145, the entire contents of which are incorporated herein by reference. In some embodiments, the biopolymer is chitosan. Chitosan is a stable, strong, and durable biopolymer that can be stored at room temperature for a long time.

[0024] Chitosan is the common name for a linear random copolymer composed of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. The molecular structure of chitosan is a linear backbone connected by glycosidic bonds. Chitosan can be obtained by deacetylating chitin, which can be obtained from one or more of crabs, shrimp, krill, and crayfish. Commercial chitosan preparations are usually prepared by chemically de-N-acetylating chitin under alkaline conditions. Depending on the source of natural chitin (e.g., extracted from crustacean shells) and its production process, the size (average molecular weight Mw) and degree of N-acetylation (% DA) of chitosan may vary. Although the poor solubility of chitosan in water and common organic solvents limits its applications, the reactive amino groups in the chitosan backbone enable chitosan to be chemically conjugated with various molecules and to tune its properties for various applications.

[0025] The degree of deacetylation of chitin (providing natural chitosan for modification) is typically in the range of about 40% - 100%, or in some embodiments 50 - 100%, which determines the charge density and makes the biopolymer amenable to reaction for modification. The charge density of chitosan is an important parameter determining its tissue adhesion properties. According to the present invention, the amount of acetylation can be adjusted by adding acetyl groups to chitosan. Thus, according to embodiments of the present invention, the modified chitosan will have free amines prepared from about 40% or more monomers, or about 50% or more monomers, or about 60% or more monomers. In various embodiments, the modified chitosan according to the present invention contains free amines in about 40% to about 75%, or about 50% to about 75%, or about 55% to about 75% of its monomers. The structure of chitosan (showing deacetylation) is shown in Formula 1:

[0026]

[0027] In various embodiments, the biopolymer is hydrophobically modified chitosan (hm-chitosan). Chitosan can be high molecular weight chitosan, or it can be medium molecular weight or low molecular weight chitosan. Generally, the molecular weight of the biopolymer (e.g., chitosan) ranges from about 25,000 to about 1,500,000 grams per mole. In various embodiments, the molecular weight of the biopolymer (e.g., chitosan) ranges from about 40,000 to about 500,000 grams per mole or more, or 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 biopolymers include low angle laser scattering (LLS) and size exclusion chromatography (SEC).

[0028] In some embodiments, the biopolymer is chitosan (before modification) having a low molecular weight of less than 150,000 Daltons. In other embodiments, the biopolymer is chitosan (before modification) having a medium molecular weight of about 150,000 to about 350,000 Daltons. In still other embodiments, the biopolymer is chitosan (before modification) having a high molecular weight of about 400,000 Daltons or higher.

[0029] The form of the natural biopolymer used may vary. For example, hm-cellulose can be formed from, but is not limited to, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, and / or hydroxyethyl methyl cellulose. Hm-chitosan can be prepared from, but is not limited to, the following chitosan salts: chitosan lactate, chitosan salicylate, chitosan pyrrolidone carboxylate, chitosan itaconate, chitosan nicotinate, chitosan oligosaccharide formate, chitosan oligosaccharide acetate, chitosan gallate, chitosan glutamate, chitosan maleate, chitosan aspartate, chitosan gluconate, and quaternary amine-substituted chitosan and its salts. Hm-alginate can be prepared from, but is not limited to, sodium alginate, potassium alginate, magnesium alginate, calcium alginate, and / or aluminum alginate.

[0030] Hydrophobic modification of the chitosan backbone is achieved by the binding of an amphiphilic compound to the chitosan amino group, such that the hydrophobic tail of the amphiphilic complex binds to the hydrophilic backbone structure. Methods for adding hydrophobic modification to the chitosan backbone have been described in U.S. Pat. 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; 11,298,517, each of which is hereby incorporated by reference in its entirety.

[0031] The hydrophobic substituents used according to the present invention can be classified as saturated hydrocarbons or unsaturated hydrocarbons. In some embodiments, the hydrophobic substituents are aliphatic, including straight-chain or branched-chain hydrocarbons, or cycloalkanes. Generally, the hydrophobic substituents are not substituted by hydrophilic groups (such as substituents containing O, S, N, or halogen atoms). For example, according to the present invention, hydroquinone (e.g., catechol) and its oxidized forms are not considered hydrophobic substituents.

[0032] In various embodiments, the hydrophobic substituents can be straight-chain or branched-chain hydrocarbons and can be saturated (e.g., completely linked by single bonds, "alkanes"), or can contain one, two, or three double bonds ("alkenes").

[0033] In some embodiments, the hydrophobic substituent is a straight or branched hydrocarbon chain. In some embodiments, the hydrophobic modification of the chitosan backbone is through the conjugation of a fatty aldehyde with the chitosan amino group such that the hydrophobic tail of the fatty aldehyde is conjugated to the hydrophilic backbone structure via an amine bond. For example, chitosan can react with an alkyl aldehyde in an aqueous solution of acetic acid and 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. Patent No. 8,932,560. The entire content thereof is incorporated herein by reference. Alternatively, the hydrophobically modified biopolymer is prepared using fatty acid anhydrides chemistry to form amide bonds with the chitosan biopolymer and the hydrocarbon chain. The amide bond formed between chitosan and the fatty acid anhydride is storage stable, even in the presence of the dilute acid required to maintain the solubility of the hydrophobically modified chitosan. Thus, the modified biopolymer can be prepared using a one-pot synthesis method without the need for strict reagents, including reducing agents. These materials can be precipitated and dried after the reaction for processing and incorporation into products, including solutions, gels, and foams. See U.S. Patent No. 11,274,194, the entire content of which is incorporated herein by reference.

[0034] In various embodiments, the hydrophobically modified biopolymer (e.g., chitosan) comprises a hydrophobic graft (i.e., a hydrophobic substituent) that is a straight or branched hydrocarbon of 6 to 18 carbon atoms. In an exemplary embodiment, the hydrophobic graft is a straight hydrocarbon and can be of uniform size or a combination of sizes. In some embodiments, the hydrophobic graft is present on from about 0.01% to about 15% or from about 0.01% to about 10% of the chitosan monomers. In some embodiments, hm-chitosan has from about 0.1% to about 5%, or from about 0.5% to about 3%, of chitosan monomers modified with a hydrocarbon chain independently selected from C6 to C18. In an exemplary embodiment, hm-chitosan has from about 1% to about 3%, or from about 1% to about 2%, of chitosan monomers modified with a hydrocarbon chain in the range independently selected from C6 to C18. In some embodiments, 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 from about 0.1% to about 5%, or from about 1% to about 3%, or from about 1% to about 2%, of the chitosan monomers.

[0035] In some embodiments, the choice of hydrophobic grafts can provide another layer to modulate hemostasis and material cohesion. For example, C8, C10, and / or C12 acyl groups that provide effective hemostasis can be used, as well as C14, C16, and / or C18 acyl groups that provide cohesion under exudate flow. Smaller hydrophobic grafts, such as C8, are fluid at room temperature and body temperature, enabling the biopolymer to effectively diffuse to the tissue surface, while larger grafts, such as C18, can hold the chains firmly together even in the presence of high exudate or blood flow. See US2020 / 0121825, the entire content of which is incorporated herein by reference.

[0036] In various embodiments, the hydrophobically modified biopolymer (e.g., hm-chitosan) can also include smaller hydrocarbon substituents (including C2, i.e., the acetyl substituent in chitin) to modulate the density of positive charges and the biodegradation rate. In some embodiments, the smaller hydrocarbon substituents are selected from C1 to C4 hydrocarbons, which allows chitosan to degrade more predictably from lysozyme activity in the body. This embodiment provides a material that can remain in the body after treating a wound or bleeding. See US2020 / 0121825, the entire content of which is incorporated herein by reference. In various embodiments, the density of C1 to C4 hydrocarbon substituents (e.g., C2) along the biopolymer backbone (e.g., the chitosan backbone) can range from about 5% to about 50% of the polymer monomers, or in some embodiments from about 20% to about 45% of the polymer monomers. In some embodiments, the hydrophobically modified polymer is hydrophobically modified chitosan, and 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.

[0037] According to the present invention, the hydrophobically modified biopolymer further includes hydroquinone groups substituted along the biopolymer backbone, and optionally, a portion of the hydroquinone groups are oxidized. For example, the hydroquinone groups can include catechol groups. The catechol moiety can be grafted onto the biopolymer using, for example, hydrocaffeic acid or L-DOPA reagents.

[0038] Adding hydroquinone groups to the hydrophobically modified biopolymer composition increases the tissue adhesion properties (e.g., mucoadhesive properties) of the hydrophobically modified composition. In addition, such hydroquinone groups added to the hydrophobically modified biopolymer help the biopolymer remain in solution and form a gel that is easy to use. The following is the chemical structure of natural chitosan, where the hydroquinone groups are added by conjugation with available amines:

[0039]

[0040] The following figure shows each monomer unit separated by parentheses (the m monomer includes a catechol moiety; the n monomer includes a free amine; the p monomer includes an acetyl group):

[0041]

[0042] The following formula shows a chitosan molecule in which the benzenediol substituents have been fully oxidized to the corresponding quinones:

[0043]

[0044] The following formula illustrates the hydrophobically modified chitosan of the present invention, which has benzenediol substituents, a portion of which are oxidized:

[0045]

[0046] As described herein, according to this formula, the hydrophobic grafts (C8 as shown above) are present with catechol and its oxidized forms in various ratios and densities. As described elsewhere herein, the modified chitosan can also contain C1-C4 groups (such as acetyl groups).

[0047] 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 having a free amine is represented by the integer n; and the amount of chitosan monomers having hydrophobic substituents is represented by the integer q:

[0048]

[0049] In various embodiments, the hydroquinone groups and their oxidized forms (m and p) are grafted onto the biopolymer at a density of from 0.1% to about 15% of the biopolymer monomers. For example, the hydrophobically modified biopolymer can be hm-chitosan, and the hydroquinone groups and their oxidized forms (m and p above) are grafted onto hm-chitosan oligosaccharide 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% of the chitosan monomers (e.g., about 0.8%). In various embodiments, about 10% to about 90% of the hydroquinone groups are oxidized (monomer p in the above formula), or about 25% to about 75% of the hydroquinone groups are oxidized, or about 30% to about 60% of the hydroquinone groups are oxidized to the corresponding quinones. In various embodiments, the ratio of unoxidized hydroquinone (e.g., catechol) to oxidized hydroquinone (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 other embodiments, the ratio can be about 2:1, about 3:1, about 4:1, or about 5:1 (e.g., in the range of about 1:2 to about 5:1, or in the range of about 1:1 to about 5:1, or in the range of about 2:1 to about 5:1). As the degree of oxidation increases, the cohesiveness of the material also increases. Similarly, the higher the content of unoxidized molecules, the greater the adhesiveness of the modified biopolymer. The biopolymer in the foregoing formula may also include monomers having acetyl groups as described above.

[0050] Partial oxidation of the hydroquinone groups allows for great tunability of the adhesion and cohesion properties of the hydrophobically modified biopolymer, for example, in the form of an aqueous hydrogel. The functionality of a hemostatic gel has two measures: cohesiveness and adhesiveness. The cohesiveness of a hemostatic gel is reflected in the ability of the gel to maintain its integrity under pressure. For example, in a burst pressure test where water flow is directed at the gel, the higher the cohesiveness of the gel, the higher the pressure required for water to penetrate the gel. On the other hand, the stickiness of the gel can be measured in a similar way. The higher the level of adhesiveness, the greater the pressure required to remove the gel from the tissue or surface to which the gel is adhered. The most functional hemostatic gels have higher cohesion values and higher adhesion values.

[0051] Although the hydrocarbon chains grafted hydrophobically also provide a framework to modulate or balance the adhesive and cohesive properties of the resulting hydrophobically modified biopolymer (as previously described), the partial oxidation of the hydroquinone groups along the backbone provides an additional layer of tunability that is independent of the hydrophobic interactions imparted by the hydrophobic groups. For example, unoxidized hydroquinone groups enhance the adhesion of the biopolymer (e.g., mucoadhesion), while oxidized hydroquinone groups enhance the cohesion of the biopolymer. In summary, the combination of hydrophobic modification, along with the addition of non-oxidized and oxidized hydroquinone groups on the biopolymer backbone, forms a highly tunable hydrogel system to optimize adhesion, cohesion, and hemostasis, thereby effectively treating bleeding in injured tissues (including moderate to severe surgical bleeding) and managing wound exudates. The cohesion of the gel can be measured according to the elastic modulus. For example, according to various embodiments, the elastic modulus of the hydrogel according to the present invention ranges from about 50 to about 5000 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.

[0052] Figure 1 Shows a comparison between hydrophobically modified chitosan further comprising 8% catechol substitution or 0.8% catechol substitution (relative to the number of monomers in the biopolymer). As Figure 1 shown, 8% catechol (right) failed to form a gel matrix but instead formed a precipitate. The 0.8% vial (left) shows a uniform and stable gel. Although the binding of catechol to chitosan has been described as exhibiting good tissue adhesion properties, surprisingly, these properties are exhibited at very low levels of catechol binding when added to hydrophobically modified chitosan. As a result, in one embodiment, the hydrophobically modified biopolymer (e.g., chitosan) has a concentration of at least about 0.1%, 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% of diol grafts (including its oxidized form) based on the monomer units. In various embodiments, the diol grafts (including its oxidized form) are present in the range of 0.2% to about 2% of the polymer monomers, or in the range of about 0.5% to about 1.5%.

[0053] The nature of the partial oxidation of catechol is unexpected and provides significant benefits to the material, specifically, its ability to modulate the adhesion and cohesion of HMC-C according to the oxidation level of the catechol groups present in the molecule. The partially oxidized catechol groups on the biopolymer backbone are quinone functional groups (i.e., the hydroxyl groups in hydroquinone are oxidized to quinone). Unoxidized hm-chitosan has no color, i.e., the gel is translucent. As Figure 3 shown, as the degree of oxidation increases, the color of the gel changes from transparent to dark orange and has less fluidity and hemostatic properties. For example, asFigure 2 As shown, ultraviolet spectroscopy can be used to monitor the oxidation level.

[0054] Exemplary methods for adding hydroquinone groups to hydrophobically modified chitosan (HMC) include the following steps. The hydrophobically modified chitosan is dissolved and / or suspended in distilled water. An acid (such as HCl) is added to the HMC to bring the solution to the desired pH value and further dissolve the HMC. A catalyst such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride and ethanol are added to the mixture. A hydroquinone source, such as 3,4-dihydroxyhydrocinnamic acid (hydrocaffeic acid) (HCA), is added to the mixture and allowed to react for about one hour. While the reaction is proceeding, an oxidant such as NaOH is added to bring the pH value to about 7.0 to about 12.5 within a few minutes. For example, the reaction can proceed for up to 10 minutes, at which point the reaction is quenched with ethanol to stop the oxidation. The higher the pH value of the solution, the higher the degree of oxidation. After the reaction is stopped, the resulting solution can be collected and processed, for example, dried and / or ground into a powder. According to the present invention, other methods for preparing materials (including powders) involving HMC containing hydroquinone groups can be employed, wherein a portion of the hydroquinone groups are oxidized.

[0055] In other aspects, the present invention provides a composition comprising the hydrophobically modified biopolymer of the present invention (e.g., having hydroquinone groups, as described, wherein a portion 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 includes water. Alternatively, the biopolymer composition can be formulated as a solid, powder, liquid, foam, or putty. For example, the biopolymer can be a solid, can be lyophilized, or can be a dehydrated solution or dehydrated foam or powder. Thus, the biopolymer can form a solid matrix. Foam formulations of the hydrophobically modified chitosan that can be used are described in US 2021 / 0353501 or US2022 / 0226625, the entire contents of which are incorporated herein by reference. In various embodiments, a propellant or CO2 generated using a dual syringe system can be used to prepare or administer the foam. In still other embodiments, the formulation is a putty as described, for example, in US 2014 / 0314706, the entire contents of which are incorporated herein by reference. Exemplary putty compositions can include a biopolymer such as polyvinyl alcohol and an ionic crosslinker such as sodium borate.

[0056] In various embodiments, based on the total weight of the composition, the concentration of the hydrophobically modified biopolymer (e.g., but not limited to ground into a powder) in the composition is from about 1 wt.% to about 5 wt.%, such as from about 1 wt.% to about 3 wt.%.

[0057] In various embodiments, the pH of the composition is from about 3.0 to about 6.0, or from about 3.5 to about 5.5, or from about 4.0 to about 5.5, or from about 4.5 to about 5.5. These pH values provide a biocompatible gel and further enable the regulation of the gel's adhesiveness and cohesiveness by adjusting the pH value within this range. In various embodiments, the solvent further includes an organic acid or an inorganic acid for adjusting the pH. In some embodiments, the organic acid or inorganic acid can 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 includes an organic acid selected from acetic acid, L-lactic acid, and combinations thereof. For example, the solvent can contain an organic acid at a concentration of about 0.03M to about 0.1M, or about 0.04 to about 0.8M, optionally about 0.05M.

[0058] In various embodiments, the composition further comprises one or more secondary polymers. In some embodiments, the secondary polymers are selected from gelatin, dextran, pectin, alginate, collagen, polyethylene oxide, gellan gum, polyvinyl alcohol, and combinations thereof. In some embodiments, the secondary polymer is in particulate or "granular" form. In some embodiments, the composition contains gelatin particles. In various embodiments, the average diameter of the particles or granules is about 10 microns to 2000 microns, or 50 microns to 1000 microns, or 100 microns to 750 microns, or 250 microns to 750 microns. In some embodiments, the concentration of the secondary polymer (e.g., gelatin particles) is 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.

[0059] In an embodiment, the composition can further contain one or more additional components, such as an anti-flocculant, an anti-foaming agent, and an antibacterial agent. For example, the composition can contain EDTA as an anti-flocculant. The concentration of EDTA can be from 1 ppm to about 1000 ppm, or about 10 ppm to about 500 ppm, or about 10 ppm to about 100 ppm. An exemplary anti-foaming agent is ethanol, and in some embodiments, the concentration of ethanol is 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.

[0060] In an exemplary embodiment, the composition contains hydrophobic modified chitosan-catechol (HMC-C) at a concentration of about 1.0 to about 4.0 wt.% (in the said embodiment), about 0.2 to about 1.0 wt.% of gelatin particles, 0.01 to 0.10 M acetic acid, about 30 to about 50 ppm EDTA; about 0.1 to about 1.0 wt.% of ethanol, and about 85 wt.% to about 98 wt.% of water.

[0061] In an exemplary embodiment, the composition comprises about 1.8 wt.% HMC-C (catechol is grafted onto medium or high molecular weight chitosan having about 35% C2 and about 1% C8 at a density of about 0.5 to about 1.5% relative to the total chitosan monomers); about 0.5 wt.% gelatin particles; about 0.05 M acetic acid; about 40 ppm EDTA; about 0.5 wt.% ethanol; and about 97% water. The exemplary composition produces a gel as shown on the left side, where the gel composition on the left side is uniformly transparent. In various embodiments, the HMC-C is partially oxidized as described. Figure 1 On the left side, where the gel composition on the left side is uniformly transparent. In various embodiments, the HMC-C is partially oxidized as described.

[0062] In other aspects, the present invention provides a method for treating bleeding or a wound, comprising applying the hydrophobic modified biopolymer described herein or the composition described herein to the bleeding or the wound. In various embodiments, the bleeding or the wound is surgical bleeding, and may also be cavity bleeding. For example, the bleeding can be arterial or venous bleeding, or in some embodiments, organ bleeding. In some embodiments, the bleeding is liver bleeding, or bleeding in the large or small intestine. In some embodiments, the bleeding is located at a position at risk of pressure or compression-related injury. Since the gel composition disclosed herein does not swell after application, the composition has an ideal safety and is suitable for sites at risk of pressure or compression-related injury. For example, such injuries may occur during surgeries selected from neurosurgery, orthopedic surgery, brain surgery, eye surgery, otolaryngology surgery, thoracic surgery, prostate surgery, thyroid surgery, cardiac surgery, vascular surgery, spinal surgery, and gynecological surgery. For example, such injuries may occur during spinal surgery, such as discectomy, laminectomy, laminotomy, lumbar decompression surgery, arthrodesis, or anterior cervical discectomy. In various embodiments, this method and the use of the composition avoid damage to one or more nerve structures, such as dural sac compression, spinal cord compression, and nerve compression.

[0063] In an embodiment, the composition is applied to a site selected from one or more of nerve structures, spinal cord, optic chiasm, spine, dural sac, peritoneal sac, blood vessels, nerves, pulmonary artery, superior vena cava, inferior vena cava, brain tissue, bladder, cavernous nerves, muscle, bone, and joints. The surgical site may include one or more types of bleeding selected from subcutaneous and muscle bleeding, bone bleeding, epidural bleeding, and large vessel bleeding. Other types of surgeries to which the method can be applied include prostatectomy, where the bleeding may involve the cavernous nerves.

[0064] In some embodiments, surgical bleeding is grade 1 to 3 bleeding (on a 5 - point scale). In some embodiments, the bleeding is at least grade 3 bleeding. In some embodiments, the bleeding is grade 4 bleeding. Generally, the amount of bleeding can be defined as: 0 (no bleeding, hemostasis); 1 (slight 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, and these factors are selected from advanced age, higher body mass index, presence of osteoporotic bone, neuromuscular scoliosis, bone metastases, and anticoagulant therapy.

[0065] Due to the cohesiveness and adhesiveness of the material and its hemostatic effect, the modified biopolymers and compositions of the present invention can even control moderate to severe bleeding.

[0066] In this context, the amount of bleeding (e.g., for skin lacerations) can be modeled as described in U.S. Patent No. 10,283,015, which is incorporated herein by reference. By pumping natural or synthetic blood into a porous surface (e.g., defined by an interchangeable plate) at different flow rates, the device can be used to simulate different scales of external bleeding.

[0067] In various embodiments, the composition can be administered in a flowable form (such as a hydrogel). Such materials are useful and easy to handle for treating surgical bleeding. In other embodiments, the modified biopolymer or its composition is coated or incorporated into other materials, such as bandages and wound dressings, especially for treating external wounds and bleeding.

[0068] As used herein, the term “about” means ± 10% of the relevant numerical value, unless the context otherwise indicates.

[0069] Example

[0070] The following examples were conducted to study hydrophobic modified chitosan materials having catechol functional groups and their oxidized forms.

[0071] Example 1

[0072] In an exemplary method, hydrophobically modified chitosan (HMC) having catechol functional groups is prepared substantially according to the following method. 10.0 g of HMC (C2 / 35, C8 / 1) is dissolved in distilled water. The HMC contains 35% of chitosan monomers having amides (i.e., non-deacetylated) and 1% of chitosan having C8 hydrocarbon grafts. Thus, the HMC has available amines on approximately 64% of the chitosan monomers. The molecular weight of the chitosan is approximately 400 kDa. After dissolution, 18.4 mL of 1 M HCL is added, which increases the viscosity and dissolves the HMC. In another container, 1.375 g of EDC is dissolved in 500 mL of ethanol and 500 mL of water. After the EDC is dissolved, 0.650 g of hydrocaffeic acid is added to the EDC solution.

[0073] In one example, the EDC hydrocaffeic acid solution is added to the HMC solution in a specified amount to prepare HMC-catechol having approximately 8% of chitosan monomers with catechol grafts. In another example, the EDC hydrocaffeic acid solution is added to the HMC solution in a specified amount ten times lower than the previous example to prepare HMC-catechol having approximately 0.8% of chitosan monomers with catechol grafts.

[0074] Figure 1 The physical properties of an aqueous solution containing 2 wt.% HMC-catechol (8%) and an aqueous solution containing 2 wt.% HMC-dibutyl phthalate (0.8%) were compared (both at pH 4.5). The composition containing 8% catechol could not form a stable gel due to lack of solubility (right). On the other hand, the vial containing 0.8% catechol on the left produced a stable gel with interesting mechanical properties.

[0075] Since catechol-modified chitosan is considered to have good mucoadhesive properties and based on the results of HMC-catechol, we investigated the ability to modulate HMC-catechol to prepare the next generation of hemostatic agents that have ideal tissue adhesion, material cohesion, and hemostatic activity.

[0076] Example 2

[0077] 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 contains 35% of chitosan monomers with amides (i.e., non-deacetylated) and 1% of chitosan grafted with C8 hydrocarbons. Thus, HMC has available amines on approximately 64% of the chitosan monomers. After dissolution, 18.4 mL of 1M HCL was added, which increased the viscosity and dissolved the HMC. In another container, 1.375 g of EDC was dissolved in 500 mL of ethanol and 500 mL of water. After the EDC was dissolved, 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, where the catechol was approximately 5 mol% (i.e., approximately 5% of the monomers had catechol substituents). Then the HMC-catechol was partially oxidized by adding 40 ml of 2M NaOH to bring the reaction pH to 12.27 for about 10 minutes. In the comparative example, the pH of the reaction was adjusted to 6.93 (Example 4). The composition with a pH of 12.27 had more oxidized catechol groups than the composition with a pH of 6.93. The reaction was quenched by adding 1 L of ethanol solution.

[0078] The composition was dried into a powder. In the range of pH 4.5 to 5.5 (i.e., approximately 5.0), the powder was redissolved to form a gel. Adding catechol groups (a portion of which was oxidized) was considered to provide a balance between the cohesiveness and mucoadhesiveness of the gel while maintaining the hemostatic properties of the HMC. As the oxidation increased, the cohesive properties of the gel also increased. If the oxidation was excessive, we estimated that if it continued for more than about 10 minutes at a pH above 12.0 before quenching with ethanol, the resulting powder would no longer dissolve.

[0079] Example 3

[0080] Hydrophobically modified chitosan with partially oxidized catechol was prepared by dissolving 10.0 g of HMC in distilled water. After dissolution, 21.4 mL of 1M HCL was added, increasing the viscosity and dissolving the HMC. In another container, 1.376 g of EDC was dissolved in 330 mL of ethanol. After the EDC was dissolved, 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. Then the HMC-catechol was partially oxidized by adding 8 mL of 2M NaOH to bring the reaction pH to 6.93. The reaction was quenched by adding 1.5 L of ethanol solution.

[0081] The tissue adhesion properties and material cohesiveness of materials prepared substantially according to this embodiment (prepared as hydrogels) were tested by testing the burst pressure. The in vivo hemostatic effect of materials prepared substantially according to this embodiment (prepared as hydrogels) was further tested using a liver bleeding test. For example, see US11274194, the entire content of which is incorporated herein by reference. The resulting materials were stickier than HMC. These materials were determined to exhibit good performance in terms of tissue adhesion, material cohesiveness, and hemostatic effect.

[0082] Analysis of HMC - partially oxidized catechol (pH 12.3) is as Figure 2 shown, which is an ultraviolet spectral analysis where two shoulder peaks 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 unoxidized catechol group. The peak at 330 corresponds to the quinone group, i.e., the oxidized catechol molecule.

[0083] Example 4

[0084] A 1% (w / v) solution of partially oxidized HMC - catechol (prepared substantially as in Example 2) was prepared in 0.05 M acetic acid in deionized water. The solution was viscous but substantially flowable. The pH of the solution was 5.4. When mixed 50 / 50% (v / v) with citrated bovine whole blood and vortexed for 1 second, the resulting mixture formed a gel that retained its own weight when the vial was inverted. See Figure 3 (top). A stock solution of the same 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) changed to dark orange and became non - flowable. This change in color from relatively transparent and slightly red to orange and then to dark orange indicates that the catechol groups attached to the hydrophobically modified chitosan backbone were completely oxidized. When mixed 50 / 50% (v / v) with citrated bovine whole blood and vortexed for 1 second, the resulting mixture did not form a gel. The blood remained freely flowing and separated from the orange gel. The orange gel retained its overall properties and did not interact substantially with the blood, except for a small amount of blood diffusing to the gel surface. As Figure 3 (bottom) shows, the gel containing fully oxidized catechol was unable to interact with the blood to form a gel complex. On the other hand, the partially oxidized HMC - catechol gel had good interaction with the blood when forming a gel complex, and also showed good tissue adhesion properties and material cohesiveness.

[0085] Example 5

[0086] A preclinical pilot study was conducted to investigate the safety and efficacy of HMC-catechol gel after epidural application using an in vivo model of sheep. This example uses HMC-catechol gel, substantially as described in Example 1. This study aimed to evaluate the local histological response to HMC-catechol gel at 30, 60, and 90 days after lumbar laminectomy, as well as the histopathological evaluation of the tissues of the spinal cord, nerve roots, and surgical vertebral bodies.

[0087] On day 0 of the study, the animals underwent lumbar laminectomy at the L3 and L5 levels, and the surgical sites were treated with HMC-catechol. In each animal, one site was considered a "clinical case" and the excess HMC-catechol not involved in clot removal was removed according to the Instructions for Use (IFU), and the second site was considered a "worst case" where the excess HMC-catechol was not removed. The animals survived for 30, 60, or 90 days postoperatively and were then euthanized. At autopsy, a postmortem laminectomy was performed at the intervention (non-surgical, L4) level for perfusion, and this level was used as a control. Tissues for histopathological and immunohistochemical (IHC) analysis were collected and fixed by immersion in 10% neutral buffered formalin (NBF).

[0088] The spinal tissues were trimmed, decalcified, processed, and embedded in paraffin blocks. The resulting blocks were sectioned by a microtome and mounted on glass slides. From each block, the glass slides were stained with hematoxylin and eosin (H&E), stained with Masson's trichrome (MT), and immunohistochemically labeled to detect ionized calcium-binding adaptor molecule 1 (IBA-1).

[0089] Under the conditions of this study, microscopic evaluation of the lumbar spine and spinal cord of three (3) sheep treated with HMC-catechol, euthanized at 30, 60, or 90 days after laminectomy at the L3 and L5 levels, showed the following significant findings. At 30, 60, and 90 days, all tissue responses observed at all laminectomy sites were similar, consistent with the expectations of this surgical model, and showed normal healing progression over the time points. At the surgical incision site (all tissues dorsal to the epidural space), healing was characterized by fibrosis, which tended to be replaced by bridging new bone, with very few residual inflammatory cells, and very little or a small amount of residual HMC-catechol, which was associated with a small number of macrophages, multinucleated giant cells, and rare lymphocytes. In this surgical model, the level of fibrosis was consistent with expectations and did not appear to be adversely affected by HMC-catechol treatment.

[0090] Importantly, the residual amount of HMC-catechol tended to decrease between each time interval, indicating that it was gradually degrading. The absorption pattern of HMC-catechol was relatively benign, associated with a small number of macrophages, multinucleated giant cells, and rare lymphocytes. There was no difference in the healing of the "standard" and "extreme operation" laminectomy sites. The changes associated with HMC-catechol were limited to low-grade lymphocyte, macrophage, and multinucleated giant cell infiltration and had no effect on healing, indicating excellent biocompatibility in the ovine model.

[0091] The tissue reaction in the epidural space was limited to fibrosis, mild residual inflammation, and occasional foci of residual HMC-catechol. The fibrosis score in the epidural space was mild to moderate, and there was no evidence of excessive fibrosis in response to HMC-catechol. Epidural HMC-catechol was detected at the 30-day and 60-day time intervals, but importantly, there was no evidence of residual HMC-catechol in the epidural space at 90 days.

[0092] The changes in the spinal cord included nerve fiber degeneration and microglial reaction, both of which were interpreted as secondary to the surgery and were not related to HMC-catechol. Another important finding was that there was no evidence of pre-mortem compression-related injury to the spinal cord. HMC-catechol was not detected in the spinal cord or subdural space. Rare and insignificant inflammatory cell infiltration or adhesions were visible in the dura mater of the subdural space. These changes were not related to the presence of HMC-catechol.

[0093] Under the conditions of this study, after laminectomy, treating the lumbar spine of sheep with HMC-catechol for a survival period of 30 days, 60 days, or 90 days showed excellent biocompatibility, good and normal healing, and no adverse findings or safety issues.

Claims

1. A hydrophobically modified biopolymer comprising hydrophobic grafts and hydroquinone groups covalently linked along the biopolymer backbone, optionally in their oxidized form.

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

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

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

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

6. The hydrophobically modified biopolymer according to 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 modified with hydrocarbon chains independently selected from C6 to C18.

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

8. The hydrophobically modified biopolymer according to any one of claims 5 to 7, wherein 5% to about 50% of the chitosan monomers comprise acetyl groups, and optionally about 10% to about 40% of the hm-chitosan monomers comprise acetyl groups; optionally, wherein the modified chitosan comprises free amines on from about 40% to about 75% of the monomers.

9. The hydrophobically modified biopolymer according to any one of claims 1 to 8, wherein the hydroquinone groups comprise a catechol moiety and optionally its oxidized form.

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

11. The hydrophobically modified biopolymer according to any one of claims 1 to 10, wherein the hydroquinone groups and optionally their oxidized form are grafted onto the biopolymer at a density of from 0.1% to about 15% of the biopolymer monomers.

12. The hydrophobically modified biopolymer according to claim 11, wherein the hydrophobically modified biopolymer is hm-chitosan and the hydroquinone groups and optionally their oxidized form are grafted onto hm-chitosan oligosaccharides at a density of from about 0.1% to about 10% of the chitosan monomers, or from about 0.1% to about 8% of the chitosan monomers, or from about 0.1% to about 5% of the chitosan monomers, or from about 0.1% to about 2% of the chitosan monomers.

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

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

15. The hydrophobically modified biopolymer according to claim 14, wherein the hydrophobically modified biopolymer is in powder form.

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

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

20. The composition according to claim 19, wherein the pH of the composition is 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. The composition according to claim 20, wherein the composition is a hydrogel.

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

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

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

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

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

27. The composition according to claim 26, wherein the secondary polymer is gelatin.

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

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

30. The composition according to any one of claims 25 to 29, wherein the concentration of the secondary polymer is 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.

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

32. The composition according to claim 31, wherein the composition comprises EDTA as the anti - flocculant.

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

34. The composition according to any one of claims 31 to 33, wherein the anti - foaming agent is ethanol.

35. The composition according to claim 34, wherein the concentration of ethanol is 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.

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

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

38. The method according to claim 37, wherein the surgical bleeding is cavity bleeding or bleeding at a site at risk of pressure or compression - related injury.

39. The method according to claim 38, wherein the surgical bleeding is an injury caused by spinal surgery, neurosurgery, or vascular surgery.

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

41. The method according to any one of claims 37 to 39, wherein the bleeding is at least grade 3 bleeding.

42. The method according to any one of claims 37 to 39, wherein the bleeding is grade 4 bleeding.

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