Preparation method and application of low-temperature cross-linked sodium hyaluronate gel particles

Abstract

The invention discloses a preparation method of low-temperature cross-linked sodium hyaluronate gel particles, which comprises the following steps: 1, mixing two sodium hyaluronates with different molecular weights, adding the mixture into a cross-linking reaction medium, and uniformly stirring and dispersing to obtain a sodium hyaluronate cross-linking reaction system mixed solution; 2, performing low-temperature standing and cross-linking reaction to obtain cross-linked sodium hyaluronate gel; 3, roughly cutting gel blocks, performing constant-volume purification, cleaning and draining to obtain cross-linked sodium hyaluronate gel; and 4, extruding and granulating to obtain the cross-linked sodium hyaluronate gel particles. In addition, the invention also discloses an application of the low-temperature cross-linked sodium hyaluronate gel particle. The mixed cross-linked gel is prepared under the conditions of sodium hyaluronates with different molecular weights and low-temperature standing, the cross-linking degree of the prepared gel can reach 20% or above, the elastic modulus can reach 400 Pa or above, the residual quantity of a cross-linking agent is smaller than 0.1 ppm, and the gel can be used for preparing a sodium hyaluronate gel product for injection and filling.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of clinical medical soft tissue injection repair and defect filling materials, and particularly relates to a preparation method and application of low-temperature cross-linked gel particles. Background Art

[0002] Sodium hyaluronate, also known as sodium hyaluronate, is commonly found in human skin, synovial fluid, vitreous humor, and soft tissue. It possesses excellent water retention, viscoelasticity, and biocompatibility, and possesses excellent physiological functionality, enhancing skin immunity and improving skin nutrient metabolism. However, due to the action of hyaluronidase in the human body, sodium hyaluronate is easily degraded after injection, resulting in a short shelf life. Therefore, in recent years, leveraging the excellent properties of sodium hyaluronate, various cross-linking agents, and various preparation methods have been employed to develop various injectable cross-linked sodium hyaluronate gel-based hydrating and moisturizing cosmetic products. Most of these products require multiple injections during use to achieve the desired hydrating and moisturizing effects. Clinical application requirements necessitate the development of an injectable filler that delivers immediate, visible wrinkle reduction and long-lasting cosmetic results with a single injection. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to address the shortcomings of the above-mentioned prior art and provide a method for preparing and applying low-temperature cross-linked gel particles. The present invention utilizes two different molecular weight sodium hyaluronates, one high and one low, and conducts a mild reaction for a long time under low-temperature static conditions in the presence of a cross-linking agent and an alkaline medium. This allows the high- and low-molecular-weight sodium hyaluronate mixture to be fully infiltrated into the reaction system mixture, thereby achieving a more uniform and complete reaction of the high- and low-molecular-weight sodium hyaluronate mixture. The resulting cross-linked sodium hyaluronate gel has a cross-linking degree exceeding 20% and an elastic modulus exceeding 400 Pa.

[0004] To solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing low-temperature cross-linked sodium hyaluronate gel particles, characterized in that it comprises the following steps:

[0005] Step 1: Add sodium hyaluronate to a cross-linking reaction medium, stir and disperse uniformly, and obtain a sodium hyaluronate cross-linking reaction system mixed solution; the cross-linking reaction medium is composed of the following components by weight: 0.5% to 1.5% of an alkaline substance, 5% to 15% of a cross-linking agent, and the balance being water for injection; the concentration of the sodium hyaluronate in the cross-linking reaction system mixed solution is 10% to 30%; the sodium hyaluronate is composed of high molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate;

[0006] Step 2: The sodium hyaluronate cross-linking reaction system mixture in step 1 is allowed to stand at 2°C to 8°C for 24 hours to 144 hours to obtain a cross-linked sodium hyaluronate gel;

[0007] Step 3: coarsely chop the cross-linked sodium hyaluronate gel described in step 2, add phosphate buffer to purify it, then wash it with water for injection, and drain the water to obtain a cross-linked sodium hyaluronate gel;

[0008] Step 4: Granulate the cross-linked sodium hyaluronate gel obtained in step 3 using a vertical high-speed screw extrusion mill to obtain cross-linked sodium hyaluronate gel particles.

[0009] The above-mentioned method for preparing low-temperature cross-linked sodium hyaluronate gel particles is characterized in that the sodium hyaluronate in step 1 is powdered sodium hyaluronate, and the viscosity average molecular weight of high molecular weight sodium hyaluronate is 1.0×10 6 ~3.0×10 6 The viscosity average molecular weight of low molecular weight sodium hyaluronate is 0.1×10 5 ~5.0×10 5 , high molecular weight sodium hyaluronate accounts for 1% to 50% of the total mass of sodium hyaluronate; the alkaline substance is sodium hydroxide, and the cross-linking agent is 1,4-butanediol diglycidyl ether.

[0010] The above-mentioned method for preparing low-temperature cross-linked sodium hyaluronate gel particles is characterized in that the stirring time in step 1 is 20 minutes to 30 minutes, and the stirring speed is 150 rpm to 200 rpm.

[0011] The above-mentioned method for preparing low-temperature cross-linked sodium hyaluronate gel particles is characterized in that the volume of the phosphate buffer in step 3 is 5 to 10 times the mass of the gel, wherein the unit of volume is mL and the unit of mass is g, and the purification time is 2 to 3 hours; the vibration frequency of the washing in step 3 is 50 Hz, the number of washings is 5 to 15 times, and each washing time is 5 to 15 minutes.

[0012] The above-mentioned method for preparing low-temperature cross-linked sodium hyaluronate gel particles is characterized in that the cutter disc aperture of the vertical high-speed screw extrusion mill in step 4 is 100 mesh and the frequency is 20 Hz; the particle size of the cross-linked sodium hyaluronate gel particles is 100 to 500 μm.

[0013] Furthermore, the present invention provides a use of low-temperature cross-linked sodium hyaluronate gel particles prepared by the above method in the preparation of sodium hyaluronate gel products for injection filling.

[0014] The above application is characterized in that the preparation method comprises the following steps:

[0015] Step 1: dispersing and dissolving sodium hyaluronate powder in phosphate buffer to obtain a non-cross-linked sodium hyaluronate solution;

[0016] Step 2: adding the non-cross-linked sodium hyaluronate solution obtained in step 1 to the low-temperature cross-linked sodium hyaluronate gel particles, and mixing them evenly using a high-speed homogenizer to obtain a mixed gel;

[0017] Step 3: Pre-fill the mixed gel in step 2 into a glass syringe after vacuuming, sterilizing and storing to obtain a sodium hyaluronate gel product for injection and filling.

[0018] The above application is characterized in that the viscosity average molecular weight of the sodium hyaluronate in step 1 is 1.0×10 6 ~3.0×10 6 The mass concentration of the non-cross-linked sodium hyaluronate solution is 1% to 5%, and the pH value of the phosphate buffer is 7.4.

[0019] The above application is characterized in that in step 2, the mass ratio of low-temperature cross-linked sodium hyaluronate gel particles to non-cross-linked sodium hyaluronate solution is (1-9):(1-9).

[0020] The above application is characterized in that in step 2, the homogenization temperature is 20°C to 23°C, the homogenizer speed is 1000rpm to 2000rpm, and the homogenization time is 10min to 30min; in step 3, a bidirectional centrifuge is used for vacuum extraction, the rotation speed is 1000rpm to 2000rpm, the time is 5min to 20min, and the vacuum degree is -0.08MPa to -0.09MPa.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. The present invention utilizes two sodium hyaluronates of different molecular weights, one high and one low, which are mixed in a specific proportion and then added to an alkaline mixed solution of a crosslinking agent to prepare a crosslinking reaction system mixture. Under low temperature and static conditions, a mild reaction is carried out for a long time under the action of the crosslinking agent and alkaline medium, allowing the high and low molecular weight sodium hyaluronate mixture to be fully infiltrated into the reaction system mixture, thereby achieving a more uniform and complete reaction of the high and low molecular weight sodium hyaluronate mixture. Because the reaction system comprises a mixture of high and low molecular weight sodium hyaluronates, the molecular chains of the high and low molecular weight sodium hyaluronates intertwine and entangle with each other. Therefore, under the mild reaction conditions of a long time, the crosslinking between the high and low molecular weight sodium hyaluronate molecular chains is enhanced, thereby improving the crosslinking degree and elastic modulus of the crosslinked sodium hyaluronate gel. The prepared crosslinked sodium hyaluronate gel has a crosslinking degree of over 20% and an elastic modulus of over 400 Pa.

[0023] 2. The present invention adopts a method for preparing cross-linked sodium hyaluronate gel under low-temperature static conditions. The process is simple, easy to operate, and the reaction conditions are mild. The reaction temperature is 2°C to 8°C, the cross-linking agent utilization rate is high, the residual amount is small, less than 1.0 ppm, and it is easy to remove.

[0024] 3. The cross-linked sodium hyaluronate gel particles of the present invention can be used to prepare sodium hyaluronate gel products for injection and filling, which can be injected using a 32G injection needle, have low pushing force, are smooth and stable, and are easy to operate clinically.

[0025] 4. The cross-linked sodium hyaluronate gel product of the present invention is suitable for repairing moderate to severe wrinkles in the maxillofacial area and filling wrinkles in the neck. It has excellent repair effects and only requires one injection. The effect at the filled area can be maintained for more than 6 months.

[0026] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the NMR image of the cross-linking degree of the sodium hyaluronate gel particles prepared in Example 1 of the present invention.

[0028] Figure 2 This is a graph of the elastic modulus of the sodium hyaluronate gel particles prepared in Example 1 of the present invention.

[0029] Figure 3 This is a gas chromatogram of the residual amount of cross-linking agent in the sodium hyaluronate gel particles prepared in Example 1 of the present invention.

[0030] Figure 4 This is a graph of the pushing force of the sodium hyaluronate gel product for injection filling prepared in Example 4 of the present invention.

[0031] Figure 5 This is a statistical chart showing a comparative test of the filling and repairing effects of the sodium hyaluronate gel product for injection prepared in Example 4 of the present invention on neck wrinkles. DETAILED DESCRIPTION

[0032] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following detailed description of the specific embodiments of the present invention is given in conjunction with the following examples. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] Example 1

[0034] The preparation method of the low-temperature cross-linked sodium hyaluronate gel particles of this embodiment comprises the following steps:

[0035] Step 1: Mix high molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate and add them to a cross-linking reaction medium, stir at 150 rpm for 30 minutes to disperse them evenly, and obtain a sodium hyaluronate cross-linking reaction system mixture; the cross-linking reaction medium is composed of the following components by weight: 0.5% sodium hydroxide, 5% 1,4-butanediol diglycidyl ether, and the balance water for injection; the mass percentage of sodium hyaluronate in the cross-linking reaction system mixture is 10%, and the mass ratio of high and low molecular weight sodium hyaluronate is 5:5; the sodium hyaluronate material is powdered sodium hyaluronate, and the viscosity-average molecular weight of the high molecular weight sodium hyaluronate is 2.0×10 6 The viscosity average molecular weight of low molecular weight sodium hyaluronate is 3.0×10 5 ;

[0036] Step 2: The sodium hyaluronate cross-linking reaction system mixture in step 1 is allowed to stand at 2° C. for 144 hours to obtain a cross-linked sodium hyaluronate gel;

[0037] Step 3: The cross-linked sodium hyaluronate gel described in step 2 is pelletized, pre-purified by adding phosphate buffer, washed with water for injection, and drained to obtain a cross-linked sodium hyaluronate gel; the volume of the phosphate buffer is 5 times the mass of the gel, where the unit of volume is mL and the unit of mass is g, and the purification time is 2 hours; the vibration frequency of the washing is 50 Hz, the number of washings is 5, and each washing time is 15 minutes;

[0038] Step 4: Granulate the cross-linked sodium hyaluronate gel obtained in step 3 using a vertical high-speed screw extrusion mill to obtain cross-linked sodium hyaluronate gel particles; the cutter disc aperture of the vertical high-speed screw extrusion mill is 100 mesh and the frequency is 20 Hz; the particle size of the cross-linked sodium hyaluronate gel particles is 100 to 500 μm.

[0039] The cross-linking degree of the cross-linked sodium hyaluronate gel particles prepared in this example was detected by nuclear magnetic resonance spectroscopy. Figure 1 As shown in the figure, after washing the cross-linked sodium hyaluronate gel with BDDE as the cross-linking agent, an appropriate amount of D2O was added and the cross-linked sodium hyaluronate gel was fully enzymatically hydrolyzed at 37°C. 1 H-NMR analysis can directly reflect the degree of modification based on the area of the characteristic signal peak, and the cross-linking degree is more than 20%.

[0040] The washed cross-linked sodium hyaluronate gel particles were placed in a rotational rheometer, and the elastic modulus of the cross-linked sodium hyaluronate gel particles prepared in this example was measured under the condition of Angular frequency ω (rad / s) of 0.1 to 100. The results are as follows: Figure 2As shown, the elastic modulus of the cross-linked sodium hyaluronate gel particles of this embodiment is above 400 Pa.

[0041] The residual amount of cross-linking agent in the cross-linked sodium hyaluronate gel particles prepared in this example was detected, and the results are shown in Tables 1 and Figure 3 The detection method is based on the gas chromatography method in Appendix E of the national industry standard YY / T 0962-2021. The detection principle is: the distribution of the components to be separated between the mobile phase and the stationary phase is different. When the two phases move relative to each other, the distribution of these components between the two phases is repeated. Even if the distribution coefficients of the components are only slightly different, there can be a significant gap with the movement of the mobile phase, and finally the components are separated. Each component enters the detector in turn, and the chromatographic signal is recorded by the data processing system to obtain a gas chromatogram. The residual amount of cross-linking agent in the cross-linked sodium hyaluronate gel particles can be calculated based on the obtained peak area.

[0042] Table 1 Cross-linking agent residual detection results

[0043]

[0044] Reference substance-1 and reference substance-2 in Table 1 are weighed standard substances BDDE, and cross-linked glue-1 and cross-linked glue-2 are tested samples. Figure 3 The retention time and peak area of the crosslinker residue in the crosslinked gel sample 1 are shown in the chromatogram. The results show that the crosslinker residues in both crosslinked gel samples 1 and 2 meet the requirements, being less than 1.0 ppm.

[0045] Example 2

[0046] The preparation method of the low-temperature cross-linked sodium hyaluronate gel particles of this embodiment comprises the following steps:

[0047] Step 1: High molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate are mixed and added to a cross-linking reaction medium, and stirred at 200 rpm for 20 minutes to uniformly disperse them to obtain a sodium hyaluronate cross-linking reaction system mixture; the cross-linking reaction medium is composed of the following components by weight: 1.5% sodium hydroxide, 15% 1,4-butanediol diglycidyl ether, and the balance water for injection; the mass percentage of sodium hyaluronate in the cross-linking reaction system mixture is 30%, and the mass ratio of high and low molecular weight sodium hyaluronate is 0.1:9.9; the sodium hyaluronate material is powdered sodium hyaluronate, and the viscosity-average molecular weight of the high molecular weight sodium hyaluronate is 1.0×10 6 The viscosity average molecular weight of low molecular weight sodium hyaluronate is 0.1×10 5 ;

[0048] Step 2: The sodium hyaluronate cross-linking reaction system mixture in step 1 is allowed to stand at 8° C. for 24 hours to obtain a cross-linked sodium hyaluronate gel;

[0049] Step 3: Pellet the cross-linked sodium hyaluronate gel described in step 2, add phosphate buffer for preliminary purification, wash with water for injection, drain the water, and obtain a cross-linked sodium hyaluronate gel; the volume of the phosphate buffer is 10 times the mass of the gel, where the unit of volume is mL and the unit of mass is g, and the purification time is 3 hours; the vibration frequency of the washing is 50 Hz, the number of washings is 15, and each washing time is 5 minutes;

[0050] Step 4: Granulate the cross-linked sodium hyaluronate gel obtained in step 3 using a vertical high-speed screw extrusion mill to obtain cross-linked sodium hyaluronate gel particles; the cutter disc aperture of the vertical high-speed screw extrusion mill is 100 mesh and the frequency is 20 Hz; the particle size of the cross-linked sodium hyaluronate gel particles is 100 to 500 μm.

[0051] The cross-linking degree, elastic modulus and residual amount of cross-linking agent of the cross-linked sodium hyaluronate gel particles prepared in this example are basically the same as those in Example 1.

[0052] Example 3

[0053] The preparation method of the low-temperature cross-linked sodium hyaluronate gel particles of this embodiment comprises the following steps:

[0054] Step 1: High molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate are mixed and added to a cross-linking reaction medium, and stirred at 180 rpm for 25 minutes to uniformly disperse them to obtain a sodium hyaluronate cross-linking reaction system mixture; the cross-linking reaction medium is composed of the following components by weight: 1% sodium hydroxide, 10% 1,4-butanediol diglycidyl ether, and the balance is water for injection; the mass percentage of sodium hyaluronate in the cross-linking reaction system mixture is 20%, and the mass ratio of high and low molecular weight sodium hyaluronate is 3:7; the sodium hyaluronate material is powdered sodium hyaluronate, and the viscosity-average molecular weight of the high molecular weight sodium hyaluronate is 3.0×10 6 The viscosity average molecular weight of low molecular weight sodium hyaluronate is 5.0×10 5 ;

[0055] Step 2: The sodium hyaluronate cross-linking reaction system mixture in step 1 is allowed to stand at 5° C. for 72 hours to obtain a cross-linked sodium hyaluronate gel;

[0056] Step 3: Pellet the cross-linked sodium hyaluronate gel described in step 2, add phosphate buffer for fixed dissolution and purification, wash with water for injection, drain the water, and obtain a cross-linked sodium hyaluronate gel; the volume of the phosphate buffer is 8 times the mass of the gel, where the unit of volume is mL and the unit of mass is g, and the purification time is 2.5 hours; the vibration frequency of the washing is 50 Hz, the number of washings is 10, and each washing time is 10 minutes;

[0057] Step 4: Granulate the cross-linked sodium hyaluronate gel obtained in step 3 using a vertical high-speed screw extrusion mill to obtain cross-linked sodium hyaluronate gel particles; the cutter disc aperture of the vertical high-speed screw extrusion mill is 100 mesh and the frequency is 20 Hz; the particle size of the cross-linked sodium hyaluronate gel particles is 100 to 500 μm.

[0058] The cross-linking degree, elastic modulus and residual amount of cross-linking agent of the cross-linked sodium hyaluronate gel particles prepared in this example are basically the same as those in Example 1.

[0059] Example 4

[0060] The low-temperature cross-linked sodium hyaluronate gel particles prepared in Example 1 are used to prepare a sodium hyaluronate gel product for injection filling. The preparation method comprises the following steps:

[0061] Step 1: Disperse and dissolve sodium hyaluronate powder in phosphate buffer to obtain a non-cross-linked sodium hyaluronate solution; the mass concentration of the non-cross-linked sodium hyaluronate solution is 3%; the viscosity-average molecular weight of the sodium hyaluronate is 1.0×10 6 , the pH value of phosphate buffer is 7.4;

[0062] Step 2: Add the non-cross-linked sodium hyaluronate solution obtained in step 1 to the low-temperature cross-linked sodium hyaluronate gel particles, and mix them evenly using a high-speed screw homogenizer to obtain a mixed gel; the mass ratio of the low-temperature cross-linked sodium hyaluronate gel particles to the non-cross-linked sodium hyaluronate solution is 5:5; the homogenization temperature is 22° C., the screw speed is 1500 rpm, the homogenization time is 10 min, and the aperture of the discharge porous plate is 80 mesh;

[0063] Step 3: Using a pre-filling method, the mixed gel described in step 2 is vacuumed using a bidirectional centrifuge, and then pre-filled into a glass syringe. After sterilization, it is stored to obtain a sodium hyaluronate gel product for injection filling; the rotation speed of the bidirectional centrifuge is 1500 rpm, the vacuuming time is 10 minutes, and the vacuum degree is -0.08 MPa to -0.09 MPa.

[0064] The propulsion force of 109 batches of sodium hyaluronate gel products for injection filling prepared according to the method of this embodiment was tested using a tensile testing machine in accordance with the YY / T 0962-2021 standard. The test range was 5, 25 mm; the test speed was 30 mm / min; the sensor was 5000 N; the needle was 32 G. The results are shown in Table 2 and Figure 4 .

[0065] Table 2 Pushing force test results

[0066]

[0067]

[0068]

[0069]

[0070] According to the statistical method, 109 samples were tested for pushing force, and the pushing force was stable with small deviation. The pushing force test curves of 109 test samples were collected into one figure ( Figure 4 ), its pushing force has slight fluctuations and is very stable.

[0071] The sodium hyaluronate gel product prepared in this example was used to fill and repair neck wrinkles. A three-month efficacy control trial was conducted with similar products. The efficacy evaluation method used was a randomized, blinded, multicenter, randomized controlled trial. The evaluation indicator was the effective rate of improvement in transverse neck wrinkles three months after injection.

[0072] See the results Figure 5 As can be seen from the figure, the sodium hyaluronate gel product for injection filling prepared in this example showed a significantly higher efficacy at three months than the control product. It is suitable for repairing moderate to severe maxillofacial wrinkles and filling neck wrinkles, demonstrating excellent repair effectiveness. With just one injection, the effect can be maintained for over six months at the filled site.

[0073] Example 5

[0074] The low-temperature cross-linked sodium hyaluronate gel particles prepared in Example 2 are used to prepare a sodium hyaluronate gel product for injection filling. The preparation method comprises the following steps:

[0075] Step 1: Disperse and dissolve sodium hyaluronate powder in phosphate buffer to obtain a non-cross-linked sodium hyaluronate solution; the mass concentration of the non-cross-linked sodium hyaluronate solution is 1%; the viscosity-average molecular weight of the sodium hyaluronate is 2.0×10 6 , the pH value of phosphate buffer is 7.4;

[0076] Step 2: Add the non-cross-linked sodium hyaluronate solution obtained in step 1 to the low-temperature cross-linked sodium hyaluronate gel particles, and mix them evenly using a high-speed screw homogenizer to obtain a mixed gel; the mass ratio of the low-temperature cross-linked sodium hyaluronate gel particles to the non-cross-linked sodium hyaluronate solution is 1:9; the homogenization temperature is 20°C, the screw speed is 1000 rpm, the homogenization time is 20 minutes, and the aperture of the discharge porous plate is 80 mesh;

[0077] Step 3: Using a pre-filling method, the mixed gel described in step 2 is vacuumed using a bidirectional centrifuge, and then pre-filled into a glass syringe. After sterilization, it is stored to obtain a sodium hyaluronate gel product for injection filling; the rotation speed of the bidirectional centrifuge is 1000 rpm, the vacuuming time is 20 minutes, and the vacuum degree is -0.08 MPa to -0.09 MPa.

[0078] The pushing force results of the sodium hyaluronate gel product for injection filling prepared in this example are basically consistent with those in Example 4.

[0079] Example 6

[0080] The low-temperature cross-linked sodium hyaluronate gel particles prepared in Example 3 are used to prepare a sodium hyaluronate gel product for injection filling. The preparation method comprises the following steps:

[0081] Step 1: Disperse and dissolve sodium hyaluronate powder in phosphate buffer to obtain a non-cross-linked sodium hyaluronate solution; the mass concentration of the non-cross-linked sodium hyaluronate solution is 5%; the viscosity-average molecular weight of the sodium hyaluronate is 3.0×10 6 , the pH value of phosphate buffer is 7.4;

[0082] Step 2: Add the non-cross-linked sodium hyaluronate solution obtained in step 1 to the low-temperature cross-linked sodium hyaluronate gel particles, and mix them evenly using a high-speed screw homogenizer to obtain a mixed gel; the feed ratio of the low-temperature cross-linked sodium hyaluronate gel particles to the non-cross-linked sodium hyaluronate solution is 9:1; the homogenization temperature is 23° C., the screw speed is 2000 rpm, the homogenization time is 30 min, and the aperture of the discharge porous plate is 80 mesh;

[0083] Step 3: Using a pre-filling method, the mixed gel described in step 2 is vacuumed using a bidirectional centrifuge, and then pre-filled into a glass syringe. After sterilization, it is stored to obtain a sodium hyaluronate gel product for injection filling; the rotation speed of the bidirectional centrifuge is 2000 rpm, the vacuuming time is 5 minutes, and the vacuum degree is -0.08 MPa to -0.09 MPa.

[0084] The pushing force results of the sodium hyaluronate gel product for injection filling prepared in this example are basically consistent with those in Example 4.

[0085] The present invention utilizes two sodium hyaluronates of different molecular weights, high and low, to undergo a gentle reaction under low-temperature, static conditions, under the action of a crosslinking agent and an alkaline medium for a relatively long time, allowing the high- and low-molecular-weight sodium hyaluronate mixture to be fully infiltrated into the reaction system mixture, thereby enabling a more uniform and complete reaction of the high- and low-molecular-weight sodium hyaluronate mixture. The cross-linked sodium hyaluronate gel particles prepared have a cross-linking degree of over 20%, an elastic modulus of over 400 Pa, a high cross-linking agent utilization rate, a low residual amount of less than 1.0 ppm, and are easily removable. The cross-linked sodium hyaluronate gel product can be used to prepare an injectable filling product. The product is injected using a 32G needle, resulting in low pushing force, smooth and stable operation, and ease of clinical operation. The cross-linked sodium hyaluronate gel product is suitable for repairing moderate to severe maxillofacial wrinkles and filling neck wrinkles, exhibiting excellent repair effects. With only a single injection, the effect at the filled site can be maintained for over six months.

[0086] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing low-temperature cross-linked sodium hyaluronate gel particles, characterized in that: The following steps are involved: Step 1: Add sodium hyaluronate to a cross-linking reaction medium, stir and disperse uniformly, and obtain a sodium hyaluronate cross-linking reaction system mixed solution; the cross-linking reaction medium is composed of the following components by weight: 0.5% to 1.5% of an alkaline substance, 5% to 15% of a cross-linking agent, and the balance being water for injection; the concentration of the sodium hyaluronate in the cross-linking reaction system mixed solution is 10% to 30%; the sodium hyaluronate is composed of high molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate; Step 2: The sodium hyaluronate cross-linking reaction system mixture in step 1 is allowed to stand at 2°C to 8°C for 24 hours to 144 hours to obtain a cross-linked sodium hyaluronate gel; Step 3: coarsely chop the cross-linked sodium hyaluronate gel described in step 2, add phosphate buffer to purify it, then wash it with water for injection, and drain the water to obtain a cross-linked sodium hyaluronate gel; Step 4: Granulate the cross-linked sodium hyaluronate gel obtained in step 3 using a vertical high-speed screw extrusion mill to obtain cross-linked sodium hyaluronate gel particles.

2. The method for preparing low-temperature cross-linked sodium hyaluronate gel particles according to claim 1, characterized in that: The sodium hyaluronate described in step 1 is powdered sodium hyaluronate, and the viscosity average molecular weight of high molecular weight sodium hyaluronate is 1.0×10 6 ~3.0×10 6 The viscosity average molecular weight of low molecular weight sodium hyaluronate is 0.1×10 5 ~5.0×10 5 , high molecular weight sodium hyaluronate accounts for 1% to 50% of the total mass of sodium hyaluronate; the alkaline substance is sodium hydroxide, and the cross-linking agent is 1,4-butanediol diglycidyl ether.

3. The method for preparing low-temperature cross-linked sodium hyaluronate gel particles according to claim 1, characterized in that: The stirring time in step 1 is 20 min to 30 min, and the stirring speed is 150 rpm to 200 rpm.

4. The method for preparing low-temperature cross-linked sodium hyaluronate gel particles according to claim 1, characterized in that: The volume of the phosphate buffer in step 3 is 5 to 10 times the mass of the gel, where the unit of volume is mL and the unit of mass is g, and the purification time is 2 h to 3 h; the vibration frequency of the washing in step 3 is 50 Hz, the number of washings is 5 to 15 times, and each washing takes 5 min to 15 min.

5. The method for preparing low-temperature cross-linked sodium hyaluronate gel particles according to claim 1, characterized in that: The cutter head of the vertical high-speed screw extrusion mill in step 4 has an aperture of 100 mesh and a frequency of 20 Hz; the particle size of the cross-linked sodium hyaluronate gel particles is 100 to 500 μm.

6. Use of low-temperature cross-linked sodium hyaluronate gel particles prepared by the method according to any one of claims 1 to 5 in preparing sodium hyaluronate gel products for injection filling.

7. The use according to claim 6, characterized in that The preparation method comprises the following steps: Step 1: dispersing and dissolving sodium hyaluronate powder in phosphate buffer to obtain a non-cross-linked sodium hyaluronate solution; Step 2: adding the non-cross-linked sodium hyaluronate solution obtained in step 1 to the low-temperature cross-linked sodium hyaluronate gel particles, and mixing them evenly using a high-speed homogenizer to obtain a mixed gel; Step 3: Pre-fill the mixed gel in step 2 into a glass syringe after vacuuming, sterilizing and storing to obtain a sodium hyaluronate gel product for injection and filling.

8. The use according to claim 7, characterized in that The viscosity average molecular weight of the sodium hyaluronate in step 1 is 1.0×10 6 ~3.0×10 6 The mass concentration of the non-cross-linked sodium hyaluronate solution is 1% to 5%, and the pH value of the phosphate buffer is 7.

4.

9. The use according to claim 7, characterized in that In step 2, the mass ratio of the low-temperature cross-linked sodium hyaluronate gel particles to the non-cross-linked sodium hyaluronate solution is (1-9):(1-9).

10. The use according to claim 7, characterized in that In step 2, the homogenization temperature is 20°C to 23°C, the homogenizer speed is 1000rpm to 2000rpm, and the homogenization time is 10min to 30min; in step 3, a bidirectional centrifuge is used for vacuum extraction, the rotation speed is 1000rpm to 2000rpm, the time is 5min to 20min, and the vacuum degree is -0.08MPa to -0.09MPa.

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