Crosslinked sodium hyaluronate and preparation method thereof

Abstract

The invention discloses cross-linked sodium hyaluronate and a preparation method thereof, and belongs to the technical field of sodium hyaluronate, the sodium hyaluronate adopts basic amino acid as a cross-linking agent, and before cross-linking reaction, a pre-cross-linking monomer is added for pre-cross-linking treatment, and the pre-cross-linking monomer contains branched lysine oligopeptide. Compared with other cross-linking agents, the alkaline amino acid has higher safety and biocompatibility, and potential safety hazards caused by residues of the cross-linking agent can be greatly reduced. In the pre-crosslinking process, a large number of reaction active sites can be additionally provided firstly, so that crosslinking points of sodium hyaluronate are improved, a crosslinked network structure which is complex and stable in structure is constructed, the compatibility between branched lysine oligopeptide and basic amino acid is good, the distance between the crosslinking points can be increased through the branched structure, and the crosslinking effect of sodium hyaluronate is improved. The cross-linked sodium hyaluronate has the advantages that the accessibility of hyaluronidase to cross-linking points is influenced, enzyme recognition sites are protected, the enzymatic degradation rate is reduced, and the enzymolysis resistance of the cross-linked sodium hyaluronate is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of sodium hyaluronate, and in particular relates to cross-linked sodium hyaluronate and a preparation method thereof. Background Art

[0002] Hyaluronic acid is a natural polysaccharide with a huge molecular weight. It exhibits unique viscoelasticity, excellent biocompatibility and degradability. It is an ideal tissue engineering material and is used in different tissue engineering fields. However, due to the poor stability of natural hyaluronic acid hydrogels, their sensitivity to hyaluronidase and free radicals, their short retention time in the body, and their poor mechanical strength, their applications are greatly limited. To this end, people chemically modify or cross-link natural hyaluronic acid to enhance the mechanical strength, stability and anti-degradation properties of the gel, so that it can be maintained in the body for a longer time, thereby expanding its application in the fields of biomedicine and tissue engineering.

[0003] The cross-linked hyaluronic acid is prepared by using one or more combined chemical cross-linking agents, utilizing the reaction between the functional groups of the cross-linking agent itself and the related polar groups on the hyaluronic acid to cross-link the hyaluronic acid molecules together, thereby increasing the molecular weight of the hyaluronic acid gel. On the basis of retaining the original biocompatibility, a cross-linked hyaluronic acid gel with a more complex molecular structure, strong viscoelasticity and significantly improved mechanical properties is obtained.

[0004] However, traditional hyaluronic acid cross-linking agents are mostly chemical reagents, which are irritating to the skin and even cytotoxic. The residues left during the cross-linking process also pose great potential hazards to users. Reducing the amount of cross-linking agent used will result in the cross-linked hyaluronic acid having poor high temperature resistance and resistance to enzymatic hydrolysis, limiting its use. Therefore, using safe and non-toxic natural cross-linking agents is an effective method. However, the cross-linked hyaluronic acid obtained by some current natural biological cross-linking agents has average mechanical strength, low degree of cross-linking, and its resistance to enzymatic hydrolysis also needs to be improved. Summary of the invention

[0005] The object of the present invention is to provide a cross-linked sodium hyaluronate and a preparation method thereof, so as to solve the problem that the cross-linked sodium hyaluronate obtained by using a natural cross-linking agent has a low degree of cross-linking and poor anti-enzymatic performance.

[0006] The purpose of the present invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing cross-linked sodium hyaluronate, comprising the following process steps: S1. adding sodium hyaluronate to an alkaline aqueous solution and stirring to dissolve to obtain a hyaluronic acid aqueous solution; S2. adding a pre-crosslinking monomer to the hyaluronic acid aqueous solution, stirring and reacting at room temperature for 1 to 3 hours to obtain a pre-reaction solution; S3. Add a crosslinking agent to the pre-reaction solution, adjust the pH value of the solution to 8 to 10, and perform a crosslinking reaction at 20 to 40° C. for 4 to 8 hours; S4. After the cross-linking reaction is completed, the cross-linked sodium hyaluronate is obtained by dialysis and freeze-drying; The pre-crosslinked monomer contains branched lysine short peptides; the crosslinking agent is a basic amino acid.

[0007] Preferably, the added amount of the pre-crosslinking monomer is 0.5 to 1.5% of the mass of sodium hyaluronate.

[0008] Preferably, the molar ratio of the cross-linking agent to sodium hyaluronate is (0.3-0.5):1.

[0009] By adopting the above technical scheme, the present invention adopts basic amino acids as cross-linking agents for hyaluronic acid. The amino groups and carboxyl groups contained in the basic amino acids can react and link with the functional groups in the hyaluronic acid to help form a cross-linked structure. The side chain functional groups can also provide additional reaction sites, thereby increasing the diversity of the cross-linking process and achieving cross-linking reactions under milder conditions. Cross-linked sodium hyaluronate with specific structures and properties can be obtained. Compared with general natural cross-linking agents, basic amino acids as cross-linking agents have better biocompatibility and lower immunogenicity. Moreover, amino acids themselves, as the basic units that constitute proteins, are safer under conditions involving human health, and can greatly reduce the potential safety hazards caused by cross-linking agent residues.

[0010] Moreover, before cross-linking, the present invention first pre-cross-links the hyaluronic acid, which can help build a relatively solid basic structure before cross-linking the basic amino acid and the hyaluronic acid, so that the three-dimensional network structure formed in the subsequent cross-linking process can be more stable, and the cross-linking degree of the cross-linked sodium hyaluronate can be improved. It can also better adjust the cross-linking density and enhance the ability of the cross-linked hyaluronic acid to fight free radicals and enzymes in the body, thereby extending the effective duration.

[0011] The pre-cross-linking monomer used in the pre-cross-linking process contains a branched lysine short peptide. The multiple reactive sites contained in the branched lysine short peptide can form covalent bond connection points with the sodium hyaluronate molecules, and each branch of the branched lysine short peptide can react as an independent active site, thereby increasing the number of cross-linking points in the cross-linking structure, thereby constructing a more complex and stable three-dimensional network structure, increasing the degree of cross-linking of subsequent cross-linking, and improving the mechanical strength and elasticity of the cross-linked sodium hyaluronate obtained. In addition, the branched lysine short peptide has good biocompatibility and does not leave toxic residues.

[0012] Its branched structure can also increase the distance between cross-linking points, and by changing the spatial conformation of the cross-linking network, it affects the accessibility of hyaluronidase to the cross-linking points, thereby slowing down the rate of hyaluronidase degradation and improving the ability of cross-linked sodium hyaluronate to resist enzymatic degradation. At the same time, it also reduces the proportion of small molecular weight fragments in the original sodium hyaluronate, thereby reducing the potential risk of inflammation caused by small molecular weight sodium hyaluronate residues.

[0013] Preferably, the raw materials of the pre-crosslinking monomer include a polyamine compound and di-tert-butoxy acyl-protected L-lysine in a molar ratio of (0.8-1):1.

[0014] Preferably, the polyamine compound is one or a combination of two of a diamine compound and a triamine compound.

[0015] More preferably, the diamine compound includes a combination of one or more of ethylenediamine, bio-based pentamethylenediamine, 1,2-propylenediamine and triethylenediamine.

[0016] More preferably, the triamine compound includes one or a combination of diethylenetriamine and triethylamine.

[0017] Preferably, the pre-crosslinking monomer is prepared according to the following method: Add di-tert-butoxyacyl protected L-lysine to a chloroform solution, adjust the temperature to 0-5°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir and mix for 1-2 hours to obtain a premixed solution; add a polyamine compound to the premixed solution, continue to stir and react at 0-5°C for 15-20 hours, and finally obtain a pre-crosslinked monomer through filtering, extraction and drying.

[0018] Preferably, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to di-tert-butoxyacyl-protected L-lysine is (0.6-0.8):1.

[0019] By adopting the above technical solution and selecting L-lysine protected by di-tert-butoxy acyl group, the chemical stability of lysine can be improved and the influence of reaction on the performance of lysine can be reduced. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is a carbodiimide condensation agent, which can help form lysine short peptides, react with the carboxyl group in L-lysine to form an acylurea intermediate that can further react with the amino group, and then further react with the amino group of the polyamine compound to form a pre-crosslinking monomer.

[0020] Pre-crosslinking the pre-crosslinked monomer containing branched lysine short peptides with sodium hyaluronate can effectively enhance the crosslinking density and uniformity between sodium hyaluronate molecules, help to form a stable crosslinked structure later, and first obtain the framework structure of crosslinked sodium hyaluronate, and then use basic amino acids for crosslinking, thereby improving the mechanical strength and stability of the crosslinked sodium hyaluronate obtained. Moreover, through the pre-crosslinking effect of the pre-crosslinked monomer, the enzyme recognition site can be protected, the rate of enzymatic degradation can be reduced, and the shelf life of the crosslinked sodium hyaluronate can be extended.

[0021] Preferably, the basic amino acids include one or a combination of lysine and arginine.

[0022] By adopting the above technical scheme, basic amino acids including lysine, arginine or a combination of the two can form electrostatic interactions with sodium hyaluronate, and the specificity of their side chains further enhances the cross-linking effect with sodium hyaluronate. The polar groups contained in the basic amino acids can form a stable structure with sodium hyaluronate, and can also form ionic bonds with the carboxyl groups in the hyaluronic acid molecules, thereby promoting the cross-linking reaction.

[0023] Basic amino acids have better biocompatibility. Using basic amino acids as cross-linking agents for sodium hyaluronate can better improve the safety of the product. However, directly using basic amino acids for cross-linking will make the properties of the resulting product unstable, especially in the solution state. Heating or long-term exposure to the outside may cause structural damage, thereby affecting the rheological properties and viscosity of the product.

[0024] The present invention adopts the form of pre-crosslinking, first pre-crosslinking sodium hyaluronate, and first constructs the obtained sodium hyaluronate cross-linked framework structure, which is not only conducive to the cross-linking effect of basic amino acids, but also can greatly improve the stability of the product. The pre-crosslinking step also introduces new cross-linking sites for sodium hyaluronate, increases the number of cross-linking points, and makes the obtained cross-linked network more compact and firm, thereby improving the mechanical strength and elastic modulus of the material. In addition, the additional cross-linking points also increase the cross-linking degree of the product, thereby effectively preventing excessive flow, and because the pre-cross-linked monomer contains branched lysine short peptides, it can retain a certain hydration state on this basis, ensuring the good injectability and plasticity of the cross-linked sodium hyaluronate.

[0025] Preferably, step S2 further comprises adding fullerol; the amount of fullerol added is 0.2 to 0.8% of the mass of sodium hyaluronate.

[0026] By adopting the above technical solution, fullerol is a fullerene derivative modified with alcohol hydroxyl group, which has better water solubility and lower cytotoxicity, and adding it to cross-linked sodium hyaluronate will not affect its safety. Most importantly, fullerol has the ability to effectively scavenge free radicals. During the pre-cross-linking process, a large number of hydroxyl functional groups in fullerol can form chemical bonds with sodium hyaluronate and pre-cross-linked monomers. The resulting cross-linked sodium hyaluronate can effectively resist the decomposition of free radicals, thereby greatly enhancing the long-term stability of cross-linked sodium hyaluronate, and can also help cross-linked sodium hyaluronate absorb and lock in moisture, forming a protective film to prevent moisture loss, and obtaining cross-linked sodium hyaluronate with superior performance.

[0027] Moreover, in the pre-cross-linking step, the abundant hydroxyl groups on the surface of fullerol can strengthen the cross-linked framework structure formed by hydrogen bonds and interactions with the branched lysine short peptides in the pre-cross-linking monomers and the polar groups in sodium hyaluronate, thereby improving the mechanical strength of the cross-linked sodium hyaluronate obtained. It can also cooperate with the pre-cross-linking monomers to adjust the fluidity and viscosity of the product, expanding the application scenarios of cross-linked sodium hyaluronate.

[0028] Beneficial effects of the present invention: 1. The present invention uses basic amino acids as cross-linking agents to better improve the safety of cross-linked sodium hyaluronate. Compared with general natural cross-linking agents, it has better biocompatibility and lower immunogenicity, and greatly reduces the safety hazards caused by cross-linking agent residues.

[0029] 2. The present invention pre-crosslinks sodium hyaluronate before the crosslinking reaction, wherein the pre-crosslinked monomer contains a branched lysine short peptide, wherein the multiple reaction sites and branching sites contained therein can help improve the crosslinking points of sodium hyaluronate, and then a more complex and stable three-dimensional network structure is constructed under the crosslinking action of basic amino acids, thereby solving the problems of unstable properties and easy destruction of product structure that may occur when crosslinking with basic amino acids alone, and can also improve the crosslinking degree and mechanical strength of the material. Its branched structure can also increase the distance between the crosslinking points, affect the accessibility of hyaluronidase to the crosslinking points, protect the enzyme recognition site, reduce the rate of enzymatic degradation, and improve the anti-enzymatic ability of crosslinked sodium hyaluronate.

[0030] 3. In the present invention, fullerol can also be added in the pre-crosslinking step, which can, on the one hand, strengthen the cross-linked network structure obtained after the pre-crosslinking treatment, and on the other hand, effectively remove free radicals, thereby effectively resisting the decomposition effect of free radicals on cross-linked sodium hyaluronate and improving the stability of cross-linked sodium hyaluronate. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Preparation Example Preparation Example 1: A pre-crosslinking monomer is prepared according to the following method: Add 0.1 mol of di-tert-butoxyacyl-protected L-lysine to 200 mL of chloroform solution, stir and mix, adjust the temperature to 0°C, add 0.7 mol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir and mix for 1 hour to obtain a premixed solution; 0.9 mol of bio-based pentamethylenediamine was added to the premixed solution, and the reaction was continued at 0° C. with stirring for 15 hours. Finally, the pre-crosslinked monomer was obtained by filtering, extracting, and drying.

[0033] Preparation Example 2, a pre-crosslinked monomer, is different from Preparation Example 1 only in that the added amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.6 mol; the added amount of bio-based pentamethylenediamine is 0.8 mol.

[0034] Preparation Example 3, a pre-crosslinked monomer, is different from Preparation Example 1 only in that the added amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.8 mol; the added amount of bio-based pentamethylenediamine is 1 mol.

[0035] Preparation Example 4, a pre-crosslinking monomer, prepared according to the following method: Add 0.1 mol of di-tert-butoxyacyl-protected L-lysine to 200 mL of chloroform solution, stir and mix, adjust the temperature to 0°C, add 0.7 mol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir and mix for 2 hours, filter, extract and dry to obtain a pre-crosslinked monomer.

[0036] Example Example 1, a cross-linked sodium hyaluronate, is prepared according to the following process steps: S1. adding 0.1 mol of sodium hyaluronate (molecular weight of 300,000 to 1,000,000 Daltons) into a 20% by mass sodium hydroxide aqueous solution and stirring to dissolve to obtain a hyaluronic acid aqueous solution; S2. Add the pre-crosslinked monomer prepared in Preparation Example 1 to the hyaluronic acid aqueous solution, wherein the amount of the pre-crosslinked monomer added is 1% of the mass of sodium hyaluronate, and stir the reaction at room temperature for 2 hours to obtain a pre-reaction solution; S3. 0.04 mol of lysine was added to the pre-reaction solution, the pH value of the solution was adjusted to 8, and the cross-linking reaction was carried out at 30° C. for 6 h; S4. After the above cross-linking reaction is completed, cross-linked sodium hyaluronate is obtained through dialysis and freeze-drying.

[0037] Example 2, a cross-linked sodium hyaluronate, is different from Example 1 only in that the added amount of the pre-cross-linked monomer prepared in Preparation Example 1 is 0.5% of the mass of the sodium hyaluronate.

[0038] Example 3, a cross-linked sodium hyaluronate, is different from Example 1 only in that the added amount of the pre-cross-linked monomer prepared in Preparation Example 1 is 1.5% of the mass of the sodium hyaluronate.

[0039] Example 4, a cross-linked sodium hyaluronate, is different from Example 1 only in that the pre-cross-linked monomer prepared in Preparation Example 1 is replaced by an equal amount of the pre-cross-linked monomer prepared in Preparation Example 2; and the amount of lysine added is 0.03 mol.

[0040] Example 5, a cross-linked sodium hyaluronate, is different from Example 1 only in that the pre-cross-linked monomer prepared in Preparation Example 1 is replaced by an equal amount of the pre-cross-linked monomer prepared in Preparation Example 3; and the amount of lysine added is 0.05 mol.

[0041] Example 6, a cross-linked sodium hyaluronate, is different from Example 1 only in that the added amount of the pre-cross-linked monomer prepared in Preparation Example 1 is 0.2% of the mass of the sodium hyaluronate.

[0042] Example 7, a cross-linked sodium hyaluronate, is different from Example 1 only in that the added amount of the pre-cross-linked monomer prepared in Preparation Example 1 is 2% of the mass of the sodium hyaluronate.

[0043] Example 8, a cross-linked sodium hyaluronate, prepared according to the following process steps: S1. adding 0.1 mol of sodium hyaluronate (molecular weight of 300,000 to 1,000,000 Daltons) into a 20% by mass sodium hydroxide aqueous solution and stirring to dissolve to obtain a hyaluronic acid aqueous solution; S2. Add the pre-crosslinked monomer prepared in Preparation Example 1 to the hyaluronic acid aqueous solution, wherein the amount of the pre-crosslinked monomer added is 1% of the mass of sodium hyaluronate, stir and mix at room temperature for 30 minutes, then add fullerol, continue stirring and reacting for 2 hours, wherein the amount of fullerol added is 0.5% of the mass of sodium hyaluronate, to obtain a pre-reaction solution; S3. 0.04 mol of lysine was added to the pre-reaction solution, the pH value of the solution was adjusted to 8, and the cross-linking reaction was carried out at 30° C. for 6 h; S4. After the above cross-linking reaction is completed, cross-linked sodium hyaluronate is obtained through dialysis and freeze-drying.

[0044] Example 9, a cross-linked sodium hyaluronate, is different from Example 8 only in that the added amount of fullerol is 0.2% of the mass of the sodium hyaluronate.

[0045] Example 10, a cross-linked sodium hyaluronate, is different from Example 8 only in that the added amount of fullerol is 0.8% of the mass of the sodium hyaluronate.

[0046] Comparative Example Comparative Example 1, a cross-linked sodium hyaluronate, is prepared according to the following process steps: S1. adding 0.1 mol of sodium hyaluronate (molecular weight of 300,000 to 1,000,000 Daltons) into a 20% by mass sodium hydroxide aqueous solution and stirring to dissolve to obtain a hyaluronic acid aqueous solution; S2. adding 0.04 mol of lysine to the hyaluronic acid aqueous solution, adjusting the pH value of the solution to 8, and performing a cross-linking reaction at 30° C. for 6 h; S3. After the above cross-linking reaction is completed, cross-linked sodium hyaluronate is obtained through dialysis and freeze-drying.

[0047] Comparative Example 2 is a cross-linked sodium hyaluronate, which is different from Example 1 only in that lysine is replaced by an equal amount of 1,4-butanediol diglycidyl ether.

[0048] Comparative Example 3, a cross-linked sodium hyaluronate, is different from Example 1 only in that the pre-cross-linked monomer prepared in Preparation Example 1 is replaced by an equal amount of the pre-cross-linked monomer prepared in Preparation Example 4.

[0049] Performance testing The cross-linked sodium hyaluronate obtained in the examples and comparative examples was added to a phosphate buffer solution to obtain a cross-linked sodium hyaluronate gel sample with a concentration of 0.85%.

[0050] 1. Viscosity performance test: The dynamic viscosity test of the gel samples of the embodiment and the comparative example was carried out using a rotational viscometer. The test results are shown in Table 1.

[0051] 2. Anti-enzymatic performance test: 30 U / mL hyaluronidase was added to the gel samples obtained in the examples and comparative examples, and the uronic acid content in the samples after 5 h and 24 h was measured to calculate the degradation percentage, which was used to characterize the anti-enzymatic performance of the cross-linked sodium hyaluronate. The test results are shown in Table 2.

[0052]

[0053]

[0054] According to Table 1 and Table 2, in combination with Example 1, Example 6, Example 7 and Comparative Example 1, it can be seen that the dynamic viscosity of Example 6, Example 7 and Comparative Example 1 has decreased, and the percentage of enzymolysis at different time periods has increased, indicating that the cross-linking performance and anti-enzymolysis performance of the cross-linked sodium hyaluronate obtained in Example 6, Example 7 and Comparative Example 1 have decreased, and the decrease in Comparative Example 1 is more obvious. The reason is that Example 6, Example 7 and Comparative Example 1 adjust the amount of pre-cross-linked monomer added in the preparation process of cross-linked sodium hyaluronate, wherein the amount of pre-cross-linked monomer added is reduced in Example 6, then the cross-linking sites in the pre-treated sodium hyaluronate are significantly reduced compared with Example 1, and the cross-linking degree of the cross-linking agent in the cross-linking process will also decrease accordingly, and the cross-linking density and stability of the obtained cross-linked sodium hyaluronate are reduced, and the lack of the effect of the pre-cross-linked monomer, the anti-enzymolysis performance is also reduced accordingly, and in Comparative Example 1, the sodium hyaluronate is not pre-cross-linked, and the basic amino acid is directly used for cross-linking, then the stability of the product is greatly reduced, and the structure is also easily damaged by external influences. In Example 7, the amount of pre-crosslinked monomer added is increased, which will result in a large number of polar groups in the sodium hyaluronate being occupied during the pre-crosslinking period, making the crosslinking density of the final cross-linked sodium hyaluronate too dense, the dynamic viscosity too high, and the mechanical strength will also decrease accordingly.

[0055] Combining Example 1 and Example 8, it can be seen that the dynamic viscosity of Example 8 has increased, and the percentage of enzymolysis at different time periods has decreased, indicating that the cross-linking performance and anti-enzymolysis performance of the cross-linked sodium hyaluronate obtained in Example 8 have increased. The reason is that in Example 8, additional fullerol is added during the pre-cross-linking process. The addition of fullerol can strengthen the cross-linked network structure and help adjust the rheological properties of the cross-linked sodium hyaluronate; and it can effectively remove free radicals in the system and improve the anti-enzymolysis ability of the cross-linked sodium hyaluronate.

[0056] Combining Example 1 and Comparative Example 2, it can be seen that the dynamic viscosity of Comparative Example 2 decreases, and the percentage of enzymolysis at different time periods increases, indicating that the cross-linking performance and anti-enzymolysis performance of the cross-linked sodium hyaluronate obtained in Comparative Example 2 are both reduced. The reason is that the conventional cross-linking agent is used to replace the basic amino acid cross-linking agent in Comparative Example 2. In addition to affecting the safety of the final product, the binding force between the conventional cross-linking agent and the pre-cross-linked sodium hyaluronate will decrease, and the binding and coordination ability with the branched lysine short peptide will decrease, which will affect the formation of the cross-linked network structure and also affect the resistance of the cross-linked sodium hyaluronate to the enzymatic reaction.

[0057] Combining Example 1 and Comparative Example 3, it can be seen that the dynamic viscosity of Comparative Example 3 decreases, and the percentage of enzymolysis at different time periods increases, indicating that the cross-linking performance and anti-enzymolysis performance of the cross-linked sodium hyaluronate obtained in Comparative Example 3 are both decreased. The reason is that the pre-cross-linked monomer added during the pre-cross-linking treatment in Comparative Example 3 does not have a large amount of branched structure, the branching sites are reduced, the stability of the three-dimensional network structure is reduced, and the distance between the cross-linking points cannot be significantly increased, thereby affecting the degradation effect of hyaluronidase on the cross-linking points. The lack of protection and support of the branched structure significantly reduces the anti-enzymolysis ability of the cross-linked sodium hyaluronate.

[0058] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0059] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing cross-linked sodium hyaluronate, characterized in that: The process steps include: S1. adding sodium hyaluronate to an alkaline aqueous solution and stirring to dissolve to obtain a hyaluronic acid aqueous solution; S2. adding a pre-crosslinking monomer to the hyaluronic acid aqueous solution, stirring and reacting at room temperature for 1 to 3 hours to obtain a pre-reaction solution; S3. Add a crosslinking agent to the pre-reaction solution, adjust the pH value of the solution to 8 to 10, and perform a crosslinking reaction at 20 to 40° C. for 4 to 8 hours; S4. After the cross-linking reaction is completed, the cross-linked sodium hyaluronate is obtained by dialysis and freeze-drying; The pre-crosslinking monomer contains a branched lysine short peptide; the crosslinking agent is a basic amino acid; The raw materials of the pre-crosslinking monomer include a polyamine compound and di-tert-butoxy acyl protected L-lysine in a molar ratio of (0.8-1):

1.

2. The method for preparing cross-linked sodium hyaluronate according to claim 1, characterized in that: The added amount of the pre-crosslinking monomer is 0.5-1.5% of the mass of sodium hyaluronate.

3. The method for preparing cross-linked sodium hyaluronate according to claim 1, characterized in that: The polyamine compound is one or both of a diamine compound and a triamine compound.

4. The method for preparing cross-linked sodium hyaluronate according to claim 1, characterized in that: The pre-crosslinked monomer is prepared according to the following method: Add di-tert-butoxyacyl protected L-lysine to a chloroform solution, adjust the temperature to 0-5°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir and mix for 1-2 hours to obtain a premixed solution; add a polyamine compound to the premixed solution, continue to stir and react at 0-5°C for 15-20 hours, and finally obtain a pre-crosslinked monomer through filtering, extraction and drying.

5. The method for preparing cross-linked sodium hyaluronate according to claim 4, characterized in that: The molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the di-tert-butoxyacyl-protected L-lysine is (0.6-0.8):

1.

6. The method for preparing cross-linked sodium hyaluronate according to claim 1, characterized in that: The basic amino acids include one or both of lysine and arginine.

7. The method for preparing cross-linked sodium hyaluronate according to claim 1, characterized in that: The molar ratio of the cross-linking agent to sodium hyaluronate is (0.3-0.5):

1.

8. The method for preparing cross-linked sodium hyaluronate according to claim 1, characterized in that: Fullerol is also added to the S2; the amount of the fullerol added is 0.2-0.8% of the mass of sodium hyaluronate.

9. A cross-linked sodium hyaluronate, characterized in that: The cross-linked sodium hyaluronate is prepared according to the preparation method of any one of claims 1 to 8.

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