A hyaluronic acid microsphere, its preparation method and application

Abstract

The present invention discloses a hyaluronic acid microsphere and its preparation method and application, belonging to the technical field of hyaluronic acid materials. The particle size distribution of the hyaluronic acid microsphere is 20 μm to 70 μm; the elastic modulus of the hyaluronic acid microsphere at a shear frequency of 5 Hz in a rheometer is 40 Pa to 800 Pa, and / or the viscous modulus at 5 Hz is 15 Pa to 1000 Pa, and / or the swelling degree is 15 to 40, and / or the pushing force is 9 N to 20 N, etc. The preparation method includes: mixing hyaluronic acid or its salt with an alkali and water, and then mixing with a cross-linking agent to obtain an aqueous phase; mixing the aqueous phase with an oil phase, performing primary emulsification, and then secondary emulsification to remove the oil phase; the primary emulsification and the secondary emulsification are respectively carried out under the conditions that the rotation speed is not lower than 1000 rpm and 4000 rpm. The hyaluronic acid microsphere can be used as a soft tissue filler, which can avoid problems such as redness, swelling, and blood vessel blockage, and improve the safety of use.

Description

Technical Field

[0001] The present invention relates to the technical field of hyaluronic acid materials, and in particular, to a hyaluronic acid microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] At present, there are various soft tissue fillers for facial aesthetic correction on the market, including autologous fat, polymethyl methacrylate, hydroxyapatite, poly-L-lactic acid, polycaprolactone, and hyaluronic acid (HA). Among them, HA is the most widely used filler, and the efficacy maintenance time is about 6 to 24 months. However, with the increase in the amount of filler used, some risks (such as bruising, swelling, infection, vascular damage, and blindness) also increase.

[0003] HA is a high molecular weight linear mucopolysaccharide with repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine. Modified HA gels have been studied for many years at home and abroad, and their application in the field of soft tissue fillers has been very mature. The preparation method of common filler products on the market is that HA reacts with a cross-linking agent under alkaline conditions to form a gel, and the obtained bulk gel is mechanically crushed or pressed into particles. However, the obtained gel has problems such as irregular particle morphology, large and uneven particle size, and may cause redness, swelling, vascular occlusion and other consequences when used for facial filling.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a hyaluronic acid microsphere, a preparation method thereof, and an application thereof to solve or improve the above technical problems.

[0006] The present invention can be realized as follows:

[0007] In a first aspect, the present invention provides a hyaluronic acid microsphere, and its particle size distribution is 20 μm to 70 μm;

[0008] The hyaluronic acid microsphere further has at least one of the following characteristics:

[0009] Characteristic 1: The elastic modulus of the hyaluronic acid microsphere at 5 Hz is 40 Pa to 800 Pa;

[0010] Characteristic 2: The viscous modulus of the hyaluronic acid microsphere at 5 Hz is 15 Pa to 1000 Pa;

[0011] Characteristic 3: The swelling degree of the hyaluronic acid microsphere is 5 to 40;

[0012] Characteristic 4: The content of hyaluronic acid in the hyaluronic acid microsphere is 5 mg / mL to 40 mg / mL;

[0013] Feature Five: The pushing force of the hyaluronic acid microspheres is 9 N to 20 N;

[0014] Feature Six: The osmotic pressure of the hyaluronic acid microspheres is 240 mOsmol / kg to 360 mOsmol / kg;

[0015] Feature Seven: The refractive index of the hyaluronic acid microspheres is 1.32 to 1.35.

[0016] In a second aspect, the present invention provides a method for preparing hyaluronic acid microspheres as described in the foregoing embodiments, comprising the following steps:

[0017] Mix hyaluronic acid or its salt with an alkali and water, and then mix with a crosslinking agent to obtain an aqueous phase;

[0018] Mix the aqueous phase with an oil phase and perform primary emulsification to obtain a dispersion;

[0019] Perform secondary emulsification on the dispersion to obtain an emulsion;

[0020] Remove the oil phase from the emulsion to obtain crosslinked hyaluronic acid microspheres;

[0021] Among them, the primary emulsification is carried out under the condition that the rotation speed is not lower than 1000 rpm, and the rotation speed of the secondary emulsification is not lower than 4000 rpm.

[0022] In an alternative embodiment, the primary emulsification is carried out at 1000 rpm to 2000 rpm for 5 min to 30 min, and the secondary emulsification is carried out at 4000 rpm to 8000 rpm for 10 min to 30 min.

[0023] In an alternative embodiment, the content of hyaluronic acid or its salt in the aqueous phase is 0.5 wt% to 20 wt%.

[0024] In an alternative embodiment, the content of the alkali in the aqueous phase is 0.5 wt% to 5 wt%.

[0025] In an alternative embodiment, the alkali includes at least one of NaOH and KOH.

[0026] In an alternative embodiment, the content of the crosslinking agent in the aqueous phase is 0.1 wt% to 10 wt%.

[0027] In an alternative embodiment, the crosslinking agent includes at least one of butanediol diglycidyl ether and divinyl sulfone.

[0028] In an alternative embodiment, the volume ratio of the oil phase to the aqueous phase is 1:1 to 10:1.

[0029] In an alternative embodiment, the oil phase comprises at least one of vegetable oil, mineral oil, silicone oil, liquid paraffin, dodecane, n-octane and cyclohexane.

[0030] In a third aspect, the present invention provides a hyaluronic acid filler, which comprises at least one of a non-crosslinked sodium hyaluronate solution and an active ingredient, and the hyaluronic acid microspheres of the foregoing embodiment;

[0031] Wherein, the active ingredient comprises at least one of amino acid and lidocaine.

[0032] In a fourth aspect, the present invention provides an application of the hyaluronic acid microspheres of the foregoing embodiment or the hyaluronic acid filler of the foregoing embodiment, for example, it can be used as a soft tissue filler.

[0033] In an alternative embodiment, the hyaluronic acid microspheres or the hyaluronic acid filler are used as a medical soft tissue filler or a cosmetic soft tissue filler.

[0034] The beneficial effects of the present invention include:

[0035] The hyaluronic acid microspheres provided by the present application have a narrow particle size distribution, which can avoid vascular embolism caused by injection into blood vessels; at the same time, the particle size of the hyaluronic acid microspheres is small, and the corresponding specific surface area is large. Even when there is overcorrection or accidental embolism during operation, it can be quickly degraded by injecting hyaluronidase at the problem site, greatly improving the safety of injection. In addition, the hyaluronic acid microspheres provided by the present application have good mechanical strength and can achieve a better filling effect after implantation, realizing the effectiveness of the filler; moreover, the hyaluronic acid microspheres also have a lower extrusion force, which is beneficial to achieving injectability.

[0036] The present invention adopts a two-step emulsification method. First, the first emulsification (pre-emulsification) is carried out to obtain a dispersion liquid, and the oil-water ratio of the emulsification can be controlled to avoid uneven emulsification caused by the high viscosity of the water phase, and better carry out the next high-speed emulsification to obtain microspheres with a narrow particle size distribution. Through the two-step cross-linking emulsification method, the number of times the microspheres are sheared by the emulsifying machine can be made more consistent, and sufficient emulsification can be obtained. In addition, by pre-emulsifying at a low speed first and then emulsifying at a high speed, the time of high-speed emulsification can be reduced, and the adhesion of microspheres and the demulsification of the emulsion caused by the temperature rise during high-speed emulsification can be avoided, and further, the particle size distribution of the microspheres can be more accurately controlled within the range of 20 μm to 50 μm. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 Microscopic photograph of the hyaluronic acid microspheres provided in Example 1 of the present invention;

[0039] Figure 2 Microscopic photograph of the hyaluronic acid microspheres provided in Comparative Example 2 of the present invention;

[0040] Figure 3 Microscopic photograph of the hyaluronic acid product provided by the existing product Runbaiyan (Bloomage Biotechnology Co., Ltd.);

[0041] Figure 4 Result graph of the animal experiment in the test example of the present invention. Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market.

[0043] The hyaluronic acid microspheres provided by the present application, their preparation methods and applications will be specifically described below.

[0044] The present application provides a kind of hyaluronic acid microspheres, and their particle size distribution is 20μm - 70μm.

[0045] The particle size distribution of the hyaluronic acid microspheres is relatively concentrated, and the particle sizes are uniform, which can avoid problems such as swelling, vascular occlusion, etc. easily caused by irregular particle morphology, large particle size and non-uniform particle size. It should be noted that if the particle size of the hyaluronic acid microspheres is less than 20μm, it is easily phagocytosed by human macrophages. If the particle size of the hyaluronic acid microspheres is greater than 70μm, it is likely to cause vascular embolism after injection into the blood vessels.

[0046] In addition, the surface of the hyaluronic acid microspheres provided by the present application is smooth, has better biocompatibility, and is not likely to cause tissue swelling and inflammation.

[0047] The elastic modulus of the hyaluronic acid microspheres provided by the present application at a shear frequency of 5 Hz of the rheometer can be 40 Pa to 800 Pa (such as 40 Pa, 50 Pa, 80 Pa, 100 Pa, 150 Pa, 200 Pa, 250 Pa, 300 Pa, 400 Pa, 500 Pa, 600 Pa, 700 Pa or 800 Pa, etc., and can also be other values within the range of 40 Pa to 800 Pa), and the viscous modulus can be 15 Pa to 1000 Pa (such as 15 Pa, 50 Pa, 80 Pa, 100 Pa, 150 Pa, 220 Pa, 250 Pa, 300 Pa, 400 Pa, 500 Pa, 600 Pa, 700 Pa, 800 Pa, 900 Pa or 1000 Pa, etc., and can also be other values within the range of 15 Pa to 1000 Pa). The hyaluronic acid microspheres with the above elastic modulus and viscous modulus can have better elasticity and viscosity, which is beneficial to improving the filling effect.

[0048] The swelling degree of the hyaluronic acid microspheres provided by the present application can be 5 to 40, such as 5, 8, 10, 12, 15, 18, 20, 25, 30, 35 or 40, etc., and can also be other values within the range of 5 to 40. The hyaluronic acid microspheres with the above swelling degree are suitable as fillers.

[0049] The content of hyaluronic acid in the hyaluronic acid microspheres provided by the present application can be 5 mg / mL to 40 mg / mL, such as 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL or 40 mg / mL, etc., and can also be other values within the range of 5 mg / mL to 40 mg / mL. The hyaluronic acid microspheres with the above hyaluronic acid content can achieve a good filling effect.

[0050] The pushing force of the hyaluronic acid microspheres provided by the present application can be 9 N to 20 N, such as 9 N, 10 N, 12 N, 15 N, 18 N or 20 N, etc., and can also be other values within the range of 9 N to 20 N. On the one hand, the hyaluronic acid microspheres with the pushing force within this range can facilitate the user's injection, and on the other hand, they can reduce the pain and discomfort brought after the product is injected.

[0051] The osmotic pressure of the hyaluronic acid microspheres provided by the present application can be 240 mOsmol / kg to 360 mOsmol / kg, such as 240 mOsmol / kg, 260 mOsmol / kg, 280 mOsmol / kg, 300 mOsmol / kg, 320 mOsmol / kg, 340 mOsmol / kg or 360 mOsmol / L, etc., or can also be other values within the range of 240 mOsmol / kg to 360 mOsmol / kg. The hyaluronic acid microspheres with the above osmotic pressure can maintain an osmotic pressure relatively consistent with that of the human body, avoiding obvious water absorption or dehydration phenomena in the injection area after being injected into the human body.

[0052] The refractive index of the hyaluronic acid microspheres provided by the present application can be 1.32 to 1.35, such as 1.32, 1.33, 1.34 or 1.35, etc., or can also be other values within the range of 1.32 to 1.35. The hyaluronic acid microspheres with the above refractive index have a high purity.

[0053] Continuing from the above, the hyaluronic acid microspheres provided by the present application have a narrow particle size distribution, which can avoid vascular embolism after being injected into blood vessels; at the same time, the particle size of the hyaluronic acid microspheres is small, and the corresponding specific surface area is large. Even when there is overcorrection or accidental embolism during operation, it can be rapidly degraded by injecting hyaluronidase at the problem site, greatly improving the safety of injection. In addition, the hyaluronic acid microspheres provided by the present application have good mechanical strength and can achieve a better filling effect after implantation, realizing the effectiveness of the filler; and, the hyaluronic acid microspheres also have a low extrusion force, which is beneficial to achieving injectability.

[0054] Correspondingly, the present invention also provides a preparation method of the above hyaluronic acid microspheres, which may include the following steps:

[0055] Mix hyaluronic acid or its salt with an alkali and water, and then mix with a cross-linking agent to obtain an aqueous phase;

[0056] Mix the aqueous phase with an oil phase and perform primary emulsification to obtain a dispersion;

[0057] Perform secondary emulsification on the dispersion to obtain an emulsion;

[0058] Remove the oil phase from the emulsion to obtain cross-linked hyaluronic acid microspheres;

[0059] Among them, the primary emulsification is carried out under the condition that the rotation speed is not lower than 1000 rpm, and the rotation speed of the secondary emulsification is not lower than 4000 rpm.

[0060] It should be noted that the conventional technology uses a single emulsification method, which is prone to the problem of uneven emulsification. The part poured into the emulsifier first will be over-emulsified to form microspheres with small particle sizes, while the part poured into the emulsifier later is prone to incomplete emulsification, forming microspheres with large particle sizes. Eventually, a microsphere product with a wide particle size distribution and poor particle size uniformity is obtained.

[0061] In the present invention, by first performing the first emulsification to obtain a dispersion through pre-emulsification, the oil-water ratio of emulsification can be accurately controlled, avoiding uneven emulsification caused by the high viscosity of the aqueous phase, and better performing the next high-speed emulsification to obtain microspheres with a narrow particle size distribution. Through the two-step cross-linking emulsification method, the number of times the microspheres are sheared by the emulsifier can be made more consistent, resulting in sufficient emulsification. In addition, by first performing low-speed pre-emulsification and then high-speed emulsification, the time of high-speed emulsification can be reduced, avoiding the adhesion of microspheres and the demulsification of the emulsion caused by the temperature rise during high-speed emulsification, and thus more accurately controlling the particle size distribution of the microspheres in the range of 20 μm to 50 μm.

[0062] For reference, the first emulsification is carried out under the conditions of 1000 rpm to 2000 rpm for 5 min to 30 min. For example, the rotation speed of this process can be 1000 rpm, 1200 rpm, 1500 rpm, 1800 rpm or 2000 rpm, etc., or any other value within the range of 1000 rpm to 2000 rpm. The emulsification time of this process can be 5 min, 10 min, 15 min, 20 min, 25 min or 30 min, etc., or other values within the range of 5 min to 30 min.

[0063] The second emulsification is carried out under the conditions of 4000 rpm to 8000 rpm for 10 min to 30 min. For example, the rotation speed of this process can be 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm or 8000 rpm, etc., or any other value within the range of 4000 rpm to 8000 rpm. The emulsification time of this process can be 10 min, 15 min, 20 min, 25 min or 30 min, etc., or other values within the range of 10 min to 30 min.

[0064] In the present invention, the hyaluronate can exemplarily but non-limitingly include sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, etc. The content of hyaluronic acid or its salt in the aqueous phase can be 0.5 wt% to 20 wt%, such as 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, 12 wt%, 15 wt%, 18 wt% or 20 wt%, etc., or other values within the range of 0.5 wt% to 20 wt%.

[0065] If the content of hyaluronic acid or its salt in the aqueous phase is too low, it will result in insufficient viscosity and elasticity of the product. If the content of hyaluronic acid or its salt in the aqueous phase is too high, it will cause problems with poor solubility.

[0066] In the present invention, the content of the base in the aqueous phase can be 0.5 wt% to 5 wt%, such as 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt% or 5 wt%, etc., or other values within the range of 0.5 wt% to 5 wt%.

[0067] Exemplarily, the base can include at least one of NaOH and KOH.

[0068] If the content of the base in the aqueous phase is too low, it will result in no cross-linking reaction occurring or incomplete cross-linking; if the content of the base in the aqueous phase is too high, it is easy to break the molecular chain of hyaluronic acid or its salt.

[0069] In the present invention, the content of the cross-linking agent in the aqueous phase can be 0.1 wt% to 10 wt%, such as 0.1 wt%, 0.2 wt%, 0.5 wt%, 1 wt%, 2 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% or 10 wt%, etc., or other values within the range of 0.1 wt% to 10 wt%.

[0070] Similarly, if the content of the cross-linking agent in the aqueous phase is too low, it will result in no cross-linking reaction occurring or incomplete cross-linking; if the content of the cross-linking agent in the aqueous phase is too high, it is easy to break the molecular chain of hyaluronic acid or its salt.

[0071] The cross-linking agent can exemplarily but non-limitingly include at least one of butanediol diglycidyl ether and divinyl sulfone.

[0072] For reference, the mixing temperature of the above materials can be 40 °C to 50 °C to enable sufficient cross-linking of hyaluronic acid or its salt.

[0073] In the present invention, the volume ratio of the oil phase to the aqueous phase can be 1:1 to 10:1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, etc., or other values within the range of 1:1 to 10:1. By mixing the aqueous phase and the oil phase, a water-in-oil system is formed, and through subsequent emulsification processes, microspheres with better morphology and size are obtained.

[0074] The above oil phase can exemplarily but non-limitingly include at least one of vegetable oil, mineral oil, silicone oil, liquid paraffin, dodecane, n-octane and cyclohexane.

[0075] As an example, the oil phase in the emulsion can be removed by filtration.

[0076] Continuing from the above, the preparation method of the hyaluronic acid microspheres provided by this application is simple, the reaction conditions are mild, the process is easy to control, and hyaluronic acid microspheres with excellent effects and high yields can be obtained.

[0077] In addition, the present invention also provides a hyaluronic acid filler, which includes at least one of a non-crosslinked sodium hyaluronate solution and an active ingredient, and the above-mentioned hyaluronic acid microspheres.

[0078] By compounding the non-crosslinked sodium hyaluronate solution with the hyaluronic acid microspheres, the shear viscosity of the product can be increased.

[0079] By compounding the active ingredient with the hyaluronic acid microspheres, other effects of the product can be increased, such as moisturizing and whitening. Exemplarily, the active ingredient may include at least one of amino acids and lidocaine.

[0080] It should be noted that regarding the compounding relationship between the non-crosslinked sodium hyaluronate solution and the active ingredient with the hyaluronic acid microspheres, it can be set as needed and will not be overly limited here.

[0081] Furthermore, the present invention also provides an application of the above-mentioned hyaluronic acid microspheres or the above-mentioned hyaluronic acid filler. For example, it can be used as a soft tissue filler.

[0082] As a reference, the hyaluronic acid microspheres or the hyaluronic acid filler can be used as a medical soft tissue filler or a cosmetic soft tissue filler.

[0083] As a reference, the hyaluronic acid microspheres or the hyaluronic acid filler can be used as a facial soft tissue filler.

[0084] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0085] Example 1

[0086] This example provides a kind of hyaluronic acid microspheres, and its preparation process includes:

[0087] S1: Mix hyaluronic acid with NaOH and water, and then add butanediol diglycidyl ether and mix at 45 °C to obtain an aqueous phase.

[0088] Among them, the content of hyaluronic acid in the aqueous phase is 10 wt%, the content of NaOH in the aqueous phase is 2 w%, and the content of butanediol diglycidyl ether in the aqueous phase is 5 wt%.

[0089] S2: Mix the aqueous phase with an oil phase and perform primary emulsification to obtain a dispersion.

[0090] Among them, the oil phase is silicone oil, and the volume ratio of the oil phase to the water phase is 5:1. The first emulsification is carried out for 20 min under the condition of 1500 rpm.

[0091] S3: Perform a second emulsification on the dispersion liquid to obtain an emulsion.

[0092] Among them, the second emulsification is carried out for 20 min under the condition of 6000 rpm.

[0093] S4: Remove the oil phase in the emulsion by filtration to obtain cross-linked hyaluronic acid microspheres.

[0094] Example 2

[0095] This example provides a kind of hyaluronic acid microspheres, and its preparation process includes:

[0096] S1: Mix hyaluronic acid with NaOH and water, and then add diglycidyl ether of butanediol and mix at 40 °C to obtain an aqueous phase.

[0097] Among them, the content of hyaluronic acid in the aqueous phase is 0.5 wt%, the content of NaOH in the aqueous phase is 0.5 w%, and the content of diglycidyl ether of butanediol in the aqueous phase is 0.1 wt%.

[0098] S2: Mix the aqueous phase with the oil phase and perform the first emulsification to obtain a dispersion liquid.

[0099] Among them, the oil phase is silicone oil, and the volume ratio of the oil phase to the aqueous phase is 1:1. The first emulsification is carried out for 30 min under the condition of 1000 rpm.

[0100] S3: Perform a second emulsification on the dispersion liquid to obtain an emulsion.

[0101] Among them, the second emulsification is carried out for 30 min under the condition of 4000 rpm.

[0102] S4: Remove the oil phase in the emulsion by filtration to obtain cross-linked hyaluronic acid microspheres.

[0103] Example 3

[0104] This example provides a kind of hyaluronic acid microspheres, and its preparation process includes:

[0105] S1: Mix hyaluronic acid with NaOH and water, and then add diglycidyl ether of butanediol and mix at 50 °C to obtain an aqueous phase.

[0106] Among them, the content of hyaluronic acid in the aqueous phase is 20 wt%, the content of NaOH in the aqueous phase is 5 w%, and the content of diglycidyl ether of butanediol in the aqueous phase is 10 wt%.

[0107] S2: Mix the aqueous phase and the oil phase and perform primary emulsification to obtain a dispersion.

[0108] Among them, the oil phase is silicone oil, and the volume ratio of the oil phase to the aqueous phase is 10:1. The primary emulsification is carried out under the condition of 2000 rpm for 5 min.

[0109] S3: Perform secondary emulsification on the dispersion to obtain an emulsion.

[0110] Among them, the secondary emulsification is carried out under the condition of 8000 rpm for 10 min.

[0111] S4: Remove the oil phase in the emulsion by filtration to obtain cross-linked hyaluronic acid microspheres.

[0112] Example 4

[0113] The difference between this example and Example 1 is that in S1, hyaluronic acid is replaced with sodium hyaluronate.

[0114] Example 5

[0115] The difference between this example and Example 1 is that in S1, butanediol diglycidyl ether is replaced with divinyl sulfone.

[0116] Example 6

[0117] The difference between this example and Example 1 is that in S2, the oil phase is mineral oil, specifically a mixture of anhydrous derivatives of D-mannitol and light mineral oil (MONTANIDE ISA51 VG).

[0118] Example 7

[0119] The difference between this example and Example 1 is that in S2, the oil phase is liquid paraffin.

[0120] Example 8

[0121] The difference between this example and Example 1 is that in S2, the oil phase is dodecane.

[0122] Example 9

[0123] The difference between this example and Example 1 is that in S2, the oil phase is n-octane.

[0124] Example 10

[0125] The difference between this example and Example 1 is that in S2, the oil phase is cyclohexane.

[0126] Comparative Example 1

[0127] The difference between this comparative example and Example 1 is that there is no S3 step, that is, directly perform S4 step after completing S2 step.

[0128] Comparative Example 2

[0129] The difference between this comparative example and Example 1 is that: there is no step S2, that is, after completing S1, the aqueous phase and the oil phase are directly mixed and then subjected to the second emulsification under the conditions in S3.

[0130] Comparative Example 3

[0131] The difference between this comparative example and Example 1 is that: in S1, the content of hyaluronic acid in the aqueous phase is 25 wt%.

[0132] Comparative Example 4

[0133] The difference between this comparative example and Example 1 is that: in S1, the content of NaOH in the aqueous phase is 10 wt%.

[0134] Comparative Example 5

[0135] The difference between this comparative example and Example 1 is that: in S1, the content of butanediol diglycidyl ether in the aqueous phase is 15 wt%.

[0136] Comparative Example 6

[0137] The difference between this comparative example and Example 1 is that: in S2, the volume ratio of the oil phase to the aqueous phase is 0.5:1.

[0138] Comparative Example 7

[0139] The difference between this comparative example and Example 1 is that: in S2, the volume ratio of the oil phase to the aqueous phase is 12:1.

[0140] Comparative Example 8

[0141] The difference between this comparative example and Example 1 is that: in S2, the rotation speed of the first emulsification is 800 rpm.

[0142] Comparative Example 9

[0143] The difference between this comparative example and Example 1 is that: in S2, the rotation speed of the first emulsification is 2200 rpm.

[0144] Comparative Example 10

[0145] The difference between this comparative example and Example 1 is that: in S3, the rotation speed of the second emulsification is 3800 rpm.

[0146] Comparative Example 11

[0147] The difference between this comparative example and Example 1 is that: in S3, the rotation speed of the second emulsification is 8200 rpm.

[0148] Comparative Example 12

[0149] The difference between this comparative example and Example 1 is that in S3, the time for the second emulsification is 5 min.

[0150] Test Example

[0151] ①. Perform performance tests on the hyaluronic acid microspheres obtained in Examples 1 to 10 and Comparative Examples 1 to 12, and the results are shown in Table 1 and Figures 1 to 3 as follows.

[0152] Among them, the particle size distribution was determined by the wet method of the third method (light scattering method) in the "Pharmacopoeia of the People's Republic of China (Part IV)" 2020 edition 0982 Particle Size and Particle Size Distribution Determination Method, and the D 50 result was recorded, and its unit is μm;

[0153] The elastic modulus and viscous modulus were determined with reference to "YY / T 0308-2015 Sodium Hyaluronate Gel for Medical Use". At 25 ± 2 °C, a frequency sweep was performed using a rheometer at a shear rate of 0.001 - 100 Hz, and a coordinate graph was plotted with the elastic modulus G' and viscous modulus G" against the frequency. The results of the elastic modulus G' and viscous modulus G" were recorded, and the units of both the elastic modulus G' and viscous modulus G" are Pa;

[0154] The swelling degree was determined with reference to "YY / T 0962-2021 Crosslinked Sodium Hyaluronate Gel for Plastic Surgery";

[0155] The pushing force was determined with reference to "YY / T 0962-2021 Crosslinked Sodium Hyaluronate Gel for Plastic Surgery", and its unit is N;

[0156] The osmotic pressure was determined with reference to "YY / T 0962-2021 Crosslinked Sodium Hyaluronate Gel for Plastic Surgery", and its unit is mOsmol / kg; the refractive index was determined with reference to "YY / T 0308-2015 Sodium Hyaluronate Gel for Medical Use".

[0157] Table 1 Test Results

[0158]

[0159]

[0160] As can be seen from Table 1, the products obtained in the examples of the present invention have the advantages of narrow particle size distribution, large specific surface area, high mechanical strength, low pushing force, moderate osmotic pressure, high purity, etc.

[0161] From Figures 1 to 3Comparisons show that spherical sodium hyaluronate gel microspheres can be prepared by the emulsification method in both Example 1 and Comparative Example 2. However, Comparative Example 2 does not have the S2 step compared to Example 1, with a wider particle size distribution and more uneven particle sizes than those in Example 1, indicating that two-step emulsification can achieve the control of a narrow particle size range for the microspheres. Figure 3 It is a listed product of Bloomage Biotechnology, and blocky irregular sodium hyaluronate gel particles are prepared without using the emulsification method.

[0162] ② Taking the hyaluronic acid microspheres prepared in the example as an example, using the commercially available product Restylane 2 as a control group, the experiment was carried out in the following manner: A syringe needle was used to pierce the left ear artery of the experimental rabbit, and 0.5 mL of Restylane 2 was injected; a syringe needle was used to pierce the right ear artery of the experimental rabbit, and 0.5 mL of the sample was injected. Observe the immediate phenomena of the rabbit's ears, as well as the phenomena within 3 weeks, and form daily observation records. The results after 3 weeks are as Figure 4 shown.

[0163] It can be Figure 4 seen that after injecting the hyaluronic acid microspheres provided in Example 1 of the present application, no blood vessel blockage or swelling occurred; however, after injecting the same type of product provided by the control group, obvious blood vessel blockage and swelling occurred.

[0164] In summary, the preparation method of the hyaluronic acid microspheres provided by the present application is simple, the reaction conditions are mild, the process is easy to control, and hyaluronic acid microspheres with excellent effects and high yields can be obtained. The obtained hyaluronic acid microspheres have a narrow particle size distribution, which can avoid blood vessel embolism after injection into blood vessels; at the same time, the hyaluronic acid microspheres have a small particle size and a relatively large specific surface area. Even when embolism occurs due to overcorrection or misoperation, it can be quickly degraded by injecting hyaluronidase at the problem site, greatly improving the safety of injection. In addition, the hyaluronic acid microspheres provided by the present application have good mechanical strength and can achieve a better filling effect after implantation, realizing the effectiveness of the filler; moreover, the hyaluronic acid microspheres also have a low extrusion force, which is beneficial to achieving injectability.

[0165] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hyaluronic acid microsphere, characterized in that, The particle size distribution of the hyaluronic acid microspheres is 20 μm to 70 μm; the swelling degree of the hyaluronic acid microspheres is 5 to 40; the hyaluronic acid microspheres also have at least one of the following characteristics: Characteristic 1: The elastic modulus of the hyaluronic acid microspheres at 5 Hz is 40 Pa to 800 Pa; Characteristic 2: The viscous modulus of the hyaluronic acid microspheres at 5 Hz is 15 Pa to 1000 Pa; Characteristic 3: The content of hyaluronic acid in the hyaluronic acid microspheres is 5 mg / mL to 40 mg / mL; Characteristic 4: The pushing force of the hyaluronic acid microspheres is 9 N to 20 N; Characteristic 5: The osmotic pressure of the hyaluronic acid microspheres is 240 mOsmol / kg to 360 mOsmol / kg; Characteristic 6: The refractive index of the hyaluronic acid microspheres is 1.32 to 1.35; The preparation method of the hyaluronic acid microspheres includes the following steps: Mix hyaluronic acid or its salt with an alkali and water, and then mix with a cross-linking agent to obtain an aqueous phase; Mix the aqueous phase with an oil phase and perform first emulsification to obtain a dispersion; Perform second emulsification on the dispersion to obtain an emulsion; Remove the oil phase from the emulsion to obtain cross-linked hyaluronic acid microspheres; The first emulsification is carried out at 1000 rpm to 2000 rpm for 5 min to 30 min, and the second emulsification is carried out at 4000 rpm to 8000 rpm for 10 min to 30 min; The content of the hyaluronic acid or its salt in the aqueous phase is 0.5 wt% to 20 wt%; the content of the alkali in the aqueous phase is 0.5 wt% to 5 wt%; the content of the cross-linking agent in the aqueous phase is 0.1 wt% to 10 wt%; the volume ratio of the oil phase to the aqueous phase is 1:1 to 10:1; The oil phase is selected from at least one of vegetable oil, mineral oil, silicone oil, liquid paraffin, dodecane, n-octane and cyclohexane.

2. A method for preparing hyaluronic acid microspheres, characterized in that, Including the following steps: Mix hyaluronic acid or its salt with an alkali and water, and then mix with a cross-linking agent to obtain an aqueous phase; Mix the aqueous phase with an oil phase and perform first emulsification to obtain a dispersion; Perform second emulsification on the dispersion to obtain an emulsion; Remove the oil phase from the emulsion to obtain cross-linked hyaluronic acid microspheres; The first emulsification is carried out at 1000 rpm to 2000 rpm for 5 min to 30 min, and the second emulsification is carried out at 4000 rpm to 8000 rpm for 10 min to 30 min; The content of the hyaluronic acid or its salt in the aqueous phase is 0.5 wt% to 20 wt%; the content of the alkali in the aqueous phase is 0.5 wt% to 5 wt%; the content of the cross-linking agent in the aqueous phase is 0.1 wt% to 10 wt%; the volume ratio of the oil phase to the aqueous phase is 1:1 to 10:1; The oil phase is selected from at least one of vegetable oil, mineral oil, silicone oil, liquid paraffin, dodecane, n-octane and cyclohexane.

3. The preparation method according to claim 2, characterized in that, The alkali includes at least one of NaOH and KOH.

4. The preparation method according to claim 2, characterized in that, The cross-linking agent includes at least one of butanediol diglycidyl ether and divinyl sulfone.

5. A hyaluronic acid filler, characterized in that, The hyaluronic acid filler includes at least one of a non-crosslinked sodium hyaluronate solution and an active ingredient, and the hyaluronic acid microspheres described in claim 1; Wherein, the active ingredient includes at least one of an amino acid and lidocaine.

6. Use of the hyaluronic acid microspheres as described in claim 1 or the hyaluronic acid filler as described in claim 5, characterized in that The hyaluronic acid microspheres or the hyaluronic acid filler are used as soft tissue fillers.

7. The application according to claim 6, wherein The hyaluronic acid microspheres or the hyaluronic acid filler are used as medical soft tissue fillers or cosmetic soft tissue fillers.

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