Method for preparing auto-crosslinkable hyaluronic acid complex using acid-base equilibrium

Abstract

Provided is a method for preparing an auto-crosslinkable hyaluronic acid complex. Provided according to one aspect of the present invention is a method for preparing a hyaluronic acid complex, the method comprising the steps of: preparing a first mixed solution containing a first hyaluronic acid component and having a pH of 2.0 to 3.0; preparing a first mixed solution containing a second hyaluronic acid component and having a pH of 5.5 to 6.5; andpreparing crosslinked hyaluronic acid complex by mixing the first mixed solution and the second mixed solution and adjusting the pH to 6.5 to 7.5,

Description

Method for producing an auto-crosslinking hyaluronic acid complex using acid-base equilibrium

[0001] The present invention relates to a method for producing an auto-crosslinking hyaluronic acid complex using acid-base equilibrium, and more specifically, to a method for producing an auto-crosslinking hyaluronic acid complex through crosslinking of high-molecular-weight and low-molecular-weight hyaluronic acid components through two-component mixing using acid-base equilibrium and oxidation-reduction processes.

[0002] Hyaluronic acid is a polysaccharide polymerized by repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. It is a widely used ingredient in various fields of the body, including skin tissue, based on its excellent biocompatibility.

[0003] In particular, hyaluronic acid, based on its excellent water content, is widely distributed in skin tissues along with collagen, a protein component. If there is not enough hyaluronic acid, the skin's water content decreases and it dries out easily, so hyaluronic acid plays an important role in the smooth functioning of skin tissues.

[0004] Considering the importance of hyaluronic acid, various methods have been attempted to supplement it externally. However, oral administration makes it difficult for hyaluronic acid introduced into the digestive tract to reach the skin. Furthermore, topical application methods struggle to ensure effective hyaluronic acid penetration into the epidermal layer, making it difficult to expect sufficient hyaluronic acid replenishment.

[0005] To improve this method of introducing hyaluronic acid, various methods for directly introducing hyaluronic acid-containing compositions into the dermal layer of the skin have been actively studied. Representative methods include introducing hyaluronic acid-containing compositions into the skin via injection, using microneedle patches, and using needle rollers. These methods are known to exhibit improved hyaluronic acid delivery capabilities compared to conventional oral administration and topical application.

[0006] While methods for delivering hyaluronic acid to the dermal layer of the skin are improving, active research is also being conducted to improve the compositions or products containing hyaluronic acid that are delivered through this method. For example, when simply introducing hyaluronic acid into the dermal layer of the skin, hyaluronic acid's high bioabsorbability and biocompatibility cause the introduced hyaluronic acid to spread throughout the body through the circulatory system beyond the location of introduction. In this case, there was a problem in that sufficient hyaluronic acid was not delivered to the areas of the skin where improvement was expected through the introduction of hyaluronic acid.

[0007] To address these issues, a method has been proposed that goes beyond providing a composition or product containing dissolved hyaluronic acid, and instead directly delivers a composition or product containing cross-linked hyaluronic acid to the skin. In this case, the cross-linked hyaluronic acid maintains a certain level of cohesiveness as a cross-linked tissue after introduction into the skin, thereby reducing the amount of hyaluronic acid lost through the circulatory system and continuously providing hyaluronic acid to the targeted skin area.

[0008] However, despite the above advantages of cross-linked hyaluronic acid, since chemical cross-linking methods have been mainly used in the past for cross-linking hyaluronic acid, there is a problem in that the cross-linking agent added for the chemical cross-linking is introduced into the skin tissue together. Most chemical cross-linking agents have been found to have negative effects on skin tissue and the body, and even chemically damage normal skin tissue or cause inflammatory reactions, so an additional purification process is required to remove the residual chemical cross-linking agent from the cross-linked hyaluronic acid, and even if the residual chemical cross-linking agent is removed through purification, there is a problem in that the release of the chemical cross-linking agent and various harmful moieties derived therefrom from the cross-linked hyaluronic acid cannot be completely prevented.

[0009] Against this backdrop, there is a continuing need for a method for producing a cross-linked hyaluronic acid complex, namely, a method for producing an acid-base equilibrium auto-cross-linking hyaluronic acid complex.

[0010] The purpose of the present invention is to provide a method for producing a cross-linked hyaluronic acid complex, which is a method for producing an auto-cross-linking hyaluronic acid complex of acid-base equilibrium.

[0011] Another object of the present invention is to provide a method for producing a hyaluronic acid complex through a two-component mixing using acid-base equilibrium and oxidation-reduction processes.

[0012] Another object of the present invention is to provide a method for producing a hyaluronic acid complex comprising high molecular weight and low molecular weight hyaluronic acid.

[0013] The purposes of the present invention are not limited to those mentioned above, and other unmentioned purposes and advantages of the present invention can be understood through the following description and will be more clearly understood through the embodiments of the present invention. Furthermore, it will be readily apparent that the purposes and advantages of the present invention can be realized by the means and combinations thereof described in the specification.

[0014] According to a first aspect of the present invention for achieving the above object, a method for producing a hyaluronic acid complex is provided, comprising: preparing a first mixed solution containing a first hyaluronic acid component and having a pH of 2.0 to 3.0; preparing a second mixed solution containing a second hyaluronic acid component and having a pH of 5.5 to 6.5; and mixing the first mixed solution and the second mixed solution and adjusting the pH to 6.5 to 7.5 to produce a cross-linked hyaluronic acid complex.

[0015] In addition, according to a second aspect of the present invention, in the first aspect, the step of preparing the first mixed solution may include the step of preparing an aqueous solution in which a first hyaluronic acid component having a number average molecular weight of 1000 to 20000 kDa is dissolved; and the step of preparing the first mixed solution by mixing 2-ethoxyethanol and Na2CO3 with the aqueous solution.

[0016] In addition, according to the third aspect of the present invention, in the second aspect, the first hyaluronic acid component included in the first mixed solution may be 25 to 35 parts by weight based on 100 parts by weight of the first mixed solution.

[0017] In addition, according to the fourth aspect of the present invention, in the third aspect, the 2-ethoxyethanol included in the first mixed solution may be 1.5 to 3.0 parts by weight based on 100 parts by weight of the first mixed solution.

[0018] In addition, according to the fifth aspect of the present invention, in the fourth aspect, the Na2CO3 included in the first mixed solution may be 2.5 to 4.0 parts by weight based on 100 parts by weight of the first mixed solution.

[0019] In addition, according to a sixth aspect of the present invention, in the first aspect, the step of preparing the second mixed solution may include the step of preparing an aqueous solution in which a second hyaluronic acid component having a number average molecular weight of 100 to 500 kDa is dissolved; and the step of preparing a second mixed solution by mixing 2-hydroxybenzaldehyde and NaHCO3 with the aqueous solution.

[0020] In addition, according to the seventh aspect of the present invention, in the sixth aspect, the second hyaluronic acid component included in the second mixed solution may be 20 to 45 parts by weight based on 100 parts by weight of the second mixed solution.

[0021] In addition, according to the eighth aspect of the present invention, in the seventh aspect, the 2-hydroxybenzaldehyde included in the second mixed solution may be 2.5 to 3.5 parts by weight based on 100 parts by weight of the second mixed solution.

[0022] In addition, according to the ninth aspect of the present invention, in the eighth aspect, the NaHCO3 included in the second mixed solution may be 2.5 to 4.0 parts by weight based on 100 parts by weight of the second mixed solution.

[0023] In addition, according to the tenth aspect of the present invention, in the fifth aspect or the ninth aspect, the step of mixing the first mixed solution and the second mixed solution and adjusting the pH to 6.5 to 7.5 may include the step of mixing the first mixed solution and the second mixed solution, then adding hydrogen peroxide and stirring; and the step of adding NaOH to the stirred mixed solution to adjust the pH to 6.5 to 7.5.

[0024] The solutions to the above problems do not enumerate all the features of the present invention. The various features of the present invention, along with their corresponding advantages and effects, can be understood in more detail by referring to the detailed description below.

[0025] According to one aspect of the present invention, an object of the present invention is to provide a method for producing a cross-linked hyaluronic acid complex, wherein a method for producing an auto-cross-linking hyaluronic acid complex of acid-base equilibrium is provided.

[0026] According to another aspect of the present invention, a method for producing a hyaluronic acid complex through a two-component mixing using acid-base equilibrium and oxidation-reduction process can be provided.

[0027] According to another aspect of the present invention, a method for producing a hyaluronic acid complex comprising high molecular weight and low molecular weight hyaluronic acid can be provided.

[0028] In addition to the aforementioned effects, the specific effects of the present invention are described below along with the specific details for implementing the invention. Furthermore, the effects of the present invention are not limited to the effects described above and can be readily achieved by the means and combinations thereof described in the specification.

[0029] FIG. 1 is a diagram showing an example of a method for manufacturing a hyaluronic acid complex according to one embodiment of the present invention.

[0030] In this specification, singular expressions include plural expressions unless the context clearly indicates otherwise.

[0031] The numerical range indicated by the term "to" in this specification refers to a numerical range that includes the values ​​described before and after the term as the lower limit and the upper limit, respectively. When multiple numerical values ​​are disclosed as the upper and lower limits of an arbitrary numerical range, the numerical range disclosed in this specification can be understood as an arbitrary numerical range that includes any one of the multiple lower limit values ​​and any one of the multiple upper limit values ​​as the lower limit and the upper limit, respectively.

[0032] As used herein, terms such as "about" or "substantially" mean a reasonable amount of variation from the term that does not significantly alter the final result. These terms may be interpreted to include a variation of at least ±5% or at least ±10%, provided that such variation does not alter the meaning of the term and render it invalid.

[0033] FIG. 1 is a diagram showing an example of a method for manufacturing a hyaluronic acid complex according to one embodiment of the present invention.

[0034] Referring to FIG. 1, according to one aspect of the present invention, a method for producing a hyaluronic acid complex is provided, comprising: preparing a first mixed solution containing a first hyaluronic acid component and having a pH of 2.0 to 3.0; preparing a second mixed solution containing a second hyaluronic acid component and having a pH of 5.5 to 6.5; and mixing the first mixed solution and the second mixed solution and adjusting the pH to 1.5 to produce a cross-linked hyaluronic acid complex.

[0035] Step (S1) of preparing a first mixed solution containing a first hyaluronic acid component

[0036] In the method for producing the above hyaluronic acid complex, the step (S1) of producing a first mixed solution containing the first hyaluronic acid component and having a pH of 2.0 to 3.0 may be a step of producing a first mixed solution as a solution by dissolving the first hyaluronic acid component in a solvent.

[0037] The first hyaluronic acid component may be sodium hyaluronate. In addition, the solvent may be a water-soluble solvent, preferably water. The first mixture exhibits an acidity of pH 2.0 to 3.0 as the first hyaluronic acid component dissolves, and it is expected that this acidity is exhibited by the hydroxyl and carboxyl groups contained in the hyaluronic acid component.

[0038] In addition, in the step (S1), the first mixture solution in which the first hyaluronic acid component is dissolved may include an additional acid component and / or a base component to have a pH of 2.0 to 3.0. The additional acid component may be an acid component including, for example, glacial acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, etc., and the additional base component may be, for example, an amine compound such as NaOH, KOH, NH3, or a metal carbonate such as sodium bicarbonate and sodium carbonate.

[0039] When the pH of the first mixed solution is less than 2.0, there is a problem that the structure of the hyaluronic acid itself may be deformed as a large amount of NaOH is used in the process of adjusting the pH to about 1.5 after the first mixed solution and the second mixed solution are mixed as the pH of the first mixed solution becomes too low, and when the pH of the first mixed solution exceeds 3.0, sufficient ion exchange may not be performed when the first mixed solution is mixed with the second mixed solution, resulting in a low degree of crosslinking. Therefore, it is preferable that the pH of the first mixed solution satisfies the above numerical range.

[0040] In addition, in the step (S1), the step of preparing the first mixed solution may be a step (S1-1) of preparing an aqueous solution in which a first hyaluronic acid component having a number average molecular weight of 1000 to 20000 kDa is dissolved. The first hyaluronic acid component used in the step (S1-1) has a relatively large number average molecular weight, so that it can perform the function of a matrix in the hyaluronic acid complex ultimately produced, and can maintain the shape of the hyaluronic acid complex when introduced by including hyaluronic acid components having a relatively small number average molecular weight through chemical bonds or intermolecular forces. In addition, the first hyaluronic acid component can first release a low-molecular-weight hyaluronic acid component after the desired usage period of the hyaluronic acid complex has elapsed, and then be biologically decomposed and moved through the circulatory system.

[0041] In addition, in the step (S1-1), the step of preparing the aqueous solution, i.e., the aqueous solution in which the first hyaluronic acid component having a number average molecular weight of 1000 to 20000 kDa is dissolved, may include a step (S1-2) of preparing a first mixture solution by mixing 2-ethoxyethanol and Na2CO3 with the aqueous solution. The aqueous solution can eliminate aggregation of the first hyaluronic acid component by including 2-ethoxyethanol and can increase miscibility with the low molecular weight hyaluronic acid component. In addition, the aqueous solution can control the reaction rate during the process in which the first mixture solution is mixed with the second mixture solution and then neutralized with NaOH by including Na2CO3, and the release rate of the hyaluronic acid component can be controlled by controlling the rate of oxidation-reduction after the hyaluronic acid complex is introduced.

[0042] In the above step (S1-2), the first hyaluronic acid component included in the first mixed solution may be 25 to 35 parts by weight based on 100 parts by weight of the first mixed solution. If the content of the first hyaluronic acid component included in the first mixed solution is less than 25 parts by weight based on 100 parts by weight of the first mixed solution, there is a problem that crosslinking does not proceed sufficiently, and if the content of the first hyaluronic acid component included in the first mixed solution is more than 35 parts by weight based on 100 parts by weight of the first mixed solution, there is a problem that mixing with the low-molecular-weight hyaluronic acid component is not sufficiently performed. Therefore, it is preferable that the content of the first hyaluronic acid component included in the first mixed solution satisfies the above numerical range.

[0043] In the above step (S1-2), the 2-ethoxyethanol included in the first mixed solution may be 1.5 to 3.0 parts by weight based on 100 parts by weight of the first mixed solution. If the content of the 2-ethoxyethanol included in the first mixed solution is less than 1.5 parts by weight based on 100 parts by weight of the first mixed solution, there is a problem that the degree of aggregation of the first hyaluronic acid component is not sufficiently reduced, and if the content of the 2-ethoxyethanol included in the first mixed solution is more than 3.0 parts by weight based on 100 parts by weight of the first mixed solution, a hyaluronic acid complex may have a negative effect on a living body when introduced. Therefore, it is preferable that the content of the 2-ethoxyethanol included in the first mixed solution satisfies the above numerical range.

[0044] In the above step (S1-2), the Na2CO3 included in the first mixed solution may be 2.5 to 4.0 parts by weight based on 100 parts by weight of the first mixed solution. If the content of the Na2CO3 included in the first mixed solution is less than 2.5 parts by weight based on 100 parts by weight of the first mixed solution, there is a problem that it is difficult to control the degree of crosslinking because the buffering effect of the hyaluronic acid complex is not sufficient, and if the content of the Na2CO3 included in the first mixed solution is more than 4.0 parts by weight based on 100 parts by weight of the first mixed solution, there is a problem that an excessive amount of a base component is required in the neutralization process of the hyaluronic acid complex. Therefore, it is preferable that the content of the Na2CO3 included in the first mixed solution satisfies the above numerical range.

[0045] Step (S2) of preparing a second mixed solution containing the second hyaluronic acid component

[0046] In the method for producing the above hyaluronic acid complex, the step (S2) of producing a second mixed solution containing the second hyaluronic acid component and having a pH of 5.5 to 6.5 may be a step of producing a second mixed solution as a solution by dissolving the second hyaluronic acid component in a solvent.

[0047] The second hyaluronic acid component may be sodium hyaluronate. In addition, the solvent may be a water-soluble solvent, preferably water. The second mixture exhibits an acidity of pH 5.5 to 6.5 as the second hyaluronic acid component dissolves, and it is expected that this acidity is exhibited by the hydroxyl and carboxyl groups contained in the hyaluronic acid component.

[0048] In addition, in the step (S2), the second mixture in which the second hyaluronic acid component is dissolved may include an additional acid component and / or a base component to have a pH of 5.5 to 6.5. The additional acid component may be an acid component including, for example, glacial acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, etc., and the additional base component may be, for example, an amine compound such as NaOH, KOH, NH3, or a metal carbonate such as sodium bicarbonate and sodium carbonate.

[0049] When the pH of the second mixed solution is less than 5.5, the pH of the second mixed solution becomes too low, so that sufficient ion exchange is not performed when mixed with the first mixed solution, resulting in a low degree of crosslinking. When the pH of the second mixed solution exceeds 6.5, the ion exchange occurring during the subsequent neutralization process using NaOH is not sufficiently performed, resulting in a low degree of crosslinking. Therefore, it is preferable that the pH of the second mixed solution satisfies the above numerical range.

[0050] In addition, in the step (S2), the step of preparing the second mixed solution may be a step (S2-1) of preparing an aqueous solution in which a second hyaluronic acid component having a number average molecular weight of 100 to 500 kDa is dissolved. The second hyaluronic acid component used in the step (S2-1) has a relatively small number average molecular weight, and thus can perform a cross-linking role between high-molecular-weight hyaluronic acid components in the hyaluronic acid complex ultimately produced, and can have an excellent degree of cross-linking by being attached to the matrix composed of the high-molecular-weight hyaluronic acid component through chemical bonding or intermolecular attraction. In addition, as the second hyaluronic acid component is released, the matrix composed of the high-molecular-weight hyaluronic acid component can be biodegraded and moved through the circulatory system after the desired retention period has ended based on biocompatibility.

[0051] In addition, in the step (S2-1), the step of preparing the aqueous solution, i.e., the aqueous solution in which the second hyaluronic acid component having a number average molecular weight of 100 to 500 kDa is dissolved, may include a step (S2-2) of preparing a second mixture solution by mixing 2-hydroxybenzaldehyde and NaHCO3 with the aqueous solution. The aqueous solution can eliminate aggregation of the second hyaluronic acid component by including 2-hydroxybenzaldehyde and can increase miscibility with the high molecular weight hyaluronic acid component. In addition, the aqueous solution can control the reaction rate during the process in which the first mixture solution is mixed with the second mixture solution and then neutralized with NaOH by including NaHCO3, and the release rate of the hyaluronic acid component can be controlled by controlling the rate of oxidation-reduction after the hyaluronic acid complex is introduced.

[0052] In the above step (S2-2), the second hyaluronic acid component included in the second mixed solution may be 20 to 45 parts by weight based on 100 parts by weight of the second mixed solution. If the content of the second hyaluronic acid component included in the second mixed solution is less than 30 parts by weight based on 100 parts by weight of the second mixed solution, there is a problem that the amount of the low-molecular-weight hyaluronic acid component released from the hyaluronic acid complex is not sufficient, and if the content of the second hyaluronic acid component included in the second mixed solution exceeds 45 parts by weight based on 100 parts by weight of the second mixed solution, there is a problem that excessive aggregation occurs between low-molecular-weight hyaluronic acid components. Therefore, it is preferable that the content of the second hyaluronic acid component included in the second mixed solution satisfies the above numerical range.

[0053] In the above step (S2-2), the 2-hydroxybenzaldehyde included in the second mixed solution may be 2.5 to 3.5 parts by weight based on 100 parts by weight of the second mixed solution. If the content of the 2-hydroxybenzaldehyde included in the second mixed solution is less than 2.5 parts by weight based on 100 parts by weight of the second mixed solution, there is a problem that the degree of inclusion of the second hyaluronic acid component in the hyaluronic acid matrix is ​​not sufficient, and thus a sufficient degree of crosslinking is not secured. In addition, if the content of the 2-hydroxybenzaldehyde included in the second mixed solution is more than 3.5 parts by weight based on 100 parts by weight of the second mixed solution, a hyaluronic acid complex may have a negative effect on a living body when introduced. Therefore, it is preferable that the content of the 2-hydroxybenzaldehyde included in the second mixed solution satisfies the above numerical range.

[0054] In the above step (S2-2), the NaHCO3 included in the second mixed solution may be 2.5 to 4.0 parts by weight based on 100 parts by weight of the second mixed solution. If the content of the NaHCO3 included in the second mixed solution is less than 2.5 parts by weight based on 100 parts by weight of the second mixed solution, there is a problem that it is difficult to control the degree of crosslinking because the buffering effect of the hyaluronic acid complex is not sufficient, and if the content of the NaHCO3 included in the second mixed solution is more than 4.0 parts by weight based on 100 parts by weight of the second mixed solution, there is a problem that an excessive amount of a base component is required in the neutralization process of the hyaluronic acid complex. Therefore, it is preferable that the content of the NaHCO3 included in the second mixed solution satisfies the above numerical range.

[0055] Step (S3) of mixing the first mixed solution and the second mixed solution and preparing a cross-linked hyaluronic acid complex

[0056] In the method for producing the above hyaluronic acid complex, the step (S3) of mixing the first mixed solution and the second mixed solution and adjusting the pH to 1.5 to produce a cross-linked hyaluronic acid complex may be a step of mixing the first mixed solution and the second mixed solution and then adding a base component to adjust the pH. The base component used in the above step (S3) may be a strong base, and for example, it is preferable to use a component in the form of an OH salt of an alkali metal such as NaOH or KOH.

[0057] In addition, in the step (S3), the step of mixing the first mixed solution and the second mixed solution and adjusting the pH to 6.5 to 7.5 may include a step (S3-1) of mixing the first mixed solution and the second mixed solution, then adding hydrogen peroxide and stirring; and a step (S3-2) of adding NaOH to the stirred mixed solution to adjust the pH to 6.5 to 7.5.

[0058] In the above step (S3-1), the hydrogen peroxide is a hydrogen peroxide aqueous solution, and may be a 20 to 70% hydrogen peroxide aqueous solution, and preferably a 30 to 50% hydrogen peroxide aqueous solution. As the hydrogen peroxide is introduced after mixing the first mixed solution and the second mixed solution, it promotes an oxidation-reduction reaction between each component included in the first mixed solution and the second mixed solution, so that the second hyaluronic acid component can be attached or bound to the matrix of the first hyaluronic acid component, and the hyaluronic acid complex can have a physical structure in which the degree of crosslinking is sufficiently secured. It may be mixed in an amount of 1 to 6 parts by weight, and preferably 2 to 4 parts by weight, based on 100 parts by weight of the total mixed first and second mixed solutions.

[0059] In the above step (S3-2), the NaOH can reduce the reactivity of the reactive functional groups of the hyaluronic acid components included in the hyaluronic acid complex by being introduced into the mixture of the first mixed solution and the second mixed solution, improve the biocompatibility of the hyaluronic acid complex finally obtained, and maintain the acid-base equilibrium of the hyaluronic acid complex so that the crosslinking degree of the hyaluronic acid complex can be sufficiently maintained even in a biological environment. The NaOH can be provided in the form of an aqueous solution, and in this case, the NaOH aqueous solution can have a concentration of 0.1 to 1.5 M, and preferably can have a concentration of 0.7 to 1.2 M. When the NaOH aqueous solution is used, the amount of the NaOH aqueous solution can be used in an amount such that the pH of the mixture to which hydrogen peroxide is applied in the mixture of the first mixed solution and the second mixed solution becomes 6.5 to 7.5.

[0060] Other ingredients

[0061] The hyaluronic acid complex according to the present invention may include other components. That is, the hyaluronic acid complex according to the present invention may be introduced into a living body and exhibit various physiological effects depending on its type, and the other components may be combined with the hyaluronic acid complex described above and provided to the body in a gel form to provide various useful effects.

[0062] Other components according to the present invention include at least one or more of proteoglycan, glycosaminoglycan, atelocollagen, beta-glucan, ubiquinone, and vitamin E, and may specifically include proteoglycan, glycosaminoglycan, atelocollagen, beta-glucan, ubiquinone, and vitamin E. The proteoglycan, glycosaminoglycan, atelocollagen, and beta-glucan are components contained in living tissues, and are known to perform the function of ensuring the elasticity of the living body and maintaining the moisture content. Specifically, when the other components are used, the natural properties of the living body can be maintained or strengthened by delivering the additional components to the surrounding soft tissues. According to some embodiments of the present invention, when the other components including all of proteoglycan, glycosaminoglycan, atelocollagen, beta-glucan, ubiquinone, and vitamin E are used, the elasticity of the living body can be further strengthened without an inhibitory effect between each component, and at the same time, the moisture content in the living body can be more effectively maintained.

[0063] In some embodiments of the present invention, the weight ratio of proteoglycan, glycosaminoglycan, atelocollagen, beta-glucan, ubiquinone, and vitamin E may be 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5, and preferably 0.8 to 1.2: 0.8 to 1.2: 0.8 to 1.2: 0.8 to 1.2: 0.8 to 1.2: 0.8 to 1.2. According to some embodiments of the present invention, when the weight ratio of the proteoglycan, glycosaminoglycan, atelocollagen, beta-glucan, ubiquinone, and vitamin E satisfies the above numerical range, the elasticity of the body can be further enhanced without an inhibitory effect between each component.

[0064] In some embodiments of the present invention, the other component may be supported in at least one of an emulsion and a liposome, and specifically, may be supported in at least one of a nanoemulsion and an elastomeric liposome. According to some embodiments of the present invention, when the other component supported in at least one of a nanoemulsion and an elastomeric liposome is used, the other component can better penetrate the body's barrier and continuously implement various physiological functions at a controlled rate. For example, the size of the elastomeric liposome may be 20 to 200 nm. For example, the nanoemulsion may be an oil-in-water (O / W) emulsion.

[0065] In some embodiments of the present invention, the hyaluronic acid complex may further include at least one of a stabilizer and a strength modifier. Specifically, the stabilizer may impart structural stability to the hyaluronic acid complex, and the strength modifier may adjust the shape and strength of the hyaluronic acid complex. For example, the stabilizer may include at least one of methylparaben, propylparaben, phenoxyethanol, and chlorphenesin. For example, the strength modifier may include at least one of guar gum, locust bean gum, carrageenan, xanthan gum, glucomannan, gellan gum, tamarind gum, tara gum, and gum arabic. For example, the content of the stabilizer and strength modifier is not particularly limited and may be variously adjusted depending on the target physical properties.

[0066] physical properties

[0067] In some embodiments of the present invention, the osmotic pressure of the hyaluronic acid complex may be 300 to 400 mOsmol / kg, preferably 350 to 390 mOsmol / kg.

[0068] In some embodiments of the present invention, the viscosity of the hyaluronic acid complex may be 17,000 to 19,000 cP at 35 degrees Celsius, and preferably 17,500 to 18,800 cP.

[0069] In some embodiments of the present invention, the storage modulus (G') of the hyaluronic acid complex at 30 degrees Celsius may be 50 to 70 Pa, preferably 55 to 65 Pa. Furthermore, in some embodiments of the present invention, the storage modulus of the hyaluronic acid complex at 40 degrees Celsius may be 40 to 60 Pa, preferably 45 to 55 Pa.

[0070] In some embodiments of the present invention, the loss modulus (G'') of the hyaluronic acid complex at 30 degrees Celsius may be 110 to 140 Pa, preferably 115 to 130 Pa. Further, in some embodiments of the present invention, the loss modulus of the hyaluronic acid complex at 40 degrees Celsius may be 80 to 110 Pa, preferably 90 to 105 Pa.

[0071] In some embodiments of the present invention, the cytotoxicity of the hyaluronic acid complex may be grade 0 or 1. For example, the cytotoxicity may be analyzed according to the criteria described in Table 8 below.

[0072] Hyaluronic acid complex

[0073] In some embodiments of the present invention, the hyaluronic acid complex can be introduced into a living body on its own, or, if necessary, in combination with various effective ingredients. The method by which the hyaluronic acid complex is introduced into a living body is not particularly limited, and any method that maintains the technical spirit of the present invention may be used.

[0074] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, this is merely an example, and the scope of the present invention is not limited by the following contents.

[0075] [Manufacturing Example: Manufacturing of Hyaluronic Acid Complex]

[0076] <Example 1: Preparation of hyaluronic acid complex according to the present invention>

[0077] As the first hyaluronic acid component, 30 g of sodium hyaluronate (number average molecular weight: 1,400 kDa) was added to 65 g of distilled water to prepare a sodium hyaluronate aqueous solution. 2.0 g of 2-ethoxyethanol and 3.0 g of Na2CO3 were added to the sodium hyaluronate aqueous solution, and the mixture was mixed for 20 minutes at a rotation speed of 300 RPM using a magnetic stirrer to prepare a first mixture solution.

[0078] As a second hyaluronic acid component, 30 g of sodium hyaluronate (number average molecular weight: 300 kDa) was added to 64 g of distilled water to prepare a sodium hyaluronate aqueous solution. 3.0 g of 2-hydroxybenzaldehyde and 3.0 g of NaHCO3 were added to the sodium hyaluronate aqueous solution, and the mixture was mixed for 20 minutes at a rotation speed of 300 RPM using a magnetic stirrer to prepare a second mixture.

[0079] The first and second mixtures were mixed using a magnetic stirrer at a rotation speed of 200 RPM for 10 minutes, then 7.5 g of a 40% aqueous hydrogen peroxide solution was added and stirred for 20 minutes. Thereafter, the mixture was neutralized using a 1.0 M aqueous NaOH solution so that the pH of the mixture became approximately 7.0. Thereafter, moisture was removed so that the concentrations of the first hyaluronic acid component and the second hyaluronic acid component were each 16 g / l, thereby finally preparing the complex of Example 1.

[0080] <Comparative Examples 1 to 10: Preparation of Comparative Hyaluronic Acid Complexes>

[0081] Using the same method as the Example 1 complex, the Examples 2 to 11 complexes were finally prepared based on the amounts of each component described in Tables 1 and 2 below.

[0082]

[0083]

[0084] *HA1: 1st hyaluronic acid component, sodium hyaluronate (number average molecular weight: 1,400 kDa)

[0085] **HA2: Second hyaluronic acid component, sodium hyaluronate (number average molecular weight: 300 kDa)

[0086] [Experimental Example 1: Osmotic Evaluation of Hyaluronic Acid Complexes]

[0087] In order to evaluate the osmotic pressure of the complex of Example 1 and the complexes of Comparative Examples 1 to 10, the osmotic pressure was measured using an osmometer (OSMOMAT 3000 BASIC) after calibration with standard solutions of osmotic pressures of 0 and 400. The results are shown in Table 3 below.

[0088]

[0089] Referring to Table 3 above, the complex of Example 1 exhibited superior osmotic pressure values ​​compared to the complexes of Comparative Examples 1 to 10, confirming that it had superior biocompatibility. In particular, it was confirmed that the complex of Example 1 could control the osmotic pressure values ​​to the levels required by various regulations by including 2-ethoxyethanol and 2-hydroxybenzaldehyde in specific numerical ranges and including Na2CO3 and NaHCO3 in specific numerical ranges.

[0090] [Experimental Example 2: Viscosity Evaluation of Hyaluronic Acid Complexes]

[0091] To confirm the viscosity of the composites of Example 1 and Comparative Examples 1 to 10, the viscosity was measured at 35°C using a rotational viscometer (BROOKFIELD, 7 rod, 20 RPM, 2 min). The results are shown in Table 4 below.

[0092]

[0093] Referring to Table 4 above, the Example 1 complex exhibited superior viscosity compared to the Comparative Examples 1 to 10 complexes, confirming that it possessed superior physical properties based on a high degree of crosslinking. In particular, the Example 1 complex was confirmed to have superior viscosity and excellent rheological properties, making it suitable for application in various applications of hyaluronic acid complexes.

[0094] [Experimental Example 3: Rheological Properties of Hyaluronic Acid Complexes]

[0095] In order to confirm the rheological properties of the composite of Example 1 and the composites of Comparative Examples 1 to 10, a rheometer was used to perform a temperature sweep (1 Hz, frequency) from 30°C to 40°C. The changes in storage modulus (G', Pa) and loss modulus (G'', Pa) according to temperature were shown in Table 5 below.

[0096]

[0097] Referring to Table 5 above, the Example 1 composite exhibits an excellent storage modulus compared to the Comparative Examples 1 to 10 composites, confirming excellent durability based on a high degree of crosslinking. In addition, the Example 1 composite exhibits an excellent loss modulus compared to the Comparative Examples 1 to 10 composites, confirming excellent shape recovery ability against deformation based on a high degree of crosslinking. In particular, since the Example 1 composite has the above characteristics in the range of 30°C to 40°C, it can be expected that the hyaluronic acid composite according to the present invention will have the above excellent characteristics when applied to a living body.

[0098] [Experimental Example 4: Evaluation of Gel Stability of Hyaluronic Acid Complexes]

[0099] The complexes of Example 1 and Comparative Examples 1 to 10 (3 mL) were placed in vials and left to stand in an incubator at 38°C. After 20 minutes, an equal amount of distilled water at the same temperature was added and left to stand in the incubator, and the distilled water was replaced daily. After 10 days, the weight loss (%) of the temperature-sensitive hydrogel compositions relative to the initial weight is shown in Table 5 below. Specifically, a lower weight loss (%) may indicate better gel stability.

[0100]

[0101] Referring to Table 6 above, it can be confirmed that the Example 1 complex exhibits a significantly lower weight loss ratio compared to the Comparative Examples 1 to 10 complexes, thereby exhibiting excellent gel stability based on a high degree of crosslinking, and further exhibiting excellent water retention properties. In particular, the Example 1 complex exhibits improved gel stability and water retention properties compared to the Comparative Example 2 complex not containing 2-ethoxyethanol and the Comparative Example 3 complex containing a low amount of 2-ethoxyethanol, thereby confirming that the hyaluronic acid complex has excellent gel stability when containing 2-ethoxyethanol within a specific numerical range. In addition, the Example 1 complex showed improved gel stability and water retention properties compared to the Comparative Example 5 complex not containing 2-hydroxybenzaldehyde and the Comparative Example 6 complex containing a low amount of 2-hydroxybenzaldehyde, confirming that the hyaluronic acid complex has excellent gel stability when containing 2-hydroxybenzaldehyde within a specific numerical range.

[0102] [Experimental Example 5: Cytotoxicity Test of Hyaluronic Acid Complex]

[0103] Human dermal fibroblasts (NHDF) were cultured at 2X10 5The cells were dispensed into a 6-well culture plate (Corning, USA) at a concentration of 10 cells / well and cultured in an incubator at 40°C. After 48 hours, the cells were in the form of a thin film (1x1cm 2 ) were placed on the center of a culture dish to contact the cells and cultured for 24 hours. Each hyaluronic acid complex film and the cultured cells were stained with hematoxylin (Hematoxylin, Sigma), and the toxicity reaction area was calculated using ImageJ software. The degree of toxicity was evaluated based on the size of the toxicity reaction area according to the standard described in Table 6 below (MCIM Amin, N. Ahmad, N. Halib, and I. Ahmad, Carbohydr. Polym., 88, 465, 2012). Here, a lower grade means lower cytotoxicity of the hydrogel. The results are shown in Table 7 below.

[0104]

[0105]

[0106] Referring to Tables 7 and 8 above, it was confirmed that the Example 1 complex exhibited significantly lower cytotoxicity compared to the Comparative Examples 1 to 10 complexes. In particular, the Example 1 complex exhibited significantly improved cytotoxicity compared to the Comparative Example 8 complex not containing Na2CO3 and NaHCO3, and exhibited improved cytotoxicity compared to the Comparative Example 9 complex containing a low content of Na2CO3 and NaHCO3, thereby confirming that when Na2CO3 and NaHCO3 are contained within a specific numerical range, the activity expression of 2-ethoxyethanol and 2-hydroxybenzaldehyde contained in the hyaluronic acid complex can be suppressed or substantially eliminated.

[0107] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims

1. A step of preparing a first mixed solution containing a first hyaluronic acid component and having a pH of 2.0 to 3.0; A step of preparing a second mixed solution having a pH of 5.5 to 6.5, including a second hyaluronic acid component; and A step of mixing the first mixed solution and the second mixed solution and adjusting the pH to 6.5 to 7.5 to prepare a cross-linked hyaluronic acid complex; A method for producing a hyaluronic acid complex, comprising:

2. A method for producing a hyaluronic acid complex, comprising: preparing an aqueous solution in which a first hyaluronic acid component having a number average molecular weight of 1000 to 20000 kDa is dissolved; and preparing a first mixed solution by mixing 2-ethoxyethanol and Na2CO3 with the aqueous solution.

3. A method for producing a hyaluronic acid complex, wherein the first hyaluronic acid component included in the first mixed solution in the second paragraph is 25 to 35 parts by weight based on 100 parts by weight of the first mixed solution.

4. A method for producing a hyaluronic acid complex, wherein, in the third paragraph, 2-ethoxyethanol included in the first mixed solution is 1.5 to 3.0 parts by weight based on 100 parts by weight of the first mixed solution.

5. A method for producing a hyaluronic acid complex, wherein, in the fourth paragraph, Na2CO3 included in the first mixed solution is 2.5 to 4.0 parts by weight based on 100 parts by weight of the first mixed solution.

6. A method for producing a hyaluronic acid complex, wherein the step of producing the second mixed solution in the first paragraph comprises the step of producing an aqueous solution in which a second hyaluronic acid component having a number average molecular weight of 100 to 500 kDa is dissolved; and the step of mixing 2-hydroxybenzaldehyde and NaHCO3 with the aqueous solution to produce the second mixed solution.

7. A method for producing a hyaluronic acid complex, wherein the second hyaluronic acid component included in the second mixed solution in the sixth paragraph is 20 to 45 parts by weight based on 100 parts by weight of the second mixed solution.

8. A method for producing a hyaluronic acid complex, wherein in paragraph 7, 2-hydroxybenzaldehyde included in the second mixed solution is 2.5 to 3.5 parts by weight based on 100 parts by weight of the second mixed solution.

9. A method for producing a hyaluronic acid complex in the 8th paragraph, wherein the amount of NaHCO3 included in the second mixed solution is 2.5 to 4.0 parts by weight based on 100 parts by weight of the second mixed solution.

10. A method for producing a hyaluronic acid complex, wherein the step of mixing the first mixed solution and the second mixed solution and adjusting the pH to 6.5 to 7.5 in the 5th or 9th paragraph comprises the steps of mixing the first mixed solution and the second mixed solution, then adding hydrogen peroxide and stirring; and the step of adding NaOH to the stirred mixed solution to adjust the pH to 6.5 to 7.5.

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