Composition for preparing biomaterial for in vivo ion-sensitive tissue repair and use thereof

Abstract

The present application relates to a composition for preparing a biomaterial for in vivo ion-sensitive tissue repair and a use thereof, and provides: a composition for preparing a solution containing a chitosan derivative in which the degree of deacetylation, the degree of substitution of alkylcarboxy groups, and the level of free amine groups are controlled, or preparing an in vivo ion-sensitive hydrogel composition comprising the solution containing the chitosan derivative; and an in vivo ion-sensitive hydrogel composition using said composition.

Description

Composition for manufacturing biomaterial for ion-sensitive tissue repair in the body and use thereof

[0001] The present invention relates to a composition for manufacturing a biomaterial for ion-sensitive tissue repair in the body and its use. This application claims priority from Republic of Korea Patent Application No. 10-2023-0118550, filed on September 6, 2024, the disclosure of which is incorporated herein by reference.

[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 rays. Facial skin aging is characterized by atrophy, sagging, and hypertrophy. Atrophy refers to a significant decrease in skin tissue thickness, while sagging subcutaneous tissue leads to excess skin and ptosis, resulting in sagging cheeks and eyelids. Hypertrophy refers to excessive weight gain, resulting in swelling of the face and neck. These changes are typically associated with loss of elasticity and a rough skin surface.

[0003] Hydrogels are materials with a polymer network structure containing a large amount of water, formed from homopolymers or copolymers. Hydrogels swell when absorbed by hydrophilic components, including water, in an aqueous solution, becoming transparent and possessing suitable mechanical properties. The swelling degree and mechanical properties of the polymer depend primarily on the properties of the polymer network structure and the formulation method. Polymers are commonly used as materials for manufacturing hydrogels, and these polymers can be categorized into natural and synthetic polymers based on their origin. Hydrogels made from natural polymers, such as agar, chitosan, carrageenan, cellulose, and xanthan gum, may convey a positive impression of natural origin to consumers. However, they are characterized by weak mechanical properties. Most of these natural polymers are formed through physical gelation reactions, making them prone to hydrogel collapse at high temperatures and mechanical strength loss at low temperatures. Moreover, even at room temperature, hydrogels can easily break or tear due to a weak external force, such as pressing with a finger. Therefore, considerable care must be taken during use and distribution. In contrast, hydrogels made from synthetic polymers possess strong bonding between molecules, such as chemical covalent bonds, through cross-linking. This allows them to exhibit strong mechanical properties, unlike natural polymers. Consequently, damage to the hydrogel form is relatively rare due to external stimuli such as temperature and external force. However, synthetic polymer-based hydrogels require improvements in biocompatibility and efficacy retention.

[0004] In particular, existing tissue repair biomaterials in the form of injectable liquid formulations have the characteristic of dissolving under acidic conditions, causing pain when injected into the skin, and the stability of the material when mixed with other materials or components under neutral conditions is reduced, and there are problems such as occasional formation of sediment within the material. Against this technological backdrop, there is a need for the development of next-generation tissue repair biomaterials that can maximize the advantages of formulations using existing natural / synthetic polymers, while complementing the existing shortcomings and achieving the ultimate goal of replacing autologous tissue (Korean Patent No. 10-0506543).

[0005] One aspect is to provide a composition for preparing an in vivo ion-sensitive hydrogel composition comprising a solution containing a chitosan derivative, each having a controlled degree of deacetylation, a degree of substitution of alkyl carboxyl groups, and a level of free amine groups.

[0006] Another aspect provides a method for preparing an in vivo ion-sensitive hydrogel composition comprising a step of mixing a solution containing a chitosan derivative, each having a controlled degree of deacetylation, a degree of substitution of alkyl carboxyl groups, and a level of free amine groups, and an aqueous solution containing phosphate ions.

[0007] Another aspect is to provide an in vivo ion-sensitive hydrogel composition prepared by the above method.

[0008] Another aspect is to provide a treatment method using the above-mentioned ion-sensitive hydrogel or tissue repair biomaterial.

[0009] One aspect provides a composition for preparing an in vivo ion-sensitive hydrogel composition comprising a solution containing a chitosan derivative satisfying the following conditions:

[0010] (a) shall have a degree of deacetylation of 70% or more;

[0011] (b) more than 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups; and

[0012] (c) More than 22% of the total amine groups in the chitosan derivative shall be free amine groups (-NH2).

[0013]

[0014] As used herein, the term "hydrogel" may refer to a three-dimensional network structure formed by cross-linking hydrophilic polymers via covalent or non-covalent bonds. Due to the hydrophilicity of the constituent materials, hydrogels absorb large amounts of water and swell in aqueous solutions and environments, but do not dissolve due to their cross-linked structure. Therefore, hydrogels with various shapes and properties can be produced depending on the constituents and manufacturing method. They generally contain a large amount of water, and thus may possess properties intermediate between those of a liquid and a solid.

[0015] As an example, the term "hydrogel" may be used interchangeably with the term "tissue repair biomaterial" or "tissue repair biomaterial composition." In one embodiment, the hydrogel may be a tissue repair biomaterial, which may be a material used for tissue repair. For example, it may refer to a soft tissue-like filling material injected into wrinkled skin or an area requiring volume, an adhesion prevention material between a surgical site and normal tissue, a tissue adhesive material, a wound dressing material for artificial skin, etc. The hydrogel may be applied to body parts such as the glabella, forehead, under-eye circles, crow's feet, nasolabial folds, cheeks, wrinkles around the mouth, and chin. The hydrogel must be biocompatible as a material directly applied to the human body. For example, when the hydrogel is filled into an area requiring volume, it is necessary to have excellent gel shape retention / persistence to form volume for a long period of time after injection. When hydrogels are used to prevent adhesion at the surgical site, they must be tissue compatible with the wound site and have little or no cytotoxicity.

[0016] As used herein, the term "tissue repair" refers to restoring the structure and function of damaged or aged tissue, and may include, but is not limited to, cosmetic filler applications, anti-adhesion agents, adhesives, wound dressings, and plastic prosthetics.

[0017] In this specification, the term "ion sensitivity" refers to a physical property in which the formulation changes depending on the surrounding ionic environment, and may refer to a property in which the formulation exists in a liquid form, i.e., a sol form, under room temperature conditions, for example, in vitro conditions, under an environment exposed to room temperature, but is converted to a gel form under in vivo conditions.

[0018] As used herein, the term "chitosan" may refer to a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine. The chitosan may include a material obtained by deacetylating chitin using an alkali or the like. More specifically, the chitosan refers to a polymer material in which the proportion of D-glucosamine generated by removing an acetyl group from N-acetyl-D-glucosamine is 70% or more, and may be represented by the following structural formula 1.

[0019] [Structural formula 1]

[0020]

[0021] As used herein, the term “chitosan derivative” refers to a substance derived from the aforementioned chitosan, and may refer to a structure in which organic chemical modifications including introduction of a specific functional group, oxidation, reduction, substitution of atoms, etc. have been introduced. Since existing chitosan derivatives in the art function as weak polyanionic polyelectrolytes, amino groups in chitosan derivatives are not protonated under neutral conditions and most carboxyl groups are insoluble, so there are limitations in their use as biomaterials. Therefore, the development of new technologies to improve the usability of chitosan derivatives as biomaterials is required. The chitosan derivative may be, for example, carboxyalkyl chitosan, and specifically, may be formed by a combination of a plurality of units represented by the following structural formula 2.

[0022] [Structural formula 2]

[0023]

[0024] In the above structural formula 2, R1 may be any one of hydrogen, an alkyl carboxy group, and an acetyl group, and R2 and R3 may independently be hydrogen or an alkyl carboxy group. Here, the alkyl carboxy group may be, for example, a methyl carboxy group or an ethyl carboxy group, and specifically, may be a methyl carboxy group.

[0025] In one specific example, the average molecular weight of the chitosan derivative may be from 50,000 to 3,000,000 Da. The average molecular weight is derived from a plurality of units represented by the structural formula 1 or structural formula 2 described above or a combination thereof, and may be appropriately changed within a conventional range known in the art.

[0026] In one specific example, the chitosan derivative may exhibit high solubility and stability under neutral conditions, and may also have the property of being gelled under in vivo conditions to form a matrix with various viscoelastic properties. To this end, the chitosan derivative may have a degree of deacetylation of 70% or more, a degree of substitution with an O-alkylcarboxyl group or an N-alkylcarboxyl group of 50% or more, and a level of free amine groups of 22% or more. Additionally, the pH of the solution or in vivo ion-sensitive hydrogel composition containing the chitosan derivative according to one embodiment may be 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 chitosan derivatives described above, the degree of substitution of the O-alkylcarboxyl group or N-alkylcarboxyl group, and the level of free amine groups are parameters that can be controlled according to techniques known in the art, and may include, but are not limited to, a series of processes for reacting chitosan powder having a certain level of deacetylation with an appropriate amount of monochloroacetic acid.

[0027] For example, the degree of deacetylation, the degree of substitution of the O-alkylcarboxyl group or the N-alkylcarboxyl group, etc. can be controlled according to the following method: In order to obtain chitosan derivatives having different deacetylation levels, 10 g of chitin can be reacted in 100 mL of a 50% NaOH solution at 100°C for 30 to 150 minutes, for example, 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, 60 to 80 minutes, or 60 to 80 minutes. In addition, in order to obtain chitosan derivatives having different levels of alkyl carboxyl groups or free amine groups, the chitosan derivative powder having the above-described controlled deacetylation level may be added to 20% NaOH at 5 w / v%, and then 1.0 to 1.5 w / v% of monochloroacetic acid may be added thereto and reacted.

[0028] In one specific example, the chitosan derivative may have a deacetylation degree of 70% to 90%. The deacetylation degree represents the level at which acetyl groups are removed from chitin, a precursor of chitosan, and may represent the percentage level of units in which R1 is hydrogen or an alkylcarboxyl group in the structural formula 2. 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%.

[0029] In one specific example, the chitosan derivative may have a degree of substitution of an O-alkylcarboxyl group or an N-alkylcarboxyl group of 50% to 90%. The degree of substitution of the O-alkylcarboxyl group or the N-alkylcarboxyl group refers to the level of substitution of hydroxyl groups and amine groups in the chitosan derivative with an -O-alkylcarboxyl group or an -N-alkylcarboxyl group, and may refer to the % level of substituents in which R2 and R3 are alkylcarboxyl groups in the structural formula 2. Here, the % level is a value calculated as the number of carboxymethyl groups per 100 anhydroglucosamine units, i.e., a concept distinct from the conventional degree of substitution that is evaluated by whether or not a carboxyl group is substituted. The degree of substitution of the O-alkylcarboxyl group or N-alkylcarboxyl group 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%. At this time, if the degree of deacetylation and the degree of substitution of the O-alkylcarboxyl group or N-alkylcarboxyl group of the chitosan derivative are outside the above range, there is a problem that the stability under neutral conditions is rapidly reduced and the number of insoluble particles that are not dissolved in the solution increases.

[0030] In one specific example, the chitosan derivative may have a free amine group level of 22% to 90%. The free amine group level refers to the level of free amine groups (-NH2) among the amine groups in the chitosan derivative, and may refer to the percentage level of substituents in which R1 is hydrogen in the structural formula 2. The free amine group level of the above chitosan derivative is 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%, 55% to 80%, 60% to 80%, It can be 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%. At this time, if the level of free amine groups of the chitosan derivative is outside the above range, there is a problem that gelation does not occur under in vivo conditions or the strength level of the formed hydrogel is very weak.

[0031] In one specific example, the chitosan derivative may have a degree of deacetylation of 70% to 90%, a degree of substitution with O-alkylcarboxyl groups or N-alkylcarboxyl groups of 50% to 90%, and a level of free amine groups of 22% to 90%, and the chitosan derivative may have, for example, a degree of deacetylation of 70% to 90%, a degree of substitution with O-alkylcarboxyl groups or N-alkylcarboxyl groups of 50% to 70%, and a level of free amine groups of 24% to 90%.

[0032] In one embodiment, 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 and the level of terminal carboxyl groups (i.e., the degree of substitution of O-alkyl carboxyl groups or N-alkyl carboxyl groups) of the chitosan derivative can 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 the level of gelation and viscoelasticity of the formed matrix under in vivo conditions. Accordingly, in this embodiment, it was confirmed that the functionality / effect as a biomaterial for tissue repair can be achieved by controlling the degree of deacetylation, the level of terminal carboxyl groups, and the level of free amine groups of the chitosan derivative, and the present invention was completed based on this.

[0033]

[0034] 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 solution, and the content of the chitosan derivative 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% based on the total volume of the chitosan derivative solution. It may be 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%.

[0035] If the content of the chitosan derivative is below the above range, there is a problem that gelation does not occur or the strength level of the formed hydrogel is very weak and the retention power is insufficient, and if the content of the chitosan derivative is above the above range, there is a problem that insoluble particles that are not dissolved in the solution increase.

[0036] In one specific example, the composition for preparing the ion-sensitive hydrogel composition in the body may further include a water-containing agent containing phosphate ions.

[0037] As used herein, the term “phosphate ion” refers to a component that binds to an amine group of a chitosan derivative to form a hydrogel under in vivo conditions or contributes to enhancing the strength of the formed hydrogel, and may be provided in the form of an aqueous solution containing phosphate ions, as an example. The aqueous solution containing the above phosphate ion is, for example, sodium phosphate dibasic (disodium hydrogen phosphate, Na2HPO4), sodium phosphate monobasic (monosodium hydrogen phosphate monobasic, NaH2PO4), ammonium hydrogen phosphate dibasic (diammonium hydrogen phosphate, (NH4)2HPO4), ammonium dihydrogen phosphate (NH4H2PO4), sodium phosphate tribasic (Na3PO4), potassium phosphate dibasic (K2HPO4), potassium phosphate monobasic (KH2PO4), dimethyl phosphate (C2H7PO4), monomagnesium phosphate (Mg(H2PO4)2), magnesium phosphate dibasic (MgHPO4), lithium It may include lithium dihydrogen phosphate (LiH2PO4), lithium phosphate (LiPO4), calcium hydrogen orthophosphate hydrate (CaHPO4·2H2O), and calcium hydrogen orthophosphate (CaHPO4), but it may be applied without limitation to any substance that can provide a phosphate group or phosphate that can bind to the amine group of the chitosan derivative.

[0038] The composition may contain an aqueous solution containing phosphate ions in an amount of 7 to 15 wt% based on the total weight of the composition, and for example, the content of the aqueous solution containing 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%.

[0039] In one specific example, the composition for preparing the in vivo ion-sensitive hydrogel composition may further comprise a liquid formulation containing glycerol.

[0040] In one specific example, the liquid formulation may be isolated in a separate space within the container from the solution containing the aforementioned chitosan derivative and / or the aqueous solution containing phosphate ions. Accordingly, the solution containing chitosan and phosphate ions and the liquid formulation containing glycerol can be maintained and stored in liquid form prior to use, and, if necessary, can also be stored frozen for long-term storage.

[0041] The above liquid formulation containing glycerol may be mixed with a solution containing the chitosan derivative and an aqueous solution containing phosphate ions to adjust the level of covalent bonding within the mixture, thereby imparting viscoelastic properties to a biomaterial for tissue repair.

[0042] The glycerol may be contained in a concentration of 0.01 v / v% to 1 v / v% based on the total volume of the liquid preparation or composition, and 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% based on the total volume of the liquid preparation or composition.

[0043]

[0044] Another aspect provides a method for preparing an in vivo ion-sensitive hydrogel composition, comprising the step of mixing a solution containing a chitosan derivative satisfying the following conditions and an aqueous solution containing phosphate ions:

[0045] (a) shall have a degree of deacetylation of 70% or more;

[0046] (b) more than 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups; and

[0047] (c) More than 22% of the total amine groups in the chitosan derivative shall be free amine groups (-NH2).

[0048] Another aspect is to provide an in vivo ion-sensitive hydrogel composition prepared by the above method.

[0049] Another aspect provides a method for administering an ion-sensitive hydrogel composition to a subject, comprising the steps of mixing a solution containing the aforementioned chitosan derivative and an aqueous solution containing phosphate ions; and injecting the mixed liquid formulation composition into the skin of a subject.

[0050] Since the method for producing the above-described in vivo ion-sensitive hydrogel composition, the in vivo ion-sensitive hydrogel composition produced by the above-described method, or the method for administering the in vivo ion-sensitive hydrogel composition directly includes or utilizes the composition for producing the above-described in vivo ion-sensitive hydrogel composition, description of common contents between the two is omitted.

[0051] According to one aspect, the in vivo ion-sensitive hydrogel composition or tissue repair biomaterial manufactured by the above method maintains high solubility of chitosan derivatives and material stability under neutral conditions, unlike existing tissue repair biomaterials that are dissolved and used under acidic conditions, thereby alleviating pain during in vivo administration and facilitating mixing with other materials or components. In addition, the in vivo ion-sensitive hydrogel composition or tissue repair biomaterial has both the characteristics of a liquid formulation that can be injected under room temperature conditions and the characteristics of being able to gel under in vivo conditions to form a matrix with viscoelasticity that matches the shape and characteristics of various tissues, and thus can be widely utilized in various fields.

[0052] The composition according to one aspect comprises a solution containing a chitosan derivative having a controlled degree of deacetylation, a degree of substitution of alkyl carboxyl groups, and a level of free amine groups, thereby enabling it to have high solubility and stability under neutral conditions, unlike conventional biomaterials for tissue repair.

[0053] The composition according to the aspect is not only stable as a material, but can also be gelled under in vivo conditions to form a matrix with various viscoelasticities, thereby providing a biomaterial for tissue repair that matches the shape and characteristics of various tissues.

[0054] Figure 1 shows the results of confirming the substitution of a methyl carboxyl group in a chitosan derivative using FT-IR spectroscopy in a solution containing a chitosan derivative according to one embodiment.

[0055] FIG. 2 shows a change in the solubility of a chitosan derivative in a neutral solvent according to the degree of substitution of a methyl carboxyl group in a solution containing a chitosan derivative having a degree of deacetylation of 50% according to one embodiment. FIG. 2A shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 0%, FIG. 2B shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 50%, and FIG. 2C shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 70%.

[0056] FIG. 3 shows a change in the solubility of a chitosan derivative in a neutral solvent according to the degree of substitution of a methyl carboxyl group in a solution containing a chitosan derivative having a degree of deacetylation of 70% according to one embodiment. FIG. 3A shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 0%, FIG. 3B shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 50%, and FIG. 3C shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 70%.

[0057] FIG. 4 shows a change in the solubility of a chitosan derivative in a neutral solvent according to the degree of substitution of a methyl carboxyl group in a solution containing a chitosan derivative having a degree of deacetylation of 90% according to one embodiment. FIG. 4A shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 0%, FIG. 4B shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 50%, and FIG. 4C shows the result of visually confirming a chitosan derivative solution having a degree of substitution of a methyl carboxyl group of 70%.

[0058] FIG. 5 shows changes in the solubility of a chitosan derivative according to changes in the pH of a solution containing a chitosan derivative having a degree of deacetylation of 90% and a degree of methylcarboxyl group substitution of 70% according to one embodiment. FIG. 5A shows the result of visually confirming a chitosan derivative solution having a pH of 6.8, FIG. 5B shows the result of visually confirming a chitosan derivative solution having a pH of 6.4, and FIG. 5C shows the result of visually confirming a chitosan derivative solution having a pH of 6.1.

[0059] Figure 6 is a result of visually confirming the characteristics of an injectable liquid formulation of an ion-sensitive hydrogel composition according to one embodiment.

[0060] Figure 7 shows the results of confirming the progress of in vivo gelation of an ion-sensitive hydrogel composition containing a chitosan derivative having a free amine group level of 21% according to one embodiment using an animal model.

[0061] FIG. 8 shows the progress of in vivo gelation of an ion-sensitive hydrogel composition comprising a chitosan derivative having 22% or more of free amine groups according to one embodiment, using an animal model. FIG. 8A shows the result of checking the progress of gelation of an ion-sensitive hydrogel composition comprising a chitosan derivative having a free amine group level of 34%, and FIG. 8B shows the result of checking the progress of gelation of an ion-sensitive hydrogel composition comprising a chitosan derivative having a free amine group level of 90%.

[0062] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0063]

[0064] [Example]

[0065] Example 1. Evaluation of the dissolution level of chitosan derivative solutions under neutral conditions.

[0066] Existing biomaterials for tissue repair, including chitosan, exhibit the characteristic of being soluble in acidic solutions, causing pain during administration in the body. In addition, there was a problem that the stability of the material rapidly deteriorated when chitosan was precipitated or mixed with other materials or components under neutral conditions. Therefore, in this example, an attempt was made to prepare a chitosan derivative solution having high solubility under neutral conditions.

[0067]

[0068] 1-1. Preparation of chitosan derivative solution for manufacturing in vivo ion-sensitive hydrogel composition

[0069] In this example, solutions containing chitosan derivatives having different degrees of deacetylation (%) and methylcarboxyl group substitution (%) were prepared as follows. As shown in Table 1 below, the chitosan derivatives were first classified into three groups based on the degrees of deacetylation (50%, 70%, and 90%), and the classified groups were reclassified into three experimental groups per group based on the degree of methylcarboxyl group substitution in the chitosan derivatives, resulting in a total of nine experimental groups. Here, the degree of deacetylation (%) represents the level at which acetyl groups are removed from chitin, which is a precursor of the chitosan derivative, and the degree of methylcarboxyl group substitution (%) represents the level at which hydroxyl groups and amine groups in the chitosan derivative are substituted with -O-methylcarboxyl groups or -N-methylcarboxyl groups. In addition, the solvent used in the chitosan derivative solution was a neutral solvent, PBS (pH 7.4).

[0070] [Table 1]

[0071]

[0072]

[0073] (1) Preparation of G1-1 experimental group

[0074] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 30 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally dried at 80°C, yielding chitosan powder with a deacetylation degree of 50%.

[0075]

[0076] (2) Preparation of G1-2 experimental group

[0077] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 30 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally obtained by drying at 80°C. Thereafter, the chitosan powder with the above 50% degree of deacetylation was added to 20% NaOH at 5 w / v%, and then monochloroacetic acid (1.0 w / v%) was added thereto and reacted at 40°C. Thereafter, the mixture was neutralized with 10% acetic acid and purified with methanol to finally obtain a chitosan derivative with a deacetylation degree of 50% and a methylcarboxyl substitution degree of 50%.

[0078]

[0079] (3) Preparation of G1-3 experimental group

[0080] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 30 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally obtained by drying at 80°C. Thereafter, the chitosan powder with the above 50% degree of deacetylation was added to 20% NaOH at 5 w / v%, and then monochloroacetic acid (1.5 w / v%) was added thereto and reacted at 40°C. Thereafter, the mixture was neutralized with 10% acetic acid and purified with methanol to finally obtain a chitosan derivative with a deacetylation degree of 50% and a methylcarboxyl substitution degree of 70%.

[0081]

[0082] (4) Preparation of G2-1 experimental group

[0083] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 60 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally dried at 80°C, yielding chitosan powder with a deacetylation degree of 70%.

[0084]

[0085] (5) Preparation of G2-2 experimental group

[0086] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 60 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally obtained by drying at 80°C. Thereafter, the chitosan powder with a deacetylation degree of 70% was added to 20% NaOH at 5 w / v%, and then monochloroacetic acid (1.0 w / v%) was added thereto and reacted at 40°C. Thereafter, the mixture was neutralized with 10% acetic acid and purified with methanol to finally obtain a chitosan derivative with a deacetylation degree of 70% and a methylcarboxyl substitution degree of 50%.

[0087]

[0088] (6) Preparation of G2-3 experimental group

[0089] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 60 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally obtained by drying at 80°C. Thereafter, the chitosan powder with a deacetylation degree of 70% was added to 20% NaOH at 5 w / v%, and then monochloroacetic acid (1.5 w / v%) was added and reacted at 40°C. Thereafter, the mixture was neutralized with 10% acetic acid and purified with methanol to finally obtain a chitosan derivative with a deacetylation degree of 70% and a methylcarboxyl substitution degree of 70%.

[0090]

[0091] (7) Preparation of G3-1 experimental group

[0092] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 150 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally dried at 80°C, yielding chitosan powder with a deacetylation degree of 90%.

[0093]

[0094] (8) Preparation of G3-2 experimental group

[0095] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 150 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally obtained by drying at 80°C. Thereafter, the chitosan powder with a deacetylation degree of 90% was added to 20% NaOH at 5 w / v%, and then monochloroacetic acid (1.0 w / v%) was added thereto and reacted at 40°C. Thereafter, the mixture was neutralized with 10% acetic acid and purified with methanol to finally obtain a chitosan derivative with a deacetylation degree of 90% and a methylcarboxyl substitution degree of 50%.

[0096]

[0097] (9) Preparation of G3-3 experimental group

[0098] 10 g of chitin was reacted in 100 mL of a 50% NaOH solution at 100°C for 150 minutes. Immediately after the reaction was completed, water was added to perform washing. The deacetylated chitosan was finally obtained by drying at 80°C. Thereafter, the chitosan powder with a deacetylation degree of 90% was added to 20% NaOH at 5 w / v%, and then monochloroacetic acid (1.5 w / v%) was added and reacted at 40°C. Thereafter, the mixture was neutralized with 10% acetic acid and purified with methanol to finally obtain a chitosan derivative with a deacetylation degree of 90% and a methylcarboxyl substitution degree of 70%.

[0099]

[0100] Thereafter, for the chitosan derivatives obtained as described above with different degrees of deacetylation and methyl carboxyl substitution, the presence or absence of methyl carboxyl substitution and the level of substitution of the chitosan derivatives were confirmed by measuring the intensity of the band using FT-IR spectroscopy (see Figure 1) and measuring the content of free amine using ninhydrin assay.

[0101]

[0102] 1-2. Evaluation of the solubility level of chitosan derivatives under neutral solvent conditions

[0103] In this example, the dissolution level of the chitosan derivative solution of Example 1-1 was evaluated to determine the influence of the degree of deacetylation and / or the degree of substitution of methyl carboxyl groups of the chitosan derivative on the dissolution level of the chitosan derivative in a neutral solvent. To this end, the dissolution levels of the chitosan derivative solutions of Example 1-1 having different degrees of deacetylation and degrees of methyl carboxyl group substitution were visually evaluated under room temperature conditions.

[0104] As a result, as shown in Fig. 2, the G1 group containing chitosan derivatives with a deacetylation degree of 50% either formed precipitates in neutral solvents or was observed as a turbid state due to the low solubility level of the chitosan derivatives. On the other hand, as shown in Figs. 3 and 4, the G2 or G3 group containing chitosan derivatives with a deacetylation degree of 70% or 90% was observed as a transparent solution due to the enhanced solubility of the chitosan derivatives in neutral solvents. In particular, this effect was confirmed from the G2-2 and G3-2 groups having a methyl carboxyl group substitution degree of 50% or more, and was observed more clearly as the methyl carboxyl group substitution degree increased.

[0105]

[0106] Example 2. Evaluation of the dissolution level of chitosan derivatives according to pH changes.

[0107] In this example, the change in the dissolution level of the chitosan derivative solution of Example 1-1 according to the pH change was evaluated to verify the difference from the existing biomaterial for tissue repair that has the characteristic of dissolving under acidic conditions. To this end, in the same manner as in Example 1, a solution containing a chitosan derivative having a degree of deacetylation of 90% and a degree of methylcarboxyl group substitution of 70% at a concentration of 2 w / v% was prepared. However, in the chitosan derivative solution, a 0.01 N HCl aqueous solution, a 0.015 N HCl aqueous solution, and a 0.02 N HCl aqueous solution were used as solvents, respectively, to adjust the pH levels of the solutions to 6.8, 6.4, and 6.1. Thereafter, in the same manner as in Example 1-2, the dissolution levels of the chitosan derivative solutions having different pHs were visually evaluated under room temperature conditions.

[0108] As a result, as shown in Fig. 5, under neutral conditions, the solution was observed as a transparent solution due to the high solubility level of the chitosan derivative, whereas under weakly acidic conditions of pH 6.5 or lower, a precipitate was formed or the solution was observed as a turbid solution due to the low solubility level of the chitosan derivative.

[0109] In summary, the above experimental results indicate that, by controlling the degree of deacetylation (70% or more) and the degree of substitution of the methyl carboxyl group (50% or more) of the chitosan derivative, a chitosan derivative solution or a biomaterial containing the same having high solubility and stability under neutral conditions, unlike existing biomaterials for tissue repair, can be produced.

[0110]

[0111] Example 3. Evaluation of ion sensitivity levels in the body according to the level of free amine groups.

[0112] To be used as a biomaterial for tissue repair, it must be available in a liquid form that can be injected at room temperature, and upon in vivo administration, it must gel to form a meaningful volume. To this end, this example aimed to determine the influence of free amine groups in chitosan derivatives on ion-sensitive behavior in vivo.

[0113]

[0114] 3-1. Preparation of an in vivo ion-sensitive hydrogel composition

[0115] In this example, solutions containing chitosan derivatives having different levels (%) of free amine groups were prepared as follows.

[0116] [Table 2]

[0117]

[0118] As shown in Table 2 above, the degree of deacetylation (%) and the degree of methyl carboxyl substitution (%) are the same as those shown in Table 1, and the free amine group (%) represents the level of free amine group (-NH2) among the amine groups in the chitosan derivative, and a neutral solvent, disodium phosphate solution, was used as the solvent in the chitosan derivative solution. In this example, the experimental groups (N1, N2, N3) were prepared by performing N, O- random carboxymethyl substitution, and the level of free amine group was controlled by controlling the degree of methyl carboxyl substitution in the same manner as in Example 1-1.

[0119] Thereafter, a 2 w / v% chitosan derivative solution and a 2 v / v% disodium hydrogen phosphate (Na2HPO4) solution were mixed to prepare a liquid ion-sensitive hydrogel composition that can be injected under room temperature conditions, as shown in Fig. 6.

[0120]

[0121] 3-2. Evaluation of in vivo ion sensitivity using animal models

[0122] In this example, the gelation of the in vivo ion-sensitive hydrogel composition of Example 3-2 was evaluated using an animal model to determine the influence of the level of free amine groups of chitosan derivatives on the in vivo ion-sensitive behavior. 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, matrix formation due to gelation was visually confirmed, and the storage modulus (G') thereof was measured.

[0123] As a result, as shown in Fig. 7, the N1 group including a chitosan derivative with a free amine group level of 21% did not form a matrix in the body. On the other hand, as shown in Fig. 8, the N2 group including a chitosan derivative with a free amine group level of 34% formed a matrix with a storage modulus of 31.97±1.58 pa, which is similar to that of a hyaluronic acid filler with a low degree of cross-linking, and the N3 group including a chitosan derivative with a free amine group level of 90% was confirmed to form a matrix with a storage modulus of 154.31±13.21 pa. By controlling the elastic modulus of the matrix in the manner proposed herein, the in vivo ion-sensitive hydrogel composition forming a matrix having a storage elastic modulus of 5 to 50 Pa can be applied to medical materials such as joint injections and skin boosters, and the in vivo ion-sensitive hydrogel composition forming a matrix having a storage elastic modulus of 100 Pa or more can be applied to medical materials for subcutaneous repair.

[0124] In summary, the above experimental results indicate that by controlling the level of free amine groups of chitosan derivatives, along with the degree of deacetylation and substitution of methyl carboxyl groups of chitosan derivatives, it is possible to not only resolve the problems of existing biomaterials for tissue repair, but also to manufacture a biomaterial that has both the properties of a liquid formulation that can be injected at room temperature and the properties of being able to gel under in vivo conditions to form a matrix with viscoelasticity that matches the shape and properties of various tissues.

[0125]

[0126] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for producing an in vivo ion-sensitive hydrogel composition comprising a solution containing a chitosan derivative satisfying the following conditions: (a) shall have a degree of deacetylation of 70% or more; (b) more than 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups; and (c) More than 22% of the total amine groups in the chitosan derivative shall be free amine groups (-NH2).

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

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

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

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

5.

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

8. A composition according to claim 1, wherein the composition further comprises an aqueous solution containing phosphate ions.

9. In claim 8, the aqueous solution containing the phosphate ion is selected from the group consisting of sodium phosphate dibasic (disodium hydrogen phosphate, Na2HPO4), sodium phosphate monobasic (monosodium hydrogen phosphate monobasic, NaH2PO4), ammonium hydrogen phosphate dibasic (diammonium hydrogen phosphate, (NH4)2HPO4), ammonium dihydrogen phosphate (NH4H2PO4), sodium phosphate tribasic (Na3PO4), potassium phosphate dibasic (K2HPO4), potassium phosphate monobasic (KH2PO4), dimethyl phosphate (C2H7PO4), monomagnesium phosphate (Mg(H2PO4)2), magnesium phosphate dibasic (magnesium phosphate dibasic, A composition comprising at least one phosphate selected from the group consisting of lithium dihydrogen phosphate (MgHPO4), lithium phosphate (LiPO4), calcium hydrogen orthophosphate hydrate (CaHPO4·2H2O), and calcium hydrogen orthophosphate (CaHPO4).

10. A composition according to claim 1, wherein the composition further comprises a liquid formulation containing glycerol.

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

12. A method for producing an in vivo ion-sensitive hydrogel composition, comprising the step of mixing a solution containing a chitosan derivative satisfying the following conditions and an aqueous solution containing phosphate ions: (a) shall have a degree of deacetylation of 70% or more; (b) more than 50% of the hydroxyl groups and amine groups in the chitosan derivative are substituted with O-alkylcarboxyl groups or N-alkylcarboxyl groups; and (c) More than 22% of the total amine groups in the chitosan derivative shall be free amine groups (-NH2).

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

5.

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

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

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