Composition for preparing biological material for in-vivo ion-sensitive tissue repair and application thereof

Abstract

The present application relates to a composition for preparing a biomaterial for repairing ion-sensitive tissue in vivo and a use thereof, and provides a solution comprising a chitosan derivative in which the degree of deacetylation, the degree of alkyl carboxyl substitution, and the free amine level are respectively adjusted, or a composition comprising a chitosan derivative in which the degree of deacetylation, the degree of alkyl carboxyl substitution, and the free amine level are respectively adjusted. The present invention relates to a chitosan derivative for preparing an in vivo ion-sensitive hydrogel composition comprising a solution comprising the chitosan derivative, and an in vivo ion-sensitive hydrogel composition in which the composition is used.

Description

Compositions for preparing biomaterials for in vivo ion-sensitive tissue repair and their applications Technical Field

[0001] This application relates to a composition for preparing biomaterials for in vivo ion-sensitive tissue repair and its use. This application claims priority to Korean Patent Application No. 10-2023-0118500, filed on September 6, 2024, the disclosure of which is incorporated herein by reference. Background Technology

[0002] Skin aging is a gradual process that occurs over time and is influenced by lifestyle factors such as alcohol consumption, smoking, and exposure to ultraviolet radiation. Facial skin aging is characterized by atrophy, sagging, and weight gain. Atrophy refers to a significant reduction in the thickness of skin tissue; subcutaneous tissue laxity leads to excess skin and drooping eyelids, resulting in sagging cheeks and eyelids. Weight gain refers to excessive weight gain due to puffiness in the face and neck. These changes are typically associated with loss of elasticity and a rough skin surface.

[0003] Hydrogels are substances containing a large amount of water and possessing a polymeric network structure, formed from homopolymers or copolymers. Hydrogels become transparent by absorbing hydrophilic components such as water into aqueous solutions, while also exhibiting moderate mechanical properties. This degree of swelling and the mechanical properties of the polymer primarily depend on the properties of the polymeric network structure and the formulation method. Polymers are commonly used as materials for preparing such hydrogels, and these polymers are classified as natural polymers and synthetic polymers based on their source. Hydrogels formed using natural polymers are characterized by their natural origin, which may give consumers a good impression, but their mechanical properties are relatively poor. These natural polymers are mostly formed through physical gelation reactions; therefore, the hydrogel form is prone to collapse at high temperatures and its mechanical strength decreases at low temperatures. Furthermore, even at room temperature, the appearance of the hydrogel can be easily broken or torn by a relatively weak external force equivalent to pressing with a finger; therefore, considerable care is required during use and distribution. In contrast, hydrogels formed using synthetic polymers exhibit strong inter-molecular bonding at the level of chemical covalent bonds through cross-linking, resulting in strong mechanical properties distinct from natural polymers. Consequently, hydrogels are less susceptible to damage from external stimuli such as temperature and force. However, improvements in biocompatibility and efficacy retention are still needed for hydrogels based on synthetic polymers.

[0004] In particular, existing tissue repair biomaterials, as injectable liquid formulations, suffer from drawbacks such as pain upon skin injection due to their solubility in acidic conditions, reduced stability when mixed with other materials or components under neutral conditions, and occasional precipitation. Against this technological backdrop, there is a need to develop a next-generation tissue repair biomaterial (Korean Patent Registration No. 10-0506543) that maximizes the advantages of existing natural / synthetic polymer formulations while overcoming the shortcomings of current technologies and ultimately achieving the goal of self-organization. Summary of the Invention

[0005] Technical issues

[0006] One aspect provides a composition for preparing an in vivo ion-sensitive hydrogel composition, comprising a solution containing a chitosan derivative, wherein the degree of deacetylation, degree of alkyl carboxyl substitution, and level of free amino groups of the chitosan derivative are adjusted respectively.

[0007] On the other hand, a method for preparing an in vivo ion-sensitive hydrogel composition is provided, comprising the step of mixing a solution containing a chitosan derivative and an aqueous solution containing phosphate ions, wherein the degree of deacetylation, the degree of alkyl carboxyl substitution, and the level of free amino groups of the chitosan derivative are adjusted respectively.

[0008] On the other hand, an in vivo ion-sensitive hydrogel composition prepared by the method is provided.

[0009] On the other hand, it provides a method of application using the aforementioned in vivo ion-sensitive hydrogel or tissue repair biomaterial.

[0010] Technical solution

[0011] One aspect provides a composition for preparing an in vivo ion-sensitive hydrogel composition, wherein the composition comprises a chitosan derivative that satisfies the following conditions: (a) having a degree of deacetylation of 70% or higher; (b) at least 50% of the hydroxyl and amino groups in the chitosan derivative are substituted with O-alkyl carboxyl groups or N-alkyl carboxyl groups; and (c) at least 22% of the entire amino group in the chitosan derivative is a free amine group (-NH2).

[0012] In this specification, the term "hydrogel" can refer to a three-dimensional network structure formed by hydrophilic polymers through covalent or non-covalent bonds. Due to the hydrophilicity of its constituent materials, it has the property of absorbing a large amount of water and swelling in aqueous solutions and aqueous environments, yet remaining insoluble due to its cross-linked structure. Therefore, hydrogels with various forms and properties can be formed depending on the constituent components and preparation methods, and, because they typically contain a large amount of water, may exhibit intermediate properties between liquids and solids.

[0013] As an example, the term "hydrogel" can be used interchangeably with the terms "biomaterial for tissue repair" or "biomaterial composition for tissue repair." In one embodiment, the hydrogel can be a biomaterial for tissue repair, i.e., a material used for tissue repair. For example, it can refer to a filler similar to soft tissue, injected into the skin where wrinkles are present or areas requiring volume, a substance used to prevent adhesion between the surgical site and normal tissue, a tissue adhesive, a wound dressing for artificial skin, etc. The hydrogel can be applied, for example, to body parts such as the glabella, forehead, under-eye area, crow's feet, nasolabial folds, cheeks, mouth wrinkles, and jawline. As a substance directly applicable to the human body, the hydrogel should be biocompatible. For example, when the hydrogel is used to fill areas requiring volume, it needs excellent shape retention / persistence to create a long-lasting sense of volume after injection. In cases where the hydrogel is used at surgical sites to prevent adhesion, it should have tissue compatibility at the wound site and low or no cytotoxicity.

[0014] In this specification, the term "for tissue repair" refers to restoring the structure and function of damaged or aged tissues, and may include, for example, applications in cosmetic fillers, adhesion prevention materials, adhesives, wound dressings, and cosmetic prostheses, but is not limited thereto.

[0015] In this specification, the term "ion sensitivity" refers to the physical property of a dosage form that changes according to the surrounding ionic environment. It can mean that it exists in a liquid dosage form, i.e., in sol form, under room temperature conditions, such as when exposed to in vitro conditions or in a normal temperature environment, but transforms into a gel form under in vivo conditions.

[0016] In this specification, the term "chitosan" can refer to a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine. The chitosan may include materials obtained by deacetylation of chitin using bases or the like. More specifically, the chitosan refers to a polymeric material in which the proportion of D-glucosamine derived from the deacetylation of N-acetyl-D-glucosamine is at least 70%, and can be represented by the following structural formula 1.

[0017] [Structure 1]

[0018] In this specification, the term "chitosan derivative" refers to a material derived from chitosan, which can refer to a structure in which organic chemical modifications are introduced, including the introduction, oxidation, reduction, and atomic substitution of specific functional groups. In the art, existing chitosan derivatives act as weak polyanionic polyelectrolytes; under neutral conditions, the amino groups within the chitosan derivative do not cationicize, and most of the carboxyl groups do not dissolve, thus limiting their use as biomaterials. Therefore, there is a need to develop a new technology to improve the practicality of chitosan derivatives as biomaterials. The chitosan derivative can be, for example, a carboxyalkyl chitosan, specifically, it can be composed of a combination of multiple monomers represented by the following structural formula 2.

[0019] [Structure 2]

[0020] In structural formula 2, R1 can be any one of hydrogen, alkyl carboxyl, and acetyl, and R2 and R3 can be hydrogen or alkyl carboxyl independently. Here, the alkyl carboxyl can be, for example, methyl carboxyl or ethyl carboxyl, and specifically methyl carboxyl.

[0021] In one embodiment, the average molecular weight of the chitosan derivative can be from 50,000 Da to 3,000,000 Da. The average molecular weight is derived from a plurality of monomers or combinations thereof represented by structural formula 1 or structural formula 2 described above, and can be suitably varied to conventional ranges known in the art.

[0022] In one embodiment, the chitosan derivative is characterized by high solubility and stability under neutral conditions while being able to gel in vivo to form a matrix with various viscoelastic properties. For this purpose, the chitosan derivative may have a degree of deacetylation of 70% or higher, a degree of O-alkyl carboxyl or N-alkyl carboxyl substitution of 50% or higher, and a level of free amines of 22% or higher. Furthermore, the pH of the solution or in vivo ion-sensitive hydrogel composition containing the chitosan derivative according to one embodiment may be from pH 6.5 to pH 7.5, and may be, 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, the degree of substitution of the above-mentioned O-alkyl carboxyl groups or N-alkyl carboxyl groups, and the level of free amino groups of the chitosan derivatives can be adjusted according to techniques known in the art. For example, it can include a series of processes that react chitosan powder with a specific degree of deacetylation with an appropriate amount of monochloroacetic acid, but is not limited thereto.

[0023] For example, the degree of deacetylation, the degree of substitution of O-alkyl carboxyl or N-alkyl carboxyl groups, etc., can be adjusted in the following way: In order to obtain chitosan derivatives with different degrees of deacetylation, 10g of chitin can be reacted in 100mL of 50% NaOH solution at 100℃ for 30 minutes to 150 minutes, for example, 30 minutes to 120 minutes, 30 minutes to 90 minutes, 30 minutes to 60 minutes, 60 minutes to 150 minutes, 60 minutes to 140 minutes, 60 minutes to 130 minutes, 60 minutes to 120 minutes, 60 minutes to 110 minutes, 60 minutes to 100 minutes, 60 minutes to 90 minutes, 60 minutes to 80 minutes, or 60 minutes to 80 minutes. Alternatively, to obtain chitosan derivatives with different levels of alkyl carboxyl groups or free amino groups, the chitosan derivative powder with the adjusted deacetylation level can be added to 20% NaOH to 5 w / v, followed by the addition of 1.0 to 1.5 w / v of monochloroacetic acid and allowed to react.

[0024] In one embodiment, the chitosan derivative may have a degree of deacetylation of 70% to 90%. The degree of deacetylation represents the level to which acetyl groups are removed from chitin, the chitosan precursor, and in structural formula 2, may represent the percentage level of monomers where R1 is hydrogen or an alkyl carboxyl 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%.

[0025] In one embodiment, the chitosan derivative may have a degree of O-alkylcarboxyl or N-alkylcarboxyl substitution of 50% to 90%. The degree of O-alkylcarboxyl or N-alkylcarboxyl substitution represents the level to which hydroxyl and amino groups within the chitosan derivative are substituted by -O-alkylcarboxyl or -N-alkylcarboxyl groups, and in structural formula 2, may represent the percentage level of substituents for alkylcarboxyl groups R2 and R3. Here, the percentage level is a concept distinct from existing substitution degrees, which are calculated as the number of carboxymethyl groups per 100 anhydrous glucosamine units and assessed based on whether carboxyl groups are substituted. The degree of O-alkyl carboxyl or N-alkyl carboxyl substitution of the chitosan derivative can 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-alkyl carboxyl or N-alkyl carboxyl substitution of the chitosan derivative exceed the aforementioned range, the stability decreases sharply under neutral conditions, and therefore, the number of undissolved insoluble particles in the solution increases.

[0026] In one embodiment, the chitosan derivative may have a free amino group level of 22% to 90%. The free amino group level represents the level of free amino groups (-NH2) in the amino groups of the chitosan derivative, or it may represent the percentage level of substituents where R1 is hydrogen in the structural formula 2. The free amino group level 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%, 65% 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% to 90%. The percentages are 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 these cases, if the free amino group level of the chitosan derivative exceeds the specified range, gelation will not occur under in vivo conditions, or the strength level of the formed hydrogel will be very low.

[0027] In one embodiment, the chitosan derivative may have a degree of deacetylation of 70% to 90%, a degree of O-alkyl carboxyl or N-alkyl carboxyl substitution of 50% to 90%, and a level of free amino groups of 22% to 90%. The chitosan derivative may, for example, have a degree of deacetylation of 70% to 90%, a degree of O-alkyl carboxyl or N-alkyl carboxyl substitution of 50% to 70%, and a level of free amino groups of 24% to 90%.

[0028] According to one embodiment, it has been confirmed that the characteristics of the in vivo ion-sensitive hydrogel composition can be adjusted according to the structure of the chitosan derivative. Specifically, the degree of deacetylation and the terminal carboxyl group level (i.e., the degree of substitution of O-alkyl carboxyl or N-alkyl carboxyl groups) of the chitosan derivative can affect the solubility and stability of the chitosan derivative under neutral conditions, and the free amino group level of the chitosan derivative can affect the viscoelasticity level of the gelled matrix under in vivo conditions. Therefore, in this embodiment, it has been confirmed that the functionality / effect as a biomaterial for tissue repair can be achieved by adjusting the degree of deacetylation, terminal carboxyl group level, and free amino group level of the chitosan derivative, and the present invention was completed based on this.

[0029] The chitosan derivative may be contained at a concentration of 1 w / v% to 5 w / v% based on the total volume of the chitosan derivative solvent, and the content of the chitosan derivative may be 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%, 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.

[0030] When the chitosan derivative content is below the specified range, gelation will not occur, or the resulting hydrogel will have very low strength and insufficient durability. When the chitosan derivative content is equal to or higher than the specified range, the number of undissolved insoluble particles in the solution will increase.

[0031] In one embodiment, the composition for preparing an in vivo ion-sensitive hydrogel composition may further comprise an aqueous solution containing phosphate ions.

[0032] The term "phosphate ion" in this specification refers to a component that binds to the amino groups of a chitosan derivative and forms a hydrogel under in vivo conditions or contributes to strengthening the strength of an already formed hydrogel, and may, as an example, be provided in the form of an aqueous solution containing phosphate ions. The aqueous solution containing phosphate ions may include, for example, disodium hydrogen phosphate (Na2HPO4), monosodium hydrogen phosphate (NaH2PO4), diammonium hydrogen phosphate ((NH4)2HPO4), ammonium dihydrogen phosphate (NH4H2PO4), trisodium phosphate (Na3PO4), dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), dimethyl phosphate (C2H7PO4), magnesium dihydrogen phosphate (Mg(H2PO4)2), magnesium hydrogen phosphate (MgHPO4), lithium dihydrogen phosphate (LiH2PO4), and lithium phosphate (LiH2PO4). It is composed of LiPO4, calcium hydrogen orthophosphate hydrate (CaHPO4·2H2O), and calcium hydrogen orthophosphate (CaHPO4). However, any substance that can provide a phosphate group or phosphate salt that can bind to the amino group of the chitosan derivative can be extended to other applications without limitation.

[0033] Based on the total weight of the composition, the composition may include an aqueous solution containing 7% to 15% phosphate ions, for example, the content of the aqueous solution containing the phosphate ions may be 7% to 9% wt, 7% to 11% wt, 7% to 13% wt, 9% to 11% wt, 9% to 13% wt, 9% to 15% wt, 11% to 13% wt, 11% to 15% wt, or 13% to 15% wt.

[0034] In one embodiment, the composition for preparing an in vivo ion-sensitive hydrogel composition may further include a liquid formulation containing glycerol.

[0035] In one embodiment, the liquid formulation can be isolated from the solution containing chitosan derivatives and / or the solution containing phosphate ions within the container's separation space. Therefore, under the conditions prior to use, the solution containing chitosan and phosphate ions and the liquid formulation containing glycerol can be maintained and preserved as liquid formulations, and, if necessary, can also be preserved in a frozen state for long-term storage.

[0036] The liquid formulation containing glycerol can be mixed with the solution containing chitosan derivatives and the aqueous solution containing phosphate ions to adjust the level of covalent bonds within the mixture, thereby imparting viscoelastic properties to the biomaterials used for tissue repair.

[0037] Based on the total volume of the liquid formulation or composition, the glycerol may be contained at a concentration of 0.01 v / v% to 1 v / v%, and based on the total volume of the liquid formulation or composition, the content of the glycerol 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.

[0038] On the other hand, a method for preparing an in vivo ion-sensitive hydrogel composition is provided, comprising the step of mixing a solution containing a chitosan derivative and an aqueous solution containing phosphate ions, wherein the chitosan derivative satisfies the following conditions: (a) having a degree of deacetylation of 70% or higher; (b) at least 50% of the hydroxyl and amino groups in the chitosan derivative are substituted with O-alkyl carboxyl groups or N-alkyl carboxyl groups; and (c) at least 22% of the entire amino group in the chitosan derivative is a free amine group (-NH2).

[0039] On the other hand, an in vivo ion-sensitive hydrogel composition prepared by the method is provided.

[0040] On the other hand, a method for applying an ion-sensitive hydrogel composition in vivo is provided, comprising: mixing the above-mentioned solution containing a chitosan derivative and an aqueous solution containing phosphate ions; and injecting the mixed liquid formulation composition into the individual's skin.

[0041] The method for preparing an in vivo ion-sensitive hydrogel composition, the in vivo ion-sensitive hydrogel composition obtained by the method, or the method for using an in vivo ion-sensitive hydrogel composition includes, as is the composition for preparing the above-mentioned in vivo ion-sensitive hydrogel composition or the use of the composition; therefore, descriptions of common content between the two will be omitted.

[0042] According to one aspect, unlike existing tissue repair biomaterials that are dissolved under acidic conditions, the in vivo ion-sensitive hydrogel composition or tissue repair biomaterial prepared by the aforementioned method maintains high solubility for chitosan derivatives and material stability under neutral conditions. This reduces pain during in vivo application and facilitates mixing with other materials or components. Furthermore, the in vivo ion-sensitive hydrogel composition or tissue repair biomaterial combines the characteristics of a liquid formulation that can be injected at room temperature with the ability to gel in vivo to form a viscoelastic matrix with various tissue shapes and properties, thus enabling its wide applicability in various fields.

[0043] Beneficial effects

[0044] According to one aspect, the composition includes a solution containing a chitosan derivative, wherein the degree of deacetylation, degree of alkyl carboxyl substitution, and level of free amino groups of the chitosan derivative are respectively adjusted, thereby exhibiting high solubility and stability under neutral conditions, unlike existing tissue repair biomaterials.

[0045] In addition to its stability as a material, the composition can also be gelled under in vivo conditions to form a matrix with various viscoelastic properties, thereby providing biomaterials for tissue repair that conform to the form and characteristics of various tissues. Attached Figure Description

[0046] Figure 1 shows the results of confirmation by Fourier transform infrared (FT-IR) spectrophotometry of the substitution of methyl carboxyl groups in a solution containing a chitosan derivative according to one embodiment.

[0047] Figure 2 shows the changes in solubility of chitosan derivatives in neutral solvents according to the degree of methyl carboxyl substitution in a solution containing a chitosan derivative with a degree of 50% deacetylation, according to one embodiment. Figure 2A shows the results of visually confirming a chitosan derivative solution with a degree of 0% methyl carboxyl substitution, Figure 2B shows the results of visually confirming a chitosan derivative solution with a degree of 50% methyl carboxyl substitution, and Figure 2C shows the results of visually confirming a chitosan derivative solution with a degree of 70% methyl carboxyl substitution.

[0048] Figure 3 shows the changes in solubility of chitosan derivatives with methyl carboxyl substitution in a neutral solvent in a solution containing a chitosan derivative with a 70% degree of deacetylation according to one embodiment. Figure 3A shows the results of visually confirming a chitosan derivative solution with a 0% degree of methyl carboxyl substitution, Figure 3B shows the results of visually confirming a chitosan derivative solution with a 50% degree of methyl carboxyl substitution, and Figure 3C shows the results of visually confirming a chitosan derivative solution with a 70% degree of methyl carboxyl substitution.

[0049] Figure 4 shows the changes in solubility of chitosan derivatives in neutral solvents according to the degree of methyl carboxyl substitution in a solution containing a chitosan derivative with a degree of 90% deacetylation, according to one embodiment. Figure 4A shows the results of a chitosan derivative solution with a degree of 0% methyl carboxyl substitution as confirmed by the naked eye, Figure 4B shows the results of a chitosan derivative solution with a degree of 50% methyl carboxyl substitution as confirmed by the naked eye, and Figure 4C shows the results of a chitosan derivative solution with a degree of 70% methyl carboxyl substitution as confirmed by the naked eye.

[0050] Figure 5 shows the change in solubility of chitosan derivatives in a solution containing a chitosan derivative having a degree of deacetylation of 90% and a degree of methyl carboxyl substitution of 70% according to one embodiment, depending on the pH of the solution. Figure 5A shows the results of visual confirmation of the chitosan derivative solution at pH 6.8, Figure 5B shows the results of visual confirmation of the chitosan derivative solution at pH 6.4, and Figure 5C shows the results of visual confirmation of the chitosan derivative solution at pH 6.1.

[0051] Figure 6 shows the results of visual confirmation of the properties of the in vivo ion-sensitive hydrogel composition according to one embodiment as an injectable liquid formulation.

[0052] Figure 7 shows the results of using an animal model to confirm whether an in vivo ion-sensitive hydrogel composition comprising a chitosan derivative with a free amine level of 21% gels in vivo according to one embodiment.

[0053] Figure 8 illustrates the use of animal models to confirm whether an in vivo ion-sensitive hydrogel composition comprising a chitosan derivative having a free amino group level of 22% or higher gels in one embodiment gels in vivo. Figure 8A shows the results of confirming whether an in vivo ion-sensitive hydrogel composition comprising a chitosan derivative having a free amino group level of 34% gels in vivo, and Figure 8B shows the results of confirming whether an in vivo ion-sensitive hydrogel composition comprising a chitosan derivative having a free amino group level of 90% gels in vivo. Detailed Implementation

[0054] The following preferred embodiments are provided to aid in understanding the present invention. However, the purpose of providing the following embodiments is merely to make the present invention clearer and more understandable, and the present invention is not limited to the following embodiments.

[0055] [Example]

[0056] Example 1: Evaluation of the solubility of chitosan derivative solutions under neutral conditions

[0057] Existing tissue repair biomaterials, including chitosan, exhibit solubility in acidic solutions, leading to pain during in vivo application. Furthermore, the material stability decreases sharply when chitosan precipitates under neutral conditions or is mixed with other materials or components. Therefore, this embodiment aims to prepare a chitosan derivative solution with high solubility under neutral conditions.

[0058] 1-1. Preparation of chitosan derivative solution for preparing in vivo ion-sensitive hydrogel compositions

[0059] In this embodiment, solutions containing chitosan derivatives with different degrees of deacetylation (%) and degrees of methyl carboxyl substitution (%) were prepared as follows. As shown in Table 1 below, the chitosan derivatives were classified into three groups based on the degree of deacetylation (50%, 70%, 90%), and each group was further classified into three experimental groups based on the degree of methyl carboxyl substitution within the chitosan derivatives, thus setting a total of 9 experimental groups. Here, the degree of deacetylation (%) represents the level of acetyl removal from chitin, the precursor of the chitosan derivative, and the degree of methyl carboxyl substitution (%) represents the level at which hydroxyl and amino groups in the chitosan derivative are replaced by -O-methyl carboxyl or -N-methyl carboxyl groups. Furthermore, the solvent used in the chitosan derivative solutions was PBS (pH 7.4), a neutral solvent.

[0060] [Table 1]

[0061] (1) Preparation of G1-1 experimental group

[0062] 10g of chitosan was reacted in 100mL of 50% NaOH solution at 100℃ for 30 minutes. After the reaction, water was added for washing. Finally, the deacetylated chitosan was dried at 80℃ to obtain chitosan powder with a degree of deacetylation of 50%.

[0063] (2) Preparation of G1-2 experimental group

[0064] 10 g of chitosan was reacted in 100 mL of 50% NaOH solution at 100 °C for 30 minutes. After the reaction, water was added for washing. Finally, the mixture was dried at 80 °C to obtain deacetylated chitosan. Then, the chitosan powder with 50% deacetylation was added to 20% NaOH to a concentration of 5 w / v, followed by the addition of monochloroacetic acid (1.0 w / v) and reaction at 40 °C. After neutralization with 10% acetic acid, the mixture was purified with methanol to finally obtain a chitosan derivative with 50% deacetylation and 50% methyl carboxyl substitution.

[0065] (3) Preparation of G1-3 experimental group

[0066] 10 g of chitosan was reacted in 100 mL of 50% NaOH solution at 100 °C for 30 minutes. After the reaction, water was added for washing. Finally, the mixture was dried at 80 °C to obtain deacetylated chitosan. Then, the chitosan powder with 50% deacetylation was added to 20% NaOH to a concentration of 5 w / v, followed by the addition of monochloroacetic acid (1.5 w / v) and reaction at 40 °C. After neutralization with 10% acetic acid, the mixture was purified with methanol to finally obtain a chitosan derivative with 50% deacetylation and 70% methyl carboxyl substitution.

[0067] (4) Preparation of G2-1 experimental group

[0068] 10g of chitosan was reacted in 100mL of 50% NaOH solution at 100℃ for 60 minutes. After the reaction, water was added for washing. Finally, the deacetylated chitosan was dried at 80℃ to obtain chitosan powder with a degree of deacetylation of 70%.

[0069] (5) Preparation of G2-2 experimental group

[0070] 10 g of chitosan was reacted in 100 mL of 50% NaOH solution at 100 °C for 60 minutes. After the reaction, water was added for washing. Finally, the mixture was dried at 80 °C to obtain deacetylated chitosan. Then, the chitosan powder with 70% deacetylation was added to 20% NaOH to a concentration of 5 w / v, followed by the addition of monochloroacetic acid (1.0 w / v) and reaction at 40 °C. After neutralization with 10% acetic acid, the mixture was purified with methanol to finally obtain a chitosan derivative with 70% deacetylation and 50% methyl carboxyl substitution.

[0071] (6) Preparation of G2-3 experimental group

[0072] 10 g of chitosan was reacted in 100 mL of 50% NaOH solution at 100 °C for 60 minutes. After the reaction, water was added for washing. Finally, the mixture was dried at 80 °C to obtain deacetylated chitosan. Then, the chitosan powder with 70% deacetylation was added to 20% NaOH to a concentration of 5 w / v, followed by the addition of monochloroacetic acid (1.5 w / v) and reaction at 40 °C. After neutralization with 10% acetic acid, the mixture was purified with methanol to finally obtain a chitosan derivative with 70% deacetylation and 70% methyl carboxyl substitution.

[0073] (7) Preparation of G3-1 experimental group

[0074] 10g of chitosan was reacted in 100mL of 50% NaOH solution at 100℃ for 150 minutes. After the reaction, water was added for washing. Finally, the deacetylated chitosan was dried at 80℃ to obtain chitosan powder with a degree of deacetylation of 90%.

[0075] (8) Preparation of G3-2 experimental group

[0076] 10 g of chitosan was reacted in 100 mL of 50% NaOH solution at 100 °C for 150 minutes. After the reaction, water was added for washing. Finally, the mixture was dried at 80 °C to obtain deacetylated chitosan. Then, the chitosan powder with 90% deacetylation was added to 20% NaOH to a concentration of 5 w / v, followed by the addition of chloroacetic acid (1.0 w / v) and reaction at 40 °C. After neutralization with 10% acetic acid, the mixture was purified with methanol to finally obtain a chitosan derivative with 90% deacetylation and 50% methyl carboxyl substitution.

[0077] (9) Preparation of G3-3 experimental group

[0078] 10 g of chitosan was reacted in 100 mL of 50% NaOH solution at 100 °C for 150 minutes. After the reaction, water was added for washing. Finally, the mixture was dried at 80 °C to obtain deacetylated chitosan. Then, the chitosan powder with 90% deacetylation was added to 20% NaOH to a concentration of 5 w / v, followed by the addition of chloroacetic acid (1.5 w / v) and reaction at 40 °C. After neutralization with 10% acetic acid, the mixture was purified with methanol to finally obtain a chitosan derivative with 90% deacetylation and 70% methyl carboxyl substitution.

[0079] Then, for the chitosan derivatives obtained above with different degrees of deacetylation and methyl carboxyl substitution, the content of free amine was measured by band intensity measurement using Fourier transform infrared spectroscopy (FT-IR) (see Figure 1) and by measuring the content of free amine using the ninhydrin assay to confirm whether the chitosan derivatives were substituted with methyl carboxyl groups and the level of substitution.

[0080] 1-2. Evaluate the solubility of chitosan derivatives under neutral solvent conditions.

[0081] This embodiment aims to confirm the effect of the degree of deacetylation and / or the degree of methyl carboxyl substitution of chitosan derivatives on the solubility of chitosan derivatives in neutral solvents by evaluating the solubility of chitosan derivative solutions from Examples 1-1. To this end, the solubility of chitosan derivative solutions from Examples 1-1 with different degrees of deacetylation and methyl carboxyl substitution was visually evaluated at room temperature.

[0082] As shown in Figure 2, the GI group, which included chitosan derivatives with a degree of deacetylation of 50%, all formed precipitates in neutral solvents or exhibited low solubility in chitosan derivatives, resulting in a turbid state. Conversely, as shown in Figures 3 and 4, the G2 or G3 groups, which included chitosan derivatives with a degree of deacetylation of 70% or 90%, showed clear solutions due to the increased solubility of chitosan derivatives in neutral solvents. This effect was particularly evident in the G2-2 and G3-2 groups, where the degree of methyl carboxyl substitution was 50% or higher, and became more pronounced with increasing methyl carboxyl substitution.

[0083] Example 2: Evaluation of the solubility of chitosan derivatives according to pH changes

[0084] This embodiment aims to examine the differences between existing tissue repair biomaterials that exhibit solubility under acidic conditions and those of the chitosan derivative solutions from Examples 1-1, by evaluating the change in solubility of the chitosan derivative solutions with pH variations. For this purpose, a solution comprising chitosan derivatives with a degree of deacetylation of 90% and a degree of methyl carboxyl substitution of 2 w / v% was prepared in the same manner as in Example 1. However, the pH levels of the chitosan derivative solutions were adjusted to 6.8, 6.4, and 6.1, respectively, using 0.01N, 0.015N, and 0.02N HCl aqueous solutions as solvents. Then, the solubility of the chitosan derivative solutions at different pH values ​​at room temperature was visually evaluated in the same manner as in Examples 1-2.

[0085] As shown in Figure 5, the solution observed under neutral conditions was transparent due to the high solubility of chitosan derivatives. Conversely, under weakly acidic conditions at pH 6.5 or lower, precipitates were formed or the solubility of chitosan derivatives was low, resulting in a turbid state.

[0086] The experimental results show that by adjusting the degree of deacetylation (70% or higher) and the degree of methyl carboxyl substitution (50% or higher) of the chitosan derivative, a chitosan derivative solution with high solubility and stability under neutral conditions can be prepared, which is different from existing tissue repair biomaterials or includes them.

[0087] Example 3: Evaluation of in vivo ion sensitivity based on free amine groups

[0088] For use as a biomaterial for tissue repair, it must exist in an injectable liquid form at room temperature and, upon in vivo application, gel to form an effective volumetric effect. Therefore, this embodiment aims to confirm the effect of free amine groups within the chitosan derivative on its in vivo ion-sensitivity behavior.

[0089] 3-1. Preparation of in vivo ion-sensitive hydrogel compositions

[0090] In this embodiment, solutions containing chitosan derivatives with different levels (%) of free amino groups were prepared as follows.

[0091] [Table 2]

[0092] As shown in Table 2, the degree of deacetylation (%) and the degree of methyl carboxyl substitution (%) are shown in Table 1. The free amine (%) represents the level of free amine (-NH2) in the chitosan derivative. In the chitosan derivative solution, disodium phosphate, a neutral solvent, was used as the solvent. In this embodiment, the experimental groups (N1, N2, N3) were prepared by performing N,O-random carboxyl methyl substitution, and the level of free amine was adjusted by adjusting the degree of methyl carboxyl substitution in the same manner as in Examples 1-1.

[0093] Then, the 2 w / v% chitosan derivative solution and 2 w / v% disodium hydrogen phosphate (Na2HPO4) were mixed to prepare an injectable, liquid, in vivo ion-sensitive hydrogel composition at room temperature.

[0094] 3-2. In vivo ion sensitivity assessment using animal models

[0095] This embodiment aims to confirm the effect of the free amino group level of chitosan derivatives on in vivo ion-sensitive behavior by evaluating whether the ion-sensitive hydrogel composition of Example 3-2 gels in vivo using an animal model. For this purpose, 0.5 ml of the ion-sensitive hydrogel composition of Example 3-1 was injected intradermally into hairless mice. One day after injection, the formation of a matrix with gelation was visually confirmed, and its storage modulus (G') was measured.

[0096] As a result, as shown in Figure 7, group N1, which included chitosan derivatives with a free amino group level of 21%, failed to form a matrix in vivo. Conversely, as shown in Figure 8, group N2, which included chitosan derivatives with a free amino group level of 34%, formed a matrix with a storage modulus similar to that of low-crosslinked hyaluronic acid fillers, namely 31.97 ± 1.58 Pa. Group N3, which included chitosan derivatives with a free amino group level of 90%, formed a matrix with a storage modulus of 154.31 ± 13.21 Pa. By adjusting the matrix modulus according to the manner suggested in this application, in vivo ion-sensitive hydrogel compositions forming matrices with storage moduli of 5-50 Pa can be applied to medical materials such as intra-articular injections and skin enhancers; in vivo ion-sensitive hydrogel compositions forming matrices with storage moduli of 100 Pa or higher can be applied to medical materials for subcutaneous repair.

[0097] The experimental results show that by adjusting the degree of deacetylation, the degree of methyl carboxyl substitution, and the level of free amino groups in the chitosan derivative, the problems of existing tissue repair biomaterials can be solved. Furthermore, a biomaterial can be prepared that combines the characteristics of an injectable liquid formulation at room temperature with the ability to gel in vivo to form a viscoelastic matrix with various tissue shapes and properties.

[0098] The description above is merely exemplary, and those skilled in the art should understand that the invention can be easily modified into other specific forms without changing the technical concept or essential features. Therefore, the embodiments described above should be understood as exemplary in all respects, and not restrictive.

Claims

1. A composition for preparing an in vivo ion-sensitive hydrogel composition, wherein, The composition comprises a chitosan derivative that satisfies the following conditions: (a) having a degree of deacetylation of 70% or higher; (b) at least 50% of the hydroxyl and amino groups in the chitosan derivative being replaced by O-alkyl carboxyl groups or N-alkyl carboxyl groups; and (c) at least 22% of the total amino groups in the chitosan derivative being free amino groups.

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, In the chitosan derivative, 50% to 90% of the hydroxyl and amino groups are replaced by O-methyl carboxyl or N-methyl carboxyl groups.

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

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

6. The composition according to claim 1, wherein, The pH of the solution is between pH 6.5 and pH 7.

5.

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

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

9. The composition according to claim 8, wherein, The aqueous solution containing phosphate ions includes at least one phosphate selected from the group consisting of disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, trisodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, dimethyl phosphate, magnesium dihydrogen phosphate, magnesium hydrogen phosphate, lithium dihydrogen phosphate, lithium phosphate, calcium hydrogen phosphate dihydrate, and calcium hydrogen phosphate.

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

11. The composition according to claim 1, which has a transparent liquid form under neutral conditions and forms a gel form in vivo.

12. A method for preparing an in vivo ion-sensitive hydrogel composition, comprising the step of mixing a solution containing a chitosan derivative and an aqueous solution containing phosphate ions, wherein, The chitosan derivative satisfies the following conditions: (a) having a degree of deacetylation of 70% or higher; (b) at least 50% of the hydroxyl and amino groups in the chitosan derivative are replaced by O-alkyl carboxyl groups or N-alkyl carboxyl groups; and (c) at least 22% of the total amino groups in the chitosan derivative are free amino groups.

13. The method according to claim 12, wherein, 50% to 90% of the hydroxyl and amino groups in the chitosan derivative are replaced by O-methyl carboxyl or N-methyl carboxyl 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 phosphate ions includes at least one phosphate selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, trisodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, dimethyl phosphate, magnesium dihydrogen phosphate, magnesium hydrogen phosphate, lithium dihydrogen phosphate, lithium phosphate, calcium hydrogen phosphate dihydrate, and calcium hydrogen phosphate.

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

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