High-performance sodium hyaluronate composite gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of high polymer materials, in particular to high-performance sodium hyaluronate composite gel as well as a preparation method and application thereof. The method comprises the following steps: uniformly mixing low-molecular-weight sodium hyaluronate and high-molecular-weight sodium hyaluronate, adding the mixture into a phosphate buffer solution, and stirring and dissolving the mixture to obtain a mixed solution; heating, performing water-bath heating and ice-water bath, repeating for 2-3 times to obtain a solution subjected to thermal cycle treatment, freezing and then unfreezing to obtain unfrozen gel; the pH value of the unfrozen gel is adjusted to 6.8-7.6, water is added, stirring treatment is conducted, and gel obtained after stirring treatment is obtained, namely the high-performance sodium hyaluronate composite gel is obtained. The gel provided by the invention is far better than a simple physically blended sample in rheological properties (such as elastic modulus and viscosity), moisturizing durability and mechanical stability, and because no chemical cross-linking agent is added, the intermolecular acting force is regulated and controlled only through a physical method, the product biocompatibility is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of high polymer materials, and in particular to a high-performance sodium hyaluronate composite gel and a preparation method and application thereof. BACKGROUND

[0002] Hyaluronic acid (HA) is a natural linear polysaccharide, which is widely used in the fields of cosmetics, medical devices and pharmaceuticals due to its excellent water retention, viscoelasticity and biocompatibility. HA with different molecular weights has different characteristics: high molecular weight HA (usually > 1000 kDa) has good film-forming property and strong water retention capacity, but it has high viscosity and poor permeability; low molecular weight HA (usually < 500 kDa) has shorter molecular chains, which can easily penetrate into the deep dermis, promote cell adhesion and proliferation, and promote collagen regeneration through inflammatory factors.

[0003] At present, in order to take into account the advantages of high and low molecular weight HA, the industry usually adopts a simple physical blending method. However, this method is only a simple superposition of the characteristics of high and low molecular weight HA, and cannot achieve synergistic enhancement of performance. There is a lack of effective intermolecular force connection between HA segments with two different molecular weights, resulting in limited mechanical strength, stability and durability of the composite system.

[0004] Therefore, it is a technical problem to be solved in the art to develop a method that can actively induce the formation of a stable and dense intermolecular crosslinking network between high and low molecular weight HA, thereby obtaining a sodium hyaluronate composite gel with excellent permeability, high mechanical strength and long-lasting water retention. SUMMARY

[0005] In order to solve the technical problems in the prior art, the purpose of the present application is to provide a high-performance sodium hyaluronate composite gel and a preparation method and application thereof to solve the above technical problems.

[0006] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions.

[0007] According to the first aspect of the present application, a high-performance sodium hyaluronate composite gel is provided, and the preparation raw materials include: low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and phosphate buffer; the molecular weight of the low molecular weight sodium hyaluronate is 35 kDa-350 kDa, and the molecular weight of the high molecular weight sodium hyaluronate is 750 kDa-1500 kDa; the mass ratio of the low molecular weight sodium hyaluronate to the high molecular weight sodium hyaluronate is (1:2)-(2:1). The mass ratio of the low molecular weight sodium hyaluronate to the high molecular weight sodium hyaluronate is (1:2)-(2:1), which is the basis for forming an effective hydrogen bond network.

[0008] In some embodiments, the pH of the phosphate buffer is 5.0-6.0; the osmotic pressure of the phosphate buffer is 300-600 mOsmol / kg. The phosphate buffer with an osmotic pressure of 300-600 mOsmol / kg can provide a high ionic strength PBS environment, which is a key prerequisite for inducing hydrogen bond formation.

[0009] In some embodiments, in the high-performance sodium hyaluronate composite gel, the total content of the low-molecular-weight sodium hyaluronate and the high-molecular-weight sodium hyaluronate is 2.8-5.5 wt%.

[0010] The preparation method provided by the present application promotes the formation of a dense hydrogen bond three-dimensional network between high and low molecular weight sodium hyaluronate through specific raw material ratio and unique thermal cycling-freezing physical crosslinking process.

[0011] According to the second aspect of the present application, the present application provides a preparation method of a high-performance sodium hyaluronate composite gel, comprising the following steps: (1) uniformly mixing low-molecular-weight sodium hyaluronate and high-molecular-weight sodium hyaluronate (raw material mixing) to obtain a mixed powder, adding the mixed powder into a phosphate buffer (PBS buffer), and stirring and dissolving (stirring and dissolving under low temperature conditions until the sodium hyaluronate (HA) is completely dissolved and uniform) to obtain a mixed solution; (2) thermal cycling treatment: heating the mixed solution (viscous solution in which sodium hyaluronate is completely dissolved) obtained in step (1) in a water bath, and then immediately transferring the solution to an ice water bath for rapid cooling; repeating the operation of step (2) 2-3 times to obtain a solution after thermal cycling treatment; (3) freezing-thawing treatment: placing the solution after thermal cycling treatment in step (2) in a refrigerator at-30℃ to-15℃ for freezing treatment to obtain a frozen product, then thawing to obtain a thawed gel; adjusting the pH of the thawed gel to 6.8-7.6 using a low-concentration sodium hydroxide solution, adding high-purity water, and stirring to obtain a stirred gel, which is the high-performance sodium hyaluronate composite gel.

[0012] In some embodiments, the temperature for stirring and dissolving in step (1) is 2-8℃.

[0013] In some embodiments, the mass ratio of the low-molecular-weight sodium hyaluronate to the high-molecular-weight sodium hyaluronate in step (1) is (1:2)-(2:1).

[0014] In some embodiments, the pH of the phosphate buffer in step (1) is 5.0-6.0; In some embodiments, the osmotic pressure of the phosphate buffer in step (1) is 300-600 mOsmol / kg. The phosphate buffer used in the present application has a high ion concentration.

[0015] In some embodiments, the content of sodium hyaluronate (the sum of the content of low-molecular-weight sodium hyaluronate and the content of high-molecular-weight sodium hyaluronate) in the mixed solution in step (1) is 6%-8% (wt%).

[0016] In some embodiments, the temperature of the water bath heating in step (2) is 80-100℃, the time of the water bath heating is 15-30 minutes; the time of the ice water bath is 15-30 minutes. This heating-cooling process is defined as one thermal cycle, which is repeated 2-3 times. The thermal cycle method used in the present application is a unique physical crosslinking process. The thermal cycle method cooperates with the subsequent freezing treatment and thawing, and is the core process step for realizing the strengthened hydrogen bond network and achieving performance breakthrough. The thermal cycle method and the freezing-thawing step are indispensable and cannot be reversed in order.

[0017] In some embodiments, the temperature of the freezing treatment in step (3) is -30℃ to -15℃, and the time of the freezing treatment is 24-48 hours; the thawing includes placing the frozen material under a temperature of 2-30℃ until complete thawing.

[0018] In some embodiments, the thawing in step (3) includes placing the frozen material under room temperature or refrigeration conditions for natural thawing.

[0019] In some embodiments, the stirring treatment in step (3) includes stirring at a pressure of 0.15-0.25 MPa and a stirring speed of 15-25 rpm for 30-60 min.

[0020] In some embodiments, the temperature of the stirring treatment in step (3) is 2-8℃. The slow dilution of the thawed gel under low temperature and pressure is aimed to maintain the fragile hydrogen bond network that has been formed not to be destroyed during the concentration reduction.

[0021] In some embodiments, the final concentration of sodium hyaluronate in the gel after the stirring treatment in step (3) is 2.8-5.5wt%.

[0022] In some embodiments, the gel after the stirring treatment in step (3) is subjected to sterilization treatment; the sterilization treatment is high-temperature steam sterilization. In some embodiments, the sterilization process includes: vacuum degassing the stirred gel, filling it into a container (vial or pre-filled syringe), heating it to 120-128°C within 10 minutes, maintaining the temperature at 120-128°C for 4-15 minutes, sterilizing it with high-temperature steam, cooling it to below 80°C within 6 minutes, and finally transferring the container containing the stirred gel to an ice-water bath for cooling for 60-90 minutes to obtain a sterilized high-performance sodium hyaluronate composite gel.

[0023] In some embodiments, step (3) of adjusting the pH of the thawed gel to 6.8-7.6 is carried out at a temperature of 2-8°C.

[0024] According to a third aspect of the present invention, the present invention provides the application of high-performance sodium hyaluronate composite gel in the preparation of cosmetics, skin care products, and medical aesthetic products.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Synergistic cross-linking network: The method provided by this invention effectively shields the electrostatic repulsion between polymer chains by limiting the molecular weight and ratio of sodium hyaluronate (HA) within a specific range and combining it with a buffer environment of high ionic strength and specific pH, thus creating optimal conditions for the formation of intermolecular hydrogen bonds. The low molecular weight HA chains act as "cross-linking bridges," filling and connecting the "backbone" composed of high molecular weight HA, forming a dense and stable three-dimensional hydrogen bond network; (2) Significantly improved performance: The method provided by this invention uses a unique "thermal cycling-freezing" physical process to "reshape" and "strengthen" the initially formed hydrogen bond network. High-temperature treatment breaks some weak hydrogen bonds, allowing the molecular chains to expand; rapid cooling "freezes" this expanded conformation and triggers an explosive and orderly recombination of hydrogen bonds. The subsequent freeze-thaw process further concentrates and compresses the HA chains through the physical displacement effect of ice crystals, greatly enhancing the hydrogen bond crosslinking density; the final gel obtained is far superior to samples with simple physical blending in terms of rheological properties (such as elastic modulus and viscosity), moisture retention, and mechanical stability.

[0026] (3) Pure and safe: The method provided by this invention does not introduce any chemical cross-linking agents in the entire preparation process, but only regulates the intermolecular forces through physical methods, resulting in products with extremely high biocompatibility. Detailed Implementation

[0027] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below.

[0028] It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0029] Example 1 The preparation method of high-performance sodium hyaluronate composite gel includes the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 350 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1500 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:2), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS buffer, pH=5.0, osmotic pressure of 310 mOsmol / kg) pre-cooled to 2 ℃ and dissolved under low-speed mechanical stirring at 2 ℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 8 wt%. (3) After sealing the obtained sodium hyaluronate solution in a container, heat it in a water bath at 100 °C for 30 minutes, and then immediately transfer it to an ice water bath at 0 °C for rapid cooling for 30 minutes. This thermal cycle process is repeated 3 times (i.e., the operation of step (3) is repeated 3 times) to obtain the solution after thermal cycle treatment. (4) The solution after thermal cycling is placed into a container and frozen in a refrigerator at -20 ℃ for 48 hours. Then it is transferred to a refrigerator at 2 ℃ to thaw until completely thawed, and a preliminary gel (the thawed gel) is obtained. (5) At 2 ℃, the pH of the thawed gel was adjusted to between 6.8 and 7.6 using a 0.1wt% sodium hydroxide solution. Then, high-purity water was added, and the gel was stirred for 1 hour at 15 rpm in a pressure stirring vessel with a pressure of 0.25 MPa to dilute it evenly to a final sodium hyaluronate concentration of 5.5wt%, thus obtaining the stirred gel, which is the high-performance sodium hyaluronate composite gel. (6) After vacuum degassing the gel after the above stirring treatment, fill it into a pre-filled syringe, and then sterilize it with high temperature steam. The sterilization temperature is 128 ℃ and the sterilization time is 4 min. The time for the sterilization treatment to rise from room temperature to 128 ℃ is controlled within 10 min (the specific heating time in Example 1 is 5 min). Within 6 min, the temperature is cooled from the sterilization temperature (128 ℃) to 80 ℃ to obtain the sterilized gel. Then, the sterilized gel is quickly placed in an ice water bath for rapid cooling (the time in the ice water bath is 90 min) to obtain the final product, namely the sterilized high-performance sodium hyaluronate composite gel.

[0030] Example 2 The preparation method of high-performance sodium hyaluronate composite gel includes the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 150 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 850 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS, pH=5.5, osmotic pressure 340 mOsmol / kg) pre-cooled to 4 ℃ and dissolved under low-speed mechanical stirring at 4 ℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 7 wt%. (3) After sealing the obtained sodium hyaluronate solution in a container, heat it in a water bath at 90 °C for 20 minutes, and then immediately transfer it to an ice water bath at 0 °C for rapid cooling for 20 minutes. Repeat this thermal cycle process 3 times (i.e. repeat step (3) 3 times) to obtain the solution after thermal cycle treatment. (4) The solution after thermal cycling is placed into a container and frozen in a refrigerator at -20 ℃ for 36 hours. Then it is moved to a refrigerator at 4 ℃ to thaw until completely thawed, and a preliminary gel (the thawed gel) is obtained. (5) At 4 ℃, the pH of the thawed gel was adjusted to between 6.8 and 7.6 using a 0.1 wt% sodium hydroxide solution. Then, high-purity water was added, and the gel was stirred for 45 min at 20 rpm in a pressure stirring vessel with a pressure of 0.2 MPa to dilute it evenly to a final sodium hyaluronate concentration of 4.8 wt%, thus obtaining the stirred gel, which is the high-performance sodium hyaluronate composite gel. (6) After vacuum degassing the gel after the above stirring treatment, fill it into a pre-filled syringe, and then sterilize it with high temperature steam. The sterilization temperature is 125 ℃ and the sterilization time is 6 min. The time for the sterilization treatment to rise from room temperature to 125 ℃ is controlled within 10 min (the specific heating time in Example 2 is 6 min). Within 6 min, the temperature is reduced from the sterilization temperature (125 ℃) to 80 ℃. Then, the sterilized gel is quickly placed in an ice water bath for rapid cooling (the time in the ice water bath is 60 min) to obtain the final product, namely the sterilized high-performance sodium hyaluronate composite gel.

[0031] Example 3 The preparation method of high-performance sodium hyaluronate composite gel includes the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 230 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1000 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was slowly added to phosphate buffer (PBS buffer, pH=5.5, osmotic pressure of 590 mOsmol / kg) that had been pre-cooled to 4 ℃ and dissolved until there were no obvious white lumps under low-speed mechanical stirring at 4 ℃ and 15 rpm to obtain a sodium hyaluronate solution with a total content of 7 wt%. (3) After sealing the obtained sodium hyaluronate solution in a container, heat it in a water bath at 90 ℃ for 20 minutes, and then immediately transfer it to an ice water bath at 0 ℃ for rapid cooling for 20 minutes. This thermal cycle process is repeated 3 times (i.e., the operation of step (3) is repeated 3 times) to obtain the solution after thermal cycle treatment. (4) The solution after thermal cycling is put into a container and placed in a refrigerator at -20 ℃ for 36 hours. Then it is moved to a refrigerator at 4 ℃ to thaw until completely thawed to obtain a preliminary gel (the thawed gel). (5) At 4 ℃, the pH of the thawed gel was adjusted to between 6.8 and 7.6 using a 0.1 wt% sodium hydroxide solution. Then, high-purity water was added, and the gel was slowly stirred for 45 min at 20 rpm in a pressure stirring vessel at 0.2 MPa to dilute it evenly to a final sodium hyaluronate concentration of 3.2 wt%, thus obtaining the stirred gel, which is the high-performance sodium hyaluronate composite gel. (6) After vacuum degassing the gel after the above stirring treatment, fill it into a pre-filled syringe, and then sterilize it with high temperature steam. The sterilization temperature is 125 ℃ and the sterilization time is 6 min. The time for the sterilization treatment to rise from room temperature to 125 ℃ is controlled within 10 min (the specific heating time in Example 3 is 5 min). Within 6 min, the temperature is reduced from the sterilization temperature (125 ℃) to 80 ℃ to obtain the sterilized gel. Then, the sterilized gel is quickly placed in an ice water bath for rapid cooling (the time in the ice water bath is 60 min) to obtain the final product, namely the sterilized high-performance sodium hyaluronate composite gel.

[0032] Example 4 The preparation method of high-performance sodium hyaluronate composite gel includes the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 35 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 750 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 2:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS, pH=6.0, osmotic pressure 600 mOsmol / kg) pre-cooled to 8 ℃ and dissolved under low-speed mechanical stirring at 8 ℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 6 wt%. (3) After sealing the obtained sodium hyaluronate solution in a container, heat it in a water bath at 80 °C for 15 minutes, and then immediately transfer it to an ice water bath at 0 °C for rapid cooling for 15 minutes. This thermal cycle process is repeated twice (i.e., the operation of step (3) is repeated twice) to obtain the solution after thermal cycle treatment. (4) The solution after thermal cycling is placed into a container and frozen in a refrigerator at -20 ℃ for 24 hours. Then it is moved to a refrigerator at 8 ℃ to thaw until completely thawed, and a preliminary gel (the thawed gel) is obtained. (5) At 8 ℃, the pH of the thawed gel was adjusted to between 6.8 and 7.6 using a 0.1wt% sodium hydroxide solution. Then, high-purity water was added, and the gel was slowly stirred for 30 min at 25 rpm in a pressure stirring vessel with a pressure of 0.15 MPa to dilute it evenly to a final sodium hyaluronate concentration of 2.8 wt%, thus obtaining the stirred gel, i.e., the high-performance sodium hyaluronate composite gel. (6) After vacuum degassing the gel after the above stirring treatment, fill it into a pre-filled syringe, and then sterilize it with high temperature steam. The sterilization temperature is 120 ℃ and the sterilization time is 15 min. The time for the sterilization treatment to rise from room temperature to 120 ℃ is controlled within 10 min (the specific heating time in Example 1 is 9 min). Within 6 min, the temperature is reduced from the sterilization temperature (120 ℃) ​​to 80 ℃ to obtain the sterilized gel. Then, the sterilized gel is quickly placed in an ice water bath for rapid cooling (the time in the ice water bath is 60 min) to obtain the final product, namely the sterilized high-performance sodium hyaluronate composite gel.

[0033] Comparative Example 1 A method for preparing a gel, comprising the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 230 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1000 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS buffer, pH=7.0, osmotic pressure 270mOsmol / kg) and dissolved under low-speed mechanical stirring at 4℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 3.2wt%. (3) After vacuum degassing the above sodium hyaluronate solution, it is filled into a pre-filled syringe and then sterilized with high-temperature steam. The sterilization temperature is 125 °C and the sterilization time is 6 min. The time for the sterilization process to rise from room temperature to 125 °C is controlled within 10 min (the specific heating time in Comparative Example 1 is 6 min). Within 6 min, the temperature is reduced from the sterilization temperature (125 °C) to 80 °C and allowed to cool naturally to obtain a gel.

[0034] Comparative Example 2 A method for preparing a gel, comprising the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 230 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1000 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS buffer, pH=5.5, osmotic pressure 590 mOsmol / kg) pre-cooled to 4 ℃ and dissolved under low-speed mechanical stirring at 4 ℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 7 wt%. (3) At 4 ℃, the pH of the sodium hyaluronate solution was adjusted to between 6.8 and 7.6 using a low concentration sodium hydroxide solution. Then, high purity water was added, and the solution was stirred for 45 min at 20 rpm in a pressure stirring vessel with a pressure of 0.2 MPa to uniformly dilute it to a final sodium hyaluronate concentration of 3.2 wt% and obtain a gel-like liquid. (4) After vacuum degassing the above gel-like liquid, fill it into a pre-filled syringe, and then sterilize it with high-temperature steam. The sterilization temperature is 125℃ and the sterilization time is 4 min. The time for the sterilization process to rise from room temperature to 125℃ is controlled within 10 min (the specific heating time is 5 min). Within 6 min, the temperature is reduced from the sterilization temperature (125℃) to 80℃ to obtain the sterilized gel. After natural cooling, the final gel is obtained.

[0035] Comparative Example 3 A method for preparing a gel, comprising the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 230 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1000 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS, pH=5.5, osmotic pressure 590 mOsmol / kg) and dissolved under low-speed mechanical stirring at 4 ℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 7wt%.

[0036] (3) After sealing the obtained sodium hyaluronate solution in a container, heat it in a water bath at 90 ℃ for 20 minutes, and then immediately transfer it to an ice water bath at 0 ℃ for rapid cooling for 20 minutes. This thermal cycle process is repeated 3 times (i.e., the operation of step (3) is repeated 3 times) to obtain the solution after thermal cycle treatment. (4) At 4 °C, the pH of the solution after thermal cycling was adjusted to between 6.8 and 7.6 using a low concentration sodium hydroxide solution. Then, high-purity water was added, and the solution was slowly stirred for 45 min at 20 rpm in a pressure stirring vessel with a pressure of 0.2 MPa to dilute it evenly to a final sodium hyaluronate concentration of 3.2 wt%, thus obtaining a gel-like liquid. (5) After vacuum degassing the above gel-like liquid, it is filled into a pre-filled syringe and then sterilized with high-temperature steam. The sterilization temperature is 125 °C and the sterilization time is 6 min. The time for the sterilization process to rise from room temperature to 125 °C is within 10 min (the heating time of Comparative Example 3 is 6 min). Within 6 min, the temperature is reduced from the sterilization temperature (125 °C) to 80 °C and allowed to cool naturally to obtain the final gel. Comparative Example 4 A method for preparing a gel, comprising the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 230 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1000 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS, pH=5.5, osmotic pressure 590 mOsmol / kg) pre-cooled to 4 ℃ and dissolved under low-speed mechanical stirring at 4 ℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 7 wt%. (3) After sealing the obtained sodium hyaluronate solution in a container, heat it in a water bath at 90 °C for 20 minutes, and then immediately transfer it to an ice water bath at 0 °C for rapid cooling for 20 minutes. Repeat this thermal cycle process 3 times (i.e. repeat step (3) 3 times) to obtain the solution after thermal cycle treatment. (4) The solution after thermal cycling is placed into a container and frozen in a refrigerator at -20 ℃ for 36 hours. Then it is moved to a refrigerator at 4 ℃ to thaw until completely thawed, and a preliminary gel (the thawed gel) is obtained. (5) At 4 ℃, the pH of the thawed gel was adjusted to between 6.8 and 7.6 using a low concentration of sodium hydroxide solution. Then, high purity water was added, and the gel was slowly stirred for 45 min at 20 rpm in a pressure stirring vessel with a pressure of 0.2 MPa to dilute it evenly to a final concentration of 3.2 wt% and obtain the stirred gel. (6) After vacuum degassing the gel after the above stirring treatment, fill it into a pre-filled syringe, and then sterilize it with high temperature steam. The sterilization temperature is 125 ℃ and the sterilization time is 6 min. The time for the sterilization treatment to rise from room temperature to 125 ℃ is controlled within 10 min (the specific heating time in Comparative Example 4 is 6 min). Within 6 min, the temperature is reduced from the sterilization temperature (125 ℃) to 80 ℃ and allowed to cool naturally to obtain the final gel.

[0037] Comparative Example 5 A method for preparing a gel, comprising the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 230 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 1000 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 1:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder and recombinant collagen fibers with a molecular weight of 55 kDa were added to phosphate buffer (PBS, pH=5.5, osmotic pressure 590 mOsmol / kg) pre-cooled to 4°C. The mass ratio of the mixed powder to the recombinant collagen fibers with a molecular weight of 55 kDa was 14:3. The solution was dissolved under low-speed mechanical stirring at 4°C and 15 rpm until no obvious white lumps were found, resulting in a composite solution with a total sodium hyaluronate content of 7 wt% and a recombinant collagen content of 1.5 wt%. The manufacturer of the recombinant collagen fibers was Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd., and the molecular weight of the recombinant collagen fibers was 55 kDa. (3) After sealing the obtained composite solution in a container, heat it in a water bath at 90 ℃ for 20 minutes, and then immediately transfer it to an ice water bath at 0 ℃ for rapid cooling for 20 minutes. Repeat this thermal cycle process 3 times (i.e., repeat step (3) 3 times) to obtain the solution after thermal cycle treatment; (4) The solution after thermal cycling is placed into a container and frozen in a refrigerator at -20 ℃ for 36 hours. Then it is moved to a refrigerator at 4 ℃ to thaw until completely thawed, and a preliminary gel (the thawed gel) is obtained. (5) At 4 ℃, the pH of the thawed gel was adjusted to between 6.8 and 7.6 using a 0.1 wt% sodium hydroxide solution. Then, high-purity water was added, and the mixture was stirred at 20 rpm for 45 min in a pressure-stirred vessel with a pressure of 0.2 MPa to make it uniformly diluted into a composite solution with a sodium hyaluronate concentration of 3.2 wt% and a recombinant collagen concentration of 0.69 wt%. (6) After vacuum degassing the above composite solution, it is filled into a pre-filled syringe and then sterilized with high-temperature steam. The sterilization temperature is 125 °C and the sterilization time is 6 min. The time for the sterilization process to rise from room temperature to 125 °C is controlled within 10 min (the specific heating time for Comparative Example 5 is 6 min). Within 6 min, the temperature is lowered from the sterilization temperature (125 °C) to 80 °C to obtain the sterilized composite solution. Then, the sterilized composite solution is rapidly cooled in an ice-water bath (the time in the ice-water bath is 60 min) to obtain the final gel.

[0038] Comparative Example 6 A method for preparing a gel, comprising the following steps: (1) Weigh low molecular weight sodium hyaluronate powder with a molecular weight of 35 kDa and high molecular weight sodium hyaluronate powder with a molecular weight of 750 kDa (the mass ratio of low molecular weight sodium hyaluronate powder to high molecular weight sodium hyaluronate powder is 2:1), mix them thoroughly in a dry state to obtain a mixed powder. (2) The above mixed powder was added to phosphate buffer (PBS buffer, pH=7.0, osmotic pressure 270mOsmol / kg) and dissolved under low-speed mechanical stirring at 8℃ and 15 rpm until there were no obvious white lumps, to obtain a sodium hyaluronate solution with a total content of 2.8 wt%. (3) After vacuum degassing the above sodium hyaluronate solution, it is filled into a pre-filled syringe and then sterilized with high-temperature steam. The sterilization temperature is 120 °C and the sterilization time is 15 min. The time for the sterilization process to rise from room temperature to 120 °C is controlled within 10 min (the specific heating time for Comparative Example 6 is 9 min). The temperature is then reduced from the sterilization temperature (125 °C) to 80 °C within 6 min and allowed to cool naturally to obtain a gel.

[0039] To verify the performance of the gels prepared in each embodiment and each comparative example, the present invention used the gels of each embodiment and each comparative example as test samples and conducted the following performance tests.

[0040] (1) Elastic modulus: Using a rotational rheometer, the oscillation test mode was selected, the test temperature was set to 25℃, and a frequency scan was performed under a strain of 5%. The modulus value corresponding to 1Hz was then recorded. The rotational rheometer was manufactured by TA Instruments, and its model was DHR-1.

[0041] (2) Shear viscosity: Using a rotational rheometer, select the flow test mode, set the test temperature to 25℃, and start at a shear rate of 0.001 s⁻¹. -1 ~1000 s -1 Perform flow scanning and take 1 second. -1 The corresponding shear viscosity value. The rotational rheometer is manufactured by TA Instruments, and its model is DHR-1.

[0042] (3) Degradation rate test: The sample was enzymatically hydrolyzed using a 1000 IU / mL hyaluronidase solution at 37 °C. After 24 h of hydrolysis, the degradation product glucuronic acid was extracted and detected. The measured glucuronic acid content was converted into the content of degraded sodium hyaluronate, and the in vitro degradation rate after 24 h was obtained by comparing it with the labeled concentration of the sample.

[0043] The specific steps include the following: Enzymatically digest the sample using a 1000 IU / mL hyaluronidase solution at 37 °C (the mass-to-volume ratio of sample to hyaluronidase solution is 1:5 g / mL, and the sample mass here is 1 g). After 24 h of digestion, add 5 mL of anhydrous ethanol to the digest and allow it to stand. Filter the supernatant after standing through a 0.45 μm filter to obtain a clear digest filtrate. Prepare a series of standard solutions using glucuronic acid standard material in test tubes, and place them together with the sample test tubes containing the clear digest filtrate in an ice-water bath. Using a pipette, add 5 mL of 0.025 mol / L sodium tetraborate sulfuric acid solution (stored in a 4 °C refrigerator for at least 2 h before use) to each tube. After addition, mix well and boil in a boiling water bath for 10 min, then remove and cool to room temperature. Add 0.20 mL of 0.125 wt% carbazole ethanol solution to each test tube, shake thoroughly, heat in a boiling water bath for 15 min, and then cool to room temperature. Using tube 0 as a control, the absorbance of each standard tube and sample tube at 530 nm was measured using a UV spectrophotometer. An absorbance-concentration standard curve was plotted using the standard tubes, and the content of glucuronic acid, a degradation product, in the sample tube was determined from the standard curve based on the sample absorbance. The measured glucuronic acid content was converted into the content of degraded sodium hyaluronate, and the in vitro degradation rate after 24 h was obtained by comparing it with the labeled concentration of the sample.

[0044] The test results are shown in Table 1 below.

[0045] Table 1 As shown in Table 1, comparing the elastic modulus, shear viscosity, and degradation rate of Examples 1, 2, 3, and 4, it can be seen that the performance of this product is mainly affected by the molecular weight and content of sodium hyaluronate. The larger the molecular weight and the higher the content, the stronger the viscoelasticity and anti-degradation properties of the final product. Comparing the data of Comparative Examples 1, 2, 3, 4, and Example 3, it can be seen that the present invention improves the viscoelasticity and anti-degradation properties of the product by increasing the ion concentration of the solvent during sodium hyaluronate dissolution, lowering the pH value; pressurizing during stirring and dilution; high and low temperature cycling after dissolution; freeze-thaw treatment after dissolution; and rapid cooling treatment after sterilization. The advantages of this process can also be seen by comparing the performance of Comparative Example 6 and Example 4. Comparing Example 3 and Comparative Example 5, it can be found that the addition of recombinant collagen does not significantly improve the product performance, but the addition of recombinant collagen increases the risk of allergies.

[0046] (4) Storage stability: After sample preparation, the intrinsic viscosity was tested and simultaneously converted to the average molecular weight. The samples were then stored at 25°C for 3 months, and the intrinsic viscosity was tested again. The intrinsic viscosity was tested according to Method II in Section 0633 of the 2025 edition of the Chinese Pharmacopoeia, Volume IV, using a 0.2 mol / L sodium chloride solution as the dilution solvent. The average molecular weight was calculated according to the conversion formula in Section 5.10.1 of YY / T 0308-2015 "Medical Sodium Hyaluronate Gel". η=0.036 M 0.78 in: η-------Intrinsic viscosity (cm) 3 / g) M ------ Average molecular weight (Da) The relevant test results are shown in Table 2 below.

[0047] Table 2 Intrinsic viscosity and corresponding molecular weight are both indicators of product stability. In existing technologies, high-temperature steam sterilization can damage the stability of sodium hyaluronate gel, specifically by reducing its molecular weight. However, data from Examples 1, 2, 3, and 4 show that the high-performance sodium hyaluronate composite gel of this invention retains high molecular weight and intrinsic viscosity even after high-temperature steam sterilization.

[0048] By comparing the intrinsic viscosity and corresponding molecular weight data of the zero-point samples and samples stored at 25 °C for 3 months in Examples 1, 2, 3, and 4, it can be seen that the preparation method of the present invention improves the product's resistance to thermal degradation and thus enhances its storage stability by increasing the ion concentration of the solvent during sodium hyaluronate dissolution, lowering the pH value, pressurizing during stirring and dilution, subjecting the product to high and low temperature cycling after dissolution, freezing and thawing after dissolution, and rapid cooling after sterilization. The advantages of the preparation method of the present invention can also be demonstrated by comparing the relevant data of Comparative Example 6 and Example 4. However, by comparing Example 3 and Comparative Example 5, it can be found that the addition of recombinant collagen does not significantly improve the product's resistance to thermal degradation (stability), but the addition of recombinant collagen increases the risk of allergies.

[0049] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A high-performance sodium hyaluronate composite gel, characterized in that, The raw materials for preparation include: low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and phosphate buffer; the molecular weight of the low molecular weight sodium hyaluronate is 35 kDa-350 kDa, and the molecular weight of the high molecular weight sodium hyaluronate is 750 kDa-1500 kDa; the mass ratio of the low molecular weight sodium hyaluronate to the high molecular weight sodium hyaluronate is (1:2)-(2:1).

2. The high-performance sodium hyaluronate composite gel according to claim 1, characterized in that, The pH of the phosphate buffer solution is 5.0-6.0; the osmotic pressure of the phosphate buffer solution is 300-600 mOsmol / kg.

3. The high-performance sodium hyaluronate composite gel according to claim 1, characterized in that, The sum of the contents of the low molecular weight sodium hyaluronate and the high molecular weight sodium hyaluronate is 2.8-5.5 wt%.

4. The method for preparing the high-performance sodium hyaluronate composite gel according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Mix low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate to obtain a mixed powder. Add the mixed powder to phosphate buffer and stir to dissolve to obtain a mixed solution. (2) Heat the mixture obtained in step (1) in a water bath, and then in an ice-water bath; repeat step (2) 2-3 times to obtain the solution after thermal cycling treatment; (3) Freeze the solution after the thermal cycling treatment in step (2) to obtain a frozen substance, and then thaw it to obtain a thawed gel; adjust the pH of the thawed gel to 6.8-7.6, add water, stir and treat to obtain a stirred gel, namely the high-performance sodium hyaluronate composite gel.

5. The method for preparing the high-performance sodium hyaluronate composite gel according to claim 4, characterized in that, The stirring and dissolving temperature in step (1) is 2-8℃; the total content of low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate in the mixture in step (1) is 6-8wt%.

6. The method for preparing the high-performance sodium hyaluronate composite gel according to claim 4, characterized in that, The water bath heating temperature in step (2) is 80-100℃, and the water bath heating time is 15-30 minutes; the ice water bath time is 15-30 minutes.

7. The method for preparing the high-performance sodium hyaluronate composite gel according to claim 4, characterized in that, The freezing temperature in step (3) is -30°C to -15°C, and the freezing time is 24-48 hours; thawing includes placing the frozen material at a temperature of 2-30°C until it is completely thawed.

8. The method for preparing the high-performance sodium hyaluronate composite gel according to claim 4, characterized in that, The stirring process in step (3) includes stirring at a speed of 15-25 rpm for 30-60 min under a pressure of 0.15-0.25 MPa.

9. The method for preparing the high-performance sodium hyaluronate composite gel according to any one of claims 4-8, characterized in that, The gel after stirring in step (3) is sterilized; the sterilization method is high-temperature steam sterilization; the sterilization process includes: vacuum degassing the gel after stirring, then filling it into a container, heating it to 120-128℃ within 10 minutes, maintaining it at 120-128℃ for 4-15 minutes, sterilizing it with high-temperature steam, then cooling it to below 80℃ within 6 minutes, and finally transferring the container containing the stirred gel to an ice-water bath to cool it for 60-90 minutes to obtain the sterilized high-performance sodium hyaluronate composite gel.

10. The application of the high-performance sodium hyaluronate composite gel according to any one of claims 1-3 in the preparation of cosmetics, skin care products, and medical aesthetic products.

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