CN114762740A

CN114762740A - Adhesive sodium hyaluronate gel for injection and preparation method thereof

Info

Publication number: CN114762740A
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: 2022-07-19
Anticipated expiration: 2041-01-12
Also published as: CN115926211A; CN114762740B

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: and dialyzing the gel M to obtain the 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, which comprises the formula (I)The compound has a structure, and n is 744-1985. The adhesive sodium hyaluronate gel for injection has the advantages of good mechanical property, easiness in injection, strong binding force with tissues, no occurrence of obvious internal displacement of the tissues, 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 sodium hyaluronate gel for injection is BDDE as a cross-linking agent, and BEED is used for simple cross-linking process, is suitable for industrial production, has clear and safe in-vivo metabolic pathway and 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 to 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 cross-linking reaction under the oxygen condition to form 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, can effectively prevent the sodium hyaluronate gel for injection from displacing after being injected into a body, and 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 disulfide bonds;

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 preparation method of the compound of the formula 1, wherein in the step of synthesizing the crosslinking agent A, cystine and 1, 4-butanediol diglycidyl ether are used as raw materials and react under alkaline conditions 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 amount ratio of the cystine to the substances of the 1, 4-butanediol diglycidyl ether is 1: 2-3, preferably 1: 2.3;

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 method according to item 1 or 2, wherein the amount ratio of the crosslinking agent A to the sodium hyaluronate repeating units in the crosslinking step is 0.03 to 0.075: 1.

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 sodium hyaluronate repeating units is 0.03-0.075: 1;

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

5. The preparation method according to any one of claims 1 to 4, wherein 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 items 1 to 5, wherein in the step of preparing the gel M, the concentration of glutathione is 10 to 30 mmol/L.

7. The method according to any one of items 1 to 6, wherein in the step of preparing the gel M, the gel L is dialyzed in an alkaline 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 6-72 h.

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

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

Preferably, a is 4 to 1117, b is 4 to 1117, and c is 65 to 5211;

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

10. The sodium hyaluronate gel according to claim 9, which is 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 view 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:

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

In the present application, "glutathione" (GSH) is a tripeptide containing a γ -amide bond and a thiol group, and is composed of glutamic acid, cysteine, and glycine, and is 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 sulfhydryl on the cysteine is an active group of the glutathione, and can perform oxidation-reduction reaction with the disulfide bond to reduce the disulfide bond into free sulfhydryl and form oxidized glutathione by itself.

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 synthesized firstly, 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 controlled through two steps of reactions respectively, 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 long-time dialysis is not needed to remove the activating agent and substances which do not participate in the reaction, no by-product exists, 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 the 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 steps are simple and efficient, and the product yield is high.

In one embodiment, the step of synthesizing the cross-linking agent A is performed by using 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 compound

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) is epoxy resin containing a 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 polycondensation 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 one embodiment, in the step of synthesizing the cross-linking agent a, the ratio of the amounts of the cystine and the 1, 4-butanediol diglycidyl ether is 1:2 to 3, and may be, for example, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.5, 1:2.7, 1:2.9, 1:3, and preferably 1: 2.3. By controlling the amount ratio of cystine to the 1, 4-butanediol diglycidyl ether in the above range, the obtained cross-linking agent A has high purity.

In a specific embodiment, in the step of synthesizing the cross-linking agent a, the synthesis temperature of the cross-linking agent a is 10 to 30 ℃, and for example, may be 10 ℃, 12 ℃, 14 ℃, 16 ℃, 18 ℃, 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃ and the like.

In a specific embodiment, in the preparation method of the present application, in the crosslinking step, the amount ratio of the crosslinking agent a to the sodium hyaluronate repeating units is 0.03 to 0.075:1, and may be, for example, 0.03:1, 0.035:1, 0.04:1, 0.045:1, 0.05:1, 0.055:1, 0.06:1, 0.065:1, 0.07:1, 0.075:1, and the like.

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, in the crosslinking step, the amount ratio of the crosslinking agent B to the sodium hyaluronate repeating units is 0.03 to 0.075:1, for example, 0.03:1, 0.035:1, 0.04:1, 0.045:1, 0.05:1, 0.055:1, 0.06:1, 0.065:1, 0.07:1, 0.075:1, etc.;

in one embodiment, in the preparation method of the present application, the ratio of the amounts of the crosslinking agent a and the crosslinking agent B is 0.5 to 1:1, and may be, for example, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, or the like.

In a specific embodiment, in the crosslinking step, the sodium hyaluronate, which has a molecular weight of 300 to 800kDa, such as 300kDa, 350kDa, 400kDa, 500kDa, 700kDa, 800kDa, etc., is too low in molecular weight of hyaluronic acid, and the gel formed after crosslinking has low mechanical strength and does not play a role of filling, is subjected to a crosslinking reaction with the crosslinking agent a and the crosslinking 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, etc.); 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 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 15 mmol. In this embodiment, the buffer may be a phosphate buffer or the like.

In a specific embodiment, in the step of treating the gel M, the gel M is dialyzed in a buffer solution with 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 a specific embodiment, 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 6-72 h, for example, 6h, 12h, 24h, 48h, 60h, 72h and the like, preferably 24-48 h.

In one embodiment, in the step of preparing the gel M, the gel L is dialyzed in an alkaline or neutral buffer solution containing glutathione, then an ester or amide local anesthetic is added, and the mixture is sieved through a 50-70-mesh sieve, 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 an ester type local anesthetic having a-COO-group in the structure, and examples of the local anesthetic belonging to this type include 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, such as 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, such as 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, such as 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 characterized in that a is 44-298, b is 44-298 and c is 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 the substances of cystine and 1, 4-butanediol diglycidyl ether 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) 939.0g of sodium hyaluronate (with the molecular weight of 800kDa and the mass of the substances of the sodium hyaluronate repeating units of 2.33mol) is dissolved in 100mL of NaOH solution with the mass fraction of 1.5%, the solution is stirred and dissolved to obtain sodium hyaluronate solution, a cross-linking agent A and BDDE are mixed and then added into the sodium hyaluronate solution, and cross-linking reaction is carried out for 8 hours at 30 ℃ to obtain cross-linked gel L, wherein the ratio of the mass of the cross-linking agent A to the mass of the BDDE is 0.5:1, and the ratio of the total mass of the cross-linking agent A and the BDDE to the mass of the substances of the sodium hyaluronate repeating units is 0.045: 1;

(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 a 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 of sodium hyaluronate (the molecular weight is 300kDa, and the amount of substances of a sodium hyaluronate repeating unit is 1.17mol) 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 crosslinked gel L, wherein the ratio of the amounts of the substances of the crosslinking agent A and the BDDE is 1:1, and the ratio of the total amount of the substances of the crosslinking agent A and the BDDE to the amount of the substances of the sodium hyaluronate repeating unit is 0.06: 1;

(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 is mixed with 0.0966mol of 1, 4-butanediol diglycidyl ether in 100mL of 0.1mol/L NaOH solutionStirring and reacting in water bath at 30 ℃ for 8 hours, and separating and purifying to obtain 0.039mol of epoxy cross-linking agent A containing disulfide bonds

Since the ratio of the amounts of cystine to BDDE was 1:2.3, the amount of crosslinker A isolated and purified in this example was higher than that in example 1.

(2) 1047.8g of sodium hyaluronate (the molecular weight is 800kDa, and the amount of substances of a sodium hyaluronate repeating unit is 2.6mol) 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 a moisture reaction is carried out for 8 hours at 55 ℃ to obtain a crosslinked gel L, wherein the amount ratio of the crosslinking agent A to the substances of the BDDE is 0.5:1, and the ratio of the total amount of the crosslinking agent A and the BDDE to the amount of the substances of the hyaluronic acid is 0.045: 1;

(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 ratio of the amounts of cystine to BDDEThe amount of the substance of the crosslinking agent A separated and purified in this example was extremely 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 0.178mol) 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 ratio of the amounts of the substances of the crosslinking agent A and the BDDE is 1:1, and the ratio of the total amount of the substances of the crosslinking agent A and the BDDE to the amount of the substances of the sodium hyaluronate repeating unit is 0.045: 1;

(3) crushing the gel L obtained in the step (2) by using mechanical force, and screening the crushed gel L through a 60-mesh screen to obtain micro-particle gel A;

(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;

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 NaOH solution with the concentration of 0.1mol/L, stirred and reacted for 8 hours in water bath at the temperature of 30 ℃, and separated and purified to obtain 0.035mol of epoxy cross-linking agent A containing disulfide bonds

(2) 939.0g of sodium hyaluronate (molecular weight 800kDa, amount of substance having sodium hyaluronate repeating unit of 2.33mol) was dissolved in 100mL of 1.5% NaOH solution with stirring to obtain sodium hyaluronate solution, and crosslinking agent A and crosslinking agent B were added to the solution

(n-11) and adding the mixture into a sodium hyaluronate solution, and carrying out crosslinking reaction at 55 ℃ for 8 hours to obtain a crosslinked gel L, wherein the mass ratio of the crosslinking agent A to the crosslinking agent B is 0.5:1, and the mass ratio of the total mass of the crosslinking agent A and the crosslinking agent B to the mass of the sodium hyaluronate is 0.045: 1;

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: 1.

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 the substance of the sodium hyaluronate repeating unit is 0.58mol, the ratio of the amount of the substance of the crosslinking agent A to the amount of the substance of BDDE is 2:1, and the ratio of the total amount of the substance of the crosslinking agent A and BDDE to the amount of the substance of the sodium hyaluronate repeating unit is 0.09: 1. 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 crosslinker a to BDDE is 0.4:1 and the ratio of the amount of total substances of crosslinker a and BDDE to the amount of substances of sodium hyaluronate repeating units is 0.21: 1. 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 for complete separation of the two slides was 2 min 13 sec.

Example 9

This example differs from example 7 in that the ratio of the amount of substances of crosslinker a to BDDE is 0.4:1 and the ratio of the total amount of substances of crosslinker a and BDDE to the amount of substances of sodium hyaluronate repeat units is 0.28: 1. 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 to obtain complete separation of the two slides was 2 minutes and 24 seconds.

The parameters and effect data for examples 1-9 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 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 better than examples 1, 3, 4 and 5. In 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 in example 6 and the ratio of the amount of the cross-linking agent B to the amount of the substance of the sodium hyaluronate repeating unit in example 4 are both smaller than those in example 4, so the adhesiveness of example 4 is better than that of example 6. In examples 7 to 9, the ratio of the amount of the crosslinking agent a to the amount of the sodium hyaluronate repeating units in examples 7 to 9 was significantly larger than that in the other examples, and the ratio of the amount of the crosslinking agent B to the amount of the sodium hyaluronate repeating units in examples 8 to 9 was significantly larger than that in the other examples, so that the adhesiveness of examples 7 to 9 was slightly better than that of example 2.

Comparative example 1

(1) 939.0g of sodium hyaluronate (with the molecular weight of 800kDa and the amount of substances of a sodium hyaluronate repeating unit of 2.33mol) is dissolved in 80mL of aqueous solution, after complete dissolution by stirring, sodium hyaluronate solution is obtained, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) which are equal to the amount of substances such as carboxyl in the sodium hyaluronate molecules are added, reaction is activated for 8 hours, and then cystine is added, and the reaction is continuously stirred for 8 hours, so that cross-linked gel is obtained, wherein the ratio of the amount of the substances of the cystine and the sodium hyaluronate repeating unit is 0.035: 1;

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

(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 25 mg/mL;

(4) And (4) sieving the gel particles obtained in the step (3) by a 60-mesh sieve, filling into a syringe, and performing 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 of sodium hyaluronate (molecular weight 800kDa, the amount of the substance of the sodium hyaluronate repeating unit is 2.33mol) is dissolved in 100mL of 1.5% NaOH solution, stirred and dissolved, 0.035mol of cystine and 0.14mol of BDDE are mixed and added into the sodium hyaluronate solution, and crosslinking reaction is carried out for 8 hours at 30 ℃ 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 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 of sodium hyaluronate (molecular weight 800kDa, the amount of the substance having a sodium hyaluronate repeating unit is 2.33mol) was dissolved in 100mL of a 1.5% NaOH solution, and the solution was stirred to dissolve the sodium hyaluronate, 0.07mol of cysteine and 0.14mol of BDDE were mixed and added to the sodium hyaluronate solution, and the mixture was subjected to a crosslinking reaction at 30 ℃ for 8 hours to obtain a sodium hyaluronate gel.

In the process of reaction, BDDE can not only react with sodium hyaluronate, but also react with amino and sulfydryl in cysteine, so that on one hand, the crosslinking degree of the crosslinked gel cannot be determined, and on the other hand, after BDDE reacts with sulfydryl in cysteine, the hyaluronic acid gel with adhesiveness cannot be obtained.

The parametric data for comparative examples 1-4 are summarized in Table 2 below:

TABLE 2

The foregoing is illustrative of the preferred embodiments of the present application and is not to be construed as limiting thereof, since other modifications and equivalents of the disclosed embodiments may be devised by those skilled in the art. 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;

2. The preparation method of claim 1, wherein in the step of synthesizing the cross-linking agent A, cystine and 1, 4-butanediol diglycidyl ether are used as raw materials and are reacted under alkaline conditions to generate the cross-linking agent A with the structural formula

The crosslinking agent A of (1);

3. The method according to claim 1 or 2, wherein the amount ratio of the cross-linking agent A to the sodium hyaluronate repeating units in the cross-linking step is 0.03-0.075: 1.

4. The method according to any one of claims 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 0.03-0.075: 1;

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

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

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

Preferably, in the step of treating the gel M, the dialysis time is 6-72 h.

8. The method according to any one of claims 1 to 7, wherein the gel M is dialyzed and then an anesthetic, preferably an ester or amide local anesthetic, is added to the gel M 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, and c is 65 to 5211;

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