Preparation method of cross-linked hyaluronic acid gel

Abstract

The invention provides a preparation method of cross-linked hyaluronic acid gel and a preparation containing microspheres and the cross-linked hyaluronic acid gel. The preparation method comprises the following steps: (1) mixing hyaluronic acid or a salt thereof, a cross-linking agent and an alkaline solution to obtain a mixed solution; (2) carrying out primary cross-linking reaction on the mixed solution to obtain first gel; (3) cutting the first gel into blocks, adding the blocks into an alkaline solution containing hyaluronic acid or salt thereof, and carrying out secondary cross-linking reaction to obtain second gel; and (4) purifying the second gel to obtain the cross-linked hyaluronic acid gel. The prepared hyaluronic acid gel has excellent cohesive force, the microspheres can be uniformly dispersed in the hyaluronic acid gel in an extremely short time, and after the hyaluronic acid gel is mixed with the microspheres, the microspheres can be uniformly distributed in the cross-linked hyaluronic acid gel for a long time and are not easy to displace, settle and agglomerate.

Description

Technical Field

[0001] This application belongs to the field of injectable facial filler technology, specifically, it relates to a method for preparing cross-linked hyaluronic acid gel and a formulation containing microspheres and cross-linked hyaluronic acid gel. Background Technology

[0002] Microsphere soft tissue fillers are a novel type of biostimulating soft tissue filler that can stimulate collagen regeneration, providing significant and lasting effects on improving facial volume loss, wrinkles, and facial contouring caused by aging. Microsphere soft tissue fillers generally consist of polymer microspheres and a dispersion. When the polymer microspheres are implanted subcutaneously, a foreign body reaction occurs, and through processes such as protein absorption, cell aggregation, and fiber encapsulation, collagen production is ultimately stimulated, achieving a lifting and firming effect on the skin.

[0003] Common dispersions include hyaluronic acid and cellulose. Hyaluronic acid is a high-molecular-weight polysaccharide widely found in human connective tissue, epithelial tissue, and nerve tissue. It possesses excellent moisturizing properties, biocompatibility, and biodegradability. In cosmetic medicine, hyaluronic acid is often used to fill wrinkles and correct facial contours.

[0004] Common polymer microspheres include poly-L-lactic acid (PLLA), polycaprolactone (PCL), polymethyl methacrylate (PMMA), and hydroxyapatite (CaHA). Unlike traditional soft tissue fillers, microsphere materials are typically hydrophobic, easily settling when mixed with low-viscosity dispersions and easily agglomerating when mixed with high-viscosity dispersions. Agglomeration or settling of microspheres prevents uniform distribution in the dispersion, affecting the safety and effectiveness of the microsphere soft tissue filler product and failing to achieve the desired filling effect. Taking CaHA microspheres as an example, CaHA is a major component of human bone and possesses excellent biocompatibility and bioactivity. Combining CaHA microspheres with hyaluronic acid gel can improve the stability and durability of the filler. After injection into the tissue, CaHA microspheres can act as a framework, promoting collagen production, while the hyaluronic acid gel provides an immediate filling effect. When preparing hyaluronic acid gels containing CaHA microspheres, it is necessary to address the issue of uniform dispersion of CaHA microspheres in the gel and to avoid microsphere settling and agglomeration during injection.

[0005] In the prior art, patent CN115282339B discloses a method for preparing cross-linked hyaluronic acid / hydroxyapatite injectable materials. This method involves mixing photocrosslinkable sodium hyaluronate and PEG-modified CaHA microspheres, followed by photocuring to obtain a cross-linked hyaluronic acid / hydroxyapatite filler. This preparation process is cumbersome, requiring the preparation of HAMA first, followed by the preparation of CaHA-modified CaHA microspheres. This not only wastes time but also makes it difficult to remove residues from each reaction. After mixing HAMA and CaHA, the high density of CaHA microspheres causes them to settle in the uncrosslinked HAMA solution, resulting in uneven distribution of microspheres. Patent application CN104853742A provides a method for preparing injectable sterile aqueous formulations based on cross-linked hyaluronic acid and hydroxyapatite for therapeutic and cosmetic applications. However, although the mixed gel obtained by this method can avoid the sedimentation of hydroxyapatite particles, the hydroxyapatite particles are difficult to distribute evenly in the mixed gel, and they are prone to displacement and accumulation during injection, affecting the injection process.

[0006] Therefore, how to ensure that polymer microspheres are uniformly distributed in the dispersion and reduce the sedimentation and aggregation of microspheres is a technical problem that urgently needs to be solved in the field of microsphere soft tissue fillers. Summary of the Invention

[0007] To address the problems existing in the prior art, this application provides a method for preparing cross-linked hyaluronic acid gel. The cross-linked hyaluronic acid gel obtained by this method, when mixed with microspheres, can ensure that the microspheres are uniformly distributed in the cross-linked hyaluronic acid gel for a long time, reducing the sedimentation and aggregation of microspheres.

[0008] Specifically, this application relates to the following aspects:

[0009] 1. A method for preparing a cross-linked hyaluronic acid gel, comprising the following steps:

[0010] Step (1): Mix hyaluronic acid or its salt, a cross-linking agent, and an alkaline solution to obtain a mixed solution;

[0011] Step (2): The mixed solution is subjected to a primary cross-linking reaction to obtain a first gel;

[0012] Step (3): Cut the first gel into pieces and add it to an alkaline solution containing hyaluronic acid or its salt to carry out a secondary cross-linking reaction to obtain the second gel;

[0013] Step (4): The second gel is purified to obtain cross-linked hyaluronic acid gel.

[0014] 2. The preparation method according to item 1, wherein the hyaluronic acid or its salt has a molecular weight of 800kDa-3000kDa.

[0015] 3. The preparation method according to item 1 or 2, wherein the mass content of the hyaluronic acid or its salt in the mixed solution is 4%-20%.

[0016] 4. The preparation method according to any one of items 1-3, wherein, by weight percentage, the amount of crosslinking agent added is 0.5%-10% of the amount of hyaluronic acid or its salt added.

[0017] 5. The preparation method according to any one of items 1-4, wherein the pH of the alkaline solution is 9-14.

[0018] 6. The preparation method according to any one of items 1-5, wherein the temperature of the crosslinking reaction in step (2) is 20-50°C and the time is 1-8h.

[0019] 7. The preparation method according to any one of items 1-6, wherein the crosslinking agent is ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, or polyglycerol polyglycidyl ether.

[0020] 8. The preparation method according to any one of items 1-7, wherein the mass content of hyaluronic acid or its salt in the alkaline solution containing hyaluronic acid or its salt in step (3) is 0.2%-2.2%.

[0021] Preferably, the temperature of the secondary crosslinking reaction in step (3) is 20-50℃ and the time is 4-15h.

[0022] 9. The cross-linked hyaluronic acid gel prepared according to any one of items 1-8.

[0023] 10. A microsphere-containing formulation comprising a cross-linked hyaluronic acid gel and microspheres prepared by the preparation method according to any one of claims 1-8, wherein the average particle size of the microspheres is 10-120 micrometers, preferably, in the formulation, the mass content of the microspheres is 6%-50% based on the total mass of the cross-linked hyaluronic acid gel and the microspheres.

[0024] More preferably, the microspheres include poly(L-lactic acid) microspheres, polycaprolactone microspheres, polymethyl methacrylate microspheres, or hydroxyapatite microspheres.

[0025] The hyaluronic acid gel prepared by the method of this application has excellent cohesive force. Microspheres can be uniformly dispersed in it in a very short time. After mixing with microspheres, it can ensure that the microspheres are uniformly distributed in the cross-linked hyaluronic acid gel for a long time and are not prone to displacement, sedimentation and aggregation.

[0026] The hyaluronic acid gel prepared by the method of this application, after being mixed with microspheres, still has microspheres that are uniformly distributed in the cross-linked hyaluronic acid gel even after being squeezed out using a syringe.

[0027] The hyaluronic acid gel prepared by the method of this application, when mixed with microspheres, exhibits centrifugal stability, ensuring that the microspheres are stably and uniformly distributed in the gel. Even after high-speed centrifugation, the microspheres do not settle or aggregate. Attached Figure Description

[0028] Figure 1 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Example 1 of this application;

[0029] Figure 2 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Example 3 of this application;

[0030] Figure 3 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Example 10 of this application;

[0031] Figure 4 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Example 11 of this application;

[0032] Figure 5 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Comparative Example 1 of this application;

[0033] Figure 6 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Comparative Example 2 of this application;

[0034] Figure 7 Microscopic images of the permeation formulation containing hydroxyapatite microspheres prepared in Comparative Example 3 of this application;

[0035] Figure 8 Microscopic images of the formulation containing hydroxyapatite microspheres prepared in Comparative Example 4 of this application;

[0036] Figure 9 This is a stability comparison diagram between Example 3 and Comparative Example 1 of this application;

[0037] Figure 10 This is a stability comparison diagram between Example 3 and Comparative Example 2 of this application;

[0038] Figure 11 This is a stability comparison chart of Example 2 and Comparative Example 3 of this application. Detailed Implementation

[0039] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0040] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0041] This application provides a method for preparing cross-linked hyaluronic acid gel, comprising the following steps:

[0042] Step (1): Mix hyaluronic acid or its salt, a cross-linking agent, and an alkaline solution to obtain a mixed solution;

[0043] Step (2): The mixed solution is subjected to a primary cross-linking reaction to obtain a first gel;

[0044] Step (3): Cut the first gel into pieces and add it to an alkaline solution containing hyaluronic acid or its salt to carry out a secondary cross-linking reaction to obtain the second gel;

[0045] Step (4): The second gel is purified to obtain cross-linked hyaluronic acid gel.

[0046] The hyaluronic acid or its salt in steps (1) and (3) may have the same molecular weight or different molecular weights.

[0047] In some specific embodiments, the molecular weight of hyaluronic acid or its salt in steps (1) and (3) is in the range of 800kDa-3000kDa, for example, it can be 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa, 2600kDa, 2700kDa, 2800kDa, 2900kDa, 3000kDa, and any value between these values.

[0048] In some specific embodiments, the hyaluronic acid or its salt in steps (1) and (3) have the same molecular weight, which is 800kDa-3000kDa. For example, it can be 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa, 2600kDa, 2700kDa, 2800kDa, 2900kDa, 3000kDa, and any value between these values.

[0049] The crosslinking agent in step (1) can be any type of crosslinking agent known in the art.

[0050] In some specific embodiments, the crosslinking agent includes ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, or polyglycerol polyglycidyl ether.

[0051] The alkaline solution in steps (1) and (3) can be a solution containing an alkaline compound known in the art. Its function is to provide alkaline reaction conditions for the crosslinking reaction, as long as it meets the pH value requirements of this application for the alkaline solution. For example, it includes sodium hydroxide solution, potassium hydroxide solution, sodium bicarbonate solution, sodium lactate solution, etc.

[0052] In some specific embodiments, the alkaline solution is a sodium hydroxide solution.

[0053] In some specific embodiments, the pH of the alkaline solution is 9-14, for example, it can be 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, and any value between these values.

[0054] In some specific embodiments, in step (1), the mass content of hyaluronic acid or its salt in the mixed solution is 4%-20%, for example, it can be 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, and any value between these values.

[0055] In some specific embodiments, in step (1), the amount of crosslinking agent added is 0.5%-10% by weight of the amount of hyaluronic acid or its salt added, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, and any value between these values.

[0056] In some specific embodiments, in step (2), the temperature of the crosslinking reaction is 20-50°C, for example, it can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and any value between these values; the crosslinking reaction time is 1-8h, for example, it can be 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, and any value between these values.

[0057] In step (3), the first gel block obtained in step (2) is cut into pieces and added to an alkaline solution containing hyaluronic acid or its salt for a secondary crosslinking reaction. The pieces can be cut according to conventional gel cutting procedures in the art, for example, into pieces with an average volume of 0.01-10 cm. 3 Small pieces, preferably 0.5-5cm 3 Small pieces.

[0058] In some specific embodiments, the mass content of hyaluronic acid or its salt in the alkaline solution containing hyaluronic acid or its salt in step (3) is 0.2%-2.2%, for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, and any value between these values.

[0059] In some specific embodiments, the temperature of the secondary crosslinking reaction in step (3) is 20-50℃, for example, it can be 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and any value between these values; the secondary crosslinking reaction time is 4-15h, for example, it can be 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, and any value between these values.

[0060] Purification in step (4) can be performed using methods known in the art, such as dialysis. In some specific embodiments, purification can be achieved by dialysis with a buffer solution of neutral pH isotonicity. In some preferred embodiments, purification can be achieved by dialysis with a phosphate-sodium chloride buffer solution of neutral pH isotonicity. In some preferred embodiments, purification can be achieved by dialysis for 24 hours with a phosphate-sodium chloride buffer solution of neutral pH isotonicity (regenerated cellulose, separation limit: molecular weight = 60 kDa).

[0061] Those skilled in the art will understand that step (4) may include steps such as pH adjustment and granulation before purification. For example, the pH of the second gel can be adjusted to neutral using 1N HCl, and then the gel can be pulverized before purification.

[0062] The neutral pH mentioned in this application refers to a pH value between 6 and 8.

[0063] In some specific embodiments, the preparation method of the cross-linked hyaluronic acid gel includes the following steps:

[0064] Step (1): Mix hyaluronic acid or its salt, crosslinking agent and alkaline solution to obtain a mixed solution, wherein the molecular weight of the hyaluronic acid or its salt is 800kDa-3000kDa, the mass content of the hyaluronic acid or its salt in the mixed solution is 4%-20%, the amount of crosslinking agent added is 0.5%-10% of the amount of hyaluronic acid or its salt added, and the pH of the alkaline solution is 9-14;

[0065] Step (2): The mixed solution is subjected to a primary crosslinking reaction to obtain a first gel, wherein the crosslinking agent includes ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether or polyglycerol polyglycidyl ether, and the temperature of the crosslinking reaction is 20-50℃ and the time is 1-8h.

[0066] Step (3): After cutting the first gel into pieces, add it to an alkaline solution containing hyaluronic acid or its salt to carry out a secondary cross-linking reaction to obtain a second gel, wherein the molecular weight of the hyaluronic acid or its salt is 800kDa-3000kDa, the mass content of the hyaluronic acid or its salt in the alkaline solution containing hyaluronic acid or its salt is 0.2%-2.2%, the temperature of the secondary cross-linking reaction is 20-50℃, and the time is 4-15h;

[0067] Step (4): The second gel is purified to obtain cross-linked hyaluronic acid gel.

[0068] This application also provides cross-linked hyaluronic acid gels obtained by any of the above preparation methods.

[0069] In some specific embodiments, the concentration of hyaluronic acid or its salt in the cross-linked hyaluronic acid gel obtained by the preparation method of this application is 10 mg / ml-30 mg / ml.

[0070] The concentration of hyaluronic acid or its salt in the cross-linked hyaluronic acid gel can be detected using methods known in the art, such as referring to Appendix C of YY / T 0962-2021 for the detection of sodium hyaluronate content in cross-linked sodium hyaluronate gel for plastic surgery.

[0071] This application also provides a microsphere-containing formulation, comprising a cross-linked hyaluronic acid gel and microspheres prepared by any of the above preparation methods, wherein the average particle size of the microspheres is 10-120 micrometers, for example, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 110 micrometers, 120 micrometers, and any value between these values.

[0072] The average particle size of the microspheres can be detected by methods known in the art, such as by scanning electron microscopy.

[0073] The microspheres mentioned therein can be various microspheres known in the art, namely the field of injectable facial filler technology, including poly-L-lactic acid microspheres, polycaprolactone microspheres, polymethyl methacrylate microspheres, or hydroxyapatite microspheres, etc.

[0074] In some specific embodiments, the microspheres described in this application can be obtained using common microsphere preparation methods in the prior art, such as solvent evaporation, spray drying, and phase separation. In some specific embodiments, polymer materials used to prepare microspheres, such as poly(L-lactic acid), polycaprolactone, polymethyl methacrylate, or hydroxyapatite, are dissolved in an organic solvent as a dispersed phase; an emulsifier is dissolved in water as a continuous phase; the dispersed phase is dispersed into the continuous phase and stirred to form droplets, then solidified and the solvent is removed to obtain microspheres. The emulsifier can be selected from one or more of polyvinyl alcohol, gelatin, Tween, and Span.

[0075] In some specific embodiments, the formulation contains 6%-50% by mass of the microspheres, based on the total mass of the cross-linked hyaluronic acid gel and the microspheres. For example, it can be 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, and any value between these values.

[0076] The hyaluronic acid gel prepared by the method of this application has excellent cohesiveness, and microspheres can be uniformly dispersed in it in a short time. After mixing with microspheres, the microspheres can be uniformly distributed in the cross-linked hyaluronic acid gel for a long time, and are not prone to displacement, sedimentation and aggregation. Even after high-speed centrifugation, no microsphere sedimentation or aggregation will occur.

[0077] The hyaluronic acid gel prepared by the method of this application, after being mixed with microspheres, still has microspheres that are uniformly distributed in the cross-linked hyaluronic acid gel even after being squeezed out using a syringe.

[0078] Example

[0079] Example 1

[0080] Preparation of cross-linked hyaluronic acid gel:

[0081] Step (1): Add 2g of sodium hyaluronate with a molecular weight of 2600kDa to a sodium hydroxide solution and stir until completely dissolved to obtain a transparent solution. The mass concentration of sodium hyaluronate is 9% and the pH of the solution is 13.4. Then add 190mg of butylene glycol glycidyl ether (BDDE) to the mixture and stir to obtain a mixed solution. The amount of BDDE added is 9.5% of the amount of sodium hyaluronate added.

[0082] Step (2): The mixed solution was placed in a water bath at 30°C for the initial cross-linking reaction. After 6 hours of reaction, the first gel was obtained.

[0083] Step (3): Cut the first gel into pieces of 0.5-5cm. 3 Small pieces of the substance were added to an alkaline solution (pH=11) containing 0.5% sodium hyaluronate (molecular weight 2600kDa) and subjected to a secondary cross-linking reaction at 28°C. After 6 hours of reaction, a second gel was obtained.

[0084] Step (4): Add 1N HCl to the second gel to adjust the pH to neutral, then crush the gel and purify it by dialyzing with phosphate-sodium chloride buffer (regenerated cellulose, separation limit: molecular weight = 60kDa) with neutral pH for 24 hours to obtain cross-linked hyaluronic acid gel.

[0085] Preparation of microsphere-containing formulations: In a centrifugal mixer (FlackTek...) Add 3g of hydroxyapatite microspheres (with an average particle size of 20-40 micrometers) and 7g of cross-linked hyaluronic acid gel to a DAC 330-100SE container. The total mass of the cross-linked hyaluronic acid gel and microspheres is 30% by mass of the microspheres. Premix with a stainless steel spatula for 1 minute, then set the rotation speed to 2000 rpm and mix for 5 minutes to obtain a uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres.

[0086] Example 2

[0087] The difference from Example 1 is that the water bath temperature for the crosslinking reaction in step (2) is 40°C and the reaction time is 2 hours.

[0088] Example 3

[0089] The difference from Example 1 is that the water bath temperature for the crosslinking reaction in step (2) is 50°C and the reaction time is 2 hours.

[0090] Example 4

[0091] The difference from Example 3 is that in step (1), the amount of sodium hyaluronate added is 1g, the concentration of sodium hyaluronate is 4.5%, and the amount of BDDE added is 95mg.

[0092] Example 5

[0093] The difference from Example 3 is that in step (1), the amount of sodium hyaluronate added is 2g, the concentration of sodium hyaluronate is 16.7%, the amount of BDDE added is 20mg, and the amount of BDDE added is 1.0% of the amount of sodium hyaluronate added.

[0094] Example 6

[0095] The difference from Example 3 is that: in step (1), the molecular weight of sodium hyaluronate is 1000 kDa, and the pH of the solution is 11; in step (3), the first gel is cut into pieces of 0.5-5 cm. 3 Small pieces of the substance were added to an alkaline solution (pH=13) containing 2% sodium hyaluronate (molecular weight is 1000kDa) and the cross-linking reaction was continued at 45℃ for 6 hours.

[0096] Example 7

[0097] The difference from Example 3 is as follows:

[0098] Preparation of the microsphere-containing formulation: 0.6 g of hydroxyapatite microspheres (with an average particle size of 20-40 μm) and 9.4 g of cross-linked hyaluronic acid gel were added to the container of a centrifuge. The total mass of the microspheres was 6% based on the total mass of the cross-linked hyaluronic acid gel and microspheres. Premixing was performed for 1 minute with a stainless steel spatula, followed by mixing at 2000 rpm for 5 minutes. A uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres was obtained.

[0099] Example 8

[0100] The difference from Example 3 is as follows:

[0101] Preparation of microsphere-containing formulations: In a centrifugal mixer (FlackTek...) Add 5g of hydroxyapatite microspheres (with an average particle size of 20-40 μm) and 5g of cross-linked hyaluronic acid gel to a DAC 330-100SE container. The total mass of the microspheres and cross-linked hyaluronic acid gel is 50% by mass. Premix with a stainless steel spatula for 1 minute, then set the rotation speed to 2000 rpm for 5 minutes. A uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres is obtained.

[0102] Example 9

[0103] The difference from Example 3 is that the secondary crosslinking reaction time in step (3) is 12h.

[0104] Example 10

[0105] The difference from Example 3 is that the average particle size of the hydroxyapatite microspheres in the preparation of the microsphere-containing formulation is 80-100 micrometers.

[0106] Example 11

[0107] The difference from Example 4 is that 3g of polycaprolactone microspheres (with an average particle size of 20-40 micrometers) were added in the preparation of the microsphere formulation.

[0108] Comparative Example 1 (no secondary crosslinking reaction was performed compared to Example 3)

[0109] Step (1): Add 2g of sodium hyaluronate with a molecular weight of 2600kDa to a sodium hydroxide solution and stir until completely dissolved to obtain a transparent solution. The concentration of sodium hyaluronate is 9% and the pH of the solution is 13.4. Then add 190mg of butylene glycol glycidyl ether (BDDE) to the mixture and stir to obtain a mixed solution. The amount of BDDE added is 9.5% of the amount of sodium hyaluronate added.

[0110] Step (2): The raw material mixture is placed in a water bath at 50°C for cross-linking reaction. After 2 hours of reaction, the first gel is obtained.

[0111] Step (3): Add 1N HCl to the first gel to adjust the pH to neutral, then crush the gel and purify it by dialyzing with phosphate-sodium chloride buffer (regenerated cellulose, separation limit: molecular weight = 60kDa) with neutral pH for 24 hours to obtain cross-linked hyaluronic acid gel.

[0112] Preparation of microsphere-containing formulations: In a centrifugal mixer (FlackTek...) Add 3g of hydroxyapatite microspheres (with an average particle size of 20-40 micrometers) and 7g of cross-linked hyaluronic acid gel to a container (DAC 330-100SE). Premix with a stainless steel spatula for 1 minute, then set the rotation speed to 2000 rpm for 5 minutes to obtain a uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres.

[0113] Comparative Example 2 (Compared to Example 3, no alkaline solution containing sodium hyaluronate was added during the secondary crosslinking)

[0114] Step (1): Add 2g of sodium hyaluronate with a molecular weight of 2600kDa to a sodium hydroxide solution and stir until completely dissolved to obtain a transparent solution. The concentration of sodium hyaluronate is 9%, and the pH of the solution is 13.4. Then add 190mg of butylene glycol glycidyl ether (BDDE) to the mixture and stir to obtain a mixed solution. The amount of BDDE added is 9.5% of the amount of sodium hyaluronate added.

[0115] Step (2): The mixed solution was placed in a water bath at 50°C for the initial cross-linking reaction. After 2 hours of reaction, the first gel was obtained.

[0116] Step (3): The first gel was transferred to 28°C to continue the cross-linking reaction. After 6 hours of reaction, the second gel was obtained.

[0117] Step (4): Add 1N HCl to the second gel to adjust the pH to neutral, then crush the gel and purify it by dialyzing with phosphate-sodium chloride buffer (regenerated cellulose, separation limit: molecular weight = 60kDa) with neutral pH for 24 hours to obtain cross-linked hyaluronic acid gel.

[0118] Preparation of microsphere-containing formulations: In a centrifugal mixer (FlackTek...) Add 3g of hydroxyapatite microspheres (with an average particle size of 20-40 micrometers) and 7g of cross-linked hyaluronic acid gel to a DAC 330-100SE container. The total mass of the cross-linked hyaluronic acid gel and microspheres is 30% by mass of the microspheres. Premix with a stainless steel spatula for 1 minute, then set the rotation speed to 2000 rpm and mix for 5 minutes to obtain a uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres.

[0119] Comparative Example 3 (Compared to Example 2, no alkaline solution containing sodium hyaluronate was added during the secondary crosslinking)

[0120] Step (1): Add 2g of sodium hyaluronate with a molecular weight of 2600kDa to a sodium hydroxide solution and stir until completely dissolved to obtain a transparent solution. The concentration of sodium hyaluronate is 9% and the pH of the solution is 13.4. Then add 190mg of butylene glycol glycidyl ether (BDDE) to the mixture and stir to obtain a mixed solution. The amount of BDDE added is 9.5% of the amount of sodium hyaluronate added.

[0121] Step (2): The mixed solution was placed in a water bath at 40°C for the initial cross-linking reaction. After 2 hours of reaction, the first gel was obtained.

[0122] Step (3): The first gel was transferred to 28°C to continue the cross-linking reaction. After 6 hours of reaction, the second gel was obtained.

[0123] Step (4): Add 1N HCl to the second gel to adjust the pH to neutral, then crush the gel and purify it by dialyzing with phosphate-sodium chloride buffer (regenerated cellulose, separation limit: molecular weight = 60kDa) with neutral pH for 24 hours to obtain cross-linked hyaluronic acid gel.

[0124] Preparation of microsphere-containing formulations: In a centrifugal mixer (FlackTek...) Add 3g of hydroxyapatite microspheres (with an average particle size of 20-40 μm) and 7g of cross-linked hyaluronic acid gel to a DAC 330-100SE container. The total mass of the microspheres is 30%. Premix with a stainless steel spatula for 1 minute, then set the rotation speed to 2000 rpm for 5 minutes. A uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres is obtained.

[0125] Comparative Example 4

[0126] Step (1): Add 2g of sodium hyaluronate with a molecular weight of 2600kDa to a sodium hydroxide solution and stir until completely dissolved to obtain a transparent solution. The concentration of sodium hyaluronate is 9% and the pH of the solution is 13.4. Then add 190mg of butylene glycol glycidyl ether (BDDE) to the mixture and stir to obtain a mixed solution. The amount of BDDE added is 9.5% of the amount of sodium hyaluronate added.

[0127] Step (2): The mixed solution was placed in a water bath at 28°C for cross-linking reaction. After 15 hours of reaction, the first gel was obtained.

[0128] Step (3): Add 1N HCl to the first gel to adjust the pH to neutral, then crush the gel and purify it by dialyzing with phosphate-sodium chloride buffer (regenerated cellulose, separation limit: molecular weight = 60kDa) with neutral pH for 24 hours to obtain cross-linked hyaluronic acid gel.

[0129] Preparation of microsphere-containing formulations: In a centrifugal mixer (FlackTek...) Add 3g of hydroxyapatite microspheres (with an average particle size of 20-40 micrometers) and 7g of cross-linked hyaluronic acid gel to a DAC 330-100SE container. The total mass of the cross-linked hyaluronic acid gel and microspheres is 30% by mass of the microspheres. Premix with a stainless steel spatula for 1 minute, then set the rotation speed to 2000 rpm and mix for 5 minutes to obtain a uniformly dispersed hyaluronic acid gel formulation containing hydroxyapatite microspheres.

[0130] The specific reaction conditions for the above embodiments and comparative examples are shown in Table 1.

[0131]

[0132]

[0133] The microsphere-containing formulations prepared in the above examples and comparative examples were tested.

[0134] Detection method:

[0135] 1. Microsphere dispersion uniformity test:

[0136] The microsphere-containing formulation was extruded onto a glass slide using a syringe equipped with a 27G needle, forming a 1 cm long formulation strip. The dispersion of the microspheres was observed under a 40× ordinary optical microscope, and the number of observed non-uniform regions (e.g., microsphere aggregation, voids, etc.) was recorded. The number of non-uniform regions in the formulation strip was counted. The test results are shown in Table 2.

[0137] Among them, uniformly distributed microspheres appear as uniform granular gray shadows, and the formulation band is a complete gray strip; if a transparent segment (cross-linked hyaluronic acid gel) is present, it indicates that the cross-linked hyaluronic acid gel and microspheres are not mixed evenly, the microspheres are aggregated, the gel is empty, and the formulation band shows an uneven area.

[0138] Optical microscope images of some formulations, such as Figure 1-8 As shown, where, Figure 1 Microscopic images of the formulation prepared in Example 1; Figure 2 Microscopic images of the formulation prepared in Example 3; Figure 3 Microscopic images of the formulation prepared in Example 10; Figure 4 Microscopic images of the formulation prepared in Example 11; Figure 5 Microscopic images of the formulation prepared for Comparative Example 1; Figure 6 Microscopic images of the formulation prepared in Comparative Example 2; Figure 7 Microscopic images of the formulation prepared in Comparative Example 3; Figure 8 Microscopic image of the formulation prepared for Comparative Example 4.

[0139] Table 2

[0140] Dispersion Number of uneven regions Example 1 Evenly dispersed none Example 2 Evenly dispersed none Example 3 Evenly dispersed none Example 4 Evenly dispersed none Example 5 Evenly dispersed none Example 6 Evenly dispersed none Example 7 Evenly dispersed none Example 8 Evenly dispersed none Example 9 Evenly dispersed none Example 10 Evenly dispersed none Example 11 Evenly dispersed none Comparative Example 1 Microspheres aggregated, stripes with gaps 12±2.6 Comparative Example 2 Microspheres aggregated, stripes with gaps 8±1 Comparative Example 3 Microspheres aggregated, stripes with gaps 5±1.8 Comparative Example 4 Microspheres aggregated, stripes with gaps 8±2.3

[0141] 2. Stability testing method:

[0142] The microsphere-containing formulations prepared in Examples 2, 3, 1, and 2 were respectively added to centrifuge tubes and centrifuged at 3000 rpm for 5 minutes. The presence of stratification was then observed. Figure 9 This is a comparison image of Example 3 and Comparative Example 1 after centrifugation. Figure 10 This is a comparison diagram of Example 3 and Comparative Example 2 after centrifugation. Figure 11 This is a comparison diagram of Example 2 and Comparative Example 3 after centrifugation.

[0143] It can be seen that the formulations prepared in Examples 2 and 3 did not show stratification after centrifugation, while the formulations prepared in Comparative Examples 1-3 showed stratification after centrifugation. This indicates that the microsphere-containing formulations prepared in the examples of this application have centrifugal stability and can stably and uniformly distribute the microspheres in the gel. Even after high-speed centrifugation, the microspheres will not settle or aggregate.

Claims

1. A method for preparing a cross-linked hyaluronic acid gel, comprising the following steps: Step (1): Mix hyaluronic acid or its salt, a cross-linking agent, and an alkaline solution to obtain a mixed solution; Step (2): The mixed solution is subjected to a primary cross-linking reaction to obtain a first gel; Step (3): Cut the first gel into pieces and add it to an alkaline solution containing hyaluronic acid or its salt to carry out a secondary cross-linking reaction to obtain the second gel; Step (4): The second gel is purified to obtain cross-linked hyaluronic acid gel.

2. The preparation method according to claim 1, wherein the hyaluronic acid or its salt has a molecular weight of 800kDa-3000kDa.

3. The preparation method according to claim 1 or 2, wherein the mass content of hyaluronic acid or its salt in the mixed solution is 4%-20%.

4. The preparation method according to any one of claims 1-3, wherein, The crosslinking agent is added at a weight percentage of 0.5%-10% of the amount of hyaluronic acid or its salt.

5. The preparation method according to any one of claims 1-4, wherein the pH of the alkaline solution is 9-14.

6. The preparation method according to any one of claims 1-5, wherein the temperature of the crosslinking reaction in step (2) is 20-50°C and the time is 1-8h.

7. The preparation method according to any one of claims 1-6, wherein the crosslinking agent is ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, or polyglycerol polyglycidyl ether.

8. The preparation method according to any one of claims 1-7, wherein the mass content of hyaluronic acid or its salt in the alkaline solution containing hyaluronic acid or its salt in step (3) is 0.2%-2.2%. Preferably, the temperature of the secondary crosslinking reaction in step (3) is 20-50℃ and the time is 4-15h.

9. The cross-linked hyaluronic acid gel prepared by the preparation method according to any one of claims 1-8.

10. A microsphere-containing formulation comprising a cross-linked hyaluronic acid gel and microspheres prepared by the preparation method according to any one of claims 1-8, wherein the average particle size of the microspheres is 10-120 micrometers, preferably, in the formulation, the mass content of the microspheres is 6%-50% based on the total mass of the cross-linked hyaluronic acid gel and the microspheres. More preferably, the microspheres include poly(L-lactic acid) microspheres, polycaprolactone microspheres, polymethyl methacrylate microspheres, or hydroxyapatite microspheres.

Images