A preparation method of hyaluronic acid microspheres for injection

Abstract

The invention discloses a preparation method of hyaluronic acid microspheres for injection, sodium hyaluronate solution is placed in an ice bath and stirred to dissolve and a cross-linking agent is added to obtain solution A; a mixed solution of an emulsifier and an oil phase is placed in an ice bath and stirred to obtain solution B, solution A and solution B are stirred and mixed to form a uniform emulsion, a cross-linking agent is added to the emulsion to carry out a cross-linking reaction, and a reaction solution is obtained; after removing the oil phase and moisture in the reaction solution, hyaluronic acid microspheres are obtained, residual cross-linking agent and emulsifier are removed and dried, the dried microspheres are added to a phosphate buffer solution and swelled, and then mixed with an equal volume of sodium hyaluronate solution to obtain hyaluronic acid microspheres for injection. The method is controlled by specific preparation steps and temperature, and it is possible to achieve uniform microsphere particle size, high yield, and high cross-linking degree, solves the problem that it is difficult to overcome swelling degree in the prior art, is difficult to be used for plasticity, and has a cross-linking agent residue.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing hyaluronic acid microspheres for injection. Background Art

[0002] Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan composed of repeating disaccharides of N-acetylglucosamine and glucuronic acid. Due to its unique physical and chemical properties, it has high clinical value and is often used as a moisturizing factor, lubricant and scaffold material. It has multiple physiological functions such as buffering pressure, filling agent and diffusion barrier, scavenging free radicals and regulating immunity. It can be widely used in medical, beauty and other fields.

[0003] Hyaluronic acid injectable fillers are currently the most widely used skin fillers. Cross-linked hyaluronic acid prepared by cross-linking can be used in the field of medical aesthetics for nasolabial fold filling, rhinoplasty, tear trough filling, and filling to improve facial wrinkles, making it an ideal material for tissue filling. Since most of the cross-linked hyaluronic acid for injection on the market currently exists in the form of gel, the preparation method mostly uses cross-linking and then cutting, crushing, and screening to obtain the hyaluronic acid gel of the required size. Observation under a microscope shows fragmented particles. The problem with this process is that the cross-linked hyaluronic acid particles obtained by cutting and crushing are completely irregular and uneven in size, which further makes it difficult to control the viscoelasticity of the product and the preparation method is inefficient. In addition, the cross-linked hyaluronic acid gels prepared by most existing methods have a low degree of cross-linking and a greater swelling degree, which cannot reach a higher final concentration, and therefore have a short maintenance time. In addition, the problem of swelling after injection is mainly due to the large swelling degree of the injected gel. After being injected into the body, it will still absorb water and increase in volume, resulting in a stiff and unnatural phenomenon. In the existing technology, a characteristic of the cross-linked hyaluronic acid gel prepared with BDDE (1,4-butanediol diglycidyl ether) as a cross-linking agent is its soft texture. However, the relatively soft texture makes most cross-linked sodium hyaluronate gels on the market only suitable for wrinkle filling, and cannot be used for shaping. Even if used for shaping, there is a problem of easy displacement and deformation.

[0004] Therefore, the problems currently faced by hyaluronic acid injection products can be summarized as: low preparation efficiency; easy metabolism in the body and short maintenance time; limited injection range, risk of displacement, and unnatural swelling, stiffness and other issues after injection.

[0005] Patent publication number CN 114369264A discloses cross-linked sodium hyaluronate microspheres and their preparation method. First, sodium hyaluronate is dissolved in an alkaline solution, spray-dried or freeze-dried in vacuum, and then ground in liquid nitrogen to prepare hydroxyl-activated sodium hyaluronate microspheres. These microspheres are then dispersed in a poor solvent for sodium hyaluronate, and a cross-linking agent is added to initiate a micro-cross-linking reaction (low-density cross-linking occurs on the surface of the microspheres). A small amount of water is then added to continue cross-linking. The cross-linking density of the microspheres can be adjusted by controlling the micro-cross-linking reaction time and the amount of water added. This simple method produces cross-linked sodium hyaluronate microspheres with good sphericity and uniform cross-linking, making them suitable for intradermal and subcutaneous injection or tissue augmentation. In addition, the invention patent with publication number CN111848991A discloses a method for preparing cross-linked hyaluronic acid microspheres. The method comprises the following steps: at low temperature, hyaluronic acid or its salt, a cross-linking agent, an alkali, and water are uniformly dispersed to obtain an aqueous phase; the aqueous phase is added to an oil phase containing an emulsifier and fully emulsified to obtain an emulsion; the emulsion is cross-linked at a cross-linking temperature, the oil phase is removed after cross-linking, and post-processing is performed to obtain cross-linked hyaluronic acid microspheres. This method adds a cross-linking agent before emulsification and suppresses the cross-linking of hyaluronic acid before emulsification by lowering the temperature. The method has the characteristics of simple preparation steps, mild reaction conditions, low amount of cross-linking agent, good cross-linking uniformity, and the prepared cross-linked hyaluronic acid microspheres have higher mechanical properties.

[0006] It can be seen that when preparing sodium hyaluronate microspheres by cross-linking with a cross-linking agent, the cross-linking reaction time, temperature, solvent and other conditions can be controlled during the reaction process to improve the cross-linking uniformity of the product, thereby improving the mechanical properties of the cross-linked hyaluronic acid microspheres, thereby extending the maintenance time of the injectable hyaluronic acid microspheres. However, the above patent still has the following defects that need to be solved urgently:

[0007] (1) CN114369264A uses spray drying or freeze drying followed by grinding to prepare hyaluronic acid microspheres. A large amount of crosslinker BDDE is used in the preparation of microspheres. Since the crosslinker has biological toxicity or potential carcinogenicity, its residual amount needs to be strictly controlled. Excessive use in the early stage will be detrimental to the subsequent impurity removal and it will be difficult to control its residual amount. Therefore, the amount of crosslinker should be reduced as much as possible during the preparation process.

[0008] (2) CN111848991A mixes hyaluronic acid with a cross-linking agent, alkali, and water at low temperature and disperses them uniformly. It can be seen that the cross-linking agent is reacted by adding all of it at once. In order to achieve uniform cross-linking, the process control is difficult, and it is difficult to achieve stable control of batch products. Summary of the Invention

[0009] The present invention aims to solve the defects in the existing cross-linking method for preparing injectable hyaluronic acid, and proposes a method for preparing injectable hyaluronic acid microspheres. During the preparation process, by reasonably controlling the temperature and adding an appropriate amount of cross-linking agent in batches, the microspheres are formed in one go during the cross-linking process, and the cross-linking degree is high, the microsphere particle size is uniform, the swelling degree is reduced, and the microspheres have strong mechanical stability. Therefore, the defect that injectable hyaluronic acid microspheres are difficult to use for plasticity is solved.

[0010] The present invention is achieved through the following technical solution: A method for preparing hyaluronic acid microspheres for injection, comprising the following steps:

[0011] S1. Prepare solution A and solution B separately,

[0012] The sodium hyaluronate solution was placed in an ice bath and stirred to dissolve, and a cross-linking agent was added thereto, stirred and mixed uniformly to prepare solution A; the mixture of the emulsifier and the oil phase was placed in an ice bath and stirred uniformly to prepare solution B.

[0013] In the solution A, the concentration of sodium hyaluronate is 16 to 320 g / L, and the concentration of the cross-linking agent is 4 to 16 g / L;

[0014] S2. Cross-linking reaction,

[0015] Stirring solution A and solution B at 2-10° C. to form a uniform emulsion, raising the temperature of the emulsion to 30° C. and continuing to stir, adding a crosslinking agent to the emulsion, stirring at 30-60° C. and performing a crosslinking reaction to prepare a reaction solution containing hyaluronic acid microspheres;

[0016] S3. Remove impurities,

[0017] After removing the oil phase and water from the reaction solution, hyaluronic acid microspheres are obtained, and after removing the residual crosslinking agent and emulsifier on the hyaluronic acid microspheres, the hyaluronic acid microspheres are dried to obtain microspheres with a particle size of 45 to 106 μm;

[0018] S4. Subsequent processing,

[0019] The microspheres are added to a phosphate buffer solution for swelling treatment, and then mixed evenly with an equal volume of sodium hyaluronate solution to obtain hyaluronic acid microspheres for injection.

[0020] Calculated by mass-to-volume ratio, the amount of hyaluronic acid microspheres added to each milliliter of phosphate buffer is 0.01-0.02 g.

[0021] In step S1 and step S4, the sodium hyaluronate solution is formed by adding sodium hyaluronate powder with a molecular weight of 200,000 to 2.2 million Da to an alkaline solution with a concentration of 0.1 to 20%, and stirring and dissolving at room temperature. The concentration of the sodium hyaluronate solution used in step S4 is controlled to be 0.03 to 0.04 g / ml.

[0022] The alkaline solution includes sodium hydroxide, potassium hydroxide, ammonia water or a mixture of at least two thereof.

[0023] In step S1 and step S4, the sodium hyaluronate solution is formed by adding sodium hyaluronate powder to an alkaline solution and stirring and dissolving the solution at room temperature. The molecular weight of the sodium hyaluronate powder used in step S1 is 200,000 to 1.5 million Da, and the molecular weight of the sodium hyaluronate powder used in step S4 is 200,000 to 2.2 million Da.

[0024] In the step S1, the mass ratio of the emulsifier to the oil phase is controlled to be 0.8-6.67:83.35.

[0025] The emulsifier includes Span 60, Span 80, a surfactant with a hydrophilic-lipophilic balance value of 3-8, or a mixture of at least two of them; the oil phase includes liquid paraffin, vegetable oil, mineral oil, silicone oil, synthetic oil, or a mixture of at least two of them.

[0026] In step S2, the amount of cross-linking agent added to each liter of emulsion is 0.78 to 3.88 g based on mass volume concentration.

[0027] In step S2, the cross-linking reaction time is controlled to be 8 to 10 hours.

[0028] In step S3, removing the oil phase and water from the reaction solution includes: adding ethyl acetate to the reaction solution under stirring, fully mixing the oil phase and ethyl acetate, allowing the mixture to stand, removing the supernatant after the hyaluronic acid microspheres are precipitated, and repeating this operation; continuing to add anhydrous ethanol to the hyaluronic acid microspheres, stirring and allowing the mixture to stand, removing the supernatant after the hyaluronic acid microspheres are precipitated, and repeating this operation.

[0029] In step S4, the swelling treatment time is controlled to be 12 to 24 hours.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) The method of the present invention can not only realize the one-time forming of hyaluronic acid microspheres during the cross-linking process by reasonably controlling the concentration of the cross-linking agent in solution A and the emulsion, but also improve the uniformity of the hyaluronic acid microsphere particles, making the pushing force of the injection more stable. The injection pushing force can be stably maintained at 6 to 8N, which can improve the handiness of clinical injection and facilitate the use of clinical physicians.

[0032] (2) The preparation process of the method of the present invention is simple. During the cross-linking reaction, the interior of the microspheres can be fully cross-linked to achieve a higher degree of cross-linking, thereby achieving a higher hyaluronic acid concentration while maintaining a lower cross-linker residue, significantly reducing the degradation rate of the same volume of hyaluronic acid, which is beneficial to reducing the injection frequency in clinical use.

[0033] (3) The method of the present invention reduces the swelling degree of hyaluronic acid microspheres and is expected to improve the problem of facial swelling after injection.

[0034] (4) The microspheres prepared by the present invention have higher mechanical strength and can solve the problem of easy displacement and deformation. Therefore, they are more suitable for plasticity. Experiments have shown that the storage modulus (G') and loss modulus (G'') of their viscoelasticity (at 37°C and 0.1 Hz) are 355 Pa and 206 Pa, respectively, and the composite viscosity is 653 Pa·s. Therefore, they have strong mechanical stability.

[0035] (5) The method of the present invention can ensure the concentration of hyaluronic acid in the microspheres due to its higher degree of cross-linking, so that the cross-linked sodium hyaluronate reaches 50 mg / mL. The uncross-linked hyaluronic acid only serves as a lubricant and dispersant, thereby prolonging its residence time in the body.

[0036] (6) The microspheres prepared by the method of the present invention also have less cross-linking agent residue. The detection result of the cross-linking agent in the finished product is 0.15±0.31μg / g, which is safer.

[0037] In summary, the present invention provides a new method for preparing injectable hyaluronic acid microspheres by cross-linking. This method, through specific preparation steps and temperature control, can not only achieve the characteristics of uniform microsphere particle size, high yield and high cross-linking degree, but also solve the problems of swelling degree and difficulty in shaping that are difficult to overcome in the prior art, as well as the problem of the neglected amount of cross-linker residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a morphological picture of the hyaluronic acid microspheres of Example 1 under a microscope.

[0039] Figure 2 This is a morphological picture of the hyaluronic acid microspheres of Example 4 under a microscope.

[0040] Figure 3 This is a pushing force curve of the hyaluronic acid microspheres in Example 1.

[0041] Figure 4 This is a rheological property curve of the hyaluronic acid microspheres of Example 1. DETAILED DESCRIPTION

[0042] The objectives, technical solutions and beneficial effects of the present invention are described in further detail below.

[0043] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0044] Hyaluronic acid microspheres for injection can be injected into the deep layer of the subcutaneous dermis to the shallow layer of the subcutaneous layer as a filler for repairing moderate to severe wrinkles or folds and facial modification. Therefore, it is necessary to meet a certain maintenance time and plasticity, and swelling will not occur after injection. Hyaluronic acid microspheres are a combination structure with a certain cross-linked state formed by cross-linking hyaluronic acid molecules using a chemically cross-linked substance. Therefore, for its cross-linking degree, the size of the microsphere particles after cross-linking, mechanical properties and the residue of the cross-linking agent, it is possible to affect the use effect of the hyaluronic acid microspheres. It is known that existing patents CN114369264A and CN111848991A disclose a cross-linking method for preparing two different operating modes of hyaluronic acid microspheres. Although certain technical guidance is given to solving the uniformity, swelling degree and mechanical properties of the microspheres, there are still risks in the preparation process. For example, the hyaluronic acid microspheres prepared by CN114369264A are prone to the residue of the cross-linking agent; CN111848991A is prone to the problem of difficulty in preparation control, etc. To this end, the present invention aims to provide a method for preparing hyaluronic acid microspheres with a simple preparation method and low control difficulty, which can not only prepare injectable hyaluronic acid microspheres that meet the needs of filling, repairing and shaping, but also achieve the stability of multiple batches of products.

[0045] The technical solution of the present invention can be summarized as follows:

[0046] Solution A: Add sodium hyaluronate powder with a molecular weight of 2 million to 2.2 million Da to an alkaline solution with a concentration of 0.1 to 20%, and stir to dissolve at room temperature to form a sodium hyaluronate solution. Then, place the sodium hyaluronate solution in an ice bath and stir to dissolve, and add a cross-linking agent thereto, stir and mix until uniform.

[0047] In the above-mentioned solution A, the concentration of sodium hyaluronate can be controlled at 16 to 320 g / L, and the concentration of the cross-linking agent can be controlled at 4 to 16 g / L. When preparing solution A, the alkaline solution used includes sodium hydroxide, potassium hydroxide, aqueous ammonia, or a mixture of at least two thereof; the cross-linking agent used includes 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone, glycerol triglycidyl ether, resorcinol diglycidyl ether, allyl glycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, or neopentyl glycol diglycidyl ether.

[0048] Solution B: Place the mixture of emulsifier and oil phase in an ice bath and stir evenly at a mass ratio of emulsifier: oil phase = 0.8~6.67:83.35 to obtain solution B.

[0049] In the above solution B, the emulsifier includes Span 60, Span 80, a surfactant with a hydrophilic-lipophilic balance value of 3-8, or a mixture of at least two of them; the oil phase includes liquid paraffin, vegetable oil, mineral oil, silicone oil, synthetic oil, or a mixture of at least two of them.

[0050] Furthermore, the above-mentioned solution A and solution B are mixed and then subjected to a cross-linking reaction. The volume ratio of solution A to solution B can be controlled to be 1-3:1-10. Specifically, solution A and solution B are stirred and mixed at 2-10°C to form a uniform emulsion. The temperature of the emulsion is then raised to 30°C and stirring is continued. A cross-linking agent is added to the emulsion. The cross-linking reaction is carried out at 30-60°C and stirred for 8-10 hours to obtain a reaction solution containing hyaluronic acid microspheres. The amount of cross-linking agent added per liter of emulsion is 0.78-3.88g based on mass volume concentration.

[0051] Continue to remove impurities from the reaction solution, including oil phase, water, cross-linking agent and emulsifier, which can also be called purification of hyaluronic acid microspheres. For example: add ethyl acetate to the reaction solution under stirring, mix the oil phase and ethyl acetate thoroughly and let it stand, remove the supernatant after the hyaluronic acid microspheres are precipitated, and repeat the operation to remove the oil phase in the reaction solution; continue to add anhydrous ethanol to the hyaluronic acid microspheres, stir and let it stand, remove the supernatant after the hyaluronic acid microspheres are precipitated, and repeat the operation to further remove the oil phase and remove water; after removing the oil phase and water from the reaction solution, hyaluronic acid microspheres are obtained, and then the residual cross-linking agent and emulsifier on the hyaluronic acid microspheres are removed, and the hyaluronic acid microspheres are dried, and screened to obtain microspheres with a particle size of 45 to 106 μm;

[0052] Finally, the purified hyaluronic acid microspheres are added to a phosphate buffer solution at a mass-to-volume ratio of 0.01 to 0.2 g per milliliter of phosphate buffer. The solution is allowed to swell for 12 to 24 hours and then mixed with an equal volume of sodium hyaluronate solution to obtain injectable hyaluronic acid microspheres. The sodium hyaluronate powder used in the solution has a molecular weight of 2.0 to 2.2 million Da, resulting in a concentration of 0.03 to 0.04 g / ml.

[0053] The structure of the hyaluronic acid microspheres of the present invention is shown in the following formula (1):

[0054] (1)

[0055] Wherein, HA is hyaluronic acid, as shown in the following formula (2):

[0056] (2)

[0057] The structure of the cross-linking agent BDDE used in the present invention is shown in the following formula (3):

[0058] (3).

[0059] The following are several typical examples to illustrate the specific implementation of the present invention. Of course, the scope of protection of the present invention is not limited to the following examples. The hyaluronic acid microspheres and sodium hyaluronate microspheres described in the present invention have the same meaning and can be replaced with each other.

[0060] Example 1:

[0061] Add 2.5 g of sodium hyaluronate powder with a molecular weight of 1.5 million Da to 31.25 ml of 1% sodium hydroxide solution and stir at room temperature for 30 minutes to dissolve to form a sodium hyaluronate solution with a concentration of 80 g / L. Then, place the above sodium hyaluronate solution in an ice bath (4°C) and continue stirring for 15 minutes. At low temperature (about 10°C), add 0.15 g of 1,4-butanediol diglycidyl ether and stir for 10 minutes to mix evenly to form solution A.

[0062] Add 3.32 g of Span-80 to 83.35 g of liquid paraffin, place in an ice bath, and stir at a low speed (about 400 rpm) to form solution B.

[0063] While stirring, slowly add Solution A to Solution B and mix at 5°C and 800 rpm for 2 h to form a uniform emulsion. Then slowly raise the temperature to 30°C. After continuing stirring for 30 min, slowly add 0.1 g of 1,4-butanediol diglycidyl ether to the emulsion and continue the reaction at 30°C and 800 rpm for 8 h.

[0064] After the reaction is completed, add 100 ml of ethyl acetate to the reaction solution under stirring. After the oil phase and ethyl acetate are fully mixed, let it stand to precipitate the hyaluronic acid microspheres. Pour off the supernatant, and then wash the oil phase thoroughly with ethyl acetate. Use 100 ml of ethyl acetate each time, repeat 3 times, and discard the supernatant.

[0065] Slowly add 150 ml of anhydrous ethanol to the washed hyaluronic acid microspheres, stir for 5 minutes and let it stand. After the microspheres are precipitated, discard the supernatant. Repeat this process three times until the microspheres appear as small white particles. Place them in a vacuum drying oven at 25°C and dry for 24 hours.

[0066] Add 200 ml of purified water to the dried microspheres, stir and swell at room temperature for 12 h, let it stand for 3 h, discard the supernatant, add 200 ml of purified water and continue stirring. After discarding the supernatant for the last time, slowly add 150 ml of anhydrous ethanol to the microspheres under rapid stirring, stir for 5 min and let it stand. After the microspheres are precipitated, discard the supernatant. Repeat 3 times until the microspheres appear as small white particles. Place them in a vacuum drying oven at 25°C and dry for 24 hours.

[0067] The dried microspheres were sieved with 325-mesh and 140-mesh sieves, and the middle part was collected to obtain microspheres with a particle size of 45 to 106 μm for use.

[0068] The above-mentioned reserved hyaluronic acid microspheres were swollen in phosphate buffer at a concentration of 0.1 g / ml for 12 hours, and then mixed evenly with an equal volume of free hyaluronic acid solution with a concentration of 0.03 g / ml and a molecular weight of 1.8 million Da to obtain hyaluronic acid microspheres for injection.

[0069] Example 2:

[0070] The preparation methods of solution A and solution B described in this example are different from those in Example 1, and the specific parameters of the cross-linking reaction are slightly different. The remaining steps are the same.

[0071] Solution A: Add 1.25 g of sodium hyaluronate powder with a molecular weight of 2.2 million Da to 31.25 ml of 5% sodium hydroxide solution and stir at room temperature for 60 minutes to dissolve to form a sodium hyaluronate solution with a concentration of 40 g / L. Then, place the above sodium hyaluronate solution in an ice bath (4°C) and continue stirring for 30 minutes. At low temperature (about 15°C), add 0.075 g of 1,4-butanediol diglycidyl ether and stir for 20 minutes to mix evenly.

[0072] Solution B: Add 6.67 g of Span-60 to a mixture of 83.35 g of liquid paraffin and vegetable oil. Stir the mixture at a low speed (about 200 rpm) in an ice bath until uniform.

[0073] Cross-linking reaction: While stirring, slowly add Solution A to Solution B. Mix at 2°C and 1500 rpm for 3 h to form a uniform emulsion. Then slowly raise the temperature to 30°C. Continue stirring for 10 min, then slowly add 0.05 g of 1,4-butanediol diglycidyl ether to the emulsion. Continue reacting at 50°C and 500 rpm for 6 h.

[0074] Example 3:

[0075] The preparation methods of solution A and solution B described in this example are different from those in Example 1, and the specific parameters of the cross-linking reaction are slightly different. The remaining steps are the same.

[0076] Solution A: Add 10 g of sodium hyaluronate powder with a molecular weight of 1.5 million Da to 31.25 ml of 20% sodium hydroxide solution, and stir at room temperature for 120 minutes to dissolve to form a sodium hyaluronate solution with a concentration of 320 g / L. Then, place the above sodium hyaluronate solution in an ice bath (4°C) and continue stirring for 5 minutes. At low temperature (about 10°C), add 0.5 g of 1,4-butanediol diglycidyl ether and stir for 20 minutes to mix evenly.

[0077] Solution B: Add 2.45 g of Span-60 to 83.35 g of liquid paraffin, place in an ice bath and stir at low speed (about 300 rpm) to obtain solution B.

[0078] Cross-linking reaction: While stirring, slowly add Solution A to Solution B. Mix at 3°C ​​and 800 rpm for 3 h to form a uniform emulsion. Then slowly raise the temperature to 30°C. Continue stirring for 15 min, then slowly add 1 g of 1,4-butanediol diglycidyl ether to the emulsion. Continue reacting at 30°C and 1000 rpm for 10 h.

[0079] Example 4:

[0080] The preparation methods of solution A and solution B described in this example are different from those in Example 1, and the specific parameters of the cross-linking reaction are slightly different. The remaining steps are the same.

[0081] Solution A: Add 2.5 g of sodium hyaluronate powder with a molecular weight of 200,000 Da to 31.25 ml of 10% sodium hydroxide solution, stir and dissolve at room temperature for 10 minutes to form a sodium hyaluronate solution with a concentration of 80 g / L. Then, place the above sodium hyaluronate solution in an ice bath (0°C) and continue stirring for 5 minutes. At low temperature (about 8°C), add 0.125 g of 1,4-butanediol diglycidyl ether (BDDE) and stir for 30 minutes to mix evenly.

[0082] Solution B: Add 0.83 g of Span-80 to 83.35 g of liquid paraffin, place in an ice bath and stir at low speed (about 300 rpm) to obtain solution B.

[0083] Cross-linking reaction: Solution A was slowly added to Solution B while stirring. Mix at 10°C and 2000 rpm for 2 h to form a uniform emulsion. The temperature was then slowly raised to 30°C. Stirring was continued for 20 min. Then, 0.125 g of 1,4-butanediol diglycidyl ether was slowly added to the emulsion. The reaction was continued at 30°C and 800 rpm for 8 h.

[0084] Example 5:

[0085] The steps of purifying hyaluronic acid microspheres in this example are different from those in Example 1, and the other steps are the same.

[0086] Add 120 ml of isopropanol to the reaction solution while stirring. After the oil phase and isopropanol are fully mixed, let it stand to precipitate the hyaluronic acid microspheres. Pour off the supernatant and then wash the oil phase thoroughly with isopropanol. Use 120 ml of isopropanol each time. Repeat 3 times and discard the supernatant.

[0087] Under rapid stirring, slowly add 200 ml of anhydrous ethanol to the washed hyaluronic acid microspheres, stir for 10 minutes and then let it stand. After the microspheres are precipitated, discard the supernatant. Repeat this process three times until the microspheres appear as small white particles. Place them in a vacuum drying oven at 40°C and dry for 12 hours.

[0088] Add 250 ml of purified water to the dried microspheres, stir and swell at room temperature for 12 hours, let it stand for 3 hours, discard the supernatant, add 250 ml of purified water again and continue stirring. After discarding the supernatant for the last time, slowly add 200 ml of anhydrous ethanol to the microspheres under rapid stirring, stir for 15 minutes and let it stand. After the microspheres are precipitated, discard the supernatant. Repeat 3 times until the microspheres appear as fine white particles, and place them in a vacuum drying oven at 40°C and dry for 24 hours.

[0089] The dried microspheres were sieved with 325-mesh and 140-mesh sieves, and the middle part was collected to obtain microspheres with a particle size of 45 to 106 μm for use.

[0090] Example 6:

[0091] The steps of purifying hyaluronic acid microspheres in this example are different from those in Example 1, and the other steps are the same.

[0092] After the reaction is completed, add 500 ml of ethyl acetate to the reaction solution under stirring. After the oil phase and ethyl acetate are fully mixed, let it stand to precipitate the hyaluronic acid microspheres. Pour off the supernatant, and then wash the oil phase thoroughly with ethyl acetate. Use 500 ml of ethyl acetate each time, repeat 3 times, and discard the supernatant.

[0093] Under rapid stirring, slowly add 500 ml of anhydrous ethanol to the washed hyaluronic acid microspheres, stir for 30 minutes and then let it stand. After the microspheres are precipitated, discard the supernatant. Repeat this process three times until the microspheres appear as fine white particles. Place them in a vacuum drying oven at 25°C and dry for 48 hours.

[0094] After drying, 500 ml of purified water was added to the microspheres, and the mixture was stirred at room temperature to swell for 24 h. The supernatant was discarded after standing for 6 h, and 500 ml of purified water was added again and the stirring was continued. After the supernatant was discarded for the last time, 500 ml of anhydrous ethanol was slowly added to the microspheres under rapid stirring. The mixture was stirred for 20 min and then stood. The supernatant was discarded after the microspheres were precipitated. This process was repeated 3 times until the microspheres became small white particles. The microspheres were placed in a vacuum drying oven at 30°C and dried for 36 h.

[0095] The dried microspheres were sieved with 325-mesh and 140-mesh sieves, and the middle part was collected to obtain microspheres with a particle size of 45 to 106 μm for use.

[0096] Comparative Example 1:

[0097] The cross-linking reaction process and specific parameters described in this comparative example are different from those in Example 1, and the remaining steps (such as the purification and preparation of hyaluronic acid microspheres for injection) are the same.

[0098] Add 2.5 g of sodium hyaluronate powder (molecular weight: 1.5 million Da) to 31.25 ml of 1% sodium hydroxide solution and dissolve with stirring at room temperature for 30 minutes to form a sodium hyaluronate solution with a concentration of 80 g / L. The sodium hyaluronate solution is then placed at 4°C for 12 hours, frozen at -20°C for 12 hours, transferred to the cold trap of a vacuum freeze-drying oven (-60°C) for another hour, and then transferred to a freeze-drying chamber for vacuum drying for 24 hours to obtain dried hyaluronic acid. The dried hyaluronic acid is ground in liquid nitrogen and passed through a 100-mesh sieve.

[0099] Then, 2 g of ground and sieved sodium hyaluronate microspheres were dispersed in an appropriate amount of acetone solution, and 0.25 g of 1,4-butanediol diglycidyl ether was added dropwise while stirring. After the addition of 1,4-butanediol diglycidyl ether was completed, stirring was continued and the cross-linking reaction was carried out at room temperature (25°C) for 6 h. Then, 2 ml of purified water was added dropwise to the above reaction solution, and the reaction was continued at room temperature for 12 h. The filtered product was then dialyzed.

[0100] Comparing this comparative example with the method of Example 1, it was found that the hyaluronic acid microspheres prepared by the method of Comparative Example 1 had the following problems:

[0101] (1) During the preparation process, this method requires freeze-drying, drying, grinding and sieving the uncross-linked microspheres. Although grinding and sieving can control the particle size, there will also be a large degree of loss, which affects the yield of this method.

[0102] (2) This method uses direct dialysis in water to remove unreacted crosslinking agent after the crosslinking reaction. However, in the actual preparation process, the hyaluronic acid inside the dialysis bag absorbs water to form a viscous gel and loses the shape of the microspheres. In this gel state, coupled with the strong acidity and hydrophilicity of hyaluronic acid itself, it is difficult to ensure that the unreacted crosslinking agent and sodium hydroxide are completely removed, which affects the performance of the microspheres.

[0103] (3) During the dialysis process, the hyaluronic acid inside the dialysis bag is always at a high concentration, and because hyaluronic acid has strong water absorption, the hyaluronic acid will only continue to absorb water and expand during dialysis. The volume of the hyaluronic acid gel in the dialysis bag continues to increase, while the concentration continues to decrease. This may cause the dialysis bag to rupture, making it difficult to proceed to the next step. In addition, the high water content will bring difficulties to the second freeze-drying, which is not conducive to practical operation.

[0104] (4) The microspheres in this method are actually cross-linked sodium hyaluronate particles formed by final grinding, and microspheres are not formed during the reaction process. The morphology of the final particles is greatly dependent on the grinding process, and there is no significant difference from the preparation method used in the prior art of first cross-linking into a gel and then crushing. In addition, grinding will cause greater losses, and the morphology of the obtained particles may also be irregular, which may cause difficulties in injection and pushing.

[0105] Comparative Example 2:

[0106] The cross-linking reaction process and specific parameters described in this comparative example are different from those in Example 1, and the remaining steps (such as the purification and preparation of hyaluronic acid microspheres for injection) are the same.

[0107] At a low temperature of 2°C, 0.25 g of 1,4-butanediol diglycidyl ether was added to an appropriate amount of water and mixed evenly. Then, 2.5 g of sodium hyaluronate powder with a molecular weight of 1.5 million Da was added and mixed evenly. Then, 31.25 ml of 1% sodium hydroxide solution was added thereto. The mixture was treated at 5000 rpm for 10 minutes using a high shear dispersing emulsification homogenizer to obtain an aqueous phase.

[0108] The aqueous phase was added to 400 mL of n-octane containing 2 wt% Span80, and treated with a high shear dispersing emulsification homogenizer at 10,000 rpm for 10 min. After the emulsification was uniform, the mixture was allowed to stand to remove bubbles.

[0109] After emulsification, the emulsion was subjected to cross-linking reaction at 30° C. for 12 h under continuous stirring.

[0110] Comparing this comparative example with the method of Example 1, it was found that the method of Comparative Example 2 had the following problems, resulting in the inability to prepare ideal hyaluronic acid microspheres:

[0111] (1) During the initial preparation of the aqueous phase, high molecular weight sodium hyaluronate is difficult to swell and dissolve in a relatively small amount of aqueous solution to form a uniform solution. In particular, the dissolution rate will be even slower under low temperature conditions. The method of this comparative example is to dissolve sodium hyaluronate in an aqueous solution of a crosslinking agent. However, the actual operation results in difficulty in uniform mixing within a short period of time. In addition, although low temperature can inhibit the crosslinking reaction to a certain extent, the crosslinking reaction may still occur slowly. In addition, due to the high concentration of the crosslinking agent, a long dissolution process may cause uneven crosslinking reaction within the aqueous phase, which is not conducive to subsequent emulsification in the oil phase and uniform crosslinking within the microspheres. Therefore, in the method of the present invention (such as Example 1), sodium hyaluronate is first dissolved at room temperature, and then a small amount of crosslinking agent is added, thereby controlling the mixing time to avoid excessive crosslinking.

[0112] (2) The method of Comparative Example 2 could not produce ideal hyaluronic acid microspheres. The reason may be that during the shear dispersion emulsification process, the hyaluronic acid had been cross-linked, and its fluidity was significantly reduced, forming colloidal lumps. In addition, the density and viscosity of the n-octane used were low, so a uniform and stable emulsion could not be formed. As a result, subsequent experiments were unable to produce hyaluronic acid microspheres with suitable morphology.

[0113] Comparative Example 3:

[0114] The method of adding the cross-linking agent in this comparative example is different from that in Example 1, and the remaining steps (such as the process of purification and preparation of hyaluronic acid microspheres for injection) are the same.

[0115] 2.5 g of sodium hyaluronate powder with a molecular weight of 1.5 million Da was added to 31.25 ml of 1% sodium hydroxide solution, and the mixture was stirred and dissolved at room temperature for 30 min to form a sodium hyaluronate solution with a concentration of 80 g / L, forming solution A.

[0116] Add 3.32 g of Span-80 to 83.35 g of liquid paraffin, place in an ice bath, and stir at a low speed (about 300 rpm) to form solution B.

[0117] Under stirring, solution A was slowly added to solution B. After stirring and mixing evenly, 0.25 g of 1,4-butanediol diglycidyl ether was added thereto, and the mixture was reacted at 30°C and 800 rpm for 8 h.

[0118] In this comparative example, the residual BDDE content in the hyaluronic acid microspheres prepared by this method was detected to be 8.1 μg / g. It can be seen that this method cannot prepare hyaluronic acid microspheres that meet the standard requirements.

[0119] Comparative Example 4:

[0120] The amount of cross-linking agent added in this comparative example is different from that in Example 1, and the remaining steps (such as the purification and preparation of hyaluronic acid microspheres for injection) are the same.

[0121] Add 2.5 g of sodium hyaluronate powder with a molecular weight of 1.5 million Da to 31.25 ml of 1% sodium hydroxide solution and stir at room temperature for 30 minutes to dissolve to form a sodium hyaluronate solution with a concentration of 80 g / L. Then, place the above sodium hyaluronate solution in an ice bath (0°C) and continue stirring for 15 minutes. At low temperature (about 8°C), add 0.0625 g of 1,4-butanediol diglycidyl ether and stir for 10 minutes to mix evenly to form solution A.

[0122] Add 3.32 g of Span-80 to 83.35 g of liquid paraffin, place in an ice bath, and stir at a low speed (about 300 rpm) to form solution B.

[0123] While stirring, slowly add Solution A to Solution B and mix at 5°C and 800 rpm for 2 h to form a uniform emulsion. Then slowly raise the temperature to 30°C. After continuing stirring for 30 min, slowly add 0.0625 g of 1,4-butanediol diglycidyl ether to the emulsion and continue the reaction at 30°C and 800 rpm for 8 h.

[0124] In this comparative example, due to insufficient addition of cross-linking agent during the preparation process, a gel was formed after swelling with water, and the swelling degree was large, indicating a low degree of cross-linking. It can be seen that this method cannot produce hyaluronic acid microspheres that meet the standard requirements.

[0125] Experimental part:

[0126] (1) Morphological observation

[0127] The hyaluronic acid microspheres for injection prepared in Example 1 and Comparative Example 4 were coated on glass slides respectively and observed under a microscope (Nikon Ts2RFL inverted microscope, 10 times, scale of 100 μm). Figure 1 and Figure 2 .

[0128] Depend on Figure 1 It can be seen that the hyaluronic acid microspheres of Example 1 are relatively round and compact individual particles with good dispersion, no aggregation, and a narrow particle size distribution range, indicating that microspheres with a high degree of cross-linking have been formed.

[0129] Depend on Figure 2 It can be seen that in the hyaluronic acid microspheres of Comparative Example 4, although some cross-linked hyaluronic acid is spherical, most of them are still irregular fragments with a relatively loose structure, indicating that microspheres with good morphology cannot be formed at this cross-linking agent concentration.

[0130] (2) Swelling degree

[0131] The hyaluronic acid microspheres for injection prepared in Example 1 and Comparative Example 4 were tested for swelling.

[0132] Swelling degree test method: Weigh the dried hyaluronic acid microspheres and record their mass. Then add sufficient purified water to the microspheres and let them stand at room temperature. When the volume of the microspheres no longer increases (more than 24 hours), aspirate the excess purified water and weigh the remaining microspheres. Calculate the swelling degree according to the following formula.

[0133]

[0134] Wherein, Q is the swelling degree of cross-linked sodium hyaluronate microspheres, m1 is the mass of cross-linked sodium hyaluronate microspheres after drying, and m2 is the mass of cross-linked sodium hyaluronate microspheres after swelling after absorbing water.

[0135] The test results are shown in Table 1 below.

[0136] Table 1 Swelling test data of Example 1 and Comparative Example 4

[0137]

[0138] It can be seen that the hyaluronic acid microspheres prepared in Example 1 have a significantly lower swelling degree than that in Comparative Example 4, and this swelling degree can improve the problem of facial swelling after injection.

[0139] (III) Enzyme resistance test

[0140] The injectable hyaluronic acid microspheres from Example 1 and Comparative Example 4 were accurately weighed, added to 1 ml of purified water, and allowed to swell at room temperature for 24 hours. These served as Experimental Groups 1 and 2. 12 mg of free sodium hyaluronate powder was accurately weighed, added to 1 ml of purified water, and allowed to swell at room temperature for 24 hours. This served as the control group. One ml of commercially available cross-linked sodium hyaluronate gel for injection (hyaluronic acid concentration: 12 mg / ml) was also used as the commercially available group.

[0141] 1 ml of hyaluronidase (3 mg / ml) was added to each of the experimental groups 1, 2, control, and commercially available solutions, and the solutions were incubated at 37°C. The time required for complete degradation of the hyaluronic acid was recorded. The sodium hyaluronate gel in both the control and commercially available groups was observed to have completely degraded into a flowing liquid within 1 hour, while the hyaluronic acid microspheres in experimental group 2 had completely degraded into a flowing liquid within 3 hours. Only the hyaluronic acid microspheres in experimental group 1 remained incompletely degraded after 84 hours, demonstrating strong resistance to enzymatic degradation and indicating a lower degradation rate. Therefore, the frequency of injections can be reduced in clinical use. For details, see Table 2 below.

[0142] (IV) Determination of residual amount of cross-linking agent BDDE

[0143] The above-mentioned experimental group 1, experimental group 2, control group and commercially available group were used as samples, and the residual amount of the cross-linking agent BDDE therein was detected according to the method in the YY / T0962-2014 industry standard for cross-linked sodium hyaluronate gel for plastic surgery, as shown in Table 2 below.

[0144] Table 2 HAase degradation time and BDDE residue data of samples

[0145]

[0146] As shown in Table 2 above, the residual amount of the cross-linking agent BDDE in the sodium hyaluronate microspheres prepared in Example 1 was only 0.15±0.31 μg / g, which is far below the 2 μg / g requirement in the standard and can be used safely.

[0147] (5) Pushing force

[0148] The hyaluronic acid microspheres prepared in Example 1 were used as samples. The push rod was pushed at a constant speed, and the sample in the syringe was pushed out through the needle to obtain an extrusion force curve. During the test, a universal material testing machine was used to measure the injection extrusion force of the hyaluronic acid microspheres.

[0149] The hyaluronic acid microspheres of Example 1 were filled into a syringe with a volume of 1 ml and a needle diameter of 0.45 mm. The syringe was then mounted on a testing device, and its pushing force curve was measured at a pushing speed of 30 mm / min. Figure 3 is the pushing force curve of the sample, such as Figure 3 It can be seen that the pushing force of the sample of Example 1 is always kept relatively stably between 6-8N, and therefore, it is easy to accurately control during injection.

[0150] (6) Rheological properties

[0151] The hyaluronic acid microspheres prepared in Example 1 were used as samples, and the changes in storage modulus G' and loss modulus G'' within the range of 0.01-10 Hz were measured using an Anton Paar MCR rheometer. A graph was drawn to investigate the viscoelasticity of the hyaluronic acid microspheres. Figure 4 is the rheological property curve of the sample, such as Figure 4 It can be seen that the sample of Example 1 exhibits higher viscoelasticity, and therefore is conducive to maintaining its own shape after injection and is not prone to displacement.

[0152] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing hyaluronic acid microspheres for injection, characterized in that: The following steps are involved: S1. Prepare solution A and solution B separately, The sodium hyaluronate solution was placed in an ice bath and stirred to dissolve, and a cross-linking agent was added thereto, stirred and mixed uniformly to prepare solution A; the mixture of the emulsifier and the oil phase was placed in an ice bath and stirred uniformly to prepare solution B. In the solution A, the concentration of sodium hyaluronate is 16 to 320 g / L, and the concentration of the cross-linking agent is 4 to 16 g / L; S2. Cross-linking reaction, Stirring solution A and solution B at 2-10° C. to form a uniform emulsion, raising the temperature of the emulsion to 30° C. and continuing to stir, adding a crosslinking agent to the emulsion, stirring at 30-60° C. and performing a crosslinking reaction to prepare a reaction solution containing hyaluronic acid microspheres; S3. Remove impurities, After removing the oil phase and water from the reaction solution, hyaluronic acid microspheres are obtained, and after removing the residual crosslinking agent and emulsifier on the hyaluronic acid microspheres, the hyaluronic acid microspheres are dried to obtain microspheres with a particle size of 45 to 106 μm; S4. Subsequent processing, The microspheres are added to a phosphate buffer solution for swelling treatment, and then mixed evenly with an equal volume of sodium hyaluronate solution to obtain hyaluronic acid microspheres for injection. Calculated by mass-to-volume ratio, the amount of hyaluronic acid microspheres added to each milliliter of phosphate buffer is 0.01-0.02 g.

2. The preparation method according to claim 1, wherein: In step S1 and step S4, the sodium hyaluronate solution is formed by adding sodium hyaluronate powder to an alkaline solution and stirring and dissolving the solution at room temperature. The molecular weight of the sodium hyaluronate powder used in step S1 is 200,000 to 1.5 million Da, and the molecular weight of the sodium hyaluronate powder used in step S4 is 200,000 to 2.2 million Da.

3. The preparation method according to claim 2, wherein: The alkaline solution includes one or a mixture of at least two of sodium hydroxide, potassium hydroxide, and ammonia water.

4. The preparation method according to claim 1, wherein: The cross-linking agent added in step S1 and step S2 is the same, and the cross-linking agent is selected from one or more of 1,4-butanediol diglycidyl ether, divinyl sulfone, glycerol triglycidyl ether, resorcinol diglycidyl ether, allyl glycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, and neopentyl glycol diglycidyl ether.

5. The preparation method according to claim 1, wherein: In the step S1, the mass ratio of the emulsifier to the oil phase is controlled to be 0.8-6.67:83.

35.

6. The preparation method according to claim 5, characterized in that: The emulsifier includes a surfactant with a hydrophilic-lipophilic balance value of 3 to 8; the oil phase includes one or a mixture of at least two of liquid paraffin, vegetable oil, mineral oil, silicone oil, and synthetic oil.

7. The preparation method according to claim 6, characterized in that: The surfactant with a hydrophilic-lipophilic balance value of 3 to 8 is selected from one of Span 60 and Span 80, or a mixture of the two.

8. The preparation method according to claim 1, wherein: In step S2, the amount of cross-linking agent added to each liter of emulsion is 0.78 to 3.88 g based on mass volume concentration.

9. The preparation method according to claim 1, wherein: In step S2, the cross-linking reaction time is controlled to be 8 to 10 hours.

10. The preparation method according to claim 1, characterized in that: In step S3, removing the oil phase and water from the reaction solution includes: adding ethyl acetate to the reaction solution under stirring, fully mixing the oil phase and ethyl acetate, allowing the mixture to stand, removing the supernatant after the hyaluronic acid microspheres are precipitated, and repeating this operation; continuing to add anhydrous ethanol to the hyaluronic acid microspheres, stirring and allowing the mixture to stand, removing the supernatant after the hyaluronic acid microspheres are precipitated, and repeating this operation.

11. The preparation method according to claim 1, characterized in that: In step S4, the swelling treatment time is controlled to be 12 to 24 hours.

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