CN114762740B

CN114762740B - Adhesive sodium hyaluronate gel for injection and preparation method thereof

Info

Publication number: CN114762740B
Application number: CN202110038748.0A
Authority: CN
Inventors: 房浩伟; 张晓鸥; 郭学平
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2023-02-17
Anticipated expiration: 2041-01-12
Also published as: CN115926211A; CN114762740A

Abstract

Disclosed is a method for preparing an adhesive sodium hyaluronate gel for injection, which comprises: synthesis of a crosslinking agent A: synthesizing a cross-linking agent A containing disulfide bonds; and (3) crosslinking: reacting sodium hyaluronate with a cross-linking agent A and a cross-linking agent B to obtain gel L; preparation of gel M: treating the gel L with glutathione to obtain a gel M; treatment of gel M: the gel M was dialyzed to obtain an adhesive sodium hyaluronate gel for injection. Also disclosed is an adhesive sodium hyaluronate gel for injection obtained by the above production method. Also discloses an adhesive sodium hyaluronate gel for injection. The adhesive sodium hyaluronate gel for injection has the advantages of good mechanical property, easy injection, strong binding force with tissues, no occurrence of obvious tissue internal displacement and good biocompatibility. The sodium hyaluronate gel has the advantages of simple preparation process, short dialysis time, low energy consumption and no by-product.

Description

Adhesive sodium hyaluronate gel for injection and preparation method thereof

Technical Field

The application belongs to the technical field of cross-linked hyaluronic acid. In particular to an adhesive sodium hyaluronate gel for injection and a preparation method thereof.

Background

Hyaluronic acid is a natural high-molecular polysaccharide, one of the components of extracellular matrix, has excellent biocompatibility and good moisture retention, is called as an excellent natural moisture retention factor, and has been widely used in the fields of medicine and beauty, tissue engineering, regenerative medicine, medical devices, and the like. At present, most of sodium hyaluronate gel for injection is sodium hyaluronate gel taking BDDE as a cross-linking agent, the BEED cross-linking process is simple, the sodium hyaluronate gel is suitable for industrial production, and the metabolic pathway in vivo is clear and safe, so that the sodium hyaluronate gel is widely adopted.

The sodium hyaluronate gel for injection used at present is mainly a gel obtained by crosslinking BDDE serving as a crosslinking agent and sodium hyaluronate serving as a raw material, has no interaction with tissues in vivo, and cannot form tight combination with the tissues; meanwhile, when the gel is injected to parts which are easy to move, such as lips, canthus and the like, the gel structure can be damaged due to the pulling of mechanical force, and the gel can slide under the action of force and is transferred to the parts which are difficult to bear force in tissues, so that the filling and beautifying functions of the injected gel on the specific parts are influenced.

In the prior art, the document "Synthesis and in vitro evaluation of hydrolyzed hyaluronic acid for a mucoadhesive drug delivery" of Krum Kafedjisiki et al discloses a crosslinked product of hyaluronic acid and cysteine ethyl ester, in the presence of activators EDAC and NHS, HA and cysteine ethyl ester amide bond are covalently grafted to form HA-Cys, the thiol content is high, and therefore, the gel adhesion is strong. And then carrying out crosslinking reaction under the oxygen condition to form the hyaluronic acid gel with adhesiveness. However, in the post-treatment process of the reaction, the activator and unreacted cystine ethyl ester need to be removed through long-time dialysis, and the reaction process is complex and is not favorable for injection filling.

Disclosure of Invention

Compared with the sodium hyaluronate gel prepared by the traditional method, the sodium hyaluronate gel for injection not only has good viscoelasticity, but also has excellent tissue adhesiveness, and can effectively prevent displacement after the sodium hyaluronate gel for injection is injected into a body, and the sodium hyaluronate gel for injection prepared by the method still keeps good biocompatibility.

The specific technical scheme of the application is as follows:

1. the preparation method of the adhesive sodium hyaluronate gel for injection is characterized by comprising the following steps:

synthesis of a crosslinking agent A: synthesizing a cross-linking agent A containing a disulfide bond;

and (3) crosslinking: reacting sodium hyaluronate with the cross-linking agent A and the cross-linking agent B to obtain gel L;

preparation of gel M: treating the gel L with glutathione to obtain a gel M;

treatment of gel M: and dialyzing the gel M to obtain the adhesive sodium hyaluronate gel for injection.

2. The method according to item 1, wherein in the step of synthesizing the crosslinking agent A, cystine and 1,4-butanediol diglycidyl ether are used as raw materials and reacted under an alkaline condition to generate the compound with the structural formula

The crosslinking agent A of (1);

preferably, in the step of synthesizing the cross-linking agent A, the ratio of the cystine to the 1,4-butanediol diglycidyl ether substance is 1:2-3, and is preferably 1.3;

preferably, in the step of synthesizing the cross-linking agent A, the synthesis temperature of the cross-linking agent A is 10-30 ℃.

3. The production method according to item 1 or 2, characterized in that, in the crosslinking step, the ratio of the amount of the crosslinking agent a to the amount of the substance of the sodium hyaluronate repeating unit is 0.03 to 0.075.

4. The production method according to any one of items 1 to 3, wherein in the crosslinking step, the crosslinking agent B is 1,4-butanediol diglycidyl ether or an ethylene glycol-like compound

Preferably, in the crosslinking step, the ratio of the amount of the crosslinking agent B to the amount of the substance of the sodium hyaluronate repeating unit is 0.03 to 0.075;

preferably, in the crosslinking step, the ratio of the amounts of the crosslinking agent A and the crosslinking agent B is 0.5 to 1:1.

5. The production method according to any one of claims 1 to 4, characterized in that, in the crosslinking step, the sodium hyaluronate is reacted with the crosslinking agent A and the crosslinking agent B at a pH of >9 to obtain the gel L;

preferably, in the crosslinking step, the molecular weight of the sodium hyaluronate is 300-800 kDa.

6. The method according to any one of claims 1 to 5, wherein the glutathione concentration in the step of preparing the gel M is 10 to 30mmol/L.

7. The process according to any one of claims 1 to 6, wherein in the step of preparing the gel M, the gel L is dialyzed against a basic or neutral buffer solution containing glutathione to obtain the gel M;

preferably, in the step of treating the gel M, the gel M is dialyzed in a buffer solution having a pH of 7.2 to 7.7 to obtain the adhesive sodium hyaluronate gel for injection.

Preferably, in the step of treating the gel M, the dialysis time is 6 to 72 hours.

8. The method according to any one of items 1 to 7, wherein in the step of treating the gel M, the gel M is dialyzed, and then an anesthetic, preferably an ester or amide local anesthetic, is added to obtain the adhesive sodium hyaluronate gel for injection.

9. An adhesive sodium hyaluronate gel for injection, which comprises a compound having a structure represented by formula (I);

preferably, a is 4 to 1117, b is 4 to 1117, c is 65 to 5211;

more preferably, a is 44 to 298, b is 45 to 298, c is 655 to 1390.

10. The sodium hyaluronate gel according to claim 9, prepared by the method according to any one of claims 1 to 8.

ADVANTAGEOUS EFFECTS OF INVENTION

The application adopts an epoxy cross-linking agent A containing disulfide bonds and 1,4-butanediol diglycidyl ether or glycol-like compounds

The adhesive sodium hyaluronate gel for injection has good mechanical property, is easy to inject, has strong binding force with tissues, can not generate obvious tissue internal displacement, and has good biocompatibility. The preparation method is simple in preparation process, short in dialysis time, low in energy consumption, free of byproducts and pollution and easy for industrial production.

Drawings

Fig. 1 is a schematic diagram of an adhesion test method of an adhesive sodium hyaluronate gel for injection according to the present application.

Detailed Description

Specific embodiments of the present application will be described in more detail below. It is to be understood that throughout the specification and claims, the terms "including" and "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "including, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

In one aspect, the present application provides a method for preparing an adhesive sodium hyaluronate gel for injection, comprising the following steps:

synthesis of a crosslinking agent A: synthesizing a cross-linking agent A containing disulfide bonds;

preparation of gel M: treating the gel L with glutathione to obtain a gel M;

Glutathione (GSH), in the present application, is a tripeptide containing a γ -amide bond and a thiol group, consisting of glutamic acid, cysteine and glycine, present in almost every cell of the body. Glutathione can help to maintain normal immune system function, and has effects of resisting oxidation and removing toxic substance. The sulfydryl on the cysteine is an active group, and can generate oxidation-reduction reaction with a disulfide bond to reduce the disulfide bond into free sulfydryl, so that oxidized glutathione is formed.

The term "dialysis" as used herein refers to a separation and purification technique for removing a crosslinking agent that has not reacted with hyaluronic acid, based on the principle that small molecules diffuse into water (or a buffer) through the gel itself.

The term "adhesion" as used herein refers to the affinity between gel and tissue, and as shown in fig. 1, the adhesion is characterized by fixing fresh pigskin on a glass slide, allowing the pigskin with subcutaneous tissue to face outward, uniformly coating the finished gel on the tissue on the inner side of the pigskin, overlapping and standing two gel-loaded glass slides for a period of time, then weighing the weight, using a stopwatch to count the time for completely separating the two glass slides, and using the separation time.

According to the preparation method, the cross-linking agent A is firstly synthesized, then the cross-linking agent A and the cross-linking agent B are utilized to carry out cross-linking reaction with the sodium hyaluronate, and the raw material proportion in each reaction is respectively controlled through two steps of reactions, so that the adhesiveness of the adhesive sodium hyaluronate gel for injection can be ensured while the cross-linking degree of the final adhesive sodium hyaluronate gel for injection is determined, the displacement after the injection into a body can be effectively prevented, and the biocompatibility is good. The preparation method of the application is adopted to prepare the adhesive sodium hyaluronate gel, so that the adhesive property of the gel can be endowed, the activating agent and substances which do not participate in the reaction do not need to be removed through long-time dialysis, byproducts do not exist, the reaction process is simple, the dialysis time is short, the preparation process time is short, and the industrial production is facilitated. In the prior art, cysteine is used for modifying hyaluronic acid, a large amount of activating agents are needed in the process, the activating agents need to be removed in the post-treatment process, the required dialysis time is long, and the condition that the thiol can be finally introduced into the sodium hyaluronate gel to endow the gel with adhesiveness cannot be ensured. The reaction mechanism of the cross-linking agent A synthesized by the method and hyaluronic acid is the same as that of 1,4-butanediol diglycidyl ether, and epoxy groups and active hydroxyl groups in the hyaluronic acid are subjected to ring-opening reaction, so that the synthesis step is simple and efficient, and the product yield is high.

In one embodiment, the method of preparation, step A of the synthetic crosslinker, comprises the step of adding cystine

And 1,4-butanediol diglycidyl ether

As raw material, under the alkaline condition, the ring-opening reaction of epoxy compound is carried out to generate the compound with the structural formula

The crosslinking agent A of (1); in the crosslinking step, the crosslinking agent B is 1,4-butanediol diglycidyl ether or glycol-like compounds

n can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, and the like.

The synthetic technical route of the cross-linking agent A is shown as follows:

the synthesis of gels L and M is as follows, and the adhesive sodium hyaluronate gel of the present application is obtained by the following synthesis:

cystine in the application is amino acid containing disulfide bonds, and can generate oxidation-reduction reaction with reducing agents such as glutathione and the like to form free sulfhydryl groups.

The 1,4-butanediol diglycidyl ether (BDDE) in the application is epoxy resin containing 1,4-butanediol ether chain, belongs to water-soluble epoxy resin, is yellow transparent liquid, has an epoxy value of 0.63-0.74 and a viscosity of 15-20 mPa & s, and is prepared by condensation polymerization of 1,4-butanediol and epoxy chloropropane.

In this embodiment, the content of thiol groups in the final sodium hyaluronate gel for injection can be controlled by the steps of crosslinking and preparing the gel M, so as to ensure the adhesiveness of the gel required for application.

In a specific embodiment, in the preparation method of the present application, in the step of synthesizing the crosslinking agent a, the ratio of the amount of the cystine to the amount of the 1,4-butanediol diglycidyl ether is 1:2-3, and may be, for example, 1:2, 1. The quantity ratio of the cystine to the 1,4-butanediol diglycidyl ether substance is controlled within the range, and the prepared cross-linking agent A has higher purity.

In one embodiment, in the preparation method of the present application, in the step of synthesizing the crosslinking agent a, the synthesis temperature of the crosslinking agent a is 10 to 30 ℃, and for example, 10 ℃, 12 ℃, 14 ℃, 16 ℃, 18 ℃, 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃ and the like can be mentioned.

In a specific embodiment, in the preparation method of the present application, the ratio of the amount of the crosslinking agent a to the amount of the sodium hyaluronate repeating units in the crosslinking step is 0.03 to 0.075, and can be, for example, 0.03.

The "sodium hyaluronate repeating unit" in the present application refers to a disaccharide repeating unit composed of D-glucuronic acid and N-acetylglucosamine in sodium hyaluronate.

In a specific embodiment, in the preparation method of the present application, the ratio of the amount of the crosslinking agent B to the amount of the sodium hyaluronate repeating units in the crosslinking step is 0.03 to 0.075, and can be, for example, 0.03;

in one embodiment, in the preparation method of the present application, the ratio of the amounts of the crosslinking agent a to the crosslinking agent B is 0.5 to 1:1, and may be, for example, 0.5.

In a specific embodiment, in the cross-linking step, the sodium hyaluronate, which has a molecular weight of 300-800 kDa, such as 300kDa, 350kDa, 400kDa, 500kDa, 700kDa, 800kDa and the like, is too low in molecular weight of hyaluronic acid, and the gel formed after cross-linking has low mechanical strength and does not play a role of filling, is subjected to cross-linking reaction with the cross-linking agent a and the cross-linking agent B at a pH >9 (for example, 9.1, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 and the like) to obtain the gel L; the molecular weight is too high, the mechanical strength of the gel formed by crosslinking is high, but the viscosity is also high, and the injection is not easy to carry out. The present invention therefore selects hyaluronic acid within the above-mentioned range.

In a specific embodiment, in the cross-linking step, the sodium hyaluronate is dissolved in a NaOH solution with a mass fraction of 1.0% to 1.5% to obtain a sodium hyaluronate solution, the cross-linking agent a and the cross-linking agent B are mixed and then added into the sodium hyaluronate solution, and a cross-linking reaction is performed at 25 to 55 ℃ for 7 to 9 hours to obtain the gel L. In one embodiment, the preparation method of the present application further comprises, after the crosslinking step and before the step of preparing the gel M, a crushing step: crushing the gel L by using mechanical force, and sieving to obtain a particulate gel A; in the step of preparing the gel M, the gel M is obtained by treating the particulate gel A with glutathione.

In one embodiment, in the step of preparing gel M, the gel L is dialyzed in a basic or neutral buffer solution containing glutathione to obtain the gel M. Glutathione can reduce disulfide bonds in the cross-linking agent A into sulfydryl, so that the final gel has adhesiveness, and the operation is as follows: transferring the gel L into a barrel with a mesh and a side cover on the side wall, adding a certain amount of buffer solution containing glutathione, stirring at low speed for 6-8h, opening the side cover, allowing the dialysate to flow away through the mesh, keeping the gel L in the barrel, continuously adding fresh buffer solution, dialyzing again, and allowing the whole dialysis process to last for 6-72h, wherein the concentration of the glutathione is 10-30 mmol, such as 10mmol, 13mmol, 15mmol, 17mmol, 20mmol, 23mmol, 25mmol, 27mmol, 30mmol, and the like, preferably 15mmol. In this embodiment, the buffer may be a phosphate buffer or the like.

In one embodiment, in the step of treating gel M, the gel M is dialyzed in a buffer solution having a pH of 7.2 to 7.7 to obtain the adhesive sodium hyaluronate gel for injection. The pH may be, for example, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, etc., preferably 7.4.

In one embodiment, in the preparation method of the present application, in the step of preparing the gel M, the dialysis time is 6 to 72 hours, for example, 6 hours, 12 hours, 24 hours, 48 hours, 60 hours, 72 hours, etc., preferably 24 to 48 hours; in the step of treating the gel M, the dialysis time is 6 to 72 hours, for example, 6 hours, 12 hours, 24 hours, 48 hours, 60 hours, 72 hours, etc., preferably 24 to 48 hours.

In one embodiment, in the step of preparing the gel M, the gel L is dialyzed in a basic or neutral buffer solution containing glutathione, then an ester or amide local anesthetic is added, and the mixture is passed through a 50-70 mesh screen, and then filled in a syringe, and is subjected to moist heat sterilization at 115-125 ℃ for 15-20 minutes, so as to obtain the adhesive sodium hyaluronate gel for injection.

A "local anesthetic" in this application consists of three moieties in chemical structure, namely an aromatic ring, a middle chain, which can be an ester chain or an amide chain, and an amine group. Local anesthetics can be divided into two classes, depending on the structure of the intermediate chain: the first type is ester local anesthetic, the structure of which has-COO-group, and the local anesthetic belonging to the first type is procaine, tetracaine and the like; the second type is an amide-based local anesthetic having a-CONH-group in its structure, and examples of the local anesthetic belonging to this type include lidocaine, bupivacaine and the like.

On the other hand, the application also provides an adhesive sodium hyaluronate gel for injection prepared by the preparation method of any one of the above mentioned items, which has the structure shown in the following formula (I).

In still another aspect, the present application also provides an adhesive sodium hyaluronate gel for injection, characterized in that it comprises a compound having a structure represented by formula (I).

In one embodiment, the adhesive sodium hyaluronate gel for injection is characterized in that a is selected from 4 to 1117, for example, 4, 10, 44, 50, 100, 200, 250, 298, 300, 400, 500, 600, 700, 800, 900, 1000, 1110, 1117 and the like, b is selected from 4 to 1117, for example, 4, 10, 44, 50, 100, 200, 250, 298, 300, 400, 500, 600, 700, 800, 900, 1000, 1110, 1117 and the like, and c is selected from 65 to 5211, for example, 65, 100, 300, 600, 655, 1000, 1390, 2000, 3000, 4000, 5000, 5211 and the like.

In one embodiment, the adhesive sodium hyaluronate gel for injection is prepared from a, 44-298 and 655-1390.

In one embodiment, the molecular weight of the sodium hyaluronate for preparing the adhesive sodium hyaluronate gel for injection is 300-800 kDa.

In one embodiment, the adhesive sodium hyaluronate gel for injection is a mixture comprising two or more different compounds having the structure represented by formula (I).

By using the preparation method, the amount of the obtained cross-linking agent A can be increased by adjusting the ratio of the amounts of cystine and 1,4-butanediol diglycidyl ether substances in the step (1), the adhesive sodium hyaluronate gel for injection prepared by the method has high adhesiveness, the time for completely separating two finally obtained glass slides is more than 1 minute and 15 seconds and can reach 2 minutes and 24 seconds at most by using the adhesiveness characterization method, and the adhesiveness of the obtained gel product can be controlled by adjusting the type of the cross-linking agent B, the ratio of the amounts of the substances of the cross-linking agent A and the sodium hyaluronate repeating unit, the ratio of the amounts of the substances of the cross-linking agent B and the sodium hyaluronate repeating unit and the ratio of the amounts of the substances of the cross-linking agent A and the cross-linking agent B.

The adhesive hyaluronic acid gel obtained by the application can be used for cosmetic filling, can also be prepared by adjusting the conditions such as hyaluronic acid composition, crosslinking mode and crosslinking degree, can be used in the fields of medical and beauty treatment, tissue adhesion, drug loading, tissue engineering, injury repair and the like with tissue adhesion requirements, can be combined with corresponding tissues more stably by improving the tissue adhesion of the material, and can effectively reduce the influence of movement on the displacement of the implanted material.

The present application will be described in detail with reference to examples. It should be understood, however, that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

Examples

Example 1

(1) 0.042mol of cystine and 0.084mol of 1, 4-butanediol diglycidyl ether are mixed in 100mL of 0.1mol/L NaOH solution, stirred and reacted in water bath at 20 ℃ for 8 hours, and separated and purified to obtain 0.035mol of epoxy cross-linking agent A containing disulfide bonds

(2) Dissolving 939.0g sodium hyaluronate (molecular weight 800kDa, amount of substance of sodium hyaluronate repeating unit is 2.33 mol) in 100mL of NaOH solution with mass fraction of 1.5%, stirring and dissolving to obtain sodium hyaluronate solution, mixing a crosslinking agent A and BDDE, adding into the sodium hyaluronate solution, and performing crosslinking reaction at 30 ℃ for 8 hours to obtain crosslinked gel L, wherein the ratio of the amount of the crosslinking agent A to the amount of the substance of BDDE is 0.5;

(3) Crushing the gel L obtained in the step (2) by using mechanical force, and sieving the crushed gel L by using a 60-mesh sieve to obtain a particulate gel A;

(4) Dialyzing the particulate gel A for 6 hours by using 10mmol of glutathione and a phosphate buffer solution with the pH value of 8 to obtain gel M;

(5) And dialyzing the gel M for 24 hours by using a phosphate buffer solution with the pH value of 7.4 to obtain a gel N, regulating and controlling the content of sodium hyaluronate in the gel N to be 25mg/mL, then sieving by using a 60-mesh sieve, filling into an injector, and carrying out moist heat sterilization at 121 ℃ for 15 minutes to obtain the adhesive sodium hyaluronate gel for injection.

The adhesion of the sodium hyaluronate gel of this example was characterized by measuring the time for complete separation of the two slides according to the following method: as shown in figure 1, fresh pigskin with the thickness of 25mm is fixed on a glass slide with the thickness of 75 mm and the thickness of the pigskin with the subcutaneous tissue is outward, 0.5mL of finished gel prepared by the technology is uniformly coated on the tissue on the inner side of the pigskin, two glass slides loaded with gel are overlapped and placed still for 2 hours, a weight of 5g is loaded, and the time for completely separating the two glass slides is counted by using a stopwatch, wherein the separation time is 1 minute and 21 seconds. The longer the separation time, the higher the adhesion.

Example 2

(1) 0.042mol of cystine and 0.084mol of 1, 4-butanediol diglycidyl ether are mixed in 100mL of 0.1mol/L NaOH solution, stirred and reacted in water bath at 10 ℃ for 8 hours, and separated and purified to obtain 0.035mol of epoxy cross-linking agent A containing disulfide bonds

(2) 471.5g sodium hyaluronate (molecular weight 300kDa, the amount of substances of a sodium hyaluronate repeating unit is 1.17 mol) is dissolved in 100mL of 1.5% NaOH solution, the solution is stirred and dissolved to obtain a sodium hyaluronate solution, a crosslinking agent A and BDDE are mixed and then added into the sodium hyaluronate solution, and crosslinking reaction is carried out for 8 hours at 25 ℃ to obtain a crosslinked gel L, wherein the amount ratio of the substances of the crosslinking agent A to the BDDE is 1:1, and the ratio of the total amount of the crosslinking agent A and the BDDE to the amount of the substances of the sodium hyaluronate repeating unit is 0.06;

(4) Dialyzing the particulate gel A for 6 hours by using a phosphate buffer solution containing 10mmol of glutathione and having a pH value of 8 to obtain gel M;

(5) And dialyzing the gel M for 24 hours by using a phosphate buffer solution with the pH value of 7.4 to obtain a gel N, regulating and controlling the content of sodium hyaluronate in the gel N to be 25mg/mL, then sieving the gel N by using a 60-mesh sieve, filling the gel N into an injector, and performing moist heat sterilization at 121 ℃ for 20 minutes to obtain the adhesive sodium hyaluronate gel for injection.

Referring to the adhesion characterization method of example 1, the time required to obtain complete separation of the two slides was 2 minutes and 6 seconds.

Example 3

(1) 0.042mol of cystine and 0.0966mol of 1, 4-butanediol diglycidyl esterMixing glycerol ether in 100mL of 0.1mol/L NaOH solution, stirring in water bath at 30 ℃ for reaction for 8 hours, separating and purifying to obtain 0.039mol of epoxy cross-linking agent A containing disulfide bonds

Since the ratio of the amount of cystine to the amount of BDDE substances is 1.3, the amount of crosslinker A substance separated and purified in the embodiment is higher than that in the embodiment 1.

(2) Dissolving 1047.8g sodium hyaluronate (molecular weight 800kDa, the amount of substances of a sodium hyaluronate repeating unit is 2.6 mol) in 100mL of a 1.5% NaOH solution, stirring and dissolving to obtain a sodium hyaluronate solution, mixing a crosslinking agent A and BDDE, adding the mixture into the sodium hyaluronate solution, and carrying out a moisture reaction at 55 ℃ for 8 hours to obtain a crosslinked gel L, wherein the ratio of the amounts of the substances of the crosslinking agent A and BDDE is 0.5;

(4) Dialyzing the particulate gel A for 6 hours by using a phosphate buffer solution containing 10mmol of glutathione and having the pH value of 8 to obtain gel M;

(5) And dialyzing the gel M for 12 hours by using a phosphate buffer solution with the pH value of 7.4 to obtain a gel N, regulating and controlling the content of sodium hyaluronate in the gel N to be 25mg/mL, then sieving the gel N by using a 60-mesh sieve, filling the gel N into an injector, and performing moist heat sterilization at 121 ℃ for 20 minutes to obtain the adhesive sodium hyaluronate gel for injection.

Referring to the adhesion characterization method of example 1, the time required for complete separation of the two slides was 1 minute and 20 seconds.

Example 4

(1) 0.021mol of cystine and 0.084mol of 1, 4-butanediol diglycidyl ether are mixed in 100mL of 0.1mol/L NaOH solution, stirred and reacted in water bath at 30 ℃ for 8 hours, and separated and purified to obtain 0.004mol of epoxy cross-linking agent A containing disulfide bonds

Since the ratio of the amounts of cystine to BDDE is 1:4, the amount of crosslinker A isolated and purified in this example is very low.

(2) Dissolving 72.5g of sodium hyaluronate (the molecular weight is 800kDa, and the amount of substances of a sodium hyaluronate repeating unit is 0.178 mol) in 100mL of 1.5% NaOH solution, stirring and dissolving to obtain a sodium hyaluronate solution, mixing a crosslinking agent A and BDDE, adding the mixture into the sodium hyaluronate solution, and carrying out a moisture reaction at 55 ℃ for 8 hours to obtain a crosslinked gel L, wherein the amount ratio of the substances of the crosslinking agent A to the BDDE is 1:1, and the amount ratio of the total substances of the crosslinking agent A and the BDDE to the amount of the substances of the sodium hyaluronate repeating unit is 0.045;

Referring to the adhesion characterization method of example 1, the time for complete separation of the two slides was 1 minute and 21 seconds.

Example 5

(1) 0.042mol of cystine and 0.084mol of 1, 4-butanediol diglycidyl ether are mixed in 100mL of 0.1mol/L NaOH solution, stirred and reacted in water bath at 30 ℃ for 8 hours, and separated and purified to obtain 0.035mol of epoxy cross-linking agent A containing disulfide bonds

(2) 939.0g sodium hyaluronate (molecular weight 800kDa, amount of substance of sodium hyaluronate repeating unit 2.33 mol) is dissolved in 100mL of 1.5% NaOH solution, and stirred to obtain sodium hyaluronate solution, and crosslinking agent is addedA and a crosslinking agent B

(n = 11) adding the mixture to a sodium hyaluronate solution, and performing a crosslinking reaction at 55 ℃ for 8 hours to obtain a crosslinked gel L, wherein the ratio of the amount of the crosslinking agent a to the amount of the crosslinking agent B is 0.5;

Referring to the adhesion characterization method of example 1, the time required for complete separation of the two slides was 1 minute and 27 seconds.

Example 6

This example differs from example 1 in that the amount of material of the sodium hyaluronate repeating units is 1.75mol, the ratio of the amount of material of the crosslinking agent a to the amount of material of BDDE is 1:1, and the ratio of the total amount of material of the crosslinking agent a and BDDE to the amount of material of the sodium hyaluronate repeating units is 0.04.

Referring to the adhesion characterization method of example 1, the time required for complete separation of the two slides was 1 minute and 4 seconds.

Example 7

This example differs from example 2 in that the amount of material of the sodium hyaluronate repeating units is 0.58mol, the ratio of the amount of material of the crosslinking agent a to the amount of material of BDDE is 2:1, and the ratio of the total amount of material of the crosslinking agent a and BDDE to the amount of material of the sodium hyaluronate repeating units is 0.09. To ensure that all disulfide bonds were reduced to sulfhydryl groups, the dialysis time was extended to 48 hours using 10mmol glutathione in pH 8 phosphate buffer.

Referring to the adhesion characterization method of example 2, the time for complete separation of the two slides was 2 min 17 sec.

Example 8

This example differs from example 7 in that the ratio of the amount of substances of crosslinking agent a to BDDE is 0.4. To ensure that all disulfide bonds were reduced to sulfhydryl groups, the dialysis time was extended to 48 hours using 10mmol glutathione in pH 8 phosphate buffer.

Referring to the adhesion characterization method of example 7, the time required for complete separation of the two slides was 2 minutes and 13 seconds.

Example 9

Referring to the adhesion characterization method of example 7, the time required to obtain complete separation of the two slides was 2 minutes and 24 seconds.

The parameters and effect data for examples 1 to 9 are summarized in table 1 below:

TABLE 1

As can be seen from the data in table 1, the ratio of the amount of the substance of the repeating unit of sodium hyaluronate to the amount of the substance of the repeating unit of sodium hyaluronate has the greatest effect, and the ratio of the amount of the substance of the repeating unit of sodium hyaluronate to the amount of the substance of the crosslinking agent B has a greater effect on the adhesion of the sodium hyaluronate. The ratio of the amount of crosslinker a to the amount of material of the sodium hyaluronate repeat units of example 2 is within the range of the present application and is superior to examples 1, 3 and 4, so the adhesion of example 2 is significantly superior to examples 1, 3, 4 and 5. Example 6 compared with example 4, the ratio of the amount of the cross-linking agent a to the amount of the substance of the sodium hyaluronate repeating unit and the ratio of the cross-linking agent B to the amount of the substance of the sodium hyaluronate repeating unit of example 6 were both smaller than those of example 4, and thus the adhesiveness of example 4 was better than that of example 6. In examples 7 to 9, the adhesion of examples 7 to 9 was slightly better than that of example 2, because the ratio of the amount of the crosslinking agent a to the amount of the substance of the sodium hyaluronate repeating unit was significantly larger in examples 7 to 9 than in other examples, and the ratio of the amount of the crosslinking agent B to the amount of the substance of the sodium hyaluronate repeating unit was significantly larger in examples 8 to 9 than in other examples.

Comparative example 1

(1) Dissolving 939.0g sodium hyaluronate (molecular weight 800kDa, and the amount of substances of a sodium hyaluronate repeating unit is 2.33 mol) in 80mL of aqueous solution, stirring and completely dissolving to obtain a sodium hyaluronate solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) which are equal to the amount of carboxyl groups in the sodium hyaluronate molecules, reacting and activating for 8 hours, then adding cystine, and continuously stirring and reacting for 8 hours to obtain a cross-linked gel, wherein the ratio of the amount of substances of the cystine and the sodium hyaluronate repeating unit is 0.035;

(2) And (2) soaking the crosslinked gel obtained in the step (1) for 8 times by using 10L of deionized water, wherein the soaking time is 6 hours each time, and after the soaking time is finished, the soaked waste liquid is replaced by 10L of deionized water again.

(3) Crushing the gel obtained in the step (2) by using mechanical force, and sieving the crushed gel with a 60-mesh sieve to obtain micro-particle gel;

(4) The particulate gel was dialyzed for 6 hours against a phosphate buffer containing 10mmol of glutathione and having a pH of 8, and the network of the particulate gel was disrupted to become a solution since all disulfide bonds in the cystine of the crosslinking agent were reduced to free thiol groups by glutathione.

By the scheme, the sodium hyaluronate gel with adhesiveness can not be obtained.

Comparative example 2

(1) Dissolving 939.0g sodium hyaluronate (molecular weight 800kDa, and the amount of substances of a sodium hyaluronate repeating unit is 2.33 mol) in 80mL of 1.5% NaOH solution, stirring and dissolving to obtain a sodium hyaluronate solution, adding BDDE into the sodium hyaluronate solution, and performing crosslinking reaction at 30 ℃ for 8 hours to obtain a crosslinked gel, wherein the ratio of the amounts of substances of the BDDE and the sodium hyaluronate repeating unit is 0.07;

(2) Crushing the crosslinked gel obtained in the step (1) by using mechanical force, and sieving the crushed crosslinked gel with a 60-mesh sieve to obtain micro-particle gel;

(3) Dialyzing the particulate gel for 24 hours by using a phosphate buffer solution with the pH value of 7.4, and regulating the content of sodium hyaluronate in the particulate gel to be 25mg/mL;

(4) And (4) sieving the gel particles obtained in the step (3) through a 60-mesh sieve, filling into a syringe, and carrying out moist heat sterilization at 121 ℃ for 18 minutes to obtain the sodium hyaluronate gel.

Referring to the adhesion characterization method of example 1, the time required for complete separation of the two slides was 9 seconds. The sodium hyaluronate gel prepared by this method has low adhesiveness.

Comparative example 3

939.0g sodium hyaluronate (molecular weight 800kDa, amount of substance of sodium hyaluronate repeating unit is 2.33 mol) is dissolved in 100mL of 1.5% NaOH solution, stirred and dissolved, 0.035mol cystine and 0.14mol BDDE are mixed and added into the sodium hyaluronate solution, and crosslinking reaction is carried out for 8 hours at 30 ℃, thus obtaining the sodium hyaluronate gel.

Referring to the adhesion characterization method of example 1, the time required for complete separation of the two slides was 33 seconds.

When the hyaluronic acid gel is prepared in such a way, the cross-linking agent BDDE can react with sodium hyaluronate and cystine at the same time, but the proportion of the two reactions cannot be guaranteed, the amount of cystine introduced into the sodium hyaluronate gel cannot be guaranteed finally, and the mercapto group can be introduced into the sodium hyaluronate gel finally to endow the gel with adhesiveness.

Comparative example 4

939.0g sodium hyaluronate (molecular weight 800kDa, amount of substance of sodium hyaluronate repeating unit is 2.33 mol) was dissolved in 100mL of 1.5% NaOH solution, stirred and dissolved, 0.07mol cysteine and 0.14mol bdde were mixed and added to the sodium hyaluronate solution, and crosslinking reaction was performed at 30 ℃ for 8 hours to obtain sodium hyaluronate gel.

When the sodium hyaluronate gel is prepared by the method, BDDE can not only react with sodium hyaluronate, but also react with amino and sulfydryl in cysteine in the reaction process, so that the crosslinking degree of the crosslinked gel cannot be determined, and the adhesive hyaluronic acid gel cannot be obtained after BDDE reacts with sulfydryl in cysteine.

The parametric data for comparative examples 1 to 4 are summarized in table 2 below:

TABLE 2

The foregoing is directed to preferred embodiments of the present application, other than the limiting examples of the present application, and variations of the present application may be made by those skilled in the art using the foregoing teachings. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Claims

preparation of gel M: treating the gel L with glutathione to obtain a gel M;

treatment of gel M: dialyzing the gel M to obtain the adhesive sodium hyaluronate gel for injection;

wherein, in the step of synthesizing the cross-linking agent A, cystine and 1,4-butanediol diglycidyl ether are used as raw materials to react under the alkaline condition to generate the compound with the structural formula of

The crosslinking agent A of (1);

in the crosslinking step, the crosslinking agent B is 1,4-butanediol diglycidyl ether or glycol-like compounds

。

2. The method according to claim 1, wherein in the step A, the ratio of the amount of the substance of cystine and 1,4-butanediol diglycidyl ether is 1 to 3.

3. The method of claim 1, wherein in the step of synthesizing crosslinker a, the ratio of the amount of cystine to the amount of 1,4-butanediol diglycidyl ether is 1.3.

4. The method according to claim 1, wherein the temperature for synthesizing the crosslinking agent A is 10 to 30 ℃.

5. The method for preparing the sodium hyaluronate according to claim 1~4, wherein in the crosslinking step, the ratio of the amount of the crosslinking agent A to the amount of the sodium hyaluronate repeating units is 0.03 to 0.075.

6. The method for preparing the sodium hyaluronate according to claim 1~4, wherein in the crosslinking step, the ratio of the amount of the crosslinking agent B to the amount of the sodium hyaluronate repeating units is 0.03 to 0.075.

7. The method for preparing the polyurethane foam according to any one of claims 1~4, wherein in the crosslinking step, the mass ratio of the crosslinking agent A to the crosslinking agent B is 0.5 to 1.

8. The method of claim 1~4 wherein said cross-linking step comprises reacting said sodium hyaluronate with said cross-linking agent A and said cross-linking agent B at a pH >9 to form said gel L.

9. The method for preparing the sodium hyaluronate according to claim 1~4, wherein the molecular weight of the sodium hyaluronate in the crosslinking step is 300 to 800kDa.

10. The method for preparing the hydrogel according to claim 1~4, wherein in the step of preparing the gel M, the concentration of glutathione is 10 to 30mmol/L.

11. The method of claim 1~4 wherein in said step of preparing gel M, said gel L is dialyzed against a basic or neutral buffer containing glutathione to obtain said gel M.

12. The method for preparing the sodium hyaluronate gel for injection according to claim 1~4, wherein the step of treating the gel M comprises dialyzing the gel M in a buffer solution with pH of 7.2 to 7.7 to obtain the sodium hyaluronate gel for injection.

13. The preparation method of any one of claims 1~4, wherein in the gel M treatment step, the dialysis time is 6 to 72h.

14. The method of claim 1~4 wherein in the step of treating gel M, the gel M is dialyzed and then an anesthetic is added to obtain the injectable sodium hyaluronate gel.

15. The method of claim 14, wherein the anesthetic is an ester or amide local anesthetic.

16. An adhesive sodium hyaluronate gel for injection, which is characterized by comprising a compound having a structure represented by formula (I),

formula (I).

17. The adhesive sodium hyaluronate gel for injection according to claim 16, wherein a is 4 to 1117, b is 4 to 1117, and c is 65 to 5211.

18. The adhesive sodium hyaluronate gel for injection according to claim 16, wherein a ranges from 44 to 298, b ranges from 45 to 298, and c ranges from 655 to 1390.

19. The adhesive sodium hyaluronate gel for injection according to claim 16, which is prepared by the method according to any one of claims 1 to 15.