The method of preparing hyaluronic acid cooperative hybrid complex gel for injection

Abstract

The present invention proposes a novel method of applying a thermal cycle to produce a cooperative complex comprising a mixture of high molecular weight (H-HA) and low molecular weight (L-HA) hyaluronic acids. This complex exhibits no change in viscosity over time and has a significantly lower viscosity than before the conventional thermal cycle. These characteristics cannot be explained simply by the thermal depolymerization of hyaluronic acid, but are attributed to cooperative interactions formed under specific conditions. This cooperative complex holds high value in medical, cosmetic, and food applications. In particular, in the medical field, it has high potential for use in therapies such as viscosupplementation for the treatment of joint inflammation or skin bioevitalization. This invention provides a new possibility for injecting or spraying high-concentration hyaluronic acid solutions with smaller needles through the low viscosity of the cooperative mixture. This enables the slow release of low and high molecular weight hyaluronic acids upon contact with the biological environment, allowing them to appropriately perform their respective biological roles.

Description

Technology Field

[0001] The present invention relates to hybrid cooperative complexes of hyaluronic acid, and describes their properties, manufacturing methods, and uses in the fields of medicine, cosmetics, and food. Background Technology

[0002] Hyaluronic acid (HA), commonly referred to as hyaluronan, is a negatively charged linear chain polysaccharide composed of repeating disaccharide units (-4GlcUAB1-3GlcNAcB1-). Here, D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) are connected by β- and β-glycosidic bonds.

[0003] Hyaluronic acid is a polysaccharide that is highly soluble in water, and HA solutions exhibit non-Newtonian viscoelastic behavior. These properties depend on molecular weight (and thus chain length, as HA is a linear polymer), concentration, pH, and ionic strength.

[0004] Weak interactions occurring between hyaluronic acid molecules, such as hydrogen bonds or hydrophobic interactions, can form very stable bonds when acting in a cooperative manner.

[0005] Cooperation develops when multiple bonds are formed simultaneously between molecules, and when weak bonds can be immediately reformed even if they are broken randomly.

[0006] Hyaluronic acid possesses high added value due to its biological properties and functions (its commercial value significantly surpasses that of other natural polysaccharides) and has diverse applications ranging from the medical field to cosmetics and nutraceuticals.

[0007] The viscoelastic properties of hyaluronic acid and the fact that it is completely non-toxic or immunogenic (the structure of HA is the same in all organisms) are the main reasons for its use in various applications.

[0008] In many applications, performance depends on the molecular weight of HA. To this end, the average molecular weight of HA and the polydispersity index (M w / M n ; Measuring the width of the molecular weight distribution curve, M n is the average molecular weight, M w Weight-average molecular weight is considered an important standard in HA production process development and application strategies.

[0009] HA induces various biological reactions depending on its molecular weight; generally, low molecular weight hyaluronic acid (hereinafter L-HA) is 5 × 10 3 ~ 5 × 10 4 High molecular weight hyaluronic acid (hereinafter H-HA) has a molecular weight of Da and is 1 × 10⁻⁶ 6 It has a molecular weight greater than or equal to Da.

[0010] L-HA and H-HA each have different aggregation modes, which significantly affect viscosity and biological properties.

[0011] In H-HA, all molecules in the solution form a three-dimensional network and maintain stable interactions, whereas in L-HA, less stable interactions are formed, resulting in lower viscosity and inconsistent results.

[0012] The viscosity of HA solutions decreases rapidly with molecular weight, which is due to differences in interaction between low molecular weight HA and high molecular weight HA. Conventional HA solutions undergo viscosity changes over time, which limits their practical applications.

[0013] In this regard, the diverse biological reactions of HA depending on molecular weight mean that low molecular weight hyaluronic acid (L-HA) and high molecular weight hyaluronic acid (H-HA) should be used according to each situation. The problem to be solved

[0014] The present invention relates to hybrid cooperative complexes of hyaluronic acid and injectable hyaluronic acid utilizing the same. Weak interactions occurring between hyaluronic acid molecules, such as hydrogen bonds or hydrophobic interactions, can form very stable bonds when acting in a cooperative manner. means of solving the problem

[0015] A hyaluronic acid complex according to an embodiment of the present invention may include the steps of: preparing an aqueous solution of low molecular weight hyaluronic acid (L-HA); preparing an aqueous solution of high molecular weight hyaluronic acid (H-HA); preparing a complex by physically centrifuging L-HA and H-HA; and heat-treating the L-HA and H-HA complex.

[0016] In one embodiment of the present invention, the molecular weight of L-HA is 1 × 10⁻⁶ 4 It can be less than or equal to Da.

[0017] In one embodiment of the present invention, the molecular weight of H-HA is 1.5 × 10⁻⁶ 6 Da to 3.5 × 10 6 It could be Da.

[0018] In one embodiment of the present invention, the mixing ratio of L-HA and H-HA may be between 0.1 and 10.

[0019] In one embodiment of the present invention, the centrifugal mixing step is performed by a physical method without adding a chemical crosslinking agent.

[0020] In one embodiment of the present invention, the heat treatment step may be performed at 121°C for 20 to 40 minutes.

[0021] In one embodiment of the present invention, the viscosity of the generated hyaluronic acid complex is lower than the viscosity of H-HA. Effects of the invention

[0022] The present invention deals with cooperative hybrid complexes of low molecular weight hyaluronic acid (L-HA) and high molecular weight hyaluronic acid (H-HA), abbreviated as "L / H-HA". The characteristics, production process, and applications of this complex in the fields of medicine, cosmetics, and food are described.

[0023] To overcome the limitation of existing technologies that could not reliably generate cooperative hybrid complexes by mixing L-HA and H-HA, we propose a method for forming stable L-HA and H-HA hybrid complexes using specific heat treatment conditions. These complexes maintain constant viscosity over time and provide improved performance compared to existing technologies. Specific details for implementing the invention

[0024] A hyaluronic acid complex according to an embodiment of the present invention may include the steps of: preparing an aqueous solution of low molecular weight hyaluronic acid (L-HA); preparing an aqueous solution of high molecular weight hyaluronic acid (H-HA); preparing a complex by physically centrifuging L-HA and H-HA; and heat-treating the L-HA and H-HA complex.

[0025] In one embodiment of the present invention, the molecular weight of L-HA is 1 × 10⁻⁶ 4 It can be less than or equal to Da.

[0026] In one embodiment of the present invention, the molecular weight of H-HA is 1.5 × 10⁻⁶ 6 Da to 3.5 × 10 6It could be Da.

[0027] In one embodiment of the present invention, the mixing ratio of L-HA and H-HA may be between 0.1 and 10.

[0028] In one embodiment of the present invention, the centrifugal mixing step is performed by a physical method without adding a chemical crosslinking agent.

[0029] In one embodiment of the present invention, the heat treatment step may be performed at 121°C for 20 to 40 minutes.

[0030] In one embodiment of the present invention, the viscosity of the generated hyaluronic acid complex is lower than the viscosity of H-HA.

[0031] Even weak bonds (hydrogen bonds and hydrophobic interactions) can form highly stable intermolecular interactions if they possess a cooperative nature. Cooperativeness develops when multiple bonds can be formed; because these bonds are weak, they can break randomly but can immediately reform.

[0032] In a solution containing a mixture of L-HA and H-HA, the following conditions are essential for cooperative intermolecular interactions to form:

[0033] 1. L-HA and H-HA must be present simultaneously in the same solution.

[0034] 2. The molecular weights of L-HA and H-HA must be appropriately set.

[0035] 3. The ratio of the two substances must be optimized.

[0036] 4. The temperature profile of the heat treatment process applied to the solution is important.

[0037] When heat treatment is performed on the mixed solution, all interchain interactions between H-HA and L-HA are broken at high temperatures, and subsequently, during the cooling phase, the molecules are randomly rearranged to form a stable hybrid system.

[0038] It has been confirmed that the physicochemical properties of this hybrid system, such as viscosity, do not change over time, which differentiates it from existing L-HA or H-HA single solutions.

[0039] 1. Principles of Cooperative Interaction

[0040] Weak bonds, such as hydrogen bonds or hydrophobic interactions, can form highly stable intermolecular interactions when they are cooperative in nature. Cooperativeness develops when multiple bonds can be formed; although these bonds may break randomly due to their weakness, they reform immediately. This allows the polymer structure to be maintained stably.

[0041] 2. Essential Conditions for L / H-HA Complex Formation

[0042] To form a cooperative hybrid complex of L-HA and H-HA, the following four conditions must be met:

[0043] 1. L-HA and H-HA must be present simultaneously in the same solution.

[0044] 2. The molecular weights of the L-HA and H-HA used must be appropriately set.

[0045] 3. The relative ratio of the two substances (L-HA / H-HA) must be optimized.

[0046] 4. The temperature and time settings of the heat treatment process are important.

[0047] 3. Formation of composite through heat treatment

[0048] When L-HA and H-HA exist in the same solution, heat treatment breaks the weak interactions between the two components and rearranges them to form a cooperative hybrid complex. The resulting complex exhibits stability, with physical properties such as viscosity remaining unchanged over time.

[0049] 4. Specific conditions for complex formation

[0050] · Heat treatment temperature: 121°C

[0051] · Cooling process: Stabilizes interactions through rapid cooling

[0052] Experimental Example 1) Production of cooperative hybrid L / HA composites at various temperatures

[0053] molecular weight 2.5 × 10 6 Da and polyvariance exponent (M w / M n A 2 wt% aqueous H / HA solution with a ) value of 1.5 and a molecular weight of 6.0 × 10 3 Prepare a 2 wt% L / HA aqueous solution with a polydispersity index of 1.8 and a Da value.

[0054] Starting the aqueous solution at 25°C, the maximum temperature (T) is reached within 10 minutes. max Heating with ) and set T max After maintaining for 20 or 40 minutes, rapidly cool to 25°C within 10 minutes.

[0055] Once the reaction is complete, viscosity is measured at a fixed shear rate (γ = 2 / s) at 25°C using an Anton Paar Physica MCR 301 rheometer, and molecular weight is measured using a size-exclusion chromatography (SEC) system.

[0056] Sample H-HA (mL) L-HA (mL) T max (°C) Processing time (minutes) Viscosity (Pas) H-HA 100 0 21.321 L-HA 0 100 0.002 L / H-HA 100 100 121 20 0.052 L / H-HA 100 100 110 20 0.083 L / H-HA 100 100 100 40 0.943

[0057] Experimental results showed that the H-HA solution alone exhibited high viscosity (21.321 Pa·s), while L-HA exhibited very low viscosity (0.002 Pa·s). When heat-treated in a mixed state of H-HA and L-HA, cooperative interactions were strengthened, forming a new complex and causing a change in viscosity. Considering that the viscosity of the L / H-HA complex was 0.052 Pa·s at 121°C for 20 minutes and 0.943 Pa·s at 100°C for 40 minutes, the viscosity of the complex showed a tendency to increase as the heat treatment temperature and time increased.

[0058] Experimental Example 2) Production of cooperative hybrid L / H-HA composites with different compositions

[0059] molecular weight 2.5 × 10 6 An aqueous H / HA solution with Da and a polydispersity index of 1.5, and a molecular weight of 6.0 × 10 3 Prepare an L / HA aqueous solution with a polydispersity index of 1.8 and set the weight ratio of L-HA to H-HA (L-HA / H-HA) to 0.0, 0.5, 1.0, and 1.5.

[0060] The mixed aqueous solution is heated to 121°C within 10 minutes starting at 25°C, maintained at that temperature for 20 minutes, and then rapidly cooled to 25°C within 10 minutes.

[0061] Viscosity and molecular weight are measured using the method of Experimental Example 1.

[0062] Sample H-HA (g) L-HA (g) L-HA / H-HA Processing time (minutes) Viscosity (Pas) H-HA 1.0 0 5.632 L / H-HA 1.0 0.5 0.5 20 0.094 L / H-HA 1.0 1.0 1.0 20 0.052 L / H-HA 1.0 1.5 1.5 20 0.048

[0063] Experimental results showed that the H-HA solution alone exhibited high viscosity (5.632 Pa·s), but when heat-treated in a mixture of H-HA and L-HA, cooperative interactions were strengthened to form a new complex, resulting in a change in viscosity. The viscosity of the L / H-HA complex was 0.094 Pa·s when the mixing ratio was 1:2, 0.052 Pa·s when it was 1:1, and 0.048 Pa·s when it was 2:1, confirming that viscosity decreases as the ratio of L-HA / H-HA increases.

[0064] As the mixing ratio of L-HA and H-HA increased, cooperative interactions were strengthened, which acted as a major factor in lowering the viscosity of the complex.

[0065] Experimental Example 3) Production of cooperative hybrid L / HA complexes using L-HA of different molecular weights

[0066] molecular weight 2.5 × 10 6 A 2 wt% aqueous H / HA solution with Da and a polydispersity index of 1.5, and a molecular weight of 6.0 × 10 3 2 wt% L / HA aqueous solution with Da and a polydispersity index of 1.8 (Sample 1) and a molecular weight of 1.2 × 10 5 Prepare a 2 wt% L / HA aqueous solution (Sample 2) with a polydispersity index of 1.7 and mix the L-HA and H-HA weight ratio (L-HA / H-HA) to 1.0.

[0067] The heat treatment process is carried out in the same manner as in Experimental Example 2, and the viscosity and molecular weight after heat treatment are measured using the method of Experimental Example 1.

[0068] Sample H-HA (mL) L-HA (mL) L-HA molecular weight (Da) Viscosity (Pas) H-HA 100 0 5.632 L-HA Sample 1 0 100 6.0 × 10 3 0.001 L-HA Sample 2 0 100 1.2 × 10 5 0.016 H-HA / Sample 1 Complex 100 100 6.0 × 10 3 0.052 H-HA / Sample 2 Complex 100 100 2.2 × 10 5 0.943

[0069] Experimental results showed that the H-HA solution alone exhibited high viscosity (5.632 Pa·s), but when heat-treated in a mixture of H-HA and L-HA, cooperative interactions were strengthened to form a new complex, resulting in a change in viscosity. The viscosity of L-HA was 0.001 Pas for Sample 1, which used L-HA with a small molecular weight, and 0.016 Pas for Sample 2, which used L-HA with a large molecular weight, confirming that viscosity increases as the molecular weight of L-HA increases.

[0070] Experimental Example 4) Kinetic analysis of cooperative hybrid L / HA composite formation depending on the presence or absence of heat treatment

[0071] Prepare and mix the aqueous hyaluronic acid solution of Experimental Example 1.

[0072] The heat treatment process is carried out in the same manner as in Experimental Example 2, but some mixed solutions are kept in a 25℃ environment and not heat-treated.

[0073] Viscosity and molecular weight are measured using the method of Experimental Example 1, and changes in viscosity over time are observed.

[0074] Time (day) Heat treatment application viscosity (Pas) Viscosity without heat treatment (Pas) 0 0.040 0.040 4 0.038 0.068 10 0.035 0.105 14 0.033 0.177 24 0.031 0.241

[0075] The initial viscosity of both the heat-treated and non-heat-treated samples was 0.040 Pas. However, the viscosity of the heat-treated sample remained largely unchanged at 0.031 Pas after 24 days, whereas the viscosity of the non-heat-treated sample increased over time to 0.241 Pas after 24 days. This is because, during heat treatment, cooperative interactions were strengthened to form a new complex, whereas in the non-heat-treated sample, H-HA and L-HA failed to form a complete cooperative bond.

[0076] Experimental Example 5) Preparation of a solid-state cooperative hybrid L / HA composite through precipitation from solution

[0077] After mixing the aqueous hyaluronic acid solution of Experimental Example 1, heat treatment is performed at 121°C for 20 minutes, and then rapidly cooled to 25°C. Anhydrous ethanol is slowly added in double the volume to the solution produced by cooling and stirred to obtain a precipitate in the form of a white powder.

[0078] The obtained precipitate is separated from the precipitation solution, partially dried, and then heated under vacuum to completely dry it, thereby obtaining an L / H-HA complex.

[0079] The yield of the L / H-HA composite obtained in the process was 99% of the theoretical value, and when the obtained composite was dissolved in water at 1% w / w, the dynamic viscosity of the solution was the same as the viscosity applied to heat treatment in Experimental Example 4.

[0080] The composite precipitated in a solid state retained its characteristics from the initial heat-treated state when dissolved in water, indicating that the composite is physicochemically stable.

[0081] Solvent selection plays a crucial role in enhancing the physical stability and purity of the complex, and anhydrous ethanol selectively precipitates the L / HA complex, allowing for the efficient collection of the solid-state complex.

[0082] Experimental Example 6) Preparation of a solid-state cooperative hybrid L / HA composite by solution freeze-drying

[0083] After mixing the aqueous solution of the hyaluronic acid from Experimental Example 1 and heat-treating it at 121°C for 20 minutes, the aqueous solution obtained is placed in a freeze-dryer to rapidly freeze it and then dried by removing moisture under vacuum to collect a sponge-shaped solid mass, and then the collected mass is mechanically processed to convert it into a white powder.

[0084] The yield of the L / H-HA composite obtained in the process was 99% of the theoretical value, and when the obtained composite was dissolved in water at 1% w / w, the dynamic viscosity of the solution was the same as the viscosity applied to heat treatment in Experimental Example 4.

[0085] The composite obtained by freeze-drying retained its characteristics from the initial heat-treated state when dissolved in water, indicating that the composite is physicochemically stable.

[0086] Experimental Example 7) Application of a cooperative hybrid L / HA complex in the field of skin biorevitalization

[0087] The L / H-HA complex obtained by freeze-drying in Experimental Example 6 is dissolved in saline at a concentration of 40 mg / mL and filled into a 1 mL syringe equipped with a 30 gauge needle to produce pharmaceutical grade hyaluronic acid for injection.

[0088] Ten volunteers with distinct signs of facial aging were injected with 1 mL of the existing product on the left side of their face and 1 mL of a syringe containing the manufactured hyaluronic acid complex on the right side of their face using the same technique to confirm the effects of wrinkle improvement and skin elasticity recovery after injection.

[0089] The experimental results showed that the skin regeneration effect lasted longer on the right side of the face, which was injected with the cooperative hybrid L / HA complex, and wrinkle improvement and skin elasticity restoration were more pronounced, demonstrating higher quality and long-term efficacy compared to existing products.

[0090] Since the complex has low viscosity, it could be easily injected even with a small gauge needle, which contributes to improved patient convenience and injection accuracy.

[0091] The L / HA complex is slowly released within the skin through the synergistic action of H-HA and L-HA, providing long-term skin regeneration effects, and it has been proven that the complex is more effective in moisturizing and restoring elasticity deep within the skin.

[0092] Experimental Example 8) Application of cooperative hybrid L / HA complexes in the field of viscosity supplementation

[0093] Pharmaceutical grade hyaluronic acid for injection is prepared using the method of Experimental Example 7.

[0094] The effects of viscosupplementation injections on the recovery of joint function and pain relief in both knees were evaluated after injection in five volunteers with bilateral knee joint disorders. Each volunteer was injected with 1 mL of the existing product into their left knee and 1 mL of a syringe containing the manufactured hyaluronic acid complex into their right knee using the same technique.

[0095] Experimental results showed that the right knee injected with the cooperative hybrid L / HA complex experienced faster pain relief and longer-lasting recovery of joint function, demonstrating higher quality and long-term efficacy compared to existing products.

[0096] Since the complex has low viscosity, it could be easily injected even with a small gauge needle, which contributes to improved patient convenience and injection accuracy.

[0097] The L / HA complex was slowly released within the skin through the synergistic action of H-HA and L-HA, providing long-term lubrication and shock absorption functions, and demonstrated that the complex is more effective in alleviating joint inflammation and tissue regeneration.

Claims

Claim 1 A method for preparing an injectable hyaluronic acid complex comprising: a step of preparing an aqueous solution of low molecular weight hyaluronic acid (L-HA); a step of preparing an aqueous solution of high molecular weight hyaluronic acid (H-HA); a step of preparing a complex by physically centrifuging L-HA and H-HA; and a step of heat-treating the L-HA and H-HA complex. Claim 2 In claim 1, the molecular weight of the L-HA is 1 × 10 4 A method for preparing an injectable hyaluronic acid complex characterized by having a value of Da or less. Claim 3 In claim 1, the molecular weight of the H-HA is 1.5 × 10⁻⁶ 6 Da to 3.5 × 10 6 A method for preparing an injectable hyaluronic acid complex characterized by being Da. Claim 4 A method for preparing an injectable hyaluronic acid complex according to claim 1, characterized in that the mixing ratio of L-HA and H-HA is between 0.1 and 10. Claim 5 A method for preparing an injectable hyaluronic acid complex, wherein, in claim 1, the centrifugal mixing step does not include a chemical crosslinking agent. Claim 6 A method for preparing an injectable hyaluronic acid complex according to claim 1, characterized in that the heat treatment step is performed at 121°C for 20 to 40 minutes. Claim 7 A method for preparing an injectable hyaluronic acid complex according to claim 1, characterized in that the viscosity of the hyaluronic acid complex is lower than the viscosity of H-HA.

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