CN113087935B

CN113087935B - Composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis and preparation method thereof

Info

Publication number: CN113087935B
Application number: CN202110543658.7A
Authority: CN
Inventors: 林莎莎; 邹圣灿; 王宝群; 姜晓文; 王君廷
Original assignee: Qingdao Chenland Marine Biotechnology Co ltd
Current assignee: Qingdao Chenland Marine Biotechnology Co ltd
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-05-27
Anticipated expiration: 2041-05-19
Also published as: CN113087935A

Abstract

The invention discloses a compound sodium hyaluronate gel for resisting hyaluronidase hydrolysis and a preparation method thereof, belonging to the technical field of compound sodium hyaluronate gel preparation. The sodium hyaluronate gel has two ways of improving the enzymolysis resistance: firstly, the sodium hyaluronate contains active functional groups and can be combined into a sodium hyaluronate structure in a grafting mode; and secondly, the sodium hyaluronate gel is added into the sodium hyaluronate gel by a direct adding mode. The gel system is constructed by the hyaluronidase activity inhibitor and the sodium hyaluronate, the gel degradation time can be delayed without excessively adding a cross-linking agent, the gel degradation time in vivo is prolonged by inhibiting the hyaluronidase activity, and a new thought is provided for facial injection of gel functional materials.

Description

Composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis and preparation method thereof

Technical Field

The invention belongs to the technical field of preparation of composite sodium hyaluronate gel, and particularly relates to composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis and a preparation method thereof.

Background

Sodium hyaluronate is a natural macromolecular polysaccharide existing in various organ tissues in the body of higher animals, and is an important component for forming tissues such as human intercellular substance, joint synovial fluid and the like. It is composed of glucuronic acid and N-acetylglucosamine, has good biocompatibility, high viscoelasticity, unique molecular structure and physicochemical properties, and shows various important physiological functions in the body.

The injection filling agent is one of the most effective ways for treating facial aging wrinkles and depressions at present, and the sodium hyaluronate products in the injection filling agent are relatively stable products. Sodium hyaluronate can be clinically used for a cosmetic injection product for most wrinkles and depressions, and is an ideal soft tissue filler. The commonly used sodium hyaluronate is basically non-animal source sodium hyaluronate obtained from a bacterial fermentation method, and the filler from the source has very obvious advantages: firstly, the structure of the vaccine is not different in different species, has no immunogenicity, and is not easy to cause immune reaction; secondly, the retention time in tissues after injection is long, and if the injection effect is poor, the hyaluronidase can be dissolved by injection. Sodium hyaluronate shows excellent organism biocompatibility when implanted into human body, and is an ideal natural biological medicine material. Sodium hyaluronate belongs to glycosaminoglycan, is very easy to degrade in vivo, and is often subjected to cross-linking modification in order to prolong the degradation time of the sodium hyaluronate in vivo. The property of the filler is influenced by the degree of crosslinking, the improvement of the degree of crosslinking is a main mode for prolonging the degradation time, a mode of adding a crosslinking agent is often adopted, any residual crosslinking agent cannot be completely removed in the prior art, the biocompatibility is reduced due to excessive crosslinking agent, and immune reaction is easily caused when the crosslinking agent is injected into a human body.

The hyaluronidase and free radicals existing in the skin can cut off the polymerized sodium hyaluronate polymer structure, the degradation of the sodium hyaluronate filling agent in vivo is generally caused by enzyme degradation, and the degradation effect of the free radicals on the sodium hyaluronate structure is relatively small, so how to inhibit the activity of the hyaluronidase becomes an important means for delaying the degradation effect of the sodium hyaluronate gel.

Disclosure of Invention

The invention aims to provide the composite sodium hyaluronate gel for resisting the hydrolysis of hyaluronidase and the preparation method thereof, and the prepared composite sodium hyaluronate gel has the advantages of low cross-linking agent residue and good enzymolysis resisting effect; in addition, the preparation method is simple and effective, and has wide application prospect.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a compound sodium hyaluronate gel for resisting hyaluronidase hydrolysis comprises the following steps:

(1) dissolving hyaluronidase inhibitor in alkali solution and stirring to obtain 1-10% (w/w) solution; adding sodium hyaluronate dry powder to make the concentration of the sodium hyaluronate dry powder be 1-20% (w/w), adding cross-linking agent to make the concentration of the sodium hyaluronate dry powder be 0.1-10% (w/w), uniformly stirring, reacting for 2-10h in an environment with the temperature of 10-90 ℃, and obtaining primary cross-linked gel after the reaction is finished; precipitating with 95% ethanol, repeatedly washing precipitate with water, and drying to obtain primary crosslinked gel powder;

(2) dissolving sodium hyaluronate dry powder in an alkali solution to prepare a solution with the concentration of 10-40% (w/w); then adding the primary crosslinked gel powder prepared in the step (1); stirring uniformly, and reacting for 2-10h at 20-50 ℃; adding a cross-linking agent after the reaction is finished to enable the concentration of the cross-linking agent to be 0.1-10% (w/w), uniformly stirring, carrying out secondary reinforced cross-linking for 2-10h at the temperature of 20-50 ℃, and obtaining composite gel after the cross-linking is finished;

(3) adding purified water with the volume 3 times that of the gel into the composite gel obtained in the step (2) for natural imbibition for 24 hours (the purified water can be replaced in the imbibition process), after the imbibition is finished, sieving the swelled gel into particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate dry powder, adding 0.5-2mL of phosphate buffer solution into 20mg of the composite sodium hyaluronate dry powder, if the buffer solution is not completely absorbed, carrying out suction drying on the residual buffer solution by using filter paper to obtain the composite sodium hyaluronate gel, adding phosphate buffer solution containing 0.05-5% of hyaluronidase inhibitor into the gel, adding 5-20% (w/w) of the phosphate buffer solution containing hyaluronidase inhibitor into the gel (the hyaluronidase inhibitor can effectively inhibit the activity), the compound sodium hyaluronate gel resisting the hydrolysis of hyaluronidase is obtained.

The hyaluronidase inhibitors in the above steps (1) and (2) may be the same or different; the hyaluronidase inhibitor is a substance with active functional groups, can be grafted to a sodium hyaluronate structure, can also be directly added into the prepared gel, and has hyaluronidase inhibition activity. Specifically, it may be selected from at least one of the following:

glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfate, etc.; polysaccharides such as chitosan, β - (1,4) -galacto-oligosaccharide, cellulose sulfate, dextran sulfate, etc.; fatty acids, such as autonomic nerve acid, oleanolic acid; antioxidants such as vitamin C, D-erythorbic acid, catechin, curcumin, tannic acid, gallic acid, procyanidin, etc.; alkaloids, such as aristolochic acid, amalin, reserpine; flavonoids such as flavone, quercetin, apigenin, luteolin, hesperidin, rutin, etc.

The grafting reaction sites of the sodium hyaluronate can occur on three groups of hydroxyl, carboxyl and N-acetamido, and the reaction of the hydroxyl is mainly divided into etherification reaction, esterification reaction and the like; the esterification reaction means that sodium hyaluronate can react with acyl chloride or acyl anhydride to generate lipid compounds, for example, acetic anhydride reacts with sodium hyaluronate. Carboxyl crosslinking often uses carbodiimides and requires reaction under acidic conditions, which can result in protonation of a portion of the amine and reduced nucleophilicity leading to incomplete reaction. Crosslinking is preferably carried out using a crosslinking agent reactive with hydroxyl groups, for example, epoxy compounds such as 1, 4-butanediol diglycidyl ether (BDDE) and dihexyl sulfone (DVS).

In the method for preparing the composite sodium hyaluronate gel resistant to hyaluronidase hydrolysis, the alkali solution in the step (1) and the step (2) is NaOH solution, and the pH range is 7-13, preferably 9-11.

Preferably, the mass ratio of the primary cross-linked gel powder to the sodium hyaluronate dry powder in the step (2) is 0.1-5: 10.

Preferably, the reaction temperature in the step (1) is 30-50 ℃; the reaction time is 3-5 h; the concentration of the hyaluronidase inhibitor is 2-5% (w/w).

Preferably, the reaction temperature in the step (2) is 30-40 ℃; the reaction time is 4-6 h.

The composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis prepared by the method.

The composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis is applied to preparation of medical filling materials.

The technical scheme of the invention has the advantages

The sodium hyaluronate gel has two ways of improving the enzymolysis resistance: firstly, the sodium hyaluronate contains active functional groups and can be combined into a sodium hyaluronate structure in a grafting mode; and secondly, the sodium hyaluronate gel is added into the sodium hyaluronate gel by a direct adding mode. The gel system is constructed by the hyaluronidase activity inhibitor and the sodium hyaluronate, the gel degradation time can be delayed without excessively adding a cross-linking agent, the gel degradation time in vivo is prolonged by inhibiting the hyaluronidase activity, and a new thought is provided for facial injection of gel functional materials.

(1) The hyaluronidase inhibitor is introduced in the preparation process, and the hyaluronidase activity inhibitor has obvious effect of inhibiting enzyme degradation. Wherein the chondroitin sulfate and other substances not only have the functions of inhibiting the activity of hyaluronidase, but also have the functions of resisting bacteria and inflammation and accelerating wound healing so as to relieve the adverse reaction of an injection part. The gel containing chondroitin sulfate is similar to natural extracellular matrix, and the biocompatibility of the gel is improved.

(2) The crosslinking degree of the sodium hyaluronate gel is improved by secondary strengthening crosslinking, and the crosslinking agent which does not react completely in the primary crosslinking can continue to react in the secondary strengthening crosslinking, so that the residue of the crosslinking agent is reduced, the enzymolysis resistance of the gel can be improved, and the immune reaction after injection can be reduced.

(3) After redissolving, the hyaluronidase activity inhibitor is directly added to strengthen the enzymolysis resistance of the gel, obviously improve the retention time of the injection gel in vivo and strengthen the treatment effect.

Drawings

FIG. 1: example 2 step preparation samples were tested for crosslinker BDDE residue at different number of ethanol washes.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

A chondroitin sulfate-sodium hyaluronate composite gel for resisting hyaluronidase hydrolysis is specifically prepared by the following steps:

1g chondroitin sulfate (molecular weight 10kDa) is weighed, dissolved in 50mL NaOH solution (pH 11) and stirred continuously, 5g sodium hyaluronate dry powder (molecular weight 800kDa) is added, and 1mL BDDE is added and stirred uniformly. Reacting for 4 hours in an environment with the temperature of 37 ℃, and obtaining the primary cross-linked gel after the reaction is finished. Precipitating the gel with 95% ethanol, repeatedly washing the precipitate with purified water, and drying to obtain gel powder.

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) and dissolving in 50mL of NaOH solution (pH is 11), then adding 2g of primary cross-linked gel dry powder, stirring uniformly, and reacting for 5h in an environment at 37 ℃. And after the reaction is finished, adding 1mL of BDDE, uniformly stirring, performing secondary reinforced crosslinking for 5 hours at the temperature of 37 ℃, and obtaining the composite gel after the crosslinking is finished. Adding purified water with the volume 3 times of that of the gel into the composite gel for natural imbibition for 24 hours, screening the swollen gel into small particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate gel dry powder, adding 20mg of the composite sodium hyaluronate gel dry powder into 1mL of phosphate buffer solution for dissolution and absorption, and if the buffer solution is not completely absorbed, carrying out suction drying on the rest buffer solution by using filter paper to obtain the composite sodium hyaluronate gel; adding phosphate buffer solution containing 2% of chondroitin sulfate, wherein the adding amount is 8% (w/w) of the composite sodium hyaluronate gel, thus obtaining the chondroitin sulfate-sodium hyaluronate composite gel product resisting hyaluronidase hydrolysis, and numbering sample 1.

Chondroitin sulfate has high biocompatibility, also has the effects of resisting bacteria and inflammation and accelerating wound healing, and can slow down inflammation when sodium hyaluronate gel is injected into skin. The chondroitin sulfate has the function of inhibiting the activity of the hyaluronidase, and the enzymolysis resistance of the sodium hyaluronate gel is improved, so that the degradation period of the sodium hyaluronate in vivo is prolonged. Research shows that the hydrogel containing chondroitin sulfate is similar to natural extracellular matrix and can effectively improve the biocompatibility of the gel, so that the gel containing chondroitin sulfate is suitable for the biomedical field.

Example 2

2g of chondroitin sulfate (molecular weight 10kDa) is weighed out, dissolved in 50mL of NaOH solution (pH 11) and stirred continuously, 5g of sodium hyaluronate dry powder (molecular weight 800kDa) is added, and 1mL of BDDE is added and stirred uniformly. Reacting for 4 hours in an environment with the temperature of 37 ℃, and obtaining the primary cross-linked gel after the reaction is finished. Precipitating the gel with 95% ethanol, repeatedly washing precipitate with purified water, and drying to obtain gel powder.

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) and dissolving in 50mL of NaOH solution (pH is 11), then adding 2g of primary cross-linked gel dry powder, stirring uniformly, and reacting for 5h in an environment at 37 ℃. And after the reaction is finished, adding 1mL of BDDE, uniformly stirring, carrying out secondary reinforced crosslinking, wherein the crosslinking time is 5h, the temperature is 37 ℃, and obtaining the composite gel after the crosslinking is finished. Adding purified water with the volume 3 times of that of the gel into the composite gel for natural imbibition for 24 hours, screening the swollen gel into small particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate gel dry powder, adding 20mg of the composite sodium hyaluronate gel dry powder into 1mL of phosphate buffer solution for dissolution, and removing excessive water on the surface of the gel by using filter paper to obtain the composite sodium hyaluronate gel; adding phosphate buffer solution containing 2% chondroitin sulfate, wherein the adding amount is 8% (w/w) of the transparent composite sodium hyaluronate gel, thus obtaining the chondroitin sulfate-sodium hyaluronate composite gel product resisting hyaluronidase hydrolysis, and numbering sample 2.

Example 3

2g of chondroitin sulfate (molecular weight 10kDa) is weighed out, dissolved in 50mL of NaOH solution (pH 11) and stirred continuously, 5g of sodium hyaluronate dry powder (molecular weight 800kDa) is added, and 1mL of BDDE is added and stirred uniformly. Reacting for 4 hours in an environment with the temperature of 37 ℃, and obtaining the primary cross-linked gel after the reaction is finished. Precipitating the gel with 95% ethanol, repeatedly washing the precipitate with purified water, and drying to obtain gel powder.

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) and dissolving in 50mL of NaOH solution (pH is 11), then adding 2g of primary cross-linked gel dry powder, stirring uniformly, and reacting for 5h in an environment at 37 ℃. And after the reaction is finished, adding 1mL of BDDE, uniformly stirring, carrying out secondary reinforced crosslinking, wherein the crosslinking time is 5h, the temperature is 37 ℃, and obtaining the composite gel after the crosslinking is finished. Adding purified water with the volume 3 times that of the gel into the composite gel for natural imbibition for 24 hours, screening the swollen gel into small particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate dry powder, adding 1mL of phosphate buffer solution into 20mg of the composite sodium hyaluronate dry powder for dissolution, removing excessive water on the surface of the gel by using filter paper to obtain a chondroitin sulfate-sodium hyaluronate composite gel product resisting hyaluronidase hydrolysis, and numbering a sample 3.

Example 4

A chitosan-sodium hyaluronate composite gel for resisting hyaluronidase hydrolysis is specifically prepared by the following steps:

1.8g of chitosan (molecular weight 50kDa) was weighed out, dissolved in 50mL of NaOH solution (pH 11) with constant stirring, and 5g of sodium hyaluronate dry powder (molecular weight 800kDa) was added to 1mL of BDDE and stirred uniformly. Reacting for 4 hours in an environment with the temperature of 37 ℃, and obtaining the primary cross-linked gel after the reaction is finished. Precipitating the gel with 95% ethanol, repeatedly washing the precipitate with purified water, and drying to obtain gel powder.

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) and dissolving in 50mL of NaOH solution (pH is 11), then adding 2g of primary cross-linked gel dry powder, stirring uniformly, and reacting for 5h in an environment at 37 ℃. And after the reaction is finished, adding 1mL of BDDE, uniformly stirring, performing secondary reinforced crosslinking, wherein the crosslinking time is 5h, the temperature is 37 ℃, and obtaining the composite gel after the crosslinking is finished. Adding purified water with the volume 3 times of that of the gel into the composite gel for natural imbibition for 24 hours, screening the swollen gel into small particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate dry powder, adding 20mg of the sodium hyaluronate dry powder into 1mL of phosphate buffer solution for dissolution and absorption, removing redundant water on the surface of the gel by using filter paper to obtain composite sodium hyaluronate gel, adding the phosphate buffer solution containing 2% of chitosan into the composite gel, wherein the adding amount is 8% (w/w) of the composite sodium hyaluronate gel, thus obtaining a chitosan-sodium hyaluronate composite gel product resisting hyaluronidase hydrolysis, and numbering a sample 4.

Chitosan is a deacetylated derivative of chitin, which mediates a variety of biological effects, including anti-angiogenic, antimicrobial and immune enhancing activities, among others. Inhibition of hyaluronidase by chitosan-NH derived from chitosan³⁺-COO of functional groups with sodium hyaluronate^-Electrostatic interactions between the groups occur, and the formation of complexes limits the substrate utilization of the enzyme.

Example 5

1.8g of chitosan (molecular weight 10kDa) was weighed out, dissolved in 50mL of NaOH solution (pH 11) with constant stirring, and 5g of sodium hyaluronate dry powder (molecular weight 800kDa) was added followed by 1mL of BDDE and stirred uniformly. Reacting for 4 hours in an environment with the temperature of 37 ℃, and obtaining the primary cross-linked gel after the reaction is finished. Precipitating the gel with 95% ethanol, repeatedly washing the precipitate with purified water, and drying to obtain gel powder.

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) and dissolving in 50mL of NaOH solution (pH is 11), then adding 2g of primary cross-linked gel dry powder, stirring uniformly, and reacting for 5h in an environment at 37 ℃. And after the reaction is finished, adding 1mL of BDDE, uniformly stirring, performing secondary reinforced crosslinking, wherein the crosslinking time is 5h, the temperature is 37 ℃, and obtaining the composite gel after the crosslinking is finished. Adding purified water with the volume 3 times that of the composite gel into the composite gel for natural swelling for 24 hours, screening the swollen gel into small particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate dry powder, adding 20mg of the composite sodium hyaluronate dry powder into 1mL of phosphate buffer solution for dissolving, removing redundant water on the surface of the gel by using filter paper, adding phosphate buffer solution containing 2% chondroitin sulfate, and adding the phosphate buffer solution in an amount of 8% (w/w) of the composite sodium hyaluronate gel to obtain a chitosan-sodium hyaluronate composite gel product resisting hyaluronidase hydrolysis, and numbering a sample 5.

Comparative example 1

5g of sodium hyaluronate dry powder (molecular weight 800kDa) is dissolved in 50mL of NaOH solution (pH 11) and stirred uniformly, 1mL of BDDE is added, and the mixture is reacted for 4 hours in an environment with the temperature of 37 ℃ to obtain primary cross-linked gel after the reaction is finished. Precipitating the gel with 95% ethanol, repeatedly washing the precipitate with purified water, and drying to obtain gel powder.

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) and dissolving in 50mL of NaOH solution (pH is 11), then adding 2g of primary cross-linked gel dry powder, stirring uniformly, and reacting for 5h in an environment at 37 ℃. And after the reaction is finished, adding 1mL of BDDE, uniformly stirring, carrying out secondary reinforced crosslinking, wherein the crosslinking time is 5h, the temperature is 37 ℃, and obtaining the composite gel after the crosslinking is finished. Adding purified water with the volume 3 times that of the gel into the gel for natural swelling for 24 hours, drying the swollen gel by using a screen to divide the swollen gel into particles with uniform particle size, carrying out alcohol precipitation, washing and drying to obtain dry powder, adding 20mg of the dry powder into 1mL of phosphate buffer solution for dissolving and absorbing, removing excessive water on the surface of the gel by using filter paper to obtain composite gel, and numbering a comparative sample 1.

Comparative example 2

Weighing 10g of sodium hyaluronate dry powder (with the molecular weight of 1500kDa) to be dissolved in 50mL of NaOH solution (with the pH value of 11), uniformly stirring, adding 1mL of BDDE, stirring, and obtaining gel after crosslinking at the temperature of 37 ℃ for 5 hours. Adding purified water with the volume 3 times that of the gel into the gel for natural swelling for 24 hours, screening the swollen gel into particles with uniform particle size by using a screen, carrying out alcohol precipitation, washing and drying to obtain dry powder, adding 20mg of the dry powder into 1mL of phosphate buffer solution for dissolving, removing excessive water on the surface of the gel by using filter paper to obtain the composite gel, and numbering a comparative sample 2.

Example 6

Samples 1-5 and

comparative samples

1 and 2 were added to the phosphate buffer of hyaluronidase (200U/mL), and samples were taken after degradation at 37 ℃ for 2, 4, 8, and 24 hours, respectively, to determine the glucuronic acid content, and the degradation rate results were calculated as shown in Table 1:

TABLE 1

According to the results in table 1, the sample prepared in example 2 has better enzymolysis resistance than that of example 1, the addition of chondroitin sulfate in the primary crosslinking process can effectively prolong the gel degradation time, and the chondroitin sulfate is grafted to the gel structure to have enzymolysis resistance; in example 3, after redissolution, chondroitin sulfate is not added, and the resistance to enzymolysis is weak compared with that in example 2, which shows that the chondroitin sulfate directly added into gel has a good effect of inhibiting enzyme activity; in example 4, chitosan was added as an enzyme activity inhibitor, and the results showed that the effect was not much different compared to chondroitin sulfate; example 5 chitosan was added to the primary crosslink, and chondroitin sulfate was added after reconstitution, and the enzyme inhibitory effect was similar to that of example 4, and the addition of the hyaluronidase inhibitor in both steps had the effect of inhibiting the enzyme activity.

Example 7

Taking samples 1-5 and

comparative samples

1 and 2 to determine the content of glucuronic acid, and calculating to obtain the content of sodium hyaluronate in the gel; the swelling degree parameter is a quantitative index for evaluating the gel structure, when the gel is dried to constant weight at 80 ℃, water is dripped until the gel swells, and the dried mass m1 and the swollen mass m2 are respectively measured. The gel swell was calculated according to the following formula:

q-swelling degree of the cross-linked sodium hyaluronate gel;

m 1-mass of crosslinked sodium hyaluronate gel after drying;

m 2-mass of crosslinked sodium hyaluronate gel after swelling.

TABLE 2

As is clear from table 2, the content of sodium hyaluronate in sample 1 is higher than that in sample 2 because chondroitin sulfate participating in the reaction of sample 1 is small, and the amount of sodium hyaluronate participating in the reaction is large in the presence of the same crosslinking agent, so the content of sodium hyaluronate measured is slightly high. Overall, samples 2-5 showed comparable sodium hyaluronate content, and the comparative sample was lower. The swelling degree is a quantitative index for evaluating the gel structure, the crosslinking degree is high under general conditions, the gel swelling degree with a compact structure is low, and the result shows that the swelling degree of the sample 1 is higher, the equivalent amount of the hyaluronidase activity inhibitor is added in the initial crosslinking process of the samples 2-5, the structure is relatively compact, the hyaluronidase activity inhibitor is not easily degraded by the hyaluronidase in vivo, and the swelling degree of a contrast sample is very high.

Example 8

Enzyme inhibitor-sodium hyaluronate complex gel crosslinker residue assay experiment:

samples were prepared as in example 2 and the residual amount of the crosslinking agent BDDE after various ethanol washes was determined. FIG. 1 shows that after 6 washes, the crosslinker residue is less than 2ppm, which meets the requirements associated with crosslinked sodium hyaluronate gel for plastic surgery.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of a compound sodium hyaluronate gel for resisting hyaluronidase hydrolysis is characterized by comprising the following steps:

(1) dissolving a hyaluronidase inhibitor in an alkali solution and continuously stirring to prepare a solution with the mass concentration of 1-20%; adding sodium hyaluronate dry powder to make its mass concentration be 1-10%, adding cross-linking agent to make its mass concentration be 0.1-10%, uniformly stirring, making reaction in the environment of 10-90 deg.C for 2-10 hr, after the reaction is completed, obtaining primary cross-linked gel; precipitating with 95% ethanol, repeatedly washing precipitate with purified water, and drying to obtain primary crosslinked gel powder;

(2) dissolving sodium hyaluronate dry powder in an alkali solution to prepare a solution with the mass concentration of 10-40%; then adding the primary crosslinked gel powder prepared in the step (1); stirring uniformly, and reacting for 2-10h at 20-50 ℃; adding a cross-linking agent after the reaction is finished, wherein the mass concentration of the cross-linking agent is 0.1-10%, uniformly stirring, carrying out secondary reinforced cross-linking, wherein the cross-linking time is 2-10h, the temperature is 20-50 ℃, and obtaining a composite gel after the cross-linking is finished;

(3) adding pure water with the volume 3 times of that of the gel into the composite gel obtained in the step (2) for natural imbibition for 24 hours, after imbibition, screening the swollen gel into small particles by using a screen, carrying out alcohol precipitation, washing and drying to obtain composite sodium hyaluronate dry powder, adding 0.5-2mL of phosphate buffer solution into 20mg of the composite sodium hyaluronate dry powder, and adding phosphate buffer solution containing 0.05-5% of hyaluronidase inhibitor to obtain the composite sodium hyaluronate gel for resisting hyaluronidase hydrolysis, wherein the addition amount of the phosphate buffer solution containing the hyaluronidase inhibitor is 5-20% of that of the composite sodium hyaluronate gel;

in the steps (1) and (2), the cross-linking agent is one of 1, 4-butanediol diglycidyl ether and divinyl sulfone.

2. The method for preparing the complex sodium hyaluronate gel resistant to hyaluronidase hydrolysis according to claim 1, wherein the hyaluronidase inhibitor is at least one of polysaccharides, fatty acids, antioxidants, and alkaloids.

3. The method of preparing a complex sodium hyaluronate gel resistant to hydrolysis by hyaluronidase according to claim 2 wherein said hyaluronidase inhibitor is: at least one of heparin, heparan sulfate, chondroitin sulfate, chitosan, beta- (1,4) -galacto-oligosaccharide, cellulose sulfate, dextran sulfate, vegetable nervonic acid, oleanolic acid, vitamin C, D-isoascorbic acid, catechin, curcumin, tannic acid, gallic acid, procyanidin, aristolochic acid, amalin, reserpine, flavone, quercetin, apigenin, luteolin, hesperidin and rutin.

4. The method for preparing the complex sodium hyaluronate gel resistant to hyaluronidase hydrolysis according to claim 1, wherein the alkali solution in the step (1) and the step (2) is NaOH solution, and the pH range is 7-13.

5. The method for preparing the composite sodium hyaluronate gel resisting hyaluronidase hydrolysis according to claim 1, wherein the mass ratio of the primary crosslinked gel powder to the sodium hyaluronate dry powder in the step (2) is 0.1-5: 10.

6. A complex sodium hyaluronate gel resistant to hydrolysis by hyaluronidase prepared by the method of any one of claims 1 to 5.

7. Use of the complex sodium hyaluronate gel resistant to hydrolysis by hyaluronidase according to claim 6 for the preparation of a medical filling material.