A method for preparing a polysaccharide sodium hyaluronate composite gel by a pre-crosslinking method

Abstract

The application relates to a method for preparing a polysaccharide sodium hyaluronate composite gel by a pre-crosslinking method, and belongs to the technical field of high polymer material preparation.The method provided by the application is characterized in that low molecular polysaccharides are pre-crosslinked, then sodium hyaluronate, a gel intermediate and a crosslinking agent are secondarily crosslinked, and finally a composite gel is prepared.The method provided by the application is simple in operation, can effectively crosslink the low molecular polysaccharides, improves the low molecular polysaccharide content in the final product, and is beneficial to improving the biological performance of the gel.

Description

Technical Field

[0001] This invention belongs to the field of polymer material preparation technology, specifically relating to a method for preparing polysaccharide sodium hyaluronate composite gel by a pre-crosslinking method. Background Technology

[0002] Hyaluronic acid (HA) is a linear high-molecular-weight polysaccharide composed of glucuronic acid and glucosamine as disaccharide units. It exists in many connective tissues such as skin, vitreous humor, cartilage, and synovial fluid, playing physiological roles in moisturizing, nourishing, repairing, and preventing damage. To date, there are numerous HA clinical products, including ophthalmic viscoelastic agents and orthopedic viscoelastic supplements, intra-articular injections for treating arthritis, postoperative adhesion prevention agents, and facial dermal fillers.

[0003] Low molecular weight glycosaminoglycans, such as chondroitin sulfate (CS), dermatan sulfate (DS), and heparin, are a class of biomaterials with excellent properties. It has been reported that low molecular weight glycosaminoglycans can reduce the viscosity of composite gels, thereby reducing injection pressure and offering significant advantages in the field of cosmetic fillers. Furthermore, sulfated glycosaminoglycans such as chondroitin sulfate also possess excellent antibacterial, anti-inflammatory, and wound-healing-promoting effects. Therefore, increasing the effective content of low molecular weight glycosaminoglycans is key to maximizing the advantages of hyaluronic acid composite gels.

[0004] Currently, there is limited attention paid to the actual cross-linking content of low molecular weight glycosaminoglycans in composite cross-linked gels, especially sulfated glycosaminoglycans. Due to the presence of sulfate groups, there are fewer active sites, greater steric hindrance, and lower cross-linking efficiency, hindering the formation of effective cross-linking and interpenetrating networks of low molecular weight glycosaminoglycans in the composite gel, thus impeding the performance synergy of the cross-linked composite gel. Existing technologies modify CS and HA (methacrylamide or aldehyde) to enable spontaneous cross-linking. However, this pretreatment is complex, and the degree and effect of modification require further investigation, as it can damage the molecular weight of HA and CS. In WO2021137837A1, chondroitin sulfate and non-cross-linked hyaluronic acid are added to cross-linked hyaluronic acid gels to promote extrusion; however, in gels obtained using this method, chondroitin sulfate is mostly in a free state, failing to exert its biological effects over a long period.

[0005] Another method is a one-step preparation of composite cross-linked gels. Patent EP2011816B1 dissolves hyaluronic acid and chondroitin sulfate simultaneously in an alkaline solution and cross-links them via BDDE. Because BDDE preferentially reacts with hyaluronic acid, which has less steric hindrance and more cross-linking sites, in an alkaline environment, most of the chondroitin sulfate remains uncross-linked and in a free state. This results in a low effective cross-linking content of chondroitin sulfate, which is detrimental to improving biocompatibility and gel retention time.

[0006] Therefore, a new method for preparing composite gels is needed to increase the content of low molecular weight glycosaminoglycans and the degree of effective cross-linking in composite gels. Summary of the Invention

[0007] In view of the above technical background, the present invention provides a method for preparing polysaccharide sodium hyaluronate composite gel. This method is a novel method suitable for industrial production. It is simple to operate and can effectively improve the effective cross-linking content of low molecular weight glycosaminoglycans in the cross-linked gel.

[0008] This invention provides a method for preparing a polysaccharide-sodium hyaluronate composite gel, which includes the following steps:

[0009] 1) Pre-crosslinking: Low molecular weight polysaccharides are added to an alkaline solution, and a portion of a crosslinking agent is added to pre-crosslink the low molecular weight polysaccharides to obtain a gel intermediate;

[0010] 2) Secondary crosslinking: Sodium hyaluronate is dissolved in an alkaline solution, and gel intermediates and remaining crosslinking agents are added to obtain a mixture. Secondary crosslinking is then performed to obtain a composite gel.

[0011] The low molecular weight polysaccharide is selected from one or more of chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratin sulfate (KS), heparin, sodium sulfated hyaluronic acid, and other sulfated glycosaminoglycans.

[0012] In some embodiments, the low molecular weight polysaccharide is preferably one or more of chondroitin sulfate, dermatin sulfate, and sodium hyaluronate sulfate.

[0013] The crosslinking agent is selected from any one of 1,4-butanediol diglycidyl ether (BDDE), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1,2-ethylenediol diglycidyl ether; preferably BDDE.

[0014] Furthermore, the alkaline solution is an aqueous solution of sodium hydroxide.

[0015] Furthermore, the mass concentration of the sodium hydroxide aqueous solution is 0.5wt% to 3wt%, preferably 0.5wt% to 2wt%.

[0016] Furthermore, the molecular weight of the low-molecular-weight polysaccharide is 1×10⁻⁶. 4 Da~5×10 5 Da, preferably 1×10 4 Da~1×10 5 Da, further preferably 1×10 4 Da~8×10 4 Da.

[0017] Furthermore, the molecular weight of the sodium hyaluronate is in the range of 1×10⁻⁶. 6 Da~3×10 6 Da, preferably 2×10 6 Da~3×10 6 Da.

[0018] Further, in step 1), the ratio of the volume (mL) of the alkaline solution to the mass (g) of the low molecular weight polysaccharide in the pre-crosslinking process is 1mL:(0.1-0.8)g, preferably 1mL:(0.2-0.6)g.

[0019] Further, the mass ratio of the crosslinking agent to the total of sodium hyaluronate and low molecular weight polysaccharide is (0.05-0.2):1, preferably (0.08-0.15):1. In step 1), the molar amount of the crosslinking agent added during pre-crosslinking accounts for 50%-100% of the total molar amount of the crosslinking agent, preferably 55%-100%.

[0020] Furthermore, the pre-crosslinking time is 1h to 9h, preferably 2h to 7h.

[0021] Furthermore, the pre-crosslinking temperature is 30℃~60℃, preferably 40℃~60℃.

[0022] Furthermore, the concentration (mass / volume, w / v) of the sodium hyaluronate in the alkaline solution is 10% to 25%, preferably 14% to 20%.

[0023] Furthermore, the mass ratio of sodium hyaluronate to low molecular weight polysaccharide is (1-10):1, preferably (1.5-5):1.

[0024] According to the present invention, the secondary crosslinking can be completed by the following methods: the mixture is first reacted at temperature A, and then crosslinked at temperature B to prepare a composite gel; or the mixture is reacted at a temperature of 40°C to 50°C to obtain a composite gel.

[0025] Furthermore, the temperature A can be 0℃ to 20℃, preferably 5℃ to 12℃.

[0026] Further, the mixture is reacted at temperature A for 24 h to 72 h, preferably 36 h to 60 h.

[0027] Further, the temperature B is 30℃~60℃, preferably 40℃~60℃, and even more preferably 50℃.

[0028] Furthermore, the mixture is reacted at temperature B for 2 to 10 hours, preferably 3 to 5 hours.

[0029] In some embodiments, the preparation method further includes: dialyzing the gel obtained after secondary crosslinking in PBS buffer. In some embodiments, the preparation method further includes: soaking the gel obtained after secondary crosslinking in PBS buffer.

[0030] In some embodiments, the preparation method further includes: cutting the gel obtained after secondary crosslinking into small pieces, soaking it in PBS buffer solution, replacing the PBS buffer solution, and soaking it thoroughly to remove unreacted crosslinking agent; then sieving the gel into small gel particles through a 160-mesh sieve, and then mechanically homogenizing it to prepare a polysaccharide sodium hyaluronate composite gel.

[0031] In some embodiments, the preparation method further includes: cutting the gel obtained after secondary crosslinking into small pieces, soaking them in a PBS buffer solution containing hydrochloric acid, replacing the PBS buffer solution, and thoroughly soaking to remove unreacted crosslinking agents; then sieving the gel into small gel particles through a 160-mesh sieve, and then mechanically homogenizing to prepare a polysaccharide sodium hyaluronate composite gel.

[0032] To obtain a sterile gel or a gel that meets the quality requirements for injection use, the composite gel can be sterilized, such as by terminal sterilization, moist heat sterilization, or irradiation sterilization. Alternatively, it can be prepared using sterile raw materials under sterile conditions to obtain an injectable composite gel.

[0033] The PBS buffer solution may be prepared from one or more of sodium chloride, potassium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate, and water. In some embodiments, the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and water. In some embodiments, the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and water.

[0034] The pH of the PBS buffer solution can be 6.5–7.5. In some embodiments, the pH of the PBS buffer solution is 6.5–6.8. In some embodiments, the pH of the PBS buffer solution is 6.8–7.4.

[0035] The second aspect of the present invention provides the role of the polysaccharide sodium hyaluronate composite gel in the preparation of a medical aesthetic injectable for separating, replacing or filling biological tissues, or increasing the volume of biological tissues, or for filling wrinkles, concealing scars, or increasing lip volume.

[0036] The advantages of the preparation method provided by this invention are mainly as follows:

[0037] 1. This invention utilizes a two-step pre-crosslinking method to prepare composite gels, which can increase the effective crosslinking content of sulfated glycosaminoglycans (GGAs) in the gel. Due to configuration and steric hindrance of sulfate groups, BDDEs typically preferentially crosslink with HAs, resulting in low crosslinking content of sulfated glycosaminoglycans in the gel, with most existing in a free state. Existing preparation methods cannot avoid the problem of difficult crosslinking of sulfated glycosaminoglycans in composite gels. This invention solves the above problem by first preparing a polysaccharide crosslinking gel intermediate and then mixing it with an HA solution for secondary crosslinking. This process ensures effective chemical crosslinking of sulfated glycosaminoglycans, improves bidirectional crosslinking with HA chains, forms an interpenetrating crosslinking network, and thus increases the sulfated glycosaminoglycan content in the final product. Compared with other methods, this scheme can increase the percentage of crosslinked sulfated glycosaminoglycans in the gel to 20%–85%, which is 2–12 times higher than other existing technologies. While increasing the effective crosslinking ratio of sulfated glycosaminoglycans, the strength of the gel is also significantly improved compared to the one-step method.

[0038] 2. This invention uses a two-step pre-crosslinking method to prepare composite gels, which offers more controllable factors compared to a one-step method, allowing for gel customization. This method is also applicable to the preparation of various sulfated glycosaminoglycans and modified polysaccharide composite crosslinked samples. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0040] Unless otherwise specified, all raw materials or reagents used in the examples are commercially available.

[0041] The room temperature described in the examples is between 20°C and 30°C.

[0042] Unless otherwise specified, the reagents described are used directly without purification. All reagents were purchased from commercial suppliers, such as Aldrich, and are ready for use without processing.

[0043] In the following examples / comparative examples, the PBS buffer solution was prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and water, with a concentration of 0.01 mol / L and a pH of 6.8.

[0044] Example 1: Effect of crosslinking time on gel properties

[0045] Dissolve 2.80 g of CS (molecular weight 46,000 Da) in 5.6 mL of 1 wt% NaOH. After the CS is completely dissolved, add 0.8350 g of BDDE according to the crosslinking agent dosage of 17.4% total crosslinking degree, and continue stirring for 0.5 h; crosslink the CS solution at 50 °C for a period of time (pre-crosslinking);

[0046] 4.80 g of HA (molecular weight 2.3 million Da) powder was slowly added to 24.4 mL of 1 wt% NaOH solution and stirred until dissolved to obtain HA solution;

[0047] The CS crosslinking solution obtained after pre-crosslinking was slowly added to the HA solution, and the mixture was stirred rapidly to ensure complete mixing (total HA concentration: 16%, w / v), followed by further stirring for 30 min. The HA / CS mixture was then allowed to crosslink at 10℃ for 48 h, followed by a second crosslinking process at 50℃. The crosslinked gel was then dialyzed in PBS buffer for 36 h, with the PBS buffer changed three times during this period. After dialyzing, the sample was homogenized using a 160-mesh sieve to obtain a composite gel.

[0048] The composite gels obtained under various experimental conditions were partially degraded and then tested. 1 H NMR was used to calculate the degree of crosslinking R (R = number of moles of BDDE / (total number of moles of HA / CS disaccharide units) through the characteristic peaks of BDDE and HA / CS disaccharide units. A portion of the gel was taken to detect the total HA content, free HA content, total CS content, and free CS content. The effective crosslinking ratio of HA and the effective crosslinking ratio of CS were calculated respectively, using the formula (1 - free content / total content) * 100%. A portion of the gel was placed on a rotational rheometer, and in plate mode, with a fixed strain of 0.01%, a frequency scan (0.01Hz to 100Hz) was selected to test the viscoelasticity of the gel at 0.1Hz.

[0049] The specific parameters and experimental results of each experiment in Example 1 are shown in Table 1.

[0050] Table 1: Process conditions and gel detection results for each experiment in Example 1

[0051]

[0052] Example 2: Effect of BDDE addition method on gel properties

[0053] 2.80 g of CS (molecular weight 46,000 Da) was dissolved in 5.6 mL of 1 wt% NaOH to obtain a CS solution; BDDE (0.6670 g, 0.5000 g, and 0.3840 g) was added to the CS solution according to the amount of crosslinking agent used, with a pre-crosslinking degree of 40%, 30%, and 23%, respectively, and crosslinking was carried out at 50 °C for 3 h.

[0054] 4.8 g of HA (molecular weight 2.3 million Da) was dissolved in 24.4 mL of 1 wt% NaOH to obtain an HA solution;

[0055] The CS crosslinking solution was then thoroughly mixed with the HA solution (total HA concentration 16%, w / v). Using 0.6670 g of BDDE as the total crosslinking agent (13.9% crosslinking degree), the remaining BDDE was added to the HA / CS mixture and stirred for 10 min. The HA / CS mixture was then allowed to crosslink at 10℃ for 48 h, followed by a further increase to 50℃ for 3 h. The crosslinked gel was then dialyzed in PBS buffer for 36 h, with the PBS buffer changed three times to remove residual small molecules and impurities. After dialyzing, the sample was homogenized using a 160-mesh sieve to obtain the composite gel.

[0056] The same method as in Example 1 was used to test various performance parameters; the specific parameters and experimental results of each experiment in Example 2 are shown in Table 2.

[0057] Table 2: Process conditions and gel detection results for each experiment in Example 2

[0058]

[0059] Based on the differences between the results of Experiment 2-1 and the results of Experiments 2-2 and 2-3, it can be determined that, under the condition of the same total crosslinking degree, adding BDDE in two steps can improve the effective crosslinking ratio and gel strength of HA compared to adding BDDE all at once.

[0060] Example 3: Effect of HA concentration on gel properties

[0061] 2.80 g of CS (molecular weight 46,000 Da) was dissolved in 5.6 mL of 1 wt% NaOH to obtain CS solution; according to the amount of crosslinking agent with a pre-crosslinking degree of 23%, 0.3840 g of BDDE was added to the CS solution, and the CS solution was crosslinked at 50 °C for 3 h.

[0062] 4.80 g of HA (molecular weight 2.3 million Da) was dissolved in 21.1 mL and 18.4 mL of 1 wt% NaOH, respectively, to obtain HA solutions;

[0063] The CS crosslinking solution and HA solution were then mixed thoroughly (total HA concentrations were 18% and 20%, w / v, respectively). The remaining BDDE was added to the HA / CS mixture, with a total crosslinking degree of 13.9% calculated as 0.6670 g of BDDE, and the mixture was stirred for 10 min. The HA / CS mixture was then allowed to crosslink at 10 °C for 48 h, followed by a further crosslinking at 50 °C for 3 h. The crosslinked gel was then dialyzed in PBS buffer for 36 h, with the PBS buffer changed three times to remove residual small molecules and impurities. After dialyzing, the sample was homogenized using a 160-mesh sieve to obtain the composite gel.

[0064] The same method as in Example 1 was used to test various performance parameters; the specific parameters and experimental results of each experiment in Example 3 are shown in Table 3.

[0065] Table 3: Process conditions and gel detection results for each experiment in Example 3

[0066]

[0067] According to the results of experiments 2-3 and 3-1, as the HA concentration increases, the HA chains become more tightly wrapped, the gel strength increases, and the effective cross-linking ratio of HA increases. In addition, HA chains can also form a denser double network structure with CS, effectively increasing the effective cross-linking ratio of CS. According to the results of experiments 3-1 and 3-2, when the HA concentration is too high, the solution hardness is high, which is not conducive to the mixing of CS and HA raw materials, and the effective cross-linking ratio of CS decreases slightly.

[0068] Example 4

[0069] 2.80 g of CS (molecular weight 46,000 Da) and 2.80 g of DS (molecular weight 35,000 Da) were dissolved in 4.7 mL of 1 wt% NaOH. After the raw materials were completely dissolved, 0.8350 g of BDDE was added, and stirring was continued for 0.5 h; the raw material solution was then crosslinked at 50 °C for 3 h.

[0070] 4.80 g of HA (molecular weight 2.3 million Da) powder was slowly added to 25.3 mL of 1 wt% NaOH solution and stirred until dissolved to obtain HA solution;

[0071] The pre-crosslinked low-molecular-weight polysaccharide gel intermediate was slowly added to the HA solution, and the mixture was stirred rapidly to ensure complete incorporation (total HA concentration: 16%, w / v), followed by continued stirring for 30 min. The HA / CS mixture was then heated to 50 °C for further crosslinking for 3 h. The crosslinked gel was then dialyzed in PBS buffer for 36 h, with the PBS buffer being changed three times during this period. After dialyzing, the sample was homogenized using a 160-mesh sieve to obtain the composite gel.

[0072] The same method as in Example 1 was used to test various performance parameters; the specific parameters and experimental results of each experiment in Example 4 are shown in Table 4.

[0073] Table 4: Process conditions and gel detection results for each experiment in Example 4

[0074]

[0075] Example 5

[0076] 2.80 g of CS (molecular weight 46,000 Da) was dissolved in 5.6 mL of 1 wt% NaOH to obtain a CS solution; 0.3840 g of BDDE was added to the CS solution with a crosslinking agent dosage of 23% for pre-crosslinking degree, and the CS solution was crosslinked at 50 °C for 3 h.

[0077] 4.80 g of HA (molecular weight 2.3 million Da and 1.5 million Da) were dissolved in 21.1 mL of 1 wt% NaOH to obtain HA solutions;

[0078] The CS crosslinking solution was then thoroughly mixed with the HA solution (total HA concentration 18%, w / v). Using 0.6670 g of BDDE as the total crosslinking agent (13.9% crosslinking degree), the remaining BDDE was added to the HA / CS mixture and stirred for 10 min. The mixture was then allowed to stand at 10℃ for 48 h for crosslinking, followed by a further increase to 50℃ for 3 h. The crosslinked gel was then immersed in PBS buffer for 40 h, with the PBS buffer changed four times to remove residual small molecules and impurities. After immersion, the sample was homogenized using a 160-mesh sieve to obtain the composite gel.

[0079] The same method as in Example 1 was used to test various performance parameters; the specific parameters and experimental results of each experiment in Example 5 are shown in Table 5.

[0080] Table 5: Process conditions and gel detection results for each experiment in Example 5

[0081]

[0082] According to the results of experiments 3-1 and 5-1, compared with dialysis purification, soaking purification can remove most of the free raw materials and increase the effective cross-linking ratio of HA and CS in the final product. According to the results of experiments 3-1 and 5-2, the gel strength decreases by about 39% when the molecular weight of HA decreases. Due to the better solubility of low molecular weight raw materials, the degree of cross-linking increases, and the effective cross-linking ratio of HA and CS increases.

[0083] Comparative Example 1: One-Step Method

[0084] 4.80 g of sodium hyaluronate powder (molecular weight 2.3 million Da) was added to 26.7 mL of 1 wt% NaOH solution. After the sodium hyaluronate dissolved evenly, 2.80 g of CS (molecular weight 46,000 Da) was slowly added and stirred until a light yellow transparent liquid was obtained. 0.6670 g of BDDE was then added, and the mixture was stirred for 30 min. The mixture was then reacted at 10 °C for 48 h, followed by a reaction at 50 °C for 3 h. The cross-linked gel was dialyzed in PBS buffer for 36 h, with the PBS buffer changed three times during this period to remove residual small molecules and impurities. After dialyzing, the sample was homogenized using a 160-mesh sieve.

[0085] The same method as in Example 1 was used to test various performance parameters; the relevant experimental parameters and test results are shown in Table 6.

[0086] Comparative Example 2CS as a Free Additive

[0087] 4.80 g of sodium hyaluronate powder (molecular weight 2.3 million Da) was added to 26.7 mL of 1 wt% NaOH solution and stirred until dissolved. 0.6670 g of BDDE was added, and the mixture was stirred for 30 min. The reaction was then carried out at 10 °C for 48 h, followed by a reaction at 50 °C for 3 h. The cross-linked gel was dialyzed in PBS buffer to remove residual small molecules and impurities. After dialyzing, the sample was homogenized using a 160-mesh sieve to obtain HA gel.

[0088] Prepare a 2wt% CS solution by dissolving CS (molecular weight 46,000 Da) in PBS buffer. Take an appropriate amount of homogenized HA gel, add the 2wt% CS solution, and stir for 30 min; control the mass ratio of HA to CS to be 1.7:1, and the total concentration of HA and CS to be between 20 mg / mL and 30 mg / mL.

[0089] The same method as in Example 1 was used to test various performance parameters; the relevant experimental parameters and test results are shown in Table 6.

[0090] Comparative examples: 3HA pre-crosslinking, CS-added secondary crosslinking

[0091] 4.80 g of sodium hyaluronate powder (molecular weight 2.3 million Da) was added to 21.1 mL of 1 wt% NaOH solution and stirred until dissolved. 0.3840 g of BDDE was weighed out as a pre-crosslinking agent (23%) and added to the HA solution. The mixture was then crosslinked at 50 °C for 3 h. 2.80 g of CS (molecular weight 46,000 Da) was weighed out and dissolved in 5.6 mL of 1 wt% NaOH solution. This solution was then added to the HA crosslinking solution and stirred until dissolved. The remaining BDDE was added to the HA / CS mixture, with a total crosslinking degree of 13.9% calculated as 0.6670 g of BDDE. The mixture was stirred for 10 min. The mixture was allowed to stand at 10 °C for 48 h for crosslinking, then heated to 50 °C for another 3 h. The crosslinked gel was then dialyzed in PBS buffer for 36 h, with the PBS buffer changed three times during this period to remove residual small molecules and impurities. After dialysis, the sample was homogenized using a 160-mesh sieve.

[0092] The same method as in Example 1 was used to test various performance parameters; the relevant experimental parameters and test results are shown in Table 6.

[0093] Table 6: Process conditions and gel detection results for each comparative example

[0094]

[0095] The experimental results show that, compared with the two-step pre-crosslinking method provided by this invention, the effective crosslinking ratio of CS in the comparative examples is relatively low. The gels prepared by the pre-crosslinking methods in Experiments 3-1 and 5-1 show significant improvements in both the effective crosslinking ratio of CS and strength compared to the gels prepared in Comparative Examples 1 and 2. Compared with Comparative Example 3, the strengths are similar, but the effective crosslinking ratio of CS is increased by nearly 30% and 60%, respectively. The gels obtained by the method provided by this invention help improve the biocompatibility of the composite gel and demonstrate the excellent effects of low-molecular-weight polysaccharides in antibacterial, anti-inflammatory, wound healing promotion, and extended efficacy cycles.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A process for the preparation of a polysaccharide sodium hyaluronate composite gel, characterized in that, Includes the following steps: 1) Pre-crosslinking: Low molecular weight polysaccharides are added to an alkaline solution, and a portion of a crosslinking agent is added to pre-crosslink the low molecular weight polysaccharides, resulting in a gel intermediate; the low molecular weight polysaccharides are one or more of chondroitin sulfate, dermatan sulfate, and sodium hyaluronate sulfate; the molecular weight range of the low molecular weight polysaccharides is 1×10⁻⁶. 4 Da~8×10 4 Da; the volume ratio of the alkaline solution to the mass of the low molecular weight polysaccharide is 1 mL : (0.2~0.6) g; the molar amount of the added cross-linking agent accounts for 55%~100% of the total molar amount of the cross-linking agent; 2) Secondary crosslinking: Sodium hyaluronate is dissolved in an alkaline solution, and a gel intermediate and remaining crosslinking agent are added to obtain a mixture. Secondary crosslinking is then performed to obtain a composite gel. The molecular weight of the sodium hyaluronate is in the range of 2 × 10⁻⁶. 6 Da~3×10 6 Da; the concentration of sodium hyaluronate dissolved in alkaline solution is 10%~25%, w / v; the mass ratio of sodium hyaluronate to low molecular weight polysaccharide is (1.5~5):1; The mass ratio of the crosslinking agent to the total of sodium hyaluronate and low molecular weight polysaccharides is (0.08~0.15):1; The secondary crosslinking includes: the mixture reacting first at temperature A and then at temperature B, or the mixture reacting at a temperature of 40℃~50℃; wherein temperature A is 0℃~20℃, the mixture reacts at temperature A for 24h~72h, temperature B is 30℃~60℃, and the mixture reacts at temperature B for 2h~10h.

2. The preparation method according to claim 1, characterized in that: The crosslinking agent is selected from any one of 1,4-butanediol diglycidyl ether, 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1,2-ethylenediol diglycidyl ether.

3. The method of claim 2, wherein: The crosslinking agent is 1,4-butanediol diglycidyl ether.

4. The method of claim 1, wherein: The alkaline solution is an aqueous solution of sodium hydroxide, and the mass concentration of the aqueous solution of sodium hydroxide is 0.5wt%~3wt%.

5. The method of claim 4, wherein: The mass concentration of the sodium hydroxide aqueous solution is 0.5wt%~2wt%.

6. The method of claim 1, wherein: The pre-crosslinking temperature is 30℃~60℃.

7. The method of claim 6, wherein: The pre-crosslinking temperature is 40℃~60℃.

8. The method of claim 7, wherein: The pre-crosslinking temperature is 50°C.

9. The method of claim 1, wherein: The pre-crosslinking time is 1h to 9h.

10. The method of claim 9, wherein: The pre-crosslinking time is 2h to 7h.

11. The method of claim 10, wherein: The pre-crosslinking time is 3 hours.

12. The preparation method according to claim 1, characterized in that, The pre-crosslinking temperature is 50°C, and the pre-crosslinking time is 3 hours; the concentration of sodium hyaluronate dissolved in the alkaline solution is 14%~20%, w / v; the temperature A is 10°C, and the mixture reacts at temperature A for 48 hours; the temperature B is 50°C, and the mixture reacts at temperature B for 3~5 hours.

13. The method of claim 1, wherein, The secondary crosslinking includes: The mixture is reacted at 50°C for 3 hours; wherein, temperature A is 5°C to 12°C, and the mixture is reacted at temperature A for 36 hours to 60 hours; and temperature B is 40°C to 60°C, and the mixture is reacted at temperature B for 3 hours to 5 hours.

14. The method of claim 13, wherein, The temperature B is 50°C.

15. The method of claim 1, wherein, The preparation method further includes: dialyzing the gel obtained after the secondary cross-linking in PBS buffer, or soaking the gel obtained after the secondary cross-linking in PBS buffer.

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