A crosslinked sodium hyaluronate and its preparation method

Abstract

The present invention discloses a 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 the cross-linking reaction, a pre-cross-linking monomer is first added to carry out pre-cross-linking treatment, wherein the pre-cross-linking monomer contains a branched lysine short peptide. Basic amino acids are specifically more secure and biocompatible than other cross-linking agents, and can greatly reduce the potential safety hazards caused by cross-linking agent residues. During the pre-cross-linking process, a large number of additional reactive sites can be provided first, thereby improving the cross-linking points of sodium hyaluronate, contributing to the construction of a complex and stable cross-linked network structure, the compatibility between its branched lysine short peptide and basic amino acids is also very good, and the branched structure can also increase the distance between the cross-linking points, affect the accessibility of hyaluronidase to the cross-linking points, protect enzyme recognition sites, reduce the rate of enzymatic degradation, and improve the anti-enzymatic ability of cross-linked sodium hyaluronate.

Description

Technical Field

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

[0002] Hyaluronic acid is a natural polysaccharide with a large molecular weight. It exhibits unique viscoelasticity, excellent biocompatibility, and biodegradability, making it an ideal tissue engineering material for various applications. However, due to the poor stability of natural hyaluronic acid hydrogels, their sensitivity to hyaluronidase and free radicals, their short survival time in the body, and their poor mechanical strength, their applications are greatly limited. To this end, natural hyaluronic acid has been chemically modified or cross-linked to enhance the mechanical strength, stability, and degradation resistance of the gel, allowing it to last longer in the body and thus expand its application in biomedicine and tissue engineering.

[0003] Cross-linked hyaluronic acid is prepared by using one or more combined chemical cross-linking agents, and 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-linkers are mostly chemical reagents, which are irritating to the skin and even cytotoxic. The residues left during the cross-linking process also pose a great potential hazard to users. Reducing the amount of cross-linker used will result in the cross-linked hyaluronic acid having poor high temperature resistance and resistance to enzymatic hydrolysis, which limits its use. Therefore, using safe and non-toxic natural cross-linkers is an effective method. However, the cross-linked hyaluronic acid obtained by some current natural biological cross-linkers 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 problems 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:

[0007] In a first aspect, the present invention provides a method for preparing cross-linked sodium hyaluronate, comprising the following steps:

[0008] S1. Sodium hyaluronate is added to an alkaline aqueous solution and stirred to dissolve to obtain a hyaluronic acid aqueous solution;

[0009] S2. Adding a pre-crosslinked monomer to the hyaluronic acid aqueous solution, stirring and reacting at room temperature for 1 to 3 hours to obtain a pre-reaction solution;

[0010] S3. Add a crosslinking agent to the pre-reaction solution, adjust the pH of the solution to 8 to 10, and perform a crosslinking reaction at 20 to 40 ° C for 4 to 8 hours;

[0011] S4. After the cross-linking reaction is completed, the cross-linked sodium hyaluronate is obtained by dialysis and freeze-drying;

[0012] The pre-crosslinked monomer contains a branched lysine short peptide; the crosslinking agent is a basic amino acid.

[0013] Preferably, the added amount of the pre-crosslinking monomer is 0.5 to 1.5% of the mass of sodium hyaluronate.

[0014] Preferably, the molar ratio of the cross-linking agent to sodium hyaluronate is (0.3-0.5):1.

[0015] By adopting the above technical solution, the present invention uses basic amino acids as cross-linking agents for hyaluronic acid. The amino and carboxyl groups contained in the basic amino acids can react and link with the functional groups in 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 of proteins, are safer under conditions involving human health, and can greatly reduce safety hazards caused by cross-linking agent residues.

[0016] Moreover, before cross-linking, the present invention pre-cross-links the hyaluronic acid, which can help build a relatively solid basic structure before cross-linking the basic amino acids and hyaluronic acid, making the three-dimensional network structure formed in the subsequent cross-linking process more stable, improving the cross-linking degree of cross-linked sodium hyaluronate, and better regulating the cross-linking density, thereby enhancing the ability of cross-linked hyaluronic acid to fight free radicals and enzymes in the body, thereby extending the effective duration.

[0017] 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-linked structure, thereby constructing a more complex and stable three-dimensional network structure, improving the degree of subsequent cross-linking, and enhancing the mechanical strength and elasticity of the cross-linked sodium hyaluronate. In addition, the branched lysine short peptide has good biocompatibility and does not leave toxic residues.

[0018] Its branched structure can also increase the distance between crosslinks, changing the spatial conformation of the crosslinked network and affecting the accessibility of hyaluronidase to the crosslinks, thereby slowing the rate of hyaluronidase degradation and improving the crosslinked sodium hyaluronate's resistance to enzymatic degradation. It also reduces the proportion of small molecular weight fragments in the original sodium hyaluronate, thereby reducing the potential risk of inflammation caused by residual small molecule sodium hyaluronate.

[0019] 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.

[0020] Preferably, the polyamine compound is one or a combination of two of a diamine compound and a triamine compound.

[0021] More preferably, the diamine compound includes a combination of one or more of ethylenediamine, bio-based pentamethylenediamine, 1,2-propylenediamine, and triethylenediamine.

[0022] More preferably, the triamine compound includes one or a combination of diethylenetriamine and triethylamine.

[0023] Preferably, the pre-crosslinking monomer is prepared according to the following method:

[0024] 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, and stir for 1-2 hours to obtain a premixed solution; add a polyamine compound to the premixed solution, continue stirring and reacting at 0-5°C for 15-20 hours, and finally obtain a pre-crosslinked monomer through filtration, extraction, and drying.

[0025] Preferably, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to bis-tert-butoxyacyl-protected L-lysine is (0.6-0.8):1.

[0026] By adopting the above technical solution and selecting L-lysine protected with a bis-tert-butoxy acyl group, the chemical stability of lysine can be improved, reducing the impact of reactions on its performance. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, a carbodiimide-based condensing agent, can help form short lysine peptides. It reacts with the carboxyl groups of L-lysine to form an acylurea intermediate that can further react with amino groups. This intermediate then reacts with the amino groups of polyamine compounds to form a pre-crosslinked monomer.

[0027] Pre-crosslinking sodium hyaluronate with a pre-crosslinking monomer containing a branched lysine peptide can effectively enhance the crosslink density and uniformity between sodium hyaluronate molecules, helping to form a stable crosslinked structure. This allows the framework structure of the cross-linked sodium hyaluronate to be first obtained, and then cross-linked with basic amino acids, thereby improving the mechanical strength and stability of the resulting cross-linked sodium hyaluronate. Furthermore, the pre-crosslinking effect of the pre-crosslinking monomer can protect enzyme recognition sites, reduce the rate of enzymatic degradation, and thus extend the shelf life of the cross-linked sodium hyaluronate.

[0028] Preferably, the basic amino acids include one or a combination of lysine and arginine.

[0029] By adopting the above technical solution, basic amino acids including lysine, arginine or a combination of the two can form electrostatic interactions with sodium hyaluronate. 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.

[0030] Basic amino acids have better biocompatibility. Using basic amino acids as cross-linkers for sodium hyaluronate can better improve the safety of the product. However, directly using basic amino acids for cross-linking will make 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.

[0031] The present invention adopts the form of pre-crosslinking, first pre-crosslinking sodium hyaluronate, and first constructing the cross-linked framework structure of sodium hyaluronate, 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, increasing the number of cross-linking points, making the resulting cross-linked network more dense and strong, thereby improving the mechanical strength and elastic modulus of the material. Moreover, the additional cross-linking points also increase the degree of cross-linking of the product, thereby effectively preventing excessive flow. Moreover, because the pre-crosslinked 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.

[0032] Preferably, step S2 further comprises adding fullerol; the amount of fullerol added is 0.2 to 0.8% of the mass of sodium hyaluronate.

[0033] By adopting the above technical solution, fullerol, a fullerene derivative modified with alcoholic hydroxyl groups, has improved water solubility and low cytotoxicity. Its addition to cross-linked sodium hyaluronate will not affect its safety. Most importantly, fullerol has the ability to effectively scavenge free radicals. During the pre-crosslinking process, the large number of hydroxyl functional groups in fullerol can form chemical bonds with sodium hyaluronate and pre-crosslinking monomers. The resulting cross-linked sodium hyaluronate can effectively resist the decomposition effects of free radicals, thereby greatly enhancing the long-term stability of the cross-linked sodium hyaluronate. It also helps the cross-linked sodium hyaluronate absorb and lock in moisture, forming a protective film to prevent water loss, resulting in a cross-linked sodium hyaluronate with even superior performance.

[0034] 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 regulate the fluidity and viscosity of the product, expanding the application scenarios of cross-linked sodium hyaluronate.

[0035] Beneficial effects of the present invention:

[0036] 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.

[0037] 2. The present invention pre-crosslinks sodium hyaluronate before the cross-linking reaction. The pre-crosslinked monomer used contains a branched lysine short peptide, and the multiple reaction sites and branching sites contained therein can help increase the cross-linking points of sodium hyaluronate. Then, under the cross-linking effect of basic amino acids, a more complex and stable three-dimensional network structure is constructed, thereby solving the problems of unstable properties and easy destruction of product structure that may occur when using basic amino acid cross-linking alone, and can also improve the cross-linking degree and mechanical strength of the material. Its branched structure can also increase the distance between cross-linking points, affect the accessibility of hyaluronidase to the cross-linking points, protect enzyme recognition sites, reduce the rate of enzymatic degradation, and improve the resistance of cross-linked sodium hyaluronate to enzymatic degradation.

[0038] 3. In the present invention, fullerol can also be added in the pre-crosslinking step. On the one hand, it can strengthen the cross-linked network structure obtained after the pre-crosslinking treatment, and on the other hand, it can effectively scavenge 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

[0039] 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 embodiments described 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0040] Preparation Example

[0041] Preparation Example 1: A pre-crosslinking monomer was prepared according to the following method:

[0042] 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, and stir and mix for 1 hour to obtain a premixed solution;

[0043] 0.9 mol of bio-based pentamethylenediamine was added to the premixed solution, and the mixture was stirred and reacted at 0°C for 15 hours. Finally, the pre-crosslinked monomer was obtained by filtration, extraction, and drying.

[0044] Preparation Example 2, a pre-crosslinked monomer, differs 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.

[0045] Preparation Example 3, a pre-crosslinked monomer, differs 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.

[0046] Preparation Example 4: A pre-crosslinked monomer was prepared according to the following method:

[0047] 0.1 mol of di-tert-butoxyacyl-protected L-lysine was added to 200 mL of chloroform solution, stirred and mixed, the temperature was adjusted to 0°C, 0.7 mol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was stirred and mixed for 2 hours, and then filtered, extracted, and dried to obtain a pre-crosslinked monomer.

[0048] Example

[0049] Example 1, a cross-linked sodium hyaluronate, is prepared according to the following process steps:

[0050] S1. Add 0.1 mol of sodium hyaluronate (molecular weight 300,000 to 1,000,000 Daltons) to a 20% by mass sodium hydroxide aqueous solution and stir to dissolve to obtain a hyaluronic acid aqueous solution;

[0051] S2. The pre-crosslinked monomer prepared in Preparation Example 1 was added to the hyaluronic acid aqueous solution, wherein the pre-crosslinked monomer was added in an amount of 1% by mass of sodium hyaluronate, and the reaction was stirred at room temperature for 2 h to obtain a pre-reaction solution;

[0052] S3. 0.04 mol of lysine was added to the pre-reaction solution, the pH of the solution was adjusted to 8, and the cross-linking reaction was carried out at 30°C for 6h;

[0053] S4. After the cross-linking reaction is completed, the cross-linked sodium hyaluronate is obtained by dialysis and freeze-drying.

[0054] Example 2, a cross-linked sodium hyaluronate, differs from Example 1 only in that the amount of the pre-cross-linked monomer prepared in Preparation Example 1 added is 0.5% of the mass of the sodium hyaluronate.

[0055] Example 3, a cross-linked sodium hyaluronate, differs 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.

[0056] Example 4, a cross-linked sodium hyaluronate, differs 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.

[0057] Example 5, a cross-linked sodium hyaluronate, differs 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.

[0058] Example 6, a cross-linked sodium hyaluronate, differs 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.

[0059] Example 7, a cross-linked sodium hyaluronate, differs 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.

[0060] Example 8, a cross-linked sodium hyaluronate, is prepared according to the following process steps:

[0061] S1. Add 0.1 mol of sodium hyaluronate (molecular weight 300,000 to 1,000,000 Daltons) to a 20% by mass sodium hydroxide aqueous solution and stir to dissolve to obtain a hyaluronic acid aqueous solution;

[0062] S2. The pre-crosslinked monomer prepared in Preparation Example 1 was added to the hyaluronic acid aqueous solution, wherein the pre-crosslinked monomer was added in an amount of 1% by weight of the sodium hyaluronate. After stirring at room temperature for 30 minutes, fullerol was added and the reaction was continued with stirring for 2 hours, wherein the fullerol was added in an amount of 0.5% by weight of the sodium hyaluronate to obtain a pre-reaction solution;

[0063] S3. 0.04 mol of lysine was added to the pre-reaction solution, the pH of the solution was adjusted to 8, and the cross-linking reaction was carried out at 30°C for 6h;

[0064] S4. After the cross-linking reaction is completed, the cross-linked sodium hyaluronate is obtained by dialysis and freeze-drying.

[0065] Example 9 is a cross-linked sodium hyaluronate, which differs from Example 8 only in that the added amount of fullerol is 0.2% of the mass of the sodium hyaluronate.

[0066] Example 10 is a cross-linked sodium hyaluronate, which differs from Example 8 only in that the added amount of fullerol is 0.8% of the mass of the sodium hyaluronate.

[0067] Comparative Example

[0068] Comparative Example 1: A cross-linked sodium hyaluronate was prepared according to the following process steps:

[0069] S1. Add 0.1 mol of sodium hyaluronate (molecular weight 300,000 to 1,000,000 Daltons) to a 20% by mass sodium hydroxide aqueous solution and stir to dissolve to obtain a hyaluronic acid aqueous solution;

[0070] S2. Add 0.04 mol of lysine to the hyaluronic acid aqueous solution, adjust the solution pH to 8, and perform a cross-linking reaction at 30°C for 6 h;

[0071] S3. After the cross-linking reaction is completed, cross-linked sodium hyaluronate is obtained through dialysis and freeze-drying.

[0072] 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.

[0073] Comparative Example 3, a cross-linked sodium hyaluronate, differs 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.

[0074] Performance testing

[0075] 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%.

[0076] 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.

[0077] 2. Anti-enzymatic Performance Test: 30 U / mL hyaluronidase was added to the gel samples obtained in the Examples and Comparative Examples. The uronic acid content in the samples was measured after 5 hours and 24 hours to calculate the degradation percentage. This was used to characterize the anti-enzymatic performance of the cross-linked sodium hyaluronate. The test results are shown in Table 2.

[0078]

[0079]

[0080] According to Tables 1 and 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 enzymatic hydrolysis at different time periods has increased, indicating that the cross-linking performance and enzymatic hydrolysis resistance of the cross-linked sodium hyaluronate obtained in Example 6, Example 7, and Comparative Example 1 have decreased, with the decrease in Comparative Example 1 being more significant. The reason for this is that the amount of pre-cross-linking monomer added during the preparation of the cross-linked sodium hyaluronate was adjusted in Example 6, Example 7, and Comparative Example 1. In particular, the amount of pre-cross-linking monomer added was reduced in Example 6. As a result, the cross-linking sites in the pre-treated sodium hyaluronate were significantly reduced compared to Example 1, and the degree of cross-linking of the cross-linking agent during the cross-linking process was also correspondingly reduced. The cross-linking density and stability of the cross-linked sodium hyaluronate obtained were both reduced. In addition, the lack of the effect of the pre-cross-linking monomer also led to a decrease in enzymatic hydrolysis resistance. In Comparative Example 1, the sodium hyaluronate was not pre-cross-linked, and basic amino acids were directly used for cross-linking. This greatly reduced the stability of the product, and the structure was also easily damaged by external influences. In Example 7, the amount of pre-crosslinking monomer added was increased, which resulted in a large number of polar groups in the sodium hyaluronate being occupied during the pre-crosslinking period, making the crosslinking density of the finally obtained cross-linked sodium hyaluronate too dense, the dynamic viscosity too high, and the mechanical strength also decreased.

[0081] Combining Examples 1 and 8, it can be seen that the dynamic viscosity of Example 8 increased, while the percentage of enzymatic degradation decreased at different time periods, indicating that the cross-linking performance and resistance to enzymatic degradation of the cross-linked sodium hyaluronate obtained in Example 8 were both improved. This is because Example 8 added additional fullerol during the pre-cross-linking process. The addition of fullerol can strengthen the cross-linked network structure, help adjust the rheological properties of the cross-linked sodium hyaluronate, and effectively scavenge free radicals in the system, improving the resistance of the cross-linked sodium hyaluronate to enzymatic degradation.

[0082] Combining Example 1 and Comparative Example 2, it can be seen that the dynamic viscosity of Comparative Example 2 decreases, and the enzymatic hydrolysis percentages at different time periods increase, indicating that the cross-linking performance and enzymatic hydrolysis resistance of the cross-linked sodium hyaluronate obtained in Comparative Example 2 are both reduced. The reason is that in Comparative Example 2, a conventional cross-linking agent is used to replace the basic amino acid cross-linking agent. 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 is reduced, and the binding and coordination ability with the branched lysine short peptide is reduced, which affects the formation of the cross-linked network structure and also affects the resistance of the cross-linked sodium hyaluronate to the enzymatic reaction.

[0083] Combining Example 1 and Comparative Example 3, it can be seen that the dynamic viscosity of Comparative Example 3 decreases, and the enzymatic hydrolysis percentages at different time periods increase, indicating that the cross-linking performance and enzymatic hydrolysis resistance of the cross-linked sodium hyaluronate obtained in Comparative Example 3 are both reduced. The reason is that the pre-cross-linking 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 enzymatic hydrolysis resistance of the cross-linked sodium hyaluronate.

[0084] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0085] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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. Sodium hyaluronate is added to an alkaline aqueous solution and stirred to dissolve to obtain a hyaluronic acid aqueous solution; S2. Adding a pre-crosslinked 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 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; Fullerol is further added to the S2; the amount of the fullerol added is 0.2 to 0.8% of the mass of the sodium hyaluronate.

2. The method for preparing cross-linked sodium hyaluronate according to claim 1, wherein: 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, wherein: 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, wherein: 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, and stir for 1-2 hours to obtain a premixed solution; add a polyamine compound to the premixed solution, continue stirring and reacting at 0-5°C for 15-20 hours, and finally obtain a pre-crosslinked monomer through filtration, 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 bis-tert-butoxyacyl-protected L-lysine is (0.6-0.8):

1.

6. The method for preparing cross-linked sodium hyaluronate according to claim 1, wherein: 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. 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 7.

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