Preparation method of two-step polymerized high-cohesiveness cross-linked sodium hyaluronate gel

Abstract

The invention relates to a preparation method of two-step polymerized high-cohesiveness cross-linked sodium hyaluronate gel, which comprises the following steps: preparing an alkaline solution, adding a cross-linking agent, and uniformly mixing to prepare an activated solution; adding high-molecular-weight sodium hyaluronate dry powder into the activated solution to obtain a first-step cross-linked sodium hyaluronate solution, adding low-molecular-weight sodium hyaluronate dry powder into the first-step cross-linked sodium hyaluronate solution to obtain cross-linked sodium hyaluronate gel, and granulating through a screen to obtain the sodium hyaluronate gel for injection. High-molecular-weight hyaluronic acid polymers are cross-linked to form a uniform three-dimensional latticed skeleton through a first reaction, and then low-molecular-weight hyaluronic acid polymers are modified on the skeleton through a second addition of monomers to form a uniform and compact three-dimensional network structure, so that the crosslinking degree is increased and the residues of a crosslinking agent are reduced; meanwhile, the viscoelasticity and cohesion of the gel are improved.

Description

Technical Field

[0001] The present invention relates to a method for preparing a two-step polymerized highly cohesive cross-linked sodium hyaluronate gel. Background Art

[0002] Hyaluronic acid (HA) is a linear macromolecular polysaccharide composed of repeated disaccharide units of D-glucuronic acid and N-acetylglucosamine. It is a natural moisturizing factor widely present in organisms, capable of retaining water 1000 times its own weight. It is also an endogenous component in the human body. With good physical and chemical properties and biocompatibility, it is widely used in fields such as ophthalmology, orthopedics, and plastic surgery. Natural hyaluronic acid is easily biodegradable and has a short retention time in the body, which limits its application in some aspects. Cross-linking hyaluronic acid with a cross-linking agent results in a stable three-dimensional network-like three-dimensional structure. This can not only effectively improve the filling effect and retention time in skin tissues but also ensure its good biocompatibility.

[0003] With the increasing number of cross-linked sodium hyaluronate gel dermal fillers on the market, people are paying more and more attention to the relationship between their different properties, such as rheology and cohesion, and clinical effects and uses. Cohesion is described as the force between particles of the same substance, manifested as the internal adhesion force that binds gel aggregates together by individual cross-linked units. That is, in materials with low cohesion, particles are easily separated, while in materials with high cohesion, it is more difficult. A dermal filler with good cohesion can be extruded from a syringe evenly and controllably and can provide a lasting correction effect by resisting shear deformation forces after being implanted into soft tissues, ensuring that irreversible deformation does not occur when stressed. Fillers that need to be injected deep to restore the volume of the mid-face and on the periosteum to correct the mandibular contour are subjected to high-frequency and high-intensity pressures and require excellent cohesion to provide sufficient stability and support.

[0004] The most similar existing technical solutions: The patent CN103146003A of Shanghai Qisheng Biological Preparation Co., Ltd. adopts a low-temperature secondary cross-linking method, using a small amount of BDDE cross-linking agent, and combines the methods of low-temperature long-time and high-temperature short-time secondary cross-linking followed by rapid dialysis to prepare a sodium hyaluronate gel with low cross-linking agent residue. In addition, there is the patent WO / 2004 / 092222 of Allergan, which combines a low-molecular-weight polymer with a high-molecular-weight polymer to prepare an injectable single-phase hydrogel with good mechanical properties under low cross-linking degrees.

[0005] Although the patent of Shanghai Qisheng can further crosslink BDDE in the system with sodium hyaluronate through the way of low-temperature secondary crosslinking, the static reaction method always makes the network inside the gel loose, and the free polymer fragments cannot dynamically combine with the framework. Moreover, using sodium hyaluronate with a single molecular weight cannot form a dense three-dimensional network, resulting in poor mechanical properties and insufficient cohesion of the gel.

[0006] The patent of Allergan uses a method of blending low-molecular-weight polymers and high-molecular-weight polymers and crosslinking with a crosslinking agent to prepare a single-phase hydrogel. However, because it blends high-molecular-weight and low-molecular-weight polymers, the high-molecular polymer and the low-molecular polymer are connected disorderly, resulting in irregular voids between the gel networks and limiting its performance. Summary of the Invention

[0007] The technical problem to be solved by the present invention is: overcoming the deficiencies of the prior art, providing a preparation method of a two-step polymerization highly cohesive crosslinked sodium hyaluronate gel, and solving the technical problems of insufficient viscoelastic properties and cohesion of the current gel.

[0008] The technical solution adopted by the present invention to solve its technical problems is:

[0009] Providing a preparation method of a two-step polymerization highly cohesive crosslinked sodium hyaluronate gel, which is characterized by including the following steps:

[0010] Step S01, preparing an alkaline solution;

[0011] Step S02, adding a crosslinking agent to the prepared alkaline solution and mixing evenly to prepare an activation solution;

[0012] Step S03, adding high-molecular-weight sodium hyaluronate dry powder to the activation solution, fully dissolving it, and standing and reacting at a lower temperature to obtain a first-step crosslinked sodium hyaluronate solution. The standing reaction temperature is 5-30 °C, and the reaction time is 8-48 h;

[0013] Step S04, adding low-molecular-weight sodium hyaluronate dry powder to the first-step crosslinked sodium hyaluronate solution, stirring until fully dissolved, and standing and reacting at a higher temperature to obtain a crosslinked sodium hyaluronate gel. The standing reaction temperature is 30-50 °C, and the reaction time is 0.5-5 h;

[0014] Step S05, cutting the crosslinked sodium hyaluronate gel into pieces, adjusting the pH and standing for dialysis;

[0015] Step S06, granulating through a sieve to obtain an injectable sodium hyaluronate gel;

[0016] The ratio of the crosslinking agent, low-molecular-weight, and high-molecular-weight sodium hyaluronate dry powder is 1:3:20-1:15:50 (W:W:W).

[0017] Further, the concentration of the alkaline solution is 0.1 - 0.5 mol / L, and the ratio of the crosslinking agent to the alkaline solution is 1:100 - 1:300 (W:W).

[0018] Further, the alkaline solution is one or a mixture of solutions of NaOH, KOH, and Na 2 CO 3 and the like.

[0019] Further, the molecular weight of the high molecular weight sodium hyaluronate dry powder is 2 - 3 million Daltons; the molecular weight of the low molecular weight sodium hyaluronate dry powder is 0.3 - 1.5 million Daltons.

[0020] Further, in step S05, a phosphate buffer solution is used to adjust the pH.

[0021] Further, in step S06, the mesh number of the sieve through which granulation is performed is 30 - 300 meshes.

[0022] Further, the crosslinking agent is 1,4 - butanediol diglycidyl ether.

[0023] The beneficial effects of the present invention are:

[0024] The preparation method of the two - step polymerization highly cohesive cross - linked sodium hyaluronate gel of the present invention makes the high molecular weight hyaluronic acid polymers cross - link with each other through the first reaction to form a uniform three - dimensional grid - like skeleton, and then by adding monomers for the second time, the low molecular weight hyaluronic acid polymers are modified on the skeleton to form a uniform and dense three - dimensional network structure, increasing the cross - linking degree and reducing the cross - linking agent residue, while improving the viscoelastic properties and cohesion of the gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below with reference to the accompanying drawings.

[0026] Figure 1 Shows the kinematic viscosity of each example and comparative example;

[0027] Figure 2 Shows the complex viscosity of each example and comparative example;

[0028] Figure 3 Shows the cohesion of each example and comparative example;

[0029] Figure 4 Shows the SEM image of the gel obtained by cross - linking involved in Example 1;

[0030] Figure 5 Shows the SEM image of the gel obtained by cross - linking involved in Example 2;

[0031] Figure 6Shows the SEM image of the gel obtained by crosslinking involved in Example 3;

[0032] Figure 7 Shows the SEM image of the gel obtained by crosslinking involved in Example 4;

[0033] Figure 8 Shows the SEM image of the gel obtained by crosslinking involved in Comparative Example 1;

[0034] Figure 9 Shows the SEM image of the gel obtained by crosslinking involved in Comparative Example 2. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] This embodiment provides a preparation method for a two-step polymerized highly cohesive crosslinked sodium hyaluronate gel, which is characterized by including the following steps:

[0037] Step S01, prepare an alkaline solution;

[0038] Step S02, add a crosslinking agent to the prepared alkaline solution and mix evenly to obtain an activated solution;

[0039] Step S03, add high-molecular-weight sodium hyaluronate dry powder to the activated solution, fully dissolve it, and then let it stand and react at a lower temperature to obtain a first-step crosslinked sodium hyaluronate solution. The standing reaction temperature is 5-30°C, and the reaction time is 8-48 h;

[0040] Step S04, add low-molecular-weight sodium hyaluronate dry powder to the first-step crosslinked sodium hyaluronate solution, stir until fully dissolved, and then let it stand and react at a higher temperature to obtain a crosslinked sodium hyaluronate gel. The standing reaction temperature is 30-50°C, and the reaction time is 0.5-5 h;

[0041] Step S05, cut the crosslinked sodium hyaluronate gel into pieces, adjust the pH and let it stand for dialysis;

[0042] Step S06, granulate through a sieve to obtain an injectable sodium hyaluronate gel;

[0043] The ratio of the crosslinking agent, low-molecular-weight, and high-molecular-weight sodium hyaluronate dry powder is 1:3:20-1:15:50 (W:W:W).

[0044] In some embodiments, specifically, the concentration of the alkaline solution is 0.1 - 0.5 mol / L, and the ratio of the crosslinking agent to the alkaline solution is 1:100 - 1:300 (W:W).

[0045] In some embodiments, specifically, the alkaline solution uses one or a mixture of solutions of NaOH, KOH, and Na 2 CO 3 and the like.

[0046] In some embodiments, specifically, the molecular weight of the high - molecular - weight sodium hyaluronate dry powder is 2 - 3 million Daltons; the molecular weight of the low - molecular - weight sodium hyaluronate dry powder is 0.3 - 1.5 million Daltons.

[0047] In some embodiments, specifically, in step S05, a phosphate buffer solution is used to adjust the pH.

[0048] In some embodiments, specifically, in step S06, the mesh number of the sieve through which granulation is performed is 30 - 300 meshes.

[0049] In some embodiments, specifically, the crosslinking agent is 1,4 - butanediol diglycidyl ether.

[0050] The present invention is described below through several embodiments: It should be noted that the description order of the following embodiments does not limit the preferred order of the embodiments of the present application. And in the following embodiments, each embodiment is described with emphasis. For parts not detailed in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0051] Example 1

[0052] A preparation method of a two - step polymerized highly cohesive cross - linked sodium hyaluronate gel, comprising the following steps:

[0053] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.1 mol / L;

[0054] Step S2, add 80 mg of 1,4 - butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0055] Step S3, add 3.5 g of high - molecular - weight sodium hyaluronate dry powder with a molecular weight of 2×10 6 Da to the activation solution; after complete dissolution, let it stand and react at a lower temperature to obtain a first - step cross - linked sodium hyaluronate solution. The standing reaction temperature is 20 °C and the reaction time is 24 h;

[0056] Step S4, add 1.0 g of low - molecular - weight sodium hyaluronate dry powder with a molecular weight of 5×105 Da, stir until completely dissolved, and let it react while standing at a relatively high temperature to obtain cross-linked sodium hyaluronate gel. The temperature for standing reaction is 40 °C and the reaction time is 3 h;

[0057] Step S5, cut the cross-linked sodium hyaluronate gel into pieces, adjust the pH with phosphate buffer solution and let it stand for dialysis;

[0058] Step S6, granulate through a sieve to obtain sodium hyaluronate gel for injection;

[0059] The mesh number of the sieve for granulation is 75 mesh.

[0060] For the sodium hyaluronate gel of this example, see Figure 4 as shown.

[0061] Example 2

[0062] A preparation method of a two-step polymerized highly cohesive cross-linked sodium hyaluronate gel, comprising the following steps:

[0063] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.1 mol / L;

[0064] Step S2, add 80 mg of 1,4-butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0065] The cross-linking agent is BDDE;

[0066] Step S3, add 4 g of high molecular weight sodium hyaluronate dry powder with a molecular weight of 2.5×10 6 Da to the activation solution; after completely dissolving, let it react while standing at a relatively low temperature to obtain the first-step cross-linked sodium hyaluronate solution. The temperature for standing reaction is 5 °C and the reaction time is 48 h;

[0067] Step S4, add 0.5 g of low molecular weight sodium hyaluronate dry powder with a molecular weight of 1×10 6 Da to the first-step cross-linked sodium hyaluronate solution, stir until completely dissolved, and let it react while standing at a relatively high temperature to obtain cross-linked sodium hyaluronate gel. The temperature for standing reaction is 30 °C and the reaction time is 5 h;

[0068] Step S5, cut the cross-linked sodium hyaluronate gel into pieces, adjust the pH with phosphate buffer solution and let it stand for dialysis;

[0069] Step S6, granulate through a sieve to obtain sodium hyaluronate gel for injection;

[0070] The mesh number of the sieve for granulation is 75 mesh.

[0071] For the sodium hyaluronate gel of this example, seeFigure 5 as shown

[0072] Example 3

[0073] A method for preparing a two-step polymerized highly cohesive cross-linked sodium hyaluronate gel, comprising the following steps:

[0074] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.2 mol / L;

[0075] Step S2, add 160 mg of 1,4-butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0076] Step S3, add 4 g of high molecular weight sodium hyaluronate dry powder with a molecular weight of 2.5×10 6 Da to the activation solution; after complete dissolution, let it stand and react at a lower temperature to obtain a first-step cross-linked sodium hyaluronate solution, with the standing reaction temperature being 30°C and the reaction time being 8 h;

[0077] Step S4, add 0.5 g of low molecular weight sodium hyaluronate dry powder with a molecular weight of 1.5×10 6 Da to the first-step cross-linked sodium hyaluronate solution, stir until completely dissolved, and let it stand and react at a higher temperature to obtain a cross-linked sodium hyaluronate gel, with the standing reaction temperature being 50°C and the reaction time being 0.5 h;

[0078] Step S5, cut the cross-linked sodium hyaluronate gel into pieces, adjust the pH with a phosphate buffer solution and let it stand for dialysis;

[0079] Step S6, granulate through a sieve to obtain an injectable sodium hyaluronate gel; the mesh number of the sieve for granulation is 75 mesh.

[0080] For the sodium hyaluronate gel of this example, see Figure 6 as shown

[0081] Example 4

[0082] A method for preparing a two-step polymerized highly cohesive cross-linked sodium hyaluronate gel, comprising the following steps:

[0083] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.5 mol / L;

[0084] Step S2, add 80 mg of 1,4-butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0085] Step S3, add 3.5 g of high molecular weight sodium hyaluronate dry powder with a molecular weight of 2.8×10 6Da; After complete dissolution, let it stand and react at a lower temperature to obtain the first-step cross-linked sodium hyaluronate solution. The standing reaction temperature is 10 °C and the reaction time is 36 h;

[0086] Step S4, add 1.0 g of low molecular weight sodium hyaluronate dry powder with a molecular weight of 5×10 5 Da to the first-step cross-linked sodium hyaluronate solution, stir until completely dissolved, and let it stand and react at a higher temperature to obtain cross-linked sodium hyaluronate gel. The standing reaction temperature is 40 °C and the reaction time is 1.5 h;

[0087] Step S5, cut the cross-linked sodium hyaluronate gel into pieces, adjust the pH with phosphate buffer solution and let it stand for dialysis;

[0088] Step S6, granulate through a sieve to obtain sodium hyaluronate gel for injection; the mesh number of the sieve for granulation is 75 mesh.

[0089] The sodium hyaluronate gel of this example is shown in Figure 7 shown.

[0090] Comparative Example 1

[0091] A preparation method of a two-step polymerized highly cohesive cross-linked sodium hyaluronate gel, comprising the following steps:

[0092] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.1 mol / L;

[0093] Step S2, add 80 mg of 1,4-butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0094] Step S3, add 3.5 g of high molecular weight sodium hyaluronate dry powder with a molecular weight of 2×10 6 Da to the activation solution; after complete dissolution, let it stand and react at a lower temperature to obtain the first-step cross-linked sodium hyaluronate solution. The standing reaction temperature is 20 °C and the reaction time is 24 h;

[0095] Step S4, continue to add 1 g of high molecular weight sodium hyaluronate dry powder to the first-step cross-linked sodium hyaluronate solution, stir until completely dissolved, and let it stand and react at a higher temperature to obtain cross-linked sodium hyaluronate gel. The standing reaction temperature is 40 °C and the reaction time is 3 h;

[0096] Step S5, cut the cross-linked sodium hyaluronate gel into pieces, adjust the pH with phosphate buffer solution and let it stand for dialysis;

[0097] Step S6, granulate through a sieve to obtain sodium hyaluronate gel for injection; the mesh number of the sieve for granulation is 75 mesh.

[0098] The sodium hyaluronate gel of this example is shown in Figure 8 as follows.

[0099] Comparative Example 2

[0100] A preparation method of a two-step polymerization highly cohesive cross-linked sodium hyaluronate gel, comprising the following steps:

[0101] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.1 mol / L;

[0102] Step S2, add 80 mg of 1,4-butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0103] Step S3, add 3.5 g of low molecular weight sodium hyaluronate dry powder with a molecular weight of 5×10 5 Da to the activation solution; after fully dissolving, let it stand and react at a lower temperature to obtain a first-step cross-linked sodium hyaluronate solution, the standing reaction temperature is 20 °C, and the reaction time is 24 h;

[0104] Step S4, continue to add 1 g of low molecular weight sodium hyaluronate dry powder to the first-step cross-linked sodium hyaluronate solution, stir until fully dissolved, and let it stand and react at a higher temperature to obtain a cross-linked sodium hyaluronate gel, the standing reaction temperature is 40 °C, and the reaction time is 3 h;

[0105] Step S5, cut the cross-linked sodium hyaluronate gel into pieces, adjust the pH with a phosphate buffer solution and let it stand for dialysis;

[0106] Step S6, granulate through a sieve to obtain an injectable sodium hyaluronate gel; the mesh number of the sieve for granulation is 75 mesh.

[0107] The sodium hyaluronate gel of this example is shown in Figure 9 as follows.

[0108] Comparative Example 3

[0109] A preparation method of a two-step polymerization highly cohesive cross-linked sodium hyaluronate gel, comprising the following steps:

[0110] Step S1, prepare an alkaline solution of 20 g of NaOH with a concentration of 0.1 mol / L;

[0111] Step S2, add 80 mg of 1,4-butanediol diglycidyl ether and mix evenly to prepare an activation solution;

[0112] Step S3, add 3.5 g of low molecular weight sodium hyaluronate dry powder with a molecular weight of 5×10 5Da; After being fully dissolved, let it stand and react at a lower temperature to obtain the first-step crosslinked sodium hyaluronate solution. The standing reaction temperature is 20 °C and the reaction time is 24 h;

[0113] Step S4, continue to add 1 g of high-molecular-weight sodium hyaluronate dry powder to the first-step crosslinked sodium hyaluronate solution. The molecular weight of the low-molecular-weight sodium hyaluronate dry powder is 2×10 6 Da; Stir until fully dissolved, let it stand and react at a higher temperature to obtain crosslinked sodium hyaluronate gel. The standing reaction temperature is 40 °C and the reaction time is 3 h;

[0114] Step S5, cut the crosslinked sodium hyaluronate gel into pieces, adjust the pH with phosphate buffer solution and let it stand for dialysis;

[0115] Step S6, granulate through a sieve to obtain sodium hyaluronate gel for injection; the mesh number of the sieve for granulation is 75 mesh.

[0116] See the following table for the performance parameters of each example and comparative example

[0117]

[0118] In Examples 1-4 of the present invention, high-molecular-weight sodium hyaluronate dry powder is added first for the first time, and low-molecular-weight sodium hyaluronate dry powder is added for the second time. In Comparative Example 1, high-molecular-weight sodium hyaluronate dry powder is added both times. In Comparative Example 2, low-molecular-weight sodium hyaluronate dry powder is added both times. In Comparative Example 3, a large amount of low-molecular-weight sodium hyaluronate dry powder is added for the first time, and a small amount of high-molecular-weight sodium hyaluronate dry powder is added for the second time. From the performance parameters obtained in the above table and Figure 1 、 2 、3, it can be seen that the viscoelastic properties and cohesion of Examples 1-4 are much higher than those of Comparative Examples 1-3.

[0119] Enlightened by the above ideal embodiments according to the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A method for preparing a two-step polymerization highly cohesive cross-linked sodium hyaluronate gel, characterized in that: The following steps are involved: Step S01, preparing alkaline solution; Step S02, adding a cross-linking agent to the prepared alkaline solution and mixing them evenly to prepare an activation solution; Step S03, adding high molecular weight sodium hyaluronate dry powder to the activation solution, and after fully dissolving, standing at a relatively low temperature for reaction to obtain the first step cross-linked sodium hyaluronate solution, the standing reaction temperature is 5 to 30° C., and the reaction time is 8 to 48 hours; Step S04, adding low molecular weight sodium hyaluronate dry powder to the first step cross-linked sodium hyaluronate solution, stirring until fully dissolved, and standing at a high temperature to react to obtain a cross-linked sodium hyaluronate gel, the standing reaction temperature is 30 to 50° C., and the reaction time is 0.5 to 5 hours; Step S05, cutting the cross-linked sodium hyaluronate gel into pieces, adjusting the pH and allowing the pieces to stand for dialysis; Step S06, granulating through a sieve to obtain sodium hyaluronate gel for injection; The ratio of the cross-linking agent, low molecular weight and high molecular weight sodium hyaluronate dry powder is 1:3:20 to 1:15:50 (W:W:W).

2. The method for preparing the two-step polymerization high cohesive cross-linked sodium hyaluronate gel according to claim 1, characterized in that: The concentration of the alkaline solution is 0.1-0.5 mol / L, and the ratio of the cross-linking agent to the alkaline solution is 1:100-1:300 (W:W).

3. The method for preparing the two-step polymerization high cohesive cross-linked sodium hyaluronate gel according to claim 1, characterized in that: The alkaline solution is a mixed solution of one or more of NaOH, KOH and Na2CO3.

4. The method for preparing the two-step polymerization high cohesive cross-linked sodium hyaluronate gel according to claim 1, characterized in that: The molecular weight of high molecular weight sodium hyaluronate dry powder is 2 to 3 million Daltons; the molecular weight of low molecular weight sodium hyaluronate dry powder is 300,000 to 1.5 million Daltons.

5. The method for preparing the two-step polymerization high cohesive cross-linked sodium hyaluronate gel according to claim 1, characterized in that: In step S05, the pH is adjusted using a phosphate buffer solution.

6. The method for preparing the two-step polymerization high cohesive cross-linked sodium hyaluronate gel according to claim 1, characterized in that: Step S06, the mesh number of the granulation screen is 30 to 300 meshes.

7. The method for preparing the two-step polymerization high cohesive cross-linked sodium hyaluronate gel according to claim 1, characterized in that: The cross-linking agent is 1,4-butanediol diglycidyl ether.

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