A preparation method of polysaccharide sodium hyaluronate composite gel

Abstract

The present invention relates to a method for preparing a polysaccharide sodium hyaluronate composite gel, and belongs to the technical field of polymer material preparation. The preparation method of the present invention prepares a polysaccharide sodium hyaluronate composite gel by first cross-linking under low temperature conditions and then cross-linking under high temperature conditions. The method provided by the present invention can obtain a gel product with good elasticity and easy injection with the simplest operation and the fastest time, has good reproducibility, is suitable for the preparation of different types of polysaccharide sodium hyaluronate composite gels, and is more conducive to industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer material preparation, and particularly relates to a preparation method of a polysaccharide sodium hyaluronate composite gel. Background Art

[0002] Hyaluronic acid is a linear high-molecular polysaccharide composed of glucuronic acid and glucosamine as disaccharide units, which exists in many connective tissues such as skin, vitreous humor of the eye, cartilage, and synovial fluid of joints, and plays physiological roles such as moisturizing, nourishing, repairing, and preventing damage therein. Hyaluronic acid is generally its sodium salt, namely sodium hyaluronate (hereinafter simply referred to as HA), which is soluble in water but insoluble in organic solvents.

[0003] Chondroitin sulfate (hereinafter simply referred to as "CS") is widely present in various animal tissues, especially rich in cartilage and connective tissues.

[0004] CN100582146C discloses a preparation method of a biocompatible crosslinked gel, which obtains a high polymer by first adding one polymer for reaction and then adding another or the same polymer for reaction. It is necessary to continuously monitor the reaction degree during the reaction process, and add another or the same polymer until a specific degree of polymerization is reached. The operation is cumbersome and not conducive to industrial production.

[0005] The present invention provides a polysaccharide sodium hyaluronate composite gel and a preparation method thereof, which has simple operation and can obtain a gel product with high gel strength and good fluidity without other additives.

[0006] Therefore, the preparation method of the polysaccharide sodium hyaluronate composite gel provided by the present invention is suitable for industrial production and has greater market value and profound practical significance. Summary of the Invention

[0007] In view of the above technical background, the present invention provides a preparation method of a polysaccharide sodium hyaluronate composite gel, which is a brand-new method suitable for industrial production.

[0008] The present invention provides a preparation method of a polysaccharide sodium hyaluronate composite gel, which comprises the following steps:

[0009] Step 1: Dissolve sodium hyaluronate in an aqueous sodium hydroxide solution, add a low-molecular polysaccharide and a crosslinking agent, first react at temperature A, and then carry out a crosslinking reaction at temperature B to prepare a polysaccharide sodium hyaluronate composite gel.

[0010] In the said Step 1, the reaction can be carried out at temperature A for 24 h to 72 h (hours), preferably 36 h to 60 h, and further preferably 48 h to 72 h.

[0011] In the step 1, it can react at temperature B for 1 h to 6 h, preferably 2 h to 5 h.

[0012] According to the present invention, the temperature A in step 1 can be 0 °C to 20 °C, preferably 5 °C to 15 °C or 8 °C to 12 °C.

[0013] According to the present invention, the temperature B in step 1 can be 25 °C to 50 °C, preferably 25 °C to 45 °C, more preferably 30 °C to 40 °C.

[0014] According to the present invention, the low molecular weight polysaccharide is selected from one or more of sodium hyaluronate (HA), chondroitin sulfate (CS), chondroitin (C), heparin, and cellulose, preferably one or more of sodium hyaluronate, chondroitin sulfate, and chondroitin.

[0015] In step 1, the mass ratio of the sodium hyaluronate to the low molecular weight polysaccharide can be (1 - 9):1, preferably (1 - 6):1.

[0016] In step 1, the molecular weight range of the sodium hyaluronate can be 1×10 6 to 3×10 6 Da, preferably 2×10 6 to 3×10 6 Da.

[0017] In step 1, the molecular weight of the low molecular weight polysaccharide can be 1×10 4 to 6×10 5 Da, preferably 2×10 4 to 4×10 5 Da.

[0018] The mass concentration of the sodium hydroxide aqueous solution can be 0.5 wt% to 3 wt%, preferably 0.5 wt% to 2 wt%;

[0019] The mass concentration of the sodium hyaluronate in the sodium hydroxide aqueous solution can be 10 wt% - 30 wt%, preferably 12 wt% - 25 wt%.

[0020] The crosslinking agent is one of 1,4 - butanediol diglycidyl ether (BDDE), 1-(2,3 - epoxypropyl)-2,3 - epoxycyclohexane, and 1,2 - ethanediol diglycidyl ether.

[0021] In step 1, the mass ratio of the crosslinking agent to the total mass of the sodium hyaluronate and the low molecular weight polysaccharide can be 1:(5 - 50), preferably 1:(5 - 40).

[0022] In some embodiments, the preparation method further includes: cutting the obtained gel into small pieces, soaking them in PBS buffer solution, and repeatedly changing the PBS buffer solution until the gel becomes colorless; then sieving the gel through a 160-mesh sieve into small gel particles, and then mechanically homogenizing to prepare a polysaccharide-sodium hyaluronate composite gel.

[0023] In some embodiments, the preparation method further includes: cutting the obtained gel into small pieces, soaking them in PBS buffer solution containing hydrochloric acid, and repeatedly changing the PBS buffer solution until the small gel pieces become colorless; then sieving the gel through a 160-mesh sieve into small gel particles, and then mechanically homogenizing to prepare a polysaccharide-sodium hyaluronate composite gel.

[0024] The PBS buffer solution can be prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid and water.

[0025] 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.

[0026] In some embodiments, the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid and water, and the pH is 6.5 - 7.0.

[0027] In some embodiments, the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and water, and the pH is 7.0 - 7.5.

[0028] According to the present invention, as a preferred embodiment, the number of times of repeatedly changing the PBS buffer solution is 4 - 8 times.

[0029] According to the present invention, in the gel, the total concentration of the polysaccharide and sodium hyaluronate can be 20 mg / mL - 30 mg / mL. In some embodiments, in the gel, the total concentration of the polysaccharide and sodium hyaluronate is 20 mg / mL - 25 mg / mL.

[0030] According to the present invention, the polysaccharide-sodium hyaluronate composite gel can be used by injection. The polysaccharide-sodium hyaluronate composite gel can be suitably used for separating, replacing or filling biological tissues, or increasing the volume of the tissues, or for aesthetic medical purposes such as filling wrinkles, covering scars, or increasing the volume of the lips.

[0031] The advantages of the preparation method provided by the present invention mainly lie in:

[0032] 1. The present invention provides a brand-new industrializable method for preparing a polysaccharide sodium hyaluronate composite gel. By adjusting factors such as alkali concentration, crosslinking agent dosage, system pH, temperature, time, etc., especially the process conditions of a multi-level crosslinking method combining cold crosslinking and heat crosslinking, the obtained gel has a more compact structure, a high crosslinking degree, better viscoelasticity, and stronger enzymatic hydrolysis resistance while maintaining excellent biocompatibility.

[0033] 2. The present invention solves the problems that long-time high-temperature crosslinking reactions are prone to cause degradation of raw materials, the final product is mainly composed of crosslinked high-molecular short chains, the gel strength is greatly reduced, and the reaction rate of low-temperature crosslinking reactions is slow, the overall strength of the obtained product is low, and the crosslinking degree is even lower.

[0034] The inventors of the present invention found through a large number of experiments that compared with a simple low-temperature crosslinked gel, the gel strength of the method of the present invention is increased by 0.4 to 3.8 times, and compared with a simple high-temperature crosslinked gel, the gel strength is increased by 2 to 17 times, showing significant advantages over conventional methods.

[0035] 3. The present invention uses a multi-level crosslinking method combining low-temperature crosslinking and high-temperature crosslinking. First, low-temperature crosslinking (0°C to 20°C) is carried out. The prepared gel is dense, has good elasticity, and low hardness. Due to the low temperature, the gelation time is long, and the substances in the polymerization system are evenly distributed, making the topological structure of the gel complete, thus having better elasticity. Low-temperature crosslinking can achieve crosslinking while reducing the degradation of raw materials, making the crosslinking network more resistant to high-temperature degradation and simultaneously achieving further crosslinking. Then, as the temperature rises, the combination rate of raw materials and crosslinking agents accelerates, increasing the crosslinking degree and the hardness of the gel. Therefore, by combining low-temperature and high-temperature crosslinking, a composite gel with good viscoelasticity can be obtained, improving the crosslinking degree and structural stability of the gel, while increasing the viscoelasticity of the gel and the supporting force after injection. (Using the process of first high-temperature and then low-temperature to prepare crosslinked gels will damage the chain length of HA macromolecular chains, causing them to lose the advantage of high molecular weight and affecting the stability and durability of the product during use.) The viscoelasticity of the composite gel prepared by the method of the present invention is increased by 31.3% to 114% compared with the gel prepared by the method of first heat crosslinking and then cold crosslinking, achieving unexpected technical effects.

[0036] 4. The inventors of the present invention found that the longer the low-temperature crosslinking reaction time, the less likely the raw materials are to degrade, and the longer the contact time with the crosslinking agent, thereby increasing the reaction degree. Superimposing high temperature further promotes the crosslinking reaction, producing a gel with high strength but a slightly reduced pushing force, which is beneficial for the injection use of the gel. At the same time, reaction conditions with mild conditions, low raw material degradation degree, and high product strength are determined through experiments, making the preparation method suitable for further industrial production implementation.

[0037] 5. The method provided by the present invention has good reproducibility and is suitable for the preparation of different types of polysaccharide sodium hyaluronate composite gels. The present invention has explored the relationship between the reaction temperature range, reaction time and the strength of the polysaccharide sodium hyaluronate composite gel, and can obtain a gel product with good elasticity and easy injection with the simplest operation and the shortest time. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a graph showing the molecular weight change of 20 wt% HA raw material in Example 5 after standing at 10 °C and 40 °C for 48 h and 3 h in sequence;

[0039] Figure 2 It is a graph showing the molecular weight change of 20 wt% HA raw material in Example 5 after standing at 40 °C for 27 h in sequence;

[0040] Figure 3 It is a graph showing the molecular weight change of 5 wt% CS raw material in Example 5 after standing at 40 °C for 22 h;

[0041] In each drawing, WDa represents ten thousand Daltons. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions.

[0043] Unless otherwise specified, the raw materials or reagents used in the embodiments are all commercially available.

[0044] The room temperature mentioned in the embodiments is 20 °C to 30 °C. Unless otherwise specified, the reagents are used directly without purification. All solvents are purchased from commercial suppliers, such as Aldrich, and can be used without treatment.

[0045] Example 1: Effects of Low Temperature, High Temperature and Reaction Time on the Viscoelasticity of HA / CS Composite Gel

[0046] Under stirring, sodium hyaluronate (HA) (molecular weight 2.3×10 6 Da, 4.0 g) powder was slowly added to 1 wt% NaOH aqueous solution (20 mL). After dissolving evenly, chondroitin sulfate (CS) (6.5×10 4 Da, 1.0 g) was slowly added, and stirred evenly until it became light yellow and transparent; 1,4-butanediol diglycidyl ether (BDDE) (0.36 g) was added to the above solution, and after stirring for 30 min, it was transferred to a Y 1 °C water bath for reaction for X 1 h; after the low-temperature reaction was completed, it was transferred to a Y 2 °C water bath for continued crosslinking for X2 h, a light yellow blocky gel was obtained.

[0047] The obtained blocky gel was cut into small pieces about 1 cm 3 in size, and the gel was soaked in PBS buffer solution containing hydrochloric acid (pH 6.8) to neutralize the sodium hydroxide therein. Then, the PBS buffer solution (pH 6.5 - 7.5) was continuously replaced until the final gel pieces became colorless. The swollen gel was sieved through a 160 - mesh sieve into gel particles, and then through mechanical homogenization, an HA / CS composite cross - linked gel product was obtained. The total concentration and cross - linking degree of HA / CS are shown in Table 1.

[0048] The prepared HA / CS cross - linked gel was extruded through a 30G needle to test the average extrusion force; after the experimental gel was degraded, 1 1H NMR was detected. The cross - linking degree was calculated through the characteristic peaks of BDDE and HA / CS disaccharide units (R = number of moles of BDDE / (total number of moles of HA / CS disaccharide units)); the experimental gel was placed on a rotational rheometer. In the plate mode, with a fixed strain of 0.01%, a frequency sweep (0.01 Hz - 100 Hz) was selected to test the viscoelasticity of the gel at 0.1 Hz. The test results are shown in Table 1.

[0049] Table 1 Process conditions and gel properties of Example 1

[0050]

[0051] It can be seen from the results in Table 1 that compared with other examples, the gel strength of Examples 1 - 5, 1 - 8 to 1 - 12 increased by 24.8% to 305%, and the average extrusion force decreased by 11.4% to 43.4%; the longer the low - temperature reaction time, the less likely the raw materials are to degrade, and the longer the contact time with the cross - linker, the higher the reaction degree. The superposition of high temperature further promotes the cross - linking reaction, making the gel strength higher and the extrusion force also decreased; under the same low - temperature reaction time, the reaction conditions of 30 °C and 5 h are milder, the degradation degree of raw materials is low, and under a longer reaction time, a gel sample with higher strength can be prepared.

[0052] Example 2: Influence of the type and molecular weight of low - molecular - weight polysaccharides on the properties of HA composite gels

[0053] Sodium hyaluronate (molecular weight 2.3×10 6 Da, 4.0 g) powder was added to 1 wt% NaOH solution (20 mL). After the sodium hyaluronate was dissolved evenly, 1 g of low - molecular - weight polysaccharide was slowly added, and stirred evenly until it became light yellow and transparent. BDDE (0.36 g) was added, and after stirring for 30 min, it was first reacted at 10 °C for 48 h, and then at 40 °C for 3 h to obtain a light yellow blocky gel. The obtained blocky gel was cut into 1 cm 3Small pieces on both sides were soaked in PBS buffer solution containing hydrochloric acid (pH 6.8) to neutralize the sodium hydroxide in them. Then, the PBS buffer solution (pH 6.5 - 7.5) was continuously changed until the final gel pieces became colorless. The swollen gel was sieved through a 160 - mesh sieve into gel particles, and then through mechanical homogenization, a high - molecular - weight HA / low - molecular - weight polysaccharide composite cross - linked gel product was obtained. The total concentration of high - molecular - weight HA / low - molecular - weight polysaccharide is shown in Table 2.

[0054] The high - molecular - weight HA / low - molecular - weight polysaccharide cross - linked gel prepared in Example 2 was extruded through a 30G needle to test the average extrusion force; after the experimental gel was degraded, 1 1H NMR was performed. The cross - linking degree was calculated through the characteristic peaks of BDDE and the disaccharide units of HA / low - molecular - weight polysaccharide (R = number of moles of BDDE / (total number of moles of HA / low - molecular - weight polysaccharide disaccharide units)); the experimental gel was placed on a rotational rheometer. In the plate mode, frequency scanning (0.01 Hz - 100 Hz) was selected to test the viscoelasticity of the gel at 0.1 Hz. The test results are shown in Table 2.

[0055] Table 2 Process conditions and gel properties of Example 2

[0056]

[0057] It can be seen from the results in Table 2 that, compared with Examples 1 - 5, under the same conditions, the introduction of low - molecular - weight HA reduces the strength of the composite cross - linked gel and increases the extrusion force; while the contributions of chondroitin and chondroitin sulfate to the properties of the composite cross - linked gel are less different. Overall, as the molecular weight of the low - molecular - weight polysaccharide increases, the gel strength and extrusion force increase; for the types of low - molecular - weight polysaccharides, compared with low - molecular - weight HA, adding C and CS slightly reduces the extrusion force of the composite cross - linked gel.

[0058] Example 3: Influence of sodium hyaluronate concentration on the properties of HA composite gel

[0059] Sodium hyaluronate (molecular weight 2.3×10 6 Da, 2.4 g) or sodium hyaluronate (molecular weight 2.3×10 6 Da, 3.2 g) powder was added into 1 wt% NaOH solution (20 mL). After the sodium hyaluronate was dissolved evenly, low - molecular - weight polysaccharide was slowly added, such as L - HA (molecular weight 4.6×10 4 Da, 0.6 g) or chondroitin sulfate (molecular weight 6.5×10 4 Da, 0.8 g). After stirring evenly until it became transparent, BDDE (0.22 g or 0.29 g) was added correspondingly. After stirring for 30 min, it was first reacted at 10 °C for 48 h, and then at 40 °C for 3 h to obtain a light - yellow massive gel. The obtained massive gel was cut into 1 cm3 Small pieces about [quantity] were soaked in PBS buffer solution containing hydrochloric acid (pH 6.5) to neutralize the sodium hydroxide therein. Then, the PBS buffer solution (pH 6.5 - 7.5) was continuously changed until the final gel pieces became colorless. The swollen gel was sieved through a 160-mesh sieve into gel particles, and then through mechanical homogenization, a high-molecular-weight HA / low-molecular-weight polysaccharide composite cross-linked gel product was obtained. The total concentration and cross-linking degree of high-molecular-weight HA / low-molecular-weight polysaccharide are shown in Table 3.

[0060] The high-molecular-weight HA / low-molecular-weight polysaccharide cross-linked gel prepared in Example 3 was extruded through a 30G needle to test the average extrusion force; after the experimental gel was degraded, 1 1H NMR was performed, and the cross-linking degree was calculated through the characteristic peaks of BDDE and the disaccharide units of HA / low-molecular-weight polysaccharide (R = number of moles of BDDE / (total number of moles of disaccharide units of HA / low-molecular-weight polysaccharide)); the experimental gel was placed on a rotational rheometer, and in the plate mode, frequency scanning (0.01 Hz - 100 Hz) was selected to test the viscoelasticity of the gel at 0.1 Hz. The test results are shown in Table 3.

[0061] Table 3 Process conditions and gel properties of Example 3

[0062]

[0063] As can be seen from the results in Table 3, as the concentration of high-molecular-weight HA increases, the gel strength increases; at a lower HA concentration, compared with L-HA, CS has a more obvious reducing effect on the extrusion force of the composite cross-linked gel and is easier to extrude during the use of the product.

[0064] Example 4: Effect of the ratio of HA to CS on the viscoelasticity of the HA / CS composite gel

[0065] Under stirring, sodium hyaluronate (4.0 g, 2.3×10 6 Da) powder was slowly added to 1 wt% NaOH solution (20 mL). After being dissolved evenly, chondroitin sulfate (CS, 6.5×10 4 Da) was slowly added. After stirring evenly until it became light yellow and transparent, BDDE was added to the above solution. After stirring evenly for 30 min, light yellow block gels were obtained at 10°C / 24 h + 30°C / 5 h and 10°C / 48 h + 40°C / 3 h respectively. The obtained block gels were cut into 1 cm 3Small pieces on the left and right were soaked in PBS buffer solution containing hydrochloric acid (pH 6.5) to neutralize the sodium hydroxide in them. Then, the PBS buffer solution (pH 6.5 - 7.5) was continuously changed until the final gel pieces became colorless. The swollen gel was sieved through a 160-mesh sieve into gel particles, and then through mechanical homogenization, an HA / CS composite cross-linked gel product was obtained. The total concentration of HA / CS is shown in Table 4.

[0066] The HA / CS cross-linked gel prepared in Example 4 was extruded through a 30G needle to test the average extrusion force; after the experimental gel was degraded, 1 HNMR was detected. The cross-linking degree was calculated through the characteristic peaks of BDDE and the disaccharide units of HA / CS (R = number of moles of BDDE / (total number of moles of HA / CS disaccharide units)); the experimental gel was placed on a rotational rheometer, and in the plate mode, frequency scanning (0.01 - 100 Hz) was selected to test the viscoelasticity of the gel at 0.1 Hz. The test results are shown in Table 4.

[0067] Table 4 Process conditions and gel properties of Example 4

[0068]

[0069]

[0070] It can be seen from the results in Table 4 that as the proportion of chondroitin sulfate increases, the gel strength gradually increases and the extrusion force gradually decreases, indicating that CS has the effect of simultaneously increasing the strength and facilitating extrusion when preparing the HA composite cross-linked gel.

[0071] Example 5: Molecular weight (unit: ten thousand daltons) change of HA raw materials under different cross-linking temperature conditions

[0072] Table 5 Molecular weight change of 20 wt% HA raw materials standing at 10°C and 40°C for 48 h and 3 h in sequence

[0073]

[0074] Table 6 Molecular weight change of 20 wt% HA raw materials standing at 40°C for 27 h

[0075]

[0076] Table 7 Molecular weight change of 5 wt% CS raw materials standing at 40°C for 22 h

[0077]

[0078] As can be seen from Table 5, within 20 h to 48 h of low-temperature static placement, the molecular weight of the HA raw material decreased less, from 2.09 million Daltons to 1.66 million Daltons and 1.46 million Daltons respectively. Notably, after the HA was placed statically at low temperature for 48 h and then transferred to high-temperature static placement, after 3 h, the molecular weight of the HA decreased even less, only decreasing to 0.91 million Daltons (38%, relative to the molecular weight after low-temperature static placement). The above results indicate that the longer the low-temperature time, the more physical entanglements occur in the raw material during static placement, and the stronger the ability to resist degradation during subsequent high-temperature crosslinking.

[0079] As can be seen from Table 6, after the HA raw material was placed statically at high temperature for 3 h, the molecular weight directly decreased to 0.82 million Daltons (61%). At this time, when low-temperature static placement was carried out again, the molecular weight would only continue to decrease slowly, with a greater degree of degradation than that of low-temperature first and then high-temperature static placement; after continuous high-temperature static placement for 27 h, the molecular weight of the HA had decreased to 0.15 million Daltons, losing its high-molecular-weight advantage.

[0080] In summary, compared with the raw materials that were first placed statically at low temperature and then at high temperature, after the HA raw materials were placed statically at high temperature, the molecular weights all decreased to 0.8 million Daltons to 0.9 million Daltons. However, the raw materials that were first placed statically at low temperature would undergo physical entanglements to varying degrees during this process, having a certain resistance to high temperature, and the situation where the high temperature directly breaks the macromolecular chains into short chains would not occur. In the case of adding a chemical crosslinking agent, it is easier to obtain physically and chemically co-crosslinked long-chain molecules, and prepare HA / CS crosslinked gels with higher stability and durability.

[0081] Comparative Example 1: Influence of Low-Temperature Crosslinking Process Conditions on the Viscoelasticity of the Inventive Gel

[0082] Operate according to Example 1, where X 1 、Y 1 、X 2 、Y 2 See Table 8.

[0083] Comparative Example 2: Influence of High-Temperature Crosslinking Process Conditions on the Viscoelasticity of the Inventive Gel

[0084] Operate according to Example 1, where X 1 、Y 1 、X 2 、Y 2 See Table 8.

[0085] Table 8 Process Conditions and Gel Properties of Comparative Examples 1 to 2

[0086]

[0087] As can be seen from Table 1 and Table 2: Compared with Comparative Examples 1-1 to 1-4, the gel strength of Examples 1-5, 1-8 to 1-12 increased by 44.1% to 376.5%; compared with Comparative Examples 2-1 to 2-4, the gel strength of Examples 1-5, 1-8 to 1-12 increased by 210.3% to 1718%. From the results of Table 2, it can be known that for the gels prepared from Comparative Examples 1-1 to 1-4, as the reaction time increased, the gel strength increased, but compared with Example 1, the overall strength was lower and the crosslinking degree was also lower. For the gels prepared from Comparative Examples 2-1 to 2-4, the crosslinking degree increased and the gel color was yellowish-brown, but the degree of gel strength reduction was more than that of Example 1.

[0088] Comparative Example 3: Preparation of HA / CS crosslinked gel using a process of high temperature first and then low temperature

[0089] In this comparative example, under the same sodium hyaluronate concentration, molecular weight, ratio of sodium hyaluronate to chondroitin sulfate, crosslinking agent dosage and alkaline conditions as in Example 1, referring to the gel preparation process of Patent CN112812330B, the change in gel viscoelasticity was investigated when the sample was first reacted at a crosslinking reaction temperature of 40°C for 3 h and then the gel was transferred to 10°C for reaction for 24 h / 48 h.

[0090] Table 9 Process conditions and gel properties of Comparative Example 3

[0091]

[0092] Preparing the HA / CS crosslinked gel using a process of high temperature first and then low temperature can improve the gel strength and crosslinking degree, but the raw material degradation data shows that high temperature first will break the chain length of the HA macromolecular chain and make it break into shorter chains. The elastic modulus of the gel prepared by this method has no advantage compared with that of the gel crosslinked at low temperature for 24 h / 48 h and then at high temperature for 3 h, but it loses the advantage of high molecular weight, which will affect the stability and durability of the product during use.

Claims

1. A method for preparing a polysaccharide sodium hyaluronate composite gel, characterized in that The method comprises the following steps: dissolving sodium hyaluronate in a sodium hydroxide aqueous solution, adding a low molecular weight polysaccharide, and then adding a cross-linking agent, first reacting at temperature A for 48 hours to 72 hours, and then cross-linking reacting at temperature B for 2 hours to 5 hours to prepare a polysaccharide sodium hyaluronate composite gel; wherein the low molecular weight polysaccharide is one or more of sodium hyaluronate, chondroitin sulfate, chondroitin, heparin and cellulose; the temperature A is 5°C-15°C; the temperature B is 25°C-45°C; the mass ratio of sodium hyaluronate to low molecular weight polysaccharide is (1-9):1; the molecular weight of sodium hyaluronate is 1×10 6 Up to 3×10 6 Da; the molecular weight of the low molecular weight polysaccharide is 1×10 4 Up to 6×10 5 Da.

2. The preparation method according to claim 1, characterized in that: The low molecular weight polysaccharide is one or more of sodium hyaluronate, chondroitin sulfate and chondroitin.

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

4. The preparation method according to claim 1, characterized in that: Include at least one of the following conditions: The mass concentration of the sodium hydroxide aqueous solution is 0.5wt%~3wt%; The mass concentration of the sodium hyaluronate in the sodium hydroxide aqueous solution is 10wt%~30wt%; The mass ratio of the cross-linking agent to the total of sodium hyaluronate and low molecular weight polysaccharides is 1:(5-50).

5. The preparation method according to claim 1, characterized in that: Include at least one of the following conditions: The mass ratio of sodium hyaluronate to low molecular weight polysaccharide is (1-6):1; The molecular weight of the sodium hyaluronate is 2×10 6 Up to 3×10 6 Da; The molecular weight of the low molecular weight polysaccharide is 2×10 4 Up to 4×10 5 Da; The mass concentration of the sodium hydroxide aqueous solution is 0.5wt%~2wt%; The mass concentration of the sodium hyaluronate in the sodium hydroxide aqueous solution is 12wt%~25wt%; The mass ratio of the cross-linking agent to the total amount of sodium hyaluronate and low-molecular polysaccharides is 1:(5-40).

6. The preparation method according to any one of claims 1 to 5, characterized in that The method also includes: cutting the obtained gel into small pieces, soaking them in a PBS buffer solution containing hydrochloric acid to neutralize the sodium hydroxide therein; then repeatedly replacing the PBS buffer solution until the gel pieces are colorless; then sieving the gel through a 160-mesh sieve, and then mechanically homogenizing to prepare a polysaccharide sodium hyaluronate composite gel product; in the gel product, the total concentration of low molecular weight polysaccharides and sodium hyaluronate is 20 mg / mL~30 mg / mL; the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid and water, and has a pH of 6.5~7.

5.

7. The preparation method according to any one of claims 1 to 3, characterized in that Include: Sodium hyaluronate is dissolved in a 0.5wt%-2wt% sodium hydroxide aqueous solution, chondroitin sulfate is added, and then a cross-linking agent 1,4-butanediol diglycidyl ether is added, and the reaction is first carried out at 5°C-15°C for 48h-72h, and then a cross-linking reaction is carried out at 25°C-45°C for 2h-5h to obtain a gel; wherein the mass concentration of the sodium hyaluronate in the sodium hydroxide aqueous solution is 12wt%-20wt%; the mass ratio of the sodium hyaluronate to the chondroitin sulfate is (1-4):1; the mass ratio of the cross-linking agent to the sum of the sodium hyaluronate and the chondroitin sulfate is 1:(10-25); the molecular weight of the sodium hyaluronate is 2×10 6 Up to 2.5×10 6 Da; the molecular weight of the chondroitin sulfate is 3×10 4 Up to 1×10 5 Da; The obtained gel is cut into small pieces, soaked in PBS buffer solution to neutralize the sodium hydroxide therein; then the PBS buffer solution is repeatedly replaced until the gel pieces are colorless; then the gel is sieved into small gel particles through a 160-mesh sieve, and then mechanically homogenized to prepare a polysaccharide sodium hyaluronate composite gel product; in the gel product, the total concentration of chondroitin sulfate and sodium hyaluronate is 20 mg / mL~30 mg / mL; the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid and water, and the pH is 6.5~7.

5.

8. The preparation method according to any one of claims 1 to 3, characterized in that Include: Sodium hyaluronate is dissolved in a 1wt% sodium hydroxide aqueous solution, chondroitin sulfate is added, and then a cross-linking agent 1,4-butanediol diglycidyl ether is added, and the reaction is first carried out at 8°C to 12°C for 48h to 72h, and then a cross-linking reaction is carried out at 30°C to 40°C for 2h to 5h to obtain a gel; wherein the mass concentration of the sodium hyaluronate in the sodium hydroxide aqueous solution is 16wt% to 20wt%; the mass ratio of the sodium hyaluronate to the chondroitin sulfate is 4:1; the mass ratio of the cross-linking agent to the sum of the sodium hyaluronate and the chondroitin sulfate is 1:(10-15); the molecular weight of the sodium hyaluronate is (2-2.5)×10 6 Da; the molecular weight of the chondroitin sulfate is (3~7)×10 4 Da; The obtained gel is cut into small pieces, and soaked in a PBS buffer solution with a pH of 6.5 to 7.0 containing hydrochloric acid to neutralize the sodium hydroxide therein; then the PBS buffer solution is repeatedly replaced until the gel pieces are colorless; then the gel is sieved into small gel particles through a 160-mesh sieve, and then mechanically homogenized to prepare a polysaccharide sodium hyaluronate composite gel product; in the gel product, the total concentration of chondroitin sulfate and sodium hyaluronate is 20 mg / mL to 25 mg / mL; the PBS buffer solution is prepared from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid and water; the pH of the replaced PBS buffer solution is 6.5 to 7.5.

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