Compositions for manufacturing biomaterials for repairing ion-sensitive tissues in the body and their uses

Abstract

This application relates to a composition for producing a biomaterial for the repair of ion-sensitive tissue in the body and its uses, and provides a composition for producing an ion-sensitive hydrogel composition containing a solution or a chitosan derivative, in which the degree of deacetylation, the degree of substitution of alkylcarboxyl groups, and the level of free amine groups are adjusted, and an ion-sensitive hydrogel composition using the composition.

Description

Technical Field

[0001] The present invention relates to a composition for producing a biomaterial for in vivo ion-sensitive tissue repair and use thereof. This application claims priority to Korean Patent Application No. 10-2023-0118550 filed on September 6, 2024, and the disclosure of the above patent application is incorporated herein by reference.

Background Art

[0002] Skin aging is a gradual phenomenon that occurs over time and is affected by lifestyle factors such as alcohol consumption, tobacco smoking, and ultraviolet exposure. Among these, facial skin aging is characterized by atrophy, sagging, and hypertrophy. Atrophy refers to a significant decrease in the thickness of skin tissue; sagging of subcutaneous tissue causes excess skin and under-eye edema, leading to sagging of the cheeks and eyelids. Furthermore, hypertrophy refers to excessive weight gain caused by swelling of the face and neck regions. These changes are typically associated with loss of elasticity and a rough skin surface condition.

[0003] A hydrogel is a substance with a polymer network structure that contains a large amount of water, and is formed from a single polymer or copolymer. Hydrogels become transparent while swelling upon absorption of water-containing hydrophilic components in an aqueous solution, and possess appropriate mechanical properties. The degree of swelling and the mechanical properties of the polymer mainly depend on the physical properties of the polymer network structure and the formulation method. Polymers are mainly used as materials that can produce such hydrogels, and these polymers are divided into natural polymers and synthetic polymers depending on the origin of the material. Hydrogels made from natural polymers are made from polymers derived from natural materials such as agar, chitosan, carrageenan, cellulose, and xanthan gum, and may give consumers a good impression of being naturally derived, but are characterized by weak mechanical properties. Since such natural polymers are mostly produced by physical gelation reactions, the hydrogel form is easily broken down under high temperature conditions, and its mechanical strength is also weak under low temperature conditions. Furthermore, even under room temperature conditions, the surface of the hydrogel can crack or be easily torn by a weak external force such as pressing with a finger, so considerable care is required during use and distribution. In contrast, hydrogels made from synthetic polymers possess strong mechanical properties due to cross-linking, which creates strong chemical covalent bonds between molecules. Unlike natural polymers, this results in relatively rare damage to the hydrogel's morphology in response to external stimuli such as temperature and external forces. However, synthetic polymer-based hydrogels require improvement in terms of biocompatibility and efficacy maintenance.

[0004] In particular, existing biomaterials for tissue repair, as injectable liquid formulations, have the problem of dissolving under acidic conditions, inducing pain during skin injection, and having reduced stability when mixed with other materials or components under neutral conditions, sometimes resulting in the formation of precipitates within the material. Against this technological backdrop, there is a need to develop next-generation biomaterials for tissue repair that maximize the advantages of existing formulations using natural / synthetic polymers, complement their existing shortcomings, and achieve the ultimate goal of replacing the body's own tissue (Korean Registered Patent No. 10-0506543). [Overview of the project] [Problems that the invention aims to solve]

[0005] One embodiment provides a composition for producing an ion-sensitive hydrogel composition for the body, comprising a solution containing a chitosan derivative in which the degree of deacetylation, the degree of substitution of alkylcarboxyl groups, and the level of free amine groups are adjusted.

[0006] Another embodiment provides a method for producing an ion-sensitive hydrogel composition in the body, comprising the step of mixing a solution containing a chitosan derivative in which the degree of deacetylation, the degree of substitution of alkylcarboxyl groups, and the level of free amine groups are controlled, with an aqueous solution containing phosphate ions.

[0007] Another embodiment is to provide an ion-sensitive hydrogel composition produced by the method described above.

[0008] Another aspect is to provide a treatment method using the aforementioned ion-sensitive hydrogel or biomaterial for tissue repair. [Means for solving the problem]

[0009] One embodiment provides a composition for producing an ion-sensitive hydrogel composition for the body, comprising a solution containing a chitosan derivative that satisfies the following conditions. (a) Having a degree of deacetylation of 70% or more (b) At least 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups. (c) At least 22% of the total amine groups in the chitosan derivative are free amino groups (-NH2).

[0010] As used herein, the term "hydrogel" refers to a three-dimensional network structure created by crosslinking hydrophilic polymers with covalent or non-covalent bonds. Due to the hydrophilicity of the constituent materials, it absorbs a large amount of water and swells in aqueous solutions and aqueous environments, but its crosslinked structure prevents it from dissolving. Therefore, depending on the constituent components and manufacturing method, hydrogels with various forms and properties can be produced, and since they generally contain a large amount of water, they have properties intermediate between liquids and solids.

[0011] For example, the term "hydrogel" may be used interchangeably with the terms "biomaterial for tissue repair" or "biomaterial composition for tissue repair." In one embodiment, the hydrogel is both a biomaterial for tissue repair and a substance used for tissue repair. For example, it refers to fillers similar to soft tissue injected into wrinkled skin or areas requiring volume, substances to prevent adhesion between surgical sites and normal tissue, tissue adhesives, wound dressings for artificial skin, etc. The hydrogel may be applied to body parts such as the glabella, forehead, under-eye bags, wrinkles around the eyes, nasolabial folds, cheeks, wrinkles around the mouth, and chin. The hydrogel is a substance applied directly to the human body and must be biocompatible. For example, when the hydrogel is used to fill areas requiring volume, it needs to have excellent retention / sustainability of its gel shape so that it can form a long-lasting sense of volume after injection. When the hydrogel is used to prevent adhesion to surgical sites, it must be tissue-compatible at the wound site and have low or no cytotoxicity.

[0012] As used herein, the term "tissue repair" refers to the restoration of the structure and function of damaged or aged tissue, and may include, but is not limited to, applications such as cosmetic fillers, anti-adhesion materials, adhesives, wound dressings, and prosthetics.

[0013] As used herein, the term "ion-sensitive" refers to the physical property of a formulation that changes in response to its surrounding ionic environment. Specifically, it refers to the property that a formulation exists as a liquid, i.e., a sol, under room temperature conditions, such as external conditions or exposure to ambient temperature, but transforms into a gel under internal conditions.

[0014] As used herein, the term "chitosan" can mean a linear polysaccharide consisting of D-glucosamine and N-acetylglucosamine. The chitosan may include a substance obtained by deacetylating chitin using an alkali or the like. More specifically, the chitosan refers to a high-molecular-weight substance in which the proportion of D-glucosamine, produced by the separation of an acetyl group from N-acetyl-D-glucosamine, is 70% or more, and can be represented by the following formula 1. [ka]

[0015] As used herein, the term "chitosan derivative" refers to a substance derived from the aforementioned chitosan, and is a structure that has undergone organic chemical modification, including the introduction of specific functional groups, oxidation, reduction, and atomic substitution. In this industry, existing chitosan derivatives have limitations as biomaterials because, as weak polyanionic polyelectrolytes, the amino groups in the chitosan derivative do not become positive under neutral conditions, and most carboxyl groups do not dissolve. Therefore, there is a need to develop new technologies to improve the biomaterial applicability of chitosan derivatives. The chitosan derivative is, for example, carboxyalkyl chitosan, and specifically, it can consist of a combination of multiple units represented by the following formula 2. [ka]

[0016] In Formula 2, R1 may be hydrogen, an alkylcarboxyl group, or an acetyl group, and R2 and R3 may independently be hydrogen or an alkylcarboxyl group. Here, the alkylcarboxyl group may be, for example, a methylcarboxyl group or an ethylcarboxyl group, and more specifically, a methylcarboxyl group.

[0017] In one specific example, the average molecular weight of the chitosan derivative may be 50,000 to 3,000,000 Da. The average molecular weight can be appropriately changed to a range known in the art, starting from a plurality of units or combinations thereof represented by Formula 1 or Formula 2 described above.

[0018] In one specific example, the chitosan derivative may exhibit high solubility and stability under neutral conditions, and may also have the property of gelling under in vivo conditions to form a matrix with various viscoelastic properties. For this purpose, the chitosan derivative may have a degree of deacetylation of 70% or more, a degree of substitution of O-alkylcarboxyl groups or N-alkylcarboxyl groups of 50% or more, and a level of free amine groups of 22% or more. Furthermore, the pH of the solution or in vivo ion-sensitive hydrogel composition containing the chitosan derivative according to one example is pH 6.5 to pH 7.5, for example, pH 6.5 to pH 7.5, pH 6.8 to pH 7.5, pH 7.1 to pH 7.5, pH 7.4 to pH 7.5, pH 6.5 to pH 7.2, pH 6.8 to pH 7.2, pH 7.1 to pH 7.2, pH 6.5 to pH 6.9, pH 6.8 to pH 6.9, or pH 6.5 to pH 6.6. The degree of deacetylation of the aforementioned chitosan derivative, the degree of substitution of the above O-alkylcarboxyl groups or N-alkylcarboxyl groups, and the level of free amine groups are parameters that can be adjusted according to known art in the art, and may include, for example, a series of steps of reacting a chitosan powder having a specific level of deacetylation with an appropriate amount of monochloroacetic acid, but is not limited thereto.

[0019] For example, the degree of deacetylation, the degree of substitution of O-alkylcarboxyl groups or N-alkylcarboxyl groups, etc., can be adjusted by the following methods. To obtain chitosan derivatives with different levels of deacetylation, 10 g of chitin can be reacted with 100 mL of 50% NaOH solution at 100°C for 30 to 120 minutes, 30 to 90 minutes, 30 to 60 minutes, 60 to 150 minutes, 60 to 140 minutes, 60 to 130 minutes, 60 to 120 minutes, 60 to 110 minutes, 60 to 100 minutes, 60 to 90 minutes, or 60 to 80 minutes, respectively. In addition, to obtain chitosan derivatives with different levels of alkylcarboxyl groups or free amine groups, the chitosan derivative powder with the adjusted level of deacetylation described above can be added to 20% NaOH at 5 w / v%, and then reacted with 1.0 to 1.5 w / v% monochloroacetic acid.

[0020] In one specific example, the chitosan derivative may have a degree of deacetylation of 70% to 90%. The degree of deacetylation indicates the level at which the acetyl group has been removed from chitin, which is a precursor of chitosan, and may indicate the percentage level of a unit in formula 2 where R1 is hydrogen or an alkylcarboxyl group. The degree of deacetylation of the chitosan derivative may be, for example, 70% to 90%, 75% to 90%, 80% to 90%, 85% to 90%, 70% to 86%, 75% to 86%, 80% to 86%, 85% to 86%, 70% to 82%, 75% to 82%, 80% to 82%, 70% to 78%, 75% to 78%, or 70% to 74%.

[0021] In one specific example, the chitosan derivative may have a degree of substitution of 50% to 90% O-alkylcarboxyl groups or N-alkylcarboxyl groups. The degree of substitution of O-alkylcarboxyl groups or N-alkylcarboxyl groups indicates the level at which hydroxyl groups and amine groups in the chitosan derivative are substituted with -O-alkylcarboxyl groups or -N-alkylcarboxyl groups, and in formula 2, it indicates the percentage level of substituents where R2 and R3 are alkylcarboxyl groups. Here, the percentage level is a concept distinct from the conventional degree of substitution, which is calculated based on the number of carboxymethyl groups per 100 anhydroglucosamine units, i.e., evaluated based on whether or not carboxyl groups are substituted. The degree of O-alkylcarboxyl group or N-alkylcarboxyl group substitution of the chitosan derivative may be, for example, 50% to 90%, 55% to 90%, 60% to 90%, 65% to 90%, 70% to 90%, 75% to 90%, 80% to 90%, 85% to 90%, 50% to 80%, 55% to 80%, 60% to 80%, 65% to 80%, 70% to 80%, 75% to 80%, 50% to 70%, 55% to 70%, 60% to 70%, 65% to 70%, 50% to 60%, or 55% to 60%. In this case, if the degree of deacetylation and the degree of O-alkylcarboxyl group or N-alkylcarboxyl group substitution of the chitosan derivative fall outside the above range, there is a problem that the stability rapidly decreases under neutral conditions, and the number of insoluble particles that do not dissolve in solution increases.

[0022] In one specific embodiment, the chitosan derivative may have a level of free amine groups of 22% to 90%. The level of said free amine groups indicates the level of free amine groups (-NH2) among the amine groups in the chitosan derivative, and in Formula 2 above, may indicate the percentage level of substituents in which R1 is hydrogen. The level of free amine groups of the chitosan derivative may be 22% to 90%, 25% to 90%, 30% to 90%, 35% to 90%, 40% to 90%, 45% to 90%, 50% to 90%, 55% to 90%, 60% to 90%, 70% to 90%, 75% to 90%, 80% to 90%, 85% to 90%, 22% to 80%, 25% to 80%, 30% to 80%, 35% to 80%, 40% to 80%, 45% to 80%, 50% to 80%, 55% to 80%, 60% to 80%, 65% to 80%, 70% to 80%, 75% to 80%, 22% to 70%, 25% to 70%, 30% to 70%, 35% to 70%, 40% to 70%, 45% to 70%, 50% to 70%, 55% to 70%, 60% to 70%, 65% to 70%, 22% to 60%, 25% to 60%, 30% to 60%, 35% to 60%, 40% to 60%, 45% to 60%, 50% to 60%, or 55% to 60%. In this case, when the level of free amine groups of the chitosan derivative is outside the above range, there are problems that gelation does not occur under in vivo conditions, or the strength level of the formed hydrogel is extremely weak.

[0023] In one specific embodiment, the chitosan derivative has a degree of deacetylation of 70% to 90%, a degree of O-alkyl carboxyl group or N-alkyl carboxyl group substitution of 50% to 90%, and a level of free amine groups of 22% to 90%. For example, the chitosan derivative may have a degree of deacetylation of 70% to 90%, a degree of O-alkyl carboxyl group or N-alkyl carboxyl group substitution of 50% to 70%, and a level of free amine groups of 24% to 90%.

[0024] According to one example, it was confirmed that the properties of the in vivo ion-sensitive hydrogel composition can be controlled depending on the structure of the chitosan derivative. Specifically, the degree of deacetylation of the chitosan derivative and the level of terminal carboxyl groups (i.e., the degree of substitution of O-alkyl carboxyl groups or N-alkyl carboxyl groups) affect the solubility and stability of the chitosan derivative under neutral conditions, and the level of free amine groups of the chitosan derivative can affect gelation under in vivo conditions and the level of viscoelasticity of the formed matrix. Therefore, in the present example, it was confirmed that by adjusting the degree of deacetylation, the level of terminal carboxyl groups, and the level of free amine groups of the chitosan derivative, functionality / effect as a biomaterial for tissue repair can be achieved, and the present invention was completed based on this.

[0025] The chitosan derivative is contained at a concentration of 1 w / v% to 5 w / v% based on the total volume of the chitosan derivative solution, and the content of the chitosan derivative, based on the total volume of the chitosan derivative solution, is 1 w / v% to 5 w / v%, 1.5 w / v% to 5 w / v%, 2 w / v% to 5 w / v%, 2.5 w / v% to 5 w / v%, 3 w / v% to 5 w / v%, 3.5 w / v% to 5 w / v%, 4 w / v% to 5 w / v%, 4.5 w / v% to 5 w / v%, 1 w / v% to 4 w / v%, 1.5 w / v% to 4 w / v%, 2 w / v% to 4 w / v%, 2.5 w / v% to 4 w / v%, 3 w / v% to 4 w / v%, 3.5 w / v% to 4 w / v%, 1 w / v% to 3 w / v%, 1.5 w / v% to 3 w / v%, 2 w / v% to 3 w / v%, or 2.5 w / v% to 3 w / v%.

[0026] When the content of the chitosan derivative is less than the above range, there are problems that gelation does not occur, or the strength level of the formed hydrogel is very low, and the retention ability is insufficient; when the content of the chitosan derivative is more than the above range, there is a problem that insoluble particles that do not dissolve in the solution increase.

[0027] In one specific example, the composition for producing the bio-sensitive hydrogel composition may further include an aqueous solution containing phosphate ions.

[0028] As used herein, the term "phosphate ion" refers to a component that binds to the amine group of a chitosan derivative to form a hydrogel under biological conditions, or contributes to strengthening the strength of the formed hydrogel, and may, for example, be provided in the form of an aqueous solution containing phosphate ions. The aqueous solutions containing the phosphate ions include, for example, disodium hydrogen phosphate (Na2HPO4), monobasic sodium hydrogen phosphate (NaH2PO4), dibasic ammonium hydrogen phosphate ((NH4)2HPO4), ammonium dihydrogen phosphate (NH4H2PO4), tribasic sodium phosphate (Na3PO4), dibasic potassium phosphate (K2HPO4), monobasic potassium phosphate (KH2PO4), dimethyl phosphate (C2H7PO4), monomagnesium phosphate (Mg(H2PO4)2), dibasic magnesium phosphate (MgHPO4), lithium dihydrogen phosphate (LiH2PO4), and lithium phosphate (Lithium This may include phosphate (LiPO4), calcium hydrogen orthophosphate hydrate (CaHPO4·2H2O), and calcium hydrogen orthophosphate (CaHPO4), but it can be applied non-limitingly to any material that can provide a phosphate group or phosphate that can bond to the amine group of the chitosan derivative.

[0029] The composition comprises an aqueous solution containing 7 to 15% by weight of phosphate ions based on the total weight of the composition. For example, the content of the aqueous solution containing phosphate ions may be 7 to 9% by weight, 7 to 11% by weight, 7 to 13% by weight, 9 to 11% by weight, 9 to 13% by weight, 9 to 15% by weight, 11 to 13% by weight, 11 to 15% by weight, or 13 to 15% by weight.

[0030] In one specific example, the composition for producing the bio-sensitive hydrogel composition may further include a liquid formulation containing glycerol.

[0031] In one specific example, the liquid formulation is also isolated in a separate space within the container from the solution containing the aforementioned chitosan derivative and / or aqueous solution containing phosphate ions. Therefore, under pre-use conditions, the solution containing chitosan and phosphate ions and the liquid formulation containing glycerol are maintained and stored in liquid formulations, and can also be stored frozen for long-term storage as needed.

[0032] The liquid formulation containing glycerol is also used to adjust the level of covalent bonding within the mixture by mixing it with a solution containing the chitosan derivative and an aqueous solution containing phosphate ions, thereby imparting viscoelastic properties to the biomaterial for tissue repair.

[0033] The glycerol is contained at a concentration of 0.01 v / v% to 1 v / v% based on the total volume of the liquid formulation or composition, and the glycerol content may be 0.01 v / v% to 1 v / v%, 0.05 v / v% to 1 v / v%, 0.1 v / v% to 1 v / v%, 0.5 v / v% to 1 v / v%, 0.01 v / v% to 0.5 v / v%, 0.05 v / v% to 0.5 v / v%, 0.1 v / v% to 0.5 v / v%, 0.01 v / v% to 0.1 v / v%, 0.05 v / v% to 0.1 v / v%, or 0.01 v / v% to 0.05 v / v% based on the total volume of the liquid formulation or composition.

[0034] Another embodiment provides a method for producing an ion-sensitive hydrogel composition, comprising the step of mixing a solution containing a chitosan derivative satisfying the following conditions with an aqueous solution containing phosphate ions. (a) Having a degree of deacetylation of 70% or more (b) At least 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups. (c) At least 22% of the total amine groups in the chitosan derivative are free amino groups (-NH2).

[0035] Another embodiment is to provide an ion-sensitive hydrogel composition produced by the method described above.

[0036] Another embodiment provides a method for administering an ion-sensitive hydrogel composition in the body, comprising the steps of mixing a solution containing the aforementioned chitosan derivative with an aqueous solution containing phosphate ions, and injecting the mixed liquid formulation composition into the skin of a solid.

[0037] The method for producing the aforementioned ion-sensitive hydrogel composition, the ion-sensitive hydrogel composition produced by the aforementioned method, or the method for administering the ion-sensitive hydrogel composition either includes the composition for producing the aforementioned ion-sensitive hydrogel composition as is, or uses it, therefore, descriptions of content common to both are omitted.

[0038] According to one embodiment, the bio-sensitive hydrogel composition or biomaterial for tissue repair produced by the above method differs from existing biomaterials for tissue repair that are dissolved and used under acidic conditions. It maintains high solubility in chitosan derivatives and material stability under neutral conditions, thus reducing pain during the in vivo administration process and facilitating mixing with other materials or components. Furthermore, the bio-sensitive hydrogel composition or biomaterial for tissue repair possesses both the characteristics of a liquid formulation that can be injected at room temperature and the ability to gel under in vivo conditions, forming a viscoelastic matrix that conforms to the morphology and characteristics of various tissues. Therefore, it can be widely utilized in various fields. [Effects of the Invention]

[0039] A composition according to one embodiment comprises a solution containing a chitosan derivative in which the degree of deacetylation, the degree of substitution of alkylcarboxyl groups, and the level of free amine groups are controlled, thereby possessing high solubility and stability under neutral conditions, unlike existing biomaterials for tissue repair.

[0040] The composition according to one embodiment not only has stability as a material, but can also gel under internal body conditions to form a matrix with various viscoelastic properties, thus providing a biomaterial for tissue repair that matches the morphology and characteristics of various tissues. [Brief explanation of the drawing]

[0041] [Figure 1] This is the result of confirming the substitution of methylcarboxyl groups in a chitosan derivative in a solution containing a chitosan derivative according to one example, using FT-IR spectroscopy. [Figure 2]In one example, the solubility of a chitosan derivative in a neutral solvent was confirmed depending on the degree of substitution of the methylcarboxyl group in a solution containing a chitosan derivative with a degree of deacetylation of 50%. Figure 2A shows the result of visual inspection of a chitosan derivative solution with a degree of substitution of methylcarboxyl group of 0%, Figure 2B shows the result of visual inspection of a chitosan derivative solution with a degree of substitution of methylcarboxyl group of 50%, and Figure 2C shows the result of visual inspection of a chitosan derivative solution with a degree of substitution of methylcarboxyl group of 70%. [Figure 3] In one example, the solubility of a chitosan derivative in a neutral solvent was confirmed depending on the degree of substitution of the methylcarboxyl group in a solution containing a chitosan derivative with a degree of deacetylation of 70%. Figure 3A shows the result of visually inspecting a chitosan derivative solution with a degree of substitution of methylcarboxyl group of 0%, Figure 3B shows the result of visually inspecting a chitosan derivative solution with a degree of substitution of methylcarboxyl group of 50%, and Figure 3C shows the result of visually inspecting a chitosan derivative solution with a degree of substitution of methylcarboxyl group of 70%. [Figure 4] In one example, the change in the solubility of a chitosan derivative in a neutral solvent depending on the degree of substitution of the methylcarboxyl group was confirmed in a solution containing a chitosan derivative with a degree of deacetylation of 90%. Figure 4A shows the result of visually inspecting a chitosan derivative solution with a degree of substitution of the methylcarboxyl group of 0%, Figure 4B shows the result of visually inspecting a chitosan derivative solution with a degree of substitution of the methylcarboxyl group of 50%, and Figure 4C shows the result of visually inspecting a chitosan derivative solution with a degree of substitution of the methylcarboxyl group of 70%. [Figure 5] In a solution containing a chitosan derivative having a degree of deacetylation of 90% and a degree of substitution of methylcarboxyl groups of 70% according to one example, the change in the solubility of the chitosan derivative due to a change in the pH of the solution was confirmed. Figure 5A shows the result of visually inspecting the chitosan derivative solution at pH 6.8, Figure 5B shows the result of visually inspecting the chitosan derivative solution at pH 6.4, and Figure 5C shows the result of visually inspecting the chitosan derivative solution at pH 6.1. [Figure 6]This is the result of visually confirming the properties of an injectable liquid formulation of an ion-sensitive hydrogel composition according to one embodiment. [Figure 7] This is a result of confirming, using an animal model, whether or not gelation occurred in the body of an ion-sensitive hydrogel composition containing a chitosan derivative with a free amine group level of 21% according to one example. [Figure 8] The results of confirming whether or not gelation occurred in the body of an ion-sensitive hydrogel composition containing a chitosan derivative having 22% or more free amine groups according to one example, were confirmed using an animal model. Figure 8A shows the results of confirming whether or not gelation occurred in an ion-sensitive hydrogel composition containing a chitosan derivative with a free amine group level of 34%, and Figure 8B shows the results of confirming whether or not gelation occurred in an ion-sensitive hydrogel composition containing a chitosan derivative with a free amine group level of 90%. [Modes for carrying out the invention]

[0042] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided to make the present invention easier to understand, and the scope of the present invention is not limited by these embodiments.

[0043] [Examples]

[0044] Example 1. Evaluation of the solubility level of chitosan derivative solution under neutral conditions.

[0045] Existing biomaterials for tissue repair containing chitosan exhibit the property of dissolving in acidic solutions, which can induce pain during the administration process. Furthermore, chitosan can precipitate under neutral conditions, and the stability of the material can rapidly decrease when mixed with other materials or components. Therefore, in this example, we aimed to produce a chitosan derivative solution that has high solubility under neutral conditions.

[0046] 1-1. Preparation of Chitosan Derivative Solution for Manufacturing In vivo Ion-Sensitive Hydrogel Compositions

[0047] In this example, solutions containing chitosan derivatives with different degrees of deacetylation (%) and methylcarboxyl group substitution (%) were prepared as described below. As shown in Table 1 below, the chitosan derivatives were first classified into three priority groups based on their degree of deacetylation (50%, 70%, 90%). These groups were then reclassified into three experimental groups based on the degree of methylcarboxyl group substitution within the chitosan derivative, resulting in a total of nine experimental groups. Here, the degree of deacetylation (%) indicates the level at which the acetyl group has been removed from chitin, the precursor of the chitosan derivative, and the degree of methylcarboxyl group substitution (%) indicates the level at which the hydroxyl group and amine group within the chitosan derivative have been substituted with either an -O-methylcarboxyl group or an -N-methylcarboxyl group. The solvent used in the chitosan derivative solutions was PBS (pH 7.4), a neutral solvent.

[0048] [Table 1] (1) Manufacturing of experimental group G1-1 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 30 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C to obtain chitosan powder with a final deacetylation degree of 50%.

[0049] (2) Manufacturing of experimental group G1-2 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 30 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C. Subsequently, the chitosan powder having a degree of 50% deacetylation was added to 20% NaOH at a rate of 5 w / v%, followed by the addition of monochloroacetic acid (1.0 w / v%), and the mixture was reacted at 40°C. After neutralization with 10% acetic acid, the mixture was purified with methanol to obtain a chitosan derivative having a degree of 50% deacetylation and a degree of 50% methylcarboxy substitution.

[0050] (3) Manufacturing of experimental group G1-3 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 30 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C. Subsequently, the chitosan powder having a degree of 50% deacetylation was added to 20% NaOH at a rate of 5 w / v%, followed by the addition of monochloroacetic acid (1.5 w / v%), and the mixture was reacted at 40°C. After neutralization with 10% acetic acid, the mixture was purified with methanol to obtain a chitosan derivative having a degree of 50% deacetylation and a degree of 70% methylcarboxy substitution.

[0051] (4) Manufacturing of the G2-1 experimental group 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 60 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C to obtain a chitosan powder with a final deacetylation degree of 70%.

[0052] (5) Manufacturing of the G2-2 experimental group 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 60 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C. Subsequently, the chitosan powder having a degree of 70% deacetylation was added to 20% NaOH at a rate of 5 w / v%, followed by the addition of monochloroacetic acid (1.0 w / v%), and the mixture was reacted at 40°C. After neutralization with 10% acetic acid, the mixture was purified with methanol to obtain a chitosan derivative having a degree of 70% deacetylation and a degree of 50% methylcarboxy substitution.

[0053] (6) Manufacturing of experimental group G2-3 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 60 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C. Subsequently, the chitosan powder having a degree of 70% deacetylation was added to 20% NaOH at a rate of 5 w / v%, followed by the addition of monochloroacetic acid (1.5 w / v%), and the mixture was reacted at 40°C. After neutralization with 10% acetic acid, the mixture was purified with methanol to obtain a chitosan derivative having a degree of 70% deacetylation and a degree of 70% methylcarboxy substitution.

[0054] (7) Manufacturing of the G3-1 experimental group 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 150 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C to obtain chitosan powder with a final deacetylation degree of 90%.

[0055] (8) Manufacturing of the G3-2 experimental group 10 g of chitin was reacted with 100 mL of a 50% NaOH solution at 100°C for 150 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C. Subsequently, the chitosan powder having a degree of 90% deacetylation was added to 20% NaOH at a rate of 5 w / v%, followed by the addition of monochloroacetic acid (1.0 w / v%), and the mixture was reacted at 40°C. After neutralization with 10% acetic acid, the mixture was purified with methanol to obtain a chitosan derivative having a degree of 90% deacetylation and a degree of 50% methylcarboxy substitution.

[0056] (9) Manufacturing of the G3-3 experimental group 10 g of chitin was reacted with 100 mL of 50% NaOH solution at 100°C for 150 minutes. Immediately after the reaction was complete, water was added and the mixture was washed. The deacetylated chitosan was finally dried at 80°C. Subsequently, the chitosan powder having a degree of 90% deacetylation was added to 20% NaOH at a rate of 5 w / v%, followed by the addition of monochloroacetic acid (1.5 w / v%), and the mixture was reacted at 40°C. After neutralization with 10% acetic acid, the mixture was purified with methanol to obtain a chitosan derivative having a degree of 90% deacetylation and a degree of 70% methylcarboxy substitution.

[0057] Subsequently, for the chitosan derivatives obtained as described above, which had different degrees of deacetylation and methylcarboxy substitution, whether or not the methylcarboxy was substituted in the chitosan derivative, and the level of substitution, was confirmed by measuring the intensity of the band using FT-IR spectroscopy (see Figure 1) and by measuring the free amine content using a ninhydrin assay.

[0058] 1-2. Evaluation of the solubility level of chitosan derivatives under neutral solvent conditions In this example, we aimed to confirm the effect of the degree of deacetylation and / or substitution of methylcarboxyl groups of a chitosan derivative on the solubility level of the chitosan derivative in a neutral solvent by evaluating the solubility level of the chitosan derivative solution of Example 1-1. To this end, the solubility levels of the chitosan derivative solutions of Example 1-1, each having a different degree of deacetylation and substitution of methylcarboxyl groups, were visually evaluated under room temperature conditions.

[0059] As a result, as shown in Figure 2, all of the G1 group, which contained chitosan derivatives with a degree of deacetylation of 50%, formed precipitates in neutral solvents or were observed as turbid due to the low solubility of the chitosan derivatives. On the other hand, as shown in Figures 3 and 4, the G2 and G3 groups, which contained chitosan derivatives with a degree of deacetylation of 70% or 90%, showed increased solubility of the chitosan derivatives in neutral solvents and were observed as clear solutions. In particular, this effect was confirmed in the G2-2 and G3-2 groups, which had a degree of substitution of methylcarboxyl groups of 50% or more, and was observed more clearly as the degree of substitution of the methylcarboxyl groups increased.

[0060] Example 2. Evaluation of the solubility level of chitosan derivatives by pH change. In this example, we attempted to verify the differences between this chitosan derivative solution and existing biomaterials for tissue repair that dissolve under acidic conditions by evaluating the change in the solubility level of the chitosan derivative solution of Example 1-1 due to pH changes. To this end, a solution containing a chitosan derivative having a degree of deacetylation of 90% and a degree of methylcarboxyl group substitution of 70% was prepared at a concentration of 2 w / v% in the same manner as in Example 1. However, in the chitosan derivative solution, 0.01N HCl aqueous solution, 0.015N HCl aqueous solution, and 0.02N HCl aqueous solution were used as solvents, and the pH levels of the solutions were adjusted to 6.8, 6.4, and 6.1, respectively. Subsequently, in the same manner as in Example 1-2, the solubility levels of the chitosan derivative solutions with different pH levels were visually evaluated under room temperature conditions.

[0061] As a result, as shown in Figure 5, under neutral conditions, the solution was observed to be clear due to the high solubility level of the chitosan derivative. On the other hand, under weakly acidic conditions of pH 6.5 or lower, a precipitate formed or the solution was observed to be turbid due to the low solubility level of the chitosan derivative.

[0062] In summary, the experimental results indicate that by adjusting the degree of deacetylation (70% or more) and the degree of substitution of methylcarboxyl groups (50% or more) of the chitosan derivative, it is possible to produce a chitosan derivative solution or a biomaterial containing it that has high solubility and stability under neutral conditions, unlike existing biomaterials for tissue repair.

[0063] Example 3. Evaluation of the level of ion sensitivity in the body based on the level of free amine groups. For use as a biomaterial for tissue repair, it must exist in a liquid form that can be injected under room temperature conditions, and after administration into the body, i.e., in vivo, it must gel to form an effective volume. Therefore, in this example, we aimed to confirm the effect of free amines in the chitosan derivative on the behavior of ion sensitivity in the body.

[0064] 3-1. Production of ion-sensitive hydrogel compositions for use in the body In this example, solutions containing chitosan derivatives with different levels (%) of free amine groups were prepared as described below.

[0065] [Table 2]

[0066] As shown in Table 2, the degree of deacetylation (%) and the degree of substitution of methylcarboxyl groups (%) are as shown in Table 1, and the percentage of free amine groups (%) indicates the level of free amine groups (-NH2) in the amine groups within the chitosan derivative. The solvent used in the chitosan derivative solution was a neutral solvent, disodium phosphate. In this example, the experimental groups (N1, N2, N3) were produced by performing N,O-random carboxymethyl substitution, and the level of free amino groups was adjusted by adjusting the degree of methylcarboxy substitution in the same manner as in Example 1-1.

[0067] Subsequently, the 2 w / v% chitosan derivative solution and 2 v / v% sodium hydrogen phosphate (Na2HPO4) were mixed to produce a liquid, bio-sensitive hydrogel composition that can be injected under room temperature conditions, as shown in Figure 6.

[0068] 3-2. Evaluation of ion sensitivity in the body using animal models In this example, we aimed to confirm the effect of the level of free amine groups in the chitosan derivative on the behavior of in vivo ion sensitivity by evaluating whether or not the in vivo ion-sensitive hydrogel composition of Example 3-2 gelled in an animal model. To this end, 0.5 ml of the in vivo ion-sensitive hydrogel composition of Example 3-1 was injected intradermally into hairless mice. One day after the injection of the hydrogel composition, we visually confirmed whether or not a matrix had formed due to the progression of gelation, and measured its storage modulus (G').

[0069] As a result, as shown in Figure 7, the N1 group, which contained a chitosan derivative with a free amine group level of 21%, was unable to form a matrix in the body. On the other hand, as shown in Figure 8, the N2 group, which contained a chitosan derivative with a free amine group level of 34%, formed a matrix with a storage modulus of 31.97 ± 1.58 Pa, similar to that of hyaluronic acid fillers with a low degree of cross-linking, and the N3 group, which contained a chitosan derivative with a free amine group level of 90%, formed a matrix with a storage modulus of 154.31 ± 13.21 Pa. By adjusting the modulus of the matrix using the method presented herein, in vivo ion-sensitive hydrogel compositions that form a matrix with a storage modulus of 5 to 50 Pa are applicable to medical materials such as intraarticular injections and skin boosters, and in vivo ion-sensitive hydrogel compositions that form a matrix with a storage modulus of 100 Pa or more are applicable to medical materials for subcutaneous repair.

[0070] In summary, the experimental results demonstrate that by adjusting the degree of deacetylation and substitution of methylcarboxyl groups in the chitosan derivative, as well as the level of free amine groups in the chitosan derivative, it is possible to not only overcome the problems of existing biomaterials for tissue repair, but also to produce a biomaterial that possesses all the necessary properties: the characteristics of a liquid formulation that can be injected at room temperature, and the ability to gel under in vivo conditions and form a viscoelastic matrix that matches the morphology and characteristics of various tissues.

[0071] The foregoing description of the present invention is illustrative, and a person with ordinary skill in the art will understand that the invention can be readily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects.

Claims

1. A composition for producing an ion-sensitive hydrogel composition for the body, comprising a solution containing a chitosan derivative that satisfies the following conditions: (a) Having a degree of deacetylation of 70% or more, (b) At least 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups, (c) More than 22% of the total amine groups in the chitosan derivative are free amino groups (free amine group, -NH ２ )

2. The composition according to claim 1, wherein the chitosan derivative has a degree of deacetylation of 70% to 90%.

3. The composition according to claim 1, wherein 50% to 90% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-methylcarboxyl groups or N-methylcarboxyl groups.

4. The composition according to claim 1, wherein 22% to 90% of the total amine groups in the chitosan derivative are free amine groups.

5. The composition according to claim 1, wherein the average molecular weight of the chitosan derivative is 50,000 to 3,000,000 Da.

6. The composition according to claim 1, wherein the solution has a pH of 6.5 to 7.

5.

7. The composition according to claim 1, wherein the chitosan derivative is contained at a concentration of 2 to 5 w / v% based on the total volume of the solution.

8. The composition according to claim 1, further comprising an aqueous solution containing phosphate ions.

9. The aqueous solution containing phosphate ions includes disodium hydrogen phosphate (Na ２ HPO ４ ), monosodium dihydrogen phosphate (monosodium hydrogen phosphate monobasic, NaH ２ PO ４ ), diammonium hydrogen phosphate (diammonium hydrogen phosphate, (NH ４ ) ２ HPO ４ ), ammonium dihydrogen phosphate (ammonium dihydrogen phosphate, NH ４ H ２ PO ４ ), trisodium phosphate (Sodium phosphate tribasic, Na ３ PO ４ ), dipotassium hydrogen phosphate (potassium phosphate dibasic, K ２ HPO ４ ), potassium dihydrogen phosphate (potassium phosphate monobasic, KH ２ PO ４ ), dimethyl phosphate (dimethyl phosphate, C ２ H ７ PO ４ ), monomagnesium phosphate (monomagnesium phosphate, Mg(H ２ PO ４ ) ２ ), magnesium hydrogen phosphate (magnesium phosphate dibasic, MgHPO ４ ), lithium dihydrogen phosphate (Lithium dihydrogen phosphate, LiH ２ PO ４ ), lithium phosphate (Lithium phosphate, LiPO ４ ), calcium hydrogen orthophosphate hydrate (calcium hydrogen orthophosphate hydrate, CaHPO ４ ·2H ２ O), Calcium hydrogen phosphate (CaHPO) ４ The composition according to claim 8, comprising at least one phosphate selected from the group consisting of ).

10. The composition according to claim 1, further comprising a liquid formulation containing glycerol.

11. The composition according to claim 1, wherein the composition is a transparent liquid formulation under neutral conditions and forms a gel formulation under in vivo conditions.

12. A method for producing an ion-sensitive hydrogel composition in the body, comprising the step of mixing a solution containing a chitosan derivative that satisfies the following conditions with an aqueous solution containing phosphate ions: (a) Having a degree of deacetylation of 70% or more, (b) At least 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups, (c) At least 22% of the total amine groups in the chitosan derivative are free amino groups (-NH2).

13. The method according to claim 12, wherein 50% to 90% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-methylcarboxyl groups or N-methylcarboxyl groups.

14. The method according to claim 12, wherein the solution containing the chitosan derivative has a pH of 6.5 to 7.

5.

15. The method according to claim 12, wherein the aqueous solution containing the phosphate ion comprises at least one phosphate selected from the group consisting of sodium dibase, sodium monobase, ammonium dibase, dihydrogen phosphate, trisodium phosphate, potassium dibase, potassium monobase, dimethyl phosphate, monomagnesium phosphate, magnesium dibase, lithium dihydrogen phosphate, lithium phosphate, calcium hydrogen phosphate hydrate, and calcium hydrogen phosphate.

16. The method according to claim 12, further comprising the step of adding and mixing a liquid formulation containing glycerol after the mixing step.

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