Preparation method of sodium chondroitin sulfate and sodium hyaluronate cross-linked hydrogel

Abstract

The invention relates to the technical field of polymer biomedical materials, and provides a preparation method of sodium chondroitin sulfate and sodium hyaluronate cross-linked hydrogel, which comprises the following steps: S1, dissolving sodium hyaluronate in a sodium hydroxide aqueous solution, then adding sodium chondroitin sulfate, then dropwise adding 1, 3-butanediol, and carrying out a reaction for 2-3 hours to obtain sodium chondroitin sulfate and sodium hyaluronate cross-linked hydrogel; the preparation method comprises the following steps: firstly, reacting a 1, 4-butanediol diglycidyl ether ethanol solution and a phase transfer catalyst at the temperature of 5-10 DEG C for 2-4 hours, and then carrying out cross-linking reaction at the temperature of 35-40 DEG C for 12-24 hours to prepare gel; and S2, cutting the gel obtained in the step S1 into small blocks, adding the small blocks into a PBS buffer solution, soaking for 20-30 minutes, replacing the PBS buffer solution, soaking until the pH value of the last washing solution is measured to be 7.4 + / -0.1, sieving, adding the small blocks into the PBS buffer solution, homogenizing for 3-5 minutes at the rotating speed of 3000-5000 rpm, and filtering to obtain the sodium chondroitin sulfate and sodium hyaluronate cross-linked hydrogel. The prepared sodium chondroitin sulfate and sodium hyaluronate cross-linked hydrogel has the advantages that the catalyst can be recycled, the enzymolysis resistance is good, and the safety is high.

Description

Technical Field

[0001] This invention relates to the field of polymer biomedical materials technology, specifically to a chondroitin sulfate sodium hyaluronate crosslinked hydrogel and its preparation method. Background Technology

[0002] Both sodium chondroitin sulfate and sodium hyaluronate are key polysaccharides in the human extracellular matrix, but they have inherent drawbacks: sodium hyaluronate is easily degraded by hyaluronidase and has weak mechanical properties, while sodium chondroitin sulfate has poor gel-forming ability and is easily hydrolyzed, making it difficult to meet the needs when used alone. The performance of single polysaccharide hydrogels is clearly limited; for example, sodium hyaluronate gels lack sufficient support, and sodium chondroitin sulfate cannot form a stable structure. However, the two have similar structures and complementary bioactivities, and their combination can simulate the extracellular matrix environment, laying a material foundation for research and development.

[0003] Early cross-linking of chondroitin sulfate sodium and sodium hyaluronate often involved physical cross-linking (ionic bonds, hydrogen bonds) or simple chemical cross-linking (glutaraldehyde, epichlorohydrin). However, this approach suffered from low cross-linking degree, weak mechanical properties, rapid degradation, and toxicity of residual cross-linking agents. 1,4-Butanediol diglycidyl ether, as a biepoxide cross-linking agent, can form stable ether bonds with the hydroxyl groups of chondroitin sulfate sodium or sodium hyaluronate, significantly improving the mechanical properties and enzymatic stability of the hydrogel. Furthermore, it exhibits low toxicity and is easily washable, making it one of the mainstream cross-linking technologies currently available.

[0004] Patent CN118546395B provides a method for preparing a polysaccharide-sodium hyaluronate composite gel, characterized by the following steps: dissolving sodium hyaluronate in an aqueous sodium hydroxide solution, adding a low-molecular-weight polysaccharide, then adding a crosslinking agent, reacting at 5-15℃ for 48-72 hours, and then reacting at 25-45℃ for 2-5 hours to obtain the polysaccharide-sodium hyaluronate composite gel. The low-molecular-weight polysaccharide is one or more of sodium hyaluronate, chondroitin sulfate, chondroitin, heparin, and cellulose; the crosslinking agent is one of 1,4-butanediol diglycidyl ether, 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1,2-ethylenediol diglycidyl ether. This invention provides a method that yields a gel product with good elasticity and easy injection in the simplest operation and fastest time, with good reproducibility, suitable for the preparation of different types of polysaccharide-sodium hyaluronate composite gels, and more conducive to industrial production. Patent CN112851988B provides a method for preparing sodium hyaluronate gel. This method includes steps such as dissolution, cross-linking reaction, pH adjustment, swelling, secondary sieving, and filling and sterilization. The method is simple and suitable for industrial production. The sodium hyaluronate gel prepared by this invention has advantages such as fine texture, good viscoelasticity, high resistance to enzymatic hydrolysis, and good biocompatibility. However, both of these preparation methods have the disadvantage of cross-linking agents easily remaining in the product, reducing product safety, and the preparation methods have long reaction times and high energy consumption.

[0005] Therefore, there is an urgent need in the market to develop a safe chondroitin sulfate sodium hyaluronate cross-linked hydrogel. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to develop a method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogels that have good safety, recyclable catalysts, and excellent resistance to enzymatic hydrolysis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel, comprising the following steps: S1. Dissolve sodium hyaluronate in a 0.5-2 wt% sodium hydroxide aqueous solution, then add sodium chondroitin sulfate, and then add dropwise a 10-30 wt% 1,4-butanediol diglycidyl ether ethanol solution and a phase transfer catalyst. First, react at a temperature of 5-10℃ for 2-4 h, and then crosslink at a temperature of 35-40℃ for 12-24 h to prepare a gel. S2. Cut the gel obtained in step S1 into small pieces, soak them in PBS buffer solution for 20-30 min, change the PBS buffer solution and soak until the pH of the last wash solution is 7.4±0.1, sieve, add it to PBS buffer solution again and homogenize at 3000-5000 rpm for 3-5 min, filter, sterilize, and obtain chondroitin sulfate sodium hyaluronate crosslinked hydrogel.

[0008] Preferably, the mass ratio of sodium hyaluronate to 0.5-2 wt% sodium hydroxide aqueous solution is 1:(5-10).

[0009] Traditional chondroitin sulfate sodium hyaluronate crosslinked hydrogels suffer from unstable crosslinking, resulting in poor resistance to enzymatic hydrolysis. Existing 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether) crosslinking agents require strict dosage control, and residual 1,4-butanediol diglycidyl ether molecules may be harmful to the human body. Therefore, this invention utilizes a phase transfer catalyst to achieve a "phase transfer-base synergistic" effect, increasing the conversion rate of 1,4-butanediol diglycidyl ether ethanol. This reduces the amount of free 1,4-butanediol diglycidyl ether ethanol in the hydrogel, thereby improving its safety.

[0010] In some embodiments, the number-average molecular weight of the sodium hyaluronate is 1 × 10⁻⁶. 6 Da~3×10 6 Da.

[0011] In some embodiments, the number-average molecular weight of the chondroitin sulfate sodium is 1 × 10⁻⁶. 4 Da~4×10 5 Da.

[0012] In some embodiments, the mass ratio of sodium hyaluronate to sodium chondroitin sulfate is 1:(0.1-0.5).

[0013] In some embodiments, the mass ratio of sodium hyaluronate to 1,4-butanediol diglycidyl ether is 1:(0.05-0.15).

[0014] This application improves the stability and safety of hydrogels in application by precisely controlling the amount of crosslinking agent and strict crosslinking reaction conditions, thereby enabling the crosslinking molecules of sodium chondroitin sulfate and sodium hyaluronate to form a more stable crosslinking structure.

[0015] In some embodiments, the method for preparing the phase transfer catalyst includes the following steps: A1. Add UiO-66-NH2 to a 0.5-2M aqueous solution of benzyltrimethylammonium chloride, stir and react at room temperature for 24-48 hours, centrifuge, wash and dry to obtain the loaded material; A2. Add the supported material obtained in step A1 to a 0.1-1M sodium hydroxide methanol solution, stir at room temperature under an inert atmosphere for 8-12 hours, wash until neutral, and dry to obtain a phase transfer catalyst.

[0016] Preferably, the mass ratio of the loading material and the 0.1-1M sodium hydroxide methanol solution in step A2 is 1:(5-15).

[0017] Preferably, the inert atmosphere is nitrogen.

[0018] This application utilizes a method where benzyltrimethylammonium chloride is first loaded onto UiO-66-NH2 and then added to a sodium hydroxide solution, causing partial replacement of -Cl and -OH groups to form benzyltrimethylammonium hydroxide. This avoids the problem of the UiO-66-NH2 structure being destroyed by strong alkalinity when directly loading benzyltrimethylammonium hydroxide onto UiO-66-NH2, thereby increasing the loading capacity of the phase transfer catalyst on UiO-66-NH2. Furthermore, benzyltrimethylammonium hydroxide exhibits better catalytic performance compared to benzyltrimethylammonium chloride. Moreover, after the phase transfer catalyst is loaded onto UiO-66-NH2, the high specific surface area and regular pores of UiO-66-NH2 ensure highly dispersed and fully exposed sites for benzyltrimethylammonium hydroxide, reducing mass transfer resistance and increasing the reaction rate at the two-phase interface, further enhancing the catalytic effect. After the reaction, the phase transfer catalyst can be recovered by filtration, washed with PBS, and reused in the next batch, reducing the consumption and residue of phase transfer catalyst and crosslinking agent while meeting the requirements of green production.

[0019] During the synthesis of hydrogels, phase transfer catalysts can lower the apparent activation energy of epoxy ring-opening, enabling the reaction to be completed at lower temperatures and faster times, thus reducing energy consumption and costs. Simultaneously, under the action of phase transfer catalysts, the cross-linked network on the hydrogel becomes more uniform, resulting in a lower swelling ratio, increased compressive strength, and further improved resistance to enzymatic hydrolysis.

[0020] In some embodiments, the mass ratio of the UiO-66-NH2 and the 0.5-2M benzyltrimethylammonium chloride aqueous solution is 1:(10-15).

[0021] In some embodiments, the mass ratio of sodium hyaluronate to phase transfer catalyst is 1:(0.10-0.25).

[0022] In some embodiments, the pH of the PBS buffer solution is 6.5-7.0.

[0023] In some embodiments, the mesh size of the sieve in the sieving operation described in step S2 is 150-180 mesh.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention develops a method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel. The method involves reacting sodium hyaluronate, sodium chondroitin sulfate, and a crosslinking agent under the catalysis of a phase transfer catalyst. After the reaction, the reactants are washed in PBS buffer and filtered to obtain the chondroitin sulfate sodium hyaluronate crosslinked hydrogel. The prepared chondroitin sulfate sodium hyaluronate crosslinked hydrogel has the advantages of catalyst recyclability, low cost, excellent resistance to enzymatic hydrolysis, and high safety.

[0025] 2. This invention first loads benzyltrimethylammonium chloride onto UiO-66-NH2 and then adds it to a sodium hydroxide solution to allow some of the -Cl and -OH groups to be replaced to form benzyltrimethylammonium hydroxide. This avoids the strong alkalinity of benzyltrimethylammonium hydroxide destroying the UiO-66-NH2 structure, thereby increasing the loading of the phase transfer catalyst on UiO-66-NH2. Furthermore, benzyltrimethylammonium hydroxide has a better catalytic effect than benzyltrimethylammonium chloride.

[0026] 3. The phase transfer catalyst prepared in this invention can reduce the apparent activation energy of epoxy ring opening during the synthesis of gel, enabling the reaction to be completed at a lower temperature and faster time, reducing energy consumption and cost. Under the action of the phase transfer catalyst, the cross-linked network on the hydrogel is more uniform, which reduces its swelling rate, increases its compressive strength, and further improves its resistance to enzymatic hydrolysis. Detailed Implementation

[0027] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0028] In the following examples, the compounds and related reagents used were all commercially available, wherein the number average molecular weight of sodium hyaluronate was 1 × 10⁻⁶. 6 Da; The number-average molecular weight of chondroitin sulfate sodium is 3 × 10⁻⁶. 4 Da;UiO-66-NH2 was prepared according to Example 1 of patent CN114471731B.

[0029] Preparation Example 1 The preparation method of phase transfer catalyst-1 includes the following steps: A1. Add 10g UiO-66-NH2 to 120g of 1M benzyltrimethylammonium chloride aqueous solution, stir and react at room temperature for 36h, centrifuge, wash with deionized water and dry to obtain the loaded material; A2. Add 10g of the supported material obtained in step A1 to 100g of 0.5M sodium hydroxide methanol solution, stir at room temperature under nitrogen for 10h, wash with deionized water until neutral, and dry to obtain phase transfer catalyst-1.

[0030] Preparation Example 2 The preparation method of phase transfer catalyst-2 is the same as that of preparation example 1, except that the amount of 1M benzyltrimethylammonium chloride aqueous solution added is 170g.

[0031] Preparation Example 3 The preparation method of phase transfer catalyst-3 includes the following steps: 10g UiO-66-NH2 is added to 120g of 1M benzyltrimethylammonium hydroxide aqueous solution, the mixture is stirred and reacted at room temperature for 36h, centrifuged, washed with deionized water, and dried to obtain phase transfer catalyst-3.

[0032] Example 1 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel includes the following steps: S1. Dissolve 10g of sodium hyaluronate in 70g of 1wt% sodium hydroxide aqueous solution, then add 3g of sodium chondroitin sulfate, and then add 5g of 20wt% 1,4-butanediol diglycidyl ether ethanol solution and 1.5g of phase transfer catalyst-1 at a rate of 2 drops / s. First, react at 7℃ for 3h, and then crosslink at 37℃ for 18h to prepare a gel. S2. Cut 10g of the gel obtained in step S1 into small pieces, add 50g of PBS buffer solution with pH 6.8 and soak for 25min. Replace the PBS buffer solution and soak until the pH of the last wash solution is 7.4. Sieve through a 160-mesh sieve, add 100g of PBS buffer solution and homogenize at 4000rpm for 4min. Filter and sterilize at 121℃ for 30min to obtain sodium chondroitin sulfate and sodium hyaluronate crosslinked hydrogel.

[0033] Example 2 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel includes the following steps: S1. Dissolve 10g of sodium hyaluronate in 50g of 2wt% sodium hydroxide aqueous solution, then add 1g of sodium chondroitin sulfate, and then add 3.34g of 30wt% 1,4-butanediol diglycidyl ether ethanol solution and 1g of phase transfer catalyst-1 at a rate of 2 drops / s. First, react at 5℃ for 4h, and then crosslink at 35℃ for 24h to prepare a gel. S2. Cut 10g of the gel obtained in step S1 into small pieces, soak them in PBS buffer solution with pH 6.5 for 20 min, change the PBS buffer solution and soak until the pH of the last wash solution is 7.3, sieve it through a 160-mesh sieve, add it to PBS buffer solution and homogenize it at 3000 rpm for 5 min, filter it, and sterilize it at 121℃ for 30 min to obtain sodium chondroitin sulfate and sodium hyaluronate crosslinked hydrogel.

[0034] Example 3 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel includes the following steps: S1. Dissolve 10g of sodium hyaluronate in 100g of 0.5wt% sodium hydroxide aqueous solution, then add 5g of sodium chondroitin sulfate, and then add 10g of 10wt% 1,4-butanediol diglycidyl ether ethanol solution and 2.5g of phase transfer catalyst-1 at a rate of 2 drops / s. First, react at 10℃ for 2h, then crosslink at 40℃ for 12h to prepare a gel. S2. Cut 10g of the gel obtained in step S1 into small pieces, add it to a PBS buffer solution with pH 7.0 and soak for 30 min. Replace the PBS buffer solution and soak until the pH of the last wash solution is 7.5. Sieve the gel through a 160-mesh sieve, add it to the PBS buffer solution and homogenize at 5000 rpm for 3 min. Filter the solution and sterilize at 121℃ for 30 min to obtain sodium chondroitin sulfate and sodium hyaluronate crosslinked hydrogel.

[0035] Example 4 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel is described, with the specific implementation method being the same as in Example 1, except that the amount of phase transfer catalyst-1 added is 3.5g.

[0036] Example 5 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel is described. The specific implementation method is the same as in Example 1, except that phase transfer catalyst-1 is replaced by phase transfer catalyst-2 in equal amounts.

[0037] Example 6 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel is described. The specific implementation method is the same as in Example 1, except that phase transfer catalyst-1 is replaced by phase transfer catalyst-3 in equal amounts.

[0038] Example 7 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel is described, with the specific implementation method being the same as in Example 1, except that phase transfer catalyst-1 is replaced by an equal amount of benzyltrimethylammonium hydroxide.

[0039] Comparative Example 1 A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel includes the following steps: S1. Dissolve 10g of sodium hyaluronate in 70g of 1wt% sodium hydroxide aqueous solution, then add 3g of sodium chondroitin sulfate, and then add 1g of 20wt% 1,4-butanediol diglycidyl ether ethanol solution at a rate of 2 drops / s. First, react at 7℃ for 3h, and then crosslink at 37℃ for 18h to prepare a gel. S2. Cut 10g of the gel obtained in step S1 into small pieces, add 50g of PBS buffer solution with pH 6.8 and soak for 25min. Replace the PBS buffer solution and soak until the pH of the last wash solution is 7.4. Sieve through a 160-mesh sieve, add 100g of PBS buffer solution and homogenize at 4000rpm for 4min. Filter and sterilize at 121℃ for 30min to obtain sodium chondroitin sulfate and sodium hyaluronate crosslinked hydrogel.

[0040] Performance testing The following performance tests were performed on the chondroitin sulfate sodium hyaluronate crosslinked hydrogels obtained in the above embodiments and comparative examples: (1) Viscoelasticity: The cross-linked hydrogel was placed on a rotational rheometer. In the flat plate mode, the frequency scan (0.01Hz~100Hz) was selected to test the viscoelasticity of the gel at 0.1Hz, namely the storage modulus and loss modulus.

[0041] (2) Enzyme resistance: The enzyme degradation rate was tested according to the [0057-0063] section of patent CN102863631B.

[0042] The test results are shown in Table 1: Table 1 As shown in Table 1, the chondroitin sulfate sodium hyaluronate crosslinked hydrogels prepared in Examples 1-3 have high storage modulus, loss modulus, and low enzymatic hydrolysis rate, indicating that they have extremely high strength, i.e., high crosslinking degree and enzymatic hydrolysis resistance. A comparison between Example 4 and Example 1 shows that changing the ratio of sodium hyaluronate to the phase transfer catalyst may create physical isolation, hindering the contact between hyaluronic acid molecular chains and the crosslinking agent, leading to uneven local crosslinking degree and the formation of "soft spots," thus resulting in decreased strength and enzymatic hydrolysis resistance of the chondroitin sulfate sodium hyaluronate crosslinked hydrogel. A comparison between Example 5 and Example 1 shows that changing the ratio of UiO-66-NH2 to benzyltrimethylammonium chloride causes excessive benzyltrimethylammonium chloride molecules to block the regular channels of UiO-66-NH2. The catalyst hinders the diffusion of reactants to internal active sites, leading to a decrease in apparent activity and consequently a deterioration in the strength and enzymatic resistance of the chondroitin sulfate-sodium hyaluronate crosslinked hydrogel. A comparison of Example 6 and Example 1 shows that using UiO-66-NH2 directly loaded with benzyltrimethylammonium hydroxide makes UiO-66-NH2 easily corroded, reducing the catalyst loading and thus worsening the strength and enzymatic resistance of the chondroitin sulfate-sodium hyaluronate crosslinked hydrogel. A comparison of Example 7 and Example 1 shows that directly adding benzyltrimethylammonium hydroxide worsens the strength and enzymatic resistance of the chondroitin sulfate-sodium hyaluronate crosslinked hydrogel. A comparison of Comparative Example 1 and Example 1 shows that without the addition of a phase transfer catalyst, the strength and enzymatic resistance of the chondroitin sulfate-sodium hyaluronate crosslinked hydrogel are relatively poor.

[0043] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a chondroitin sulfate sodium hyaluronate crosslinked hydrogel, characterized in that, Includes the following steps: S1. Dissolve sodium hyaluronate in a 0.5-2 wt% sodium hydroxide aqueous solution, then add sodium chondroitin sulfate, and then add dropwise a 10-30 wt% 1,4-butanediol diglycidyl ether ethanol solution and a phase transfer catalyst. First, react at a temperature of 5-10℃ for 2-4 h, and then crosslink at a temperature of 35-40℃ for 12-18 h to prepare a gel. S2. Cut the gel obtained in step S1 into small pieces, soak them in PBS buffer solution for 20-30 min, change the PBS buffer solution and soak until the pH of the last wash solution is 7.4±0.1, sieve, add it to PBS buffer solution again and homogenize at 3000-5000 rpm for 3-5 min, filter, sterilize, and obtain chondroitin sulfate sodium hyaluronate crosslinked hydrogel.

2. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The number-average molecular weight of the sodium hyaluronate is 1×10⁻⁶. 6 Da~3×10 6 Da.

3. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The number-average molecular weight of the chondroitin sulfate sodium is 1×10⁻⁶. 4 Da~4×10 5 Da.

4. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The mass ratio of sodium hyaluronate to sodium chondroitin sulfate is 1:(0.1-0.5).

5. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The mass ratio of sodium hyaluronate to 1,4-butanediol diglycidyl ether is 1:(0.05-0.15).

6. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The preparation method of the phase transfer catalyst includes the following steps: A1. Add UiO-66-NH2 to a 0.5-2M aqueous solution of benzyltrimethylammonium chloride, stir and react at room temperature for 24-48 hours, centrifuge, wash and dry to obtain the loaded material; A2. Add the supported material obtained in step A1 to a 0.1-1M sodium hydroxide methanol solution, stir at room temperature under an inert atmosphere for 8-12 hours, wash until neutral, and dry to obtain a phase transfer catalyst.

7. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 6, characterized in that, The mass ratio of the UiO-66-NH2 and the 0.5-2M benzyltrimethylammonium chloride aqueous solution is 1:(10-15).

8. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The mass ratio of sodium hyaluronate to phase transfer catalyst is 1:(0.10-0.25).

9. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, The pH of the PBS buffer solution is 6.5-7.

0.

10. The method for preparing chondroitin sulfate sodium hyaluronate crosslinked hydrogel according to claim 1, characterized in that, In step S2, the sieve mesh size is 150-180 mesh.

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