A method for preparing amide bond cross-linked sodium hyaluronate hydrogel

Abstract

The present invention relates to a technical field of biomedical materials, and in particular to a method for preparing an amide-bond cross-linked sodium hyaluronate hydrogel. The method comprises the following steps: mixing a cross-linking agent containing two or more active primary amines with sodium hyaluronate in a buffer solution, and adding a proton-trapping agent; adding a coupling agent to the buffer solution to activate the carboxyl group of sodium hyaluronate, and synthesizing an amide with the activated carboxyl group and the amino group in the cross-linking agent in one pot; adding an alkali solution for hydrolysis, and then neutralizing; and finally dialyzing, lyophilizing, and rehydrating to obtain an amide-bond cross-linked sodium hyaluronate hydrogel. The present invention helps N-methylmorpholine regeneration by adding a proton-trapping agent in advance before the coupling agent activates the carboxyl group, and competes with the esterification reaction, thereby effectively reducing the formation of ester bonds. The sodium hyaluronate hydrogel prepared by the present invention has good thermal stability and degradation resistance, and has broad application prospects in the fields of soft tissue filling and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a method for preparing an amide bond cross-linked sodium hyaluronate hydrogel. Background Art

[0002] Cross-linked sodium hyaluronate is one of the most widely used filler materials in the medical beauty industry, especially in facial filling and wrinkle removal. Sodium hyaluronate itself is a natural polysaccharide with good biocompatibility and biodegradability, but because its natural form is rapidly degraded in the body, it usually needs to be chemically modified to improve its stability and durability in the body.

[0003] The sites that can be modified on sodium hyaluronate include hydroxyl, carboxyl and acetamide. Currently, the main modification sites are concentrated on hydroxyl and carboxyl groups. Judging from the cross-linked sodium hyaluronate fillers approved for sale in the domestic and foreign markets, the cross-linking sites are mainly concentrated on hydroxyl groups, and the mainstream cross-linking agents are still 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS). However, the ether bond formed at this cross-linking site is still not enough to resist degradation, such as high-temperature degradation of the final product by high-temperature steam sterilization and enzymatic degradation of active enzymes in the body. In addition, both cross-linking agents have certain biosafety risks. Studies have pointed out that BDDE may cause cytotoxicity and induce inflammatory responses, while DVS is listed as a controlled highly toxic drug.

[0004] In the modification of the carboxyl end, natural amino acids and their salts can be selected as cross-linking agents. The reaction is mainly that the carboxyl group is activated by the coupling agent, and then the amino group attacks the activated adduct as a nucleophilic group to form a cross-linked bond without an activator. For example, the patent disclosed in the patent application publication number CN 105713211A discloses a method for preparing a new type of skin filler, using 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) as a coupling agent and lysine as a cross-linking agent to react with the activated carboxyl group of sodium hyaluronate to form an amide bond cross-linking to prepare a sodium hyaluronate hydrogel. In this preparation method, the cross-linking agent lysine has better biosafety, but in this preparation method, the activated carboxyl group of sodium hyaluronate not only undergoes an amidation reaction with the amino group of lysine, but also undergoes an esterification reaction with the hydroxyl group inside the molecule. The ester bond has poor thermal stability, which will cause the gel to undergo significant degradation after high-temperature steam sterilization, and the physical properties of the obtained sodium hyaluronate hydrogel are poor. Summary of the invention

[0005] The present invention aims to overcome the defects in the prior art that when the modification site of sodium hyaluronate is a carboxyl group, the activated carboxyl group in the reaction process is easy to form an ester bond, resulting in the prepared sodium hyaluronate hydrogel being easy to hydrolyze and having poor stability. A method for preparing an amide bond cross-linked sodium hyaluronate hydrogel is provided to overcome the above defects.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: A method for preparing an amide bond cross-linked sodium hyaluronate hydrogel, characterized in that it comprises the following steps: S1. Mixing a cross-linking agent containing two or more active primary amines with sodium hyaluronate in a buffer solution, and adding a proton capture agent; S2. Adding a coupling agent to the above buffer solution to activate the carboxyl groups of sodium hyaluronate, the activated carboxyl groups and the amino groups in the cross-linking agent synthesize amides in one pot to obtain an amide-bond cross-linked sodium hyaluronate stock solution; S3, adding alkaline solution to the sodium hyaluronate stock solution for hydrolysis, and then neutralizing to obtain an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The amide-bond cross-linked sodium hyaluronate preparatory solution is dialyzed, freeze-dried, and rehydrated to obtain an amide-bond cross-linked sodium hyaluronate hydrogel.

[0007] In the coupling reaction of sodium hyaluronate, the activated sodium hyaluronate active intermediate ester is highly active and is easily attacked by other nucleophilic reagents in the reaction medium. In addition to reacting with amino groups to form amides, it can also react with hydroxyl groups to form esters. The hydroxyl group is closer to the activated carboxyl group and is more likely to undergo esterification. The ester bond is thermally unstable and easily hydrolyzed, which has a significant effect on the mechanical properties of the cross-linked sodium hyaluronate gel.

[0008] To reduce the ester bonds in the cross-linked sodium hyaluronate gel, the ester bonds can be removed by alkaline hydrolysis or high temperature cracking after the ester bonds are formed, or the reaction of forming ester bonds can be hindered during the cross-linking reaction. However, if the cross-linked sodium hyaluronate has formed more ester bonds, even if the ester bonds are hydrolyzed at this time, the performance of the cross-linked sodium hyaluronate hydrogel obtained is still poor because the structure of the cross-linked sodium hyaluronate is destroyed to a certain extent. Therefore, to minimize the ester bonds, it is necessary to suppress the esterification reaction in advance during the one-pot synthesis. Before adding the coupling agent, the present invention first adds a proton capture agent to the buffer solution mixed with the cross-linking agent and sodium hyaluronate. When the coupling agent is added and activates the carboxyl group of sodium hyaluronate, the morpholine group in the molecular structure will be left. At this time, the proton capture agent in the reaction medium can help N-methylmorpholine (NMM) regenerate, and compete with the esterification reaction, reducing the formation of ester bonds while significantly improving the efficiency of the amide reaction.

[0009] Preferably, the proton-trapping agent in step S1 is a non-nucleophilic base. The non-nucleophilic base can provide a lone pair of electrons on the nitrogen atom to remove protons. The inventor accidentally discovered that adding an appropriate amount of non-nucleophilic base during the cross-linking reaction can reduce the occurrence of esterification reaction to a certain extent. Using a non-nucleophilic base as a proton-trapping agent can absorb protons in the chemical reaction of the present invention, but will not react with other nucleophiles, improve the regeneration of NMM, and compete with side reactions to reduce the formation of ester bonds. Through subsequent experiments, it was determined that the cross-linked sodium hyaluronate gel prepared using a non-nucleophilic base as a proton-trapping agent has excellent thermal stability and degradation resistance.

[0010] As further preferred, the non-nucleophilic base is one or both of triethylamine and sodium bicarbonate. In practice, sodium bicarbonate is preferred.

[0011] Preferably, the pH of the buffer solution in step S1 is between 7 and 9. When the pH in the buffer solution is too high, the competitive ability of the proton capture agent and the esterification reaction decreases, and when the sodium hyaluronate undergoes a subsequent cross-linking reaction, the process of ester bond formation and hydrolysis will seriously affect the thermal stability of the prepared sodium hyaluronate hydrogel.

[0012] Preferably, the cross-linking agent in step S1 is one or more of lysine, arginine, lysine hydrochloride, and lysine acetate. Using natural amino acids as cross-linking agents can reduce potential biosafety issues relative to the currently widely used BDDE and DVS. In addition, the amino acid structure is relatively simple, which is conducive to controlling the accuracy of the cross-linking reaction. In practice, the molar equivalent ratio of the cross-linking agent to the disaccharide unit of sodium hyaluronate is preferably 0.1 to 1.

[0013] As further preferred, the chirality of the cross-linking agent is L-form. The above amino acids may be in D-form and / or L-form, and L-lysine and its salts are preferred in the present invention.

[0014] Preferably, the coupling agent in step S2 is one or both of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine tetrafluoroborate (DMTMM BF4) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM). DMTMM is the reaction product of the triazine ring derivative 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM), and is very easy to react with nucleophilic reagents. In the coupling reaction, DMTMM can be used to synthesize amides in a simple one-step manner and activate carboxyl groups as effectively as the traditional two-step method. DMTMM is easily soluble in water, which enables it to participate in the reaction better and can be completely removed from the reaction product. Compared with EDC / NHS, the aqueous solution of DMTMM is more stable, has high reaction activity, is less dependent on the pH of the reaction medium, has fewer by-products, and the morpholine derivatives that do not participate in the reaction do not react with sodium hyaluronate and are easy to remove. In practice, the molar equivalent ratio of the coupling agent to the disaccharide unit of sodium hyaluronate is preferably 0.2-2.

[0015] Preferably, the alkali solution in step S3 is one or both of sodium hydroxide solution and potassium hydroxide solution. In practice, too high a temperature for hydrolysis of the alkali solution may easily lead to hydrolysis of the amide bond, and the alkali solution hydrolysis temperature is preferably 2°C to 8°C.

[0016] Preferably, the reaction temperature in step S2 is between 35° C. and 60° C. A higher temperature promotes the formation of more crosslinking points, thereby increasing the crosslinking density. However, if the temperature is too high, it may cause excessive crosslinking reaction, forming an overly dense network structure, and affecting the physical properties of the hydrogel.

[0017] Preferably, the sodium hyaluronate dissolution mass concentration described in step S1 is 5%-15%, and the molecular weight of the sodium hyaluronate is 100000 Dalton-800000 Dalton. The sodium hyaluronate molecular weight and mass concentration selected by the present invention play a certain role in realizing the preparation of amide bond cross-linked sodium hyaluronate hydrogel. Generally, the length of the sodium hyaluronate molecular chain and the distance between molecules will affect the contact between the amino group of the cross-linking agent and the active intermediate ester of sodium hyaluronate. Due to the effects of hydrogen bonds and covalent cross-linking, the molecules of sodium hyaluronate can be entangled with each other to form a network structure. Longer molecular chains are more prone to intermolecular entanglement than shorter molecular chains, and the higher the mass concentration, the denser the network structure, which is very conducive to chemical modification of small molecule cross-linking agents. However, the higher the molecular weight and mass concentration, the greater the friction between molecules, the viscosity will rise sharply, which will cause uneven dissolution of sodium hyaluronate and affect the subsequent cross-linking reaction. In practice, the dissolved mass concentration of sodium hyaluronate is preferably 6%-13%, and the molecular weight of sodium hyaluronate is preferably 200,000 Daltons-600,000 Daltons.

[0018] Therefore, the present invention has the following beneficial effects: (1) The present invention can effectively inhibit the esterification reaction of sodium hyaluronate by adding a proton capture agent, avoid the influence of unstable ester bonds on the performance of sodium hyaluronate hydrogel, and effectively improve the efficiency of the amide reaction.

[0019] (2) The present invention uses a non-nucleophilic base as an esterification inhibitor, and confirms through experiments that the cross-linked sodium hyaluronate gel finally obtained has excellent thermal stability and degradation resistance.

[0020] (3) The present invention further optimizes the preferred scheme and proposes a feasible method for preparing sodium hyaluronate hydrogel with good thermal stability and degradation resistance, which has broad application prospects in the fields of soft tissue filling and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of coupling agent activation of carboxyl group of sodium hyaluronate.

[0022] Figure 2 Schematic diagram of the cross-linking reaction principle.

[0023] Figure 3 It is a comparison chart of rheological properties between Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further clearly and completely described below through specific examples and in combination with the accompanying drawings. It should be noted that the embodiments described in the present invention are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but they are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in 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. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the embodiments, unless otherwise specified, can be obtained from commercial channels.

[0025] The reaction substances added in the following examples are all based on the molar number of disaccharide units of sodium hyaluronate. Figure 1 Explanation (taking DMTMM as an example). The carboxyl group of sodium hyaluronate first performs a nucleophilic attack on the triazine part of DMTMM, a nucleophilic aromatic substitution reaction occurs, an ester bond is formed between sodium hyaluronate and DMTMM, and the morpholine group NMM is removed to obtain an active intermediate ester.

[0026] The cross-linking reaction can be Figure 2Explanation (taking L-lysine as an example), in the figure HA = sodium hyaluronate, the α-amino group at the N-terminus of L-lysine and the ε-amino group in the side chain carry out nucleophilic attack on the active intermediate ester (cross-linking reaction).

[0027] Example 1 S1. Add 0.5 equivalents of L-lysine and sodium hyaluronate with a molecular weight of 500,000 Daltons to a phosphate buffer, and then add 0.1 equivalents of sodium bicarbonate solution, the mass concentration of sodium hyaluronate is 9%, and adjust the pH of the solution to 7.2; S2, adding 0.5 equivalent ratio of DMTMM, adjusting the reaction temperature to 40°C, and reacting for 4 hours to obtain an amide bond cross-linked sodium hyaluronate stock solution; S3, adding 1% sodium hydroxide solution for hydrolysis for 6 hours, neutralizing the pH with hydrochloric acid, and obtaining an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The prepared solution was dialyzed with a large amount of phosphate buffer for 4 days to remove reaction impurities, and then freeze-dried and rehydrated to a cross-linked sodium hyaluronate concentration of 20 mg / mL to obtain an amide bond cross-linked sodium hyaluronate hydrogel.

[0028] Example 2 S1. Add 0.7 equivalents of L-lysine and sodium hyaluronate with a molecular weight of 500,000 Daltons to a phosphate buffer, and then add 0.1 equivalents of sodium bicarbonate solution, the mass concentration of sodium hyaluronate is 9%, and adjust the pH of the solution to 7.8; S2, adding 0.5 equivalent ratio of DMTMM, adjusting the reaction temperature to 40°C, and reacting for 4 hours to obtain an amide bond cross-linked sodium hyaluronate stock solution; S3, adding 1% sodium hydroxide solution for hydrolysis for 6 hours, neutralizing the pH with hydrochloric acid, and obtaining an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The prepared solution was dialyzed with a large amount of phosphate buffer for 4 days to remove reaction impurities, and then freeze-dried and rehydrated to a cross-linked sodium hyaluronate concentration of 20 mg / mL to obtain an amide bond cross-linked sodium hyaluronate hydrogel.

[0029] Example 3 S1. Add 0.5 equivalents of L-lysine and sodium hyaluronate with a molecular weight of 500,000 Daltons to a phosphate buffer, and then add 0.1 equivalents of sodium bicarbonate solution, the mass concentration of sodium hyaluronate is 9%, and adjust the pH of the solution to 8.0; S2, adding 0.5 equivalent ratio of DMTMM, adjusting the reaction temperature to 40°C, and reacting for 4 hours to obtain an amide bond cross-linked sodium hyaluronate stock solution; S3, adding 1% sodium hydroxide solution for hydrolysis for 6 hours, neutralizing the pH with hydrochloric acid, and obtaining an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The prepared solution was dialyzed with a large amount of phosphate buffer for 4 days to remove reaction impurities, and then freeze-dried and rehydrated to a cross-linked sodium hyaluronate concentration of 20 mg / mL to obtain an amide bond cross-linked sodium hyaluronate hydrogel.

[0030] Example 4 S1. Add 0.5 equivalents of L-lysine and sodium hyaluronate with a molecular weight of 500,000 Daltons to a phosphate buffer, and then add 0.1 equivalents of sodium bicarbonate solution, the mass concentration of sodium hyaluronate is 9%, and adjust the pH of the solution to 7.2; S2, adding 0.8 equivalent ratio of DMTMM, adjusting the reaction temperature to 40°C, and reacting for 4 hours to obtain amide bond cross-linked sodium hyaluronate stock solution; S3, adding 1% sodium hydroxide solution for hydrolysis for 6 hours, neutralizing the pH with hydrochloric acid, and obtaining an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The prepared solution was dialyzed with a large amount of phosphate buffer for 4 days to remove reaction impurities, and then freeze-dried and rehydrated to a cross-linked sodium hyaluronate concentration of 20 mg / mL to obtain an amide bond cross-linked sodium hyaluronate hydrogel.

[0031] Comparative Example 1 S1. Add 0.5 equivalents of L-lysine and sodium hyaluronate with a molecular weight of 500,000 Daltons into a phosphate buffer solution, wherein the mass concentration of the sodium hyaluronate is 9%, and the pH of the solution is adjusted to 7.2; S2, adding 0.5 equivalent ratio of DMTMM, adjusting the reaction temperature to 40°C, and reacting for 4 hours to obtain an amide bond cross-linked sodium hyaluronate stock solution; S3, adding 1% sodium hydroxide solution for hydrolysis for 6 hours, neutralizing the pH with hydrochloric acid, and obtaining an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The prepared solution was dialyzed with a large amount of phosphate buffer for 4 days to remove reaction impurities, and then freeze-dried and rehydrated to a cross-linked sodium hyaluronate concentration of 20 mg / mL to obtain an amide bond cross-linked sodium hyaluronate hydrogel.

[0032] Comparative Example 2 S1. Add 0.5 equivalents of L-lysine and sodium hyaluronate with a molecular weight of 500,000 Daltons to a phosphate buffer, and then add 0.1 equivalents of sodium bicarbonate solution, the mass concentration of sodium hyaluronate is 9%, and adjust the pH of the solution to 7.2; S2, adding 0.5 equivalent ratio of DMTMM, adjusting the reaction temperature to 40°C, and reacting for 4 hours to obtain an amide bond cross-linked sodium hyaluronate preparatory solution; S3. The prepared solution was dialyzed with a large amount of phosphate buffer for 4 days to remove reaction impurities, and then freeze-dried and rehydrated to a cross-linked sodium hyaluronate concentration of 20 mg / mL to obtain an amide bond cross-linked sodium hyaluronate hydrogel.

[0033] Thermal stability test of amide bond cross-linked sodium hyaluronate hydrogel: The hydrogels prepared in Examples 1 to 4 and Comparative Examples 1 to 3 were sterilized by steam at 121° C. for 15 min, and the elastic modulus (Elastic modulus, G′ ) percentage loss. The lower the percentage loss value, the better the thermal stability of the hydrogel, and it can be considered that the degradation resistance is stronger. At 25±0.2°C, a rheometer was used to perform a frequency scan at a shear rate of 0.01Hz-10Hz under a shear strain of 1%, and a logarithmic coordinate graph of the logarithm of the viscous modulus and elastic modulus and the frequency was plotted. The corresponding elastic modulus value under 0.1Hz was taken to obtain Table 1. The coordinate graph of the test results of Example 1 and Comparative Example 1 is shown in Figure 3 shown.

[0034] Table 1, rheological test data of examples and comparative examples Example / Comparative Example L-Lysine molar equivalent ratio DMTMM molar equivalent ratio pH of reaction medium Before sterilization After sterilization Percentage of loss Example 1 0.5 0.5 7.2 689 Pa 636 Pa 7.8% Example 2 0.7 0.5 7.7 609 Pa 576 Pa 5.4% Example 3 0.5 0.5 8.0 686 Pa 657 Pa 4.2% Example 4 0.5 0.8 7.2 925 Pa 908 Pa 1.8% Comparative Example 1 0.5 0.5 7.2 544 Pa 271 Pa 50.2% Comparative Example 2 0.5 0.5 7.2 863 Pa 608 Pa 29.5% As shown in Table 1, Figure 3 As shown, it can be seen that the embodiments can still maintain a high elastic modulus after high-temperature sterilization, and the loss percentage of the elastic modulus is extremely low, which can indicate that the embodiments have good thermal stability. Compared with Example 1, Comparative Example 1 has a lower elastic modulus before sterilization due to the lack of the addition of a proton capture agent, which results in more ester bonds in the hydrogel, and a significant decrease in performance after high-temperature sterilization; Comparative Example 2 is compared with Example 1, and no alkaline hydrolysis is performed after the cross-linking reaction, and the residual ester bonds in the hydrogel make the hydrogel thermally unstable. Combining the test results of Comparative Example 1 and Comparative Example 2, it can be seen that the ability of adding a proton capture agent to improve the thermal stability of the hydrogel is significantly stronger than alkaline hydrolysis.

[0035] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing an amide bond cross-linked sodium hyaluronate hydrogel, characterized in that: The steps include: S1. Mixing a cross-linking agent containing two or more active primary amines with sodium hyaluronate in a buffer solution, and adding a proton capture agent; S2. Adding a coupling agent to the above buffer solution to activate the carboxyl groups of sodium hyaluronate, the activated carboxyl groups and the amino groups in the cross-linking agent synthesize amides in one pot to obtain an amide-bond cross-linked sodium hyaluronate stock solution; S3, adding alkaline solution to the sodium hyaluronate stock solution for hydrolysis, and then neutralizing to obtain an amide bond cross-linked sodium hyaluronate preparatory solution; S4. The amide-bond cross-linked sodium hyaluronate preparatory solution is dialyzed, freeze-dried, and rehydrated to obtain an amide-bond cross-linked sodium hyaluronate hydrogel.

2. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The proton capture agent in step S1 is a non-nucleophilic base.

3. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 2, characterized in that: The non-nucleophilic base is one or both of triethylamine and sodium bicarbonate.

4. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The pH of the buffer solution in step S1 is between 7 and 9.

5. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The cross-linking agent in step S1 is one or more of lysine, arginine, lysine hydrochloride, and lysine acetate.

6. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 5, characterized in that: The chirality of the cross-linking agent is L form.

7. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The coupling agent in step S2 is one or both of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine tetrafluoroborate and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride.

8. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The alkali solution in step S3 is one or both of sodium hydroxide solution and potassium hydroxide solution.

9. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The reaction temperature in step S2 is between 35°C and 60°C.

10. The method for preparing an amide bond cross-linked sodium hyaluronate hydrogel according to claim 1, characterized in that: The mass concentration of the sodium hyaluronate dissolved in step S1 is 5%-15%, and the molecular weight of the sodium hyaluronate is 100,000 Daltons-800,000 Daltons.

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