Preparation method of dextran-sodium hyaluronate gel with anti-enzymatic degradation performance

Abstract

The present invention provides a method for preparing a dextran-sodium hyaluronate gel having anti-dextranase and anti-hyaluronidase enzymatic hydrolysis properties. The preparation method of the present invention includes steps such as dissolution, cross-linking reaction, pH adjustment, swelling and dialysis, sieving, and filling and sterilization. The method is simple and is conducive to industrial production. The gel prepared by the method of the present invention has high safety performance and good anti-dextranase and anti-hyaluronidase enzymatic hydrolysis properties.

Description

Technical Field

[0001] The present invention relates to the fields of materials science and clinical medicine. Specifically, it relates to a method for preparing a dextran-sodium hyaluronate gel with anti-enzymatic degradation properties. Background Art

[0002] Hyaluronic acid is a linear high-molecular polysaccharide composed of disaccharide units formed by alternating D-glucuronic acid and N-acetyl-D-glucosamine, which are connected by glycosidic bonds. Generally, hyaluronic acid exists in the form of its sodium salt, sodium hyaluronate, and is widely distributed in the extracellular matrix of various tissues in the human body, such as the skin, vitreous body, and joints. Sodium hyaluronate has excellent hydrophilicity. After chemical covalent cross-linking with a cross-linking agent, it can form a three-dimensional network hydrogel structure with high viscoelasticity and excellent biocompatibility. Based on these properties, sodium hyaluronate and its cross-linked forms have been widely used in the fields of biomedicine and medical aesthetics and have become important components of various filling materials.

[0003] Sodium hyaluronate is easily degraded by hyaluronidase and free radicals in the human body. Chemical cross-linking can cause the polysaccharide chains to form a hydrogel structure, enhancing the stability of the molecules. Commonly used cross-linking agents containing diepoxy groups such as 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, and 1,3-diepoxybutane are used. Under alkaline conditions, the epoxy group undergoes ring opening and is nucleophilically attacked by the hydroxyl group on the sugar unit of sodium hyaluronate to form a stable ether bond. This three-dimensional hydrogel structure can improve the physical and chemical properties of the material and enhance the mechanical strength, thereby extending its persistence in the skin to a certain extent, but this improvement is still limited. Therefore, beauty seekers usually need to be injected multiple times within one year to maintain their ideal cosmetic filling effect.

[0004] Patent CN108250462A discloses a method for preparing anti-enzymatic degradation cross-linked sodium hyaluronate. This method cross-links high-molecular-weight and low-molecular-weight sodium hyaluronate to enhance the stability of the three-dimensional network structure. However, this method does not change the bonding mode between the polysaccharide chain and the cross-linking agent, so the improvement of its anti-enzymatic degradation performance is still limited.

[0005] Patent CN111012953B enhances the anti-enzymatic degradation performance of the composite material by adding indomethacin-poly(lactic acid) microspheres to the gel and utilizing the inhibitory effect of indomethacin on hyaluronidase. However, dichloromethane is used in the preparation process of the microspheres, which introduces the risk of residual toxic organic reagents. In addition, since the combination of the gel and the poly(lactic acid) microspheres is mainly used for injection in the deep dermis layer, its application scenario is relatively limited.

[0006] Dextran is a high-molecular polysaccharide with a branched structure formed by the linkage of glucose units through glycosidic bonds. Both dextran and sodium hyaluronate are polysaccharide chains formed by six-membered ring sugar units through glycosidic bonds, but they differ in the type of glycosidic bond and the side chain groups of the polysaccharide backbone. Dextran can be divided into α-dextran and β-dextran according to the type of glycosidic bond. α-Dextran, also known as dextran, has long been widely used as a plasma substitute due to its excellent molecular stability and biosecurity. In addition, hydrogel materials based on crosslinked dextran have been used as drug delivery carriers for decades due to their excellent drug release characteristics and biocompatibility. β-Dextran has attracted attention due to its immunomodulatory and anti-inflammatory effects, and many liquid wound dressings based on β-dextran have been developed at home and abroad for promoting the recovery of skin wounds.

[0007] Patent CN105377898A discloses a preparation method of a binary crosslinked hydrogel, in which dextran and sodium hyaluronate are crosslinked through (poly)phosphodiester bonds, and the formed hydrogel shows stronger resistance to hyaluronidase degradation than the gel crosslinked with sodium hyaluronate alone. Patent CN106661133A discloses a dextran grafting method for a crosslinked hydrogel, in which small molecule dextran first diffuses freely into the gel and then is grafted onto the sodium hyaluronate backbone through amide bonds. Compared with before grafting, the swelling degree of the gel decreases and the resistance to hyaluronidase increases. Both of these methods introduce dextran with resistance to hyaluronidase degradation through chemical crosslinking, realizing the covalent bond connection between dextran and sodium hyaluronate. The newly formed binary crosslinked product has an additional bonding mode compared with the single network of crosslinked sodium hyaluronate, and the steric hindrance provided by the dextran sugar chain further limits the enzymatic hydrolysis efficiency of hyaluronidase.

[0008] However, as a biodegradable high-molecular polysaccharide, dextran is also easily degraded by glycosidases and free radicals in vivo. Although the aforementioned binary composite crosslinking method enhances the network structure of the original sodium hyaluronate gel, no crosslinking chemical bonds are formed between the dextran sugar chains to obtain a structure reinforcement similar to that of sodium hyaluronate. Secondly, the technological processes of the aforementioned two composite crosslinking methods are complex, and the intermediate steps all involve compounds that are irritating to the skin. Therefore, these methods have not been widely applied in the fields of biomedicine or medical beauty. Summary of the Invention

[0009] The purpose of the present invention is to provide a preparation method for a multi-crosslinking combination of dextran-sodium hyaluronate based on the optimization of anti-enzymatic degradation performance.

[0010] The purpose of the present invention is also to provide a hydrogel with a simple technological process and no irritation to the skin.

[0011] In the first aspect of the present invention, a method for preparing a dextran-sodium hyaluronate gel with anti-dextranase and hyaluronidase enzymatic hydrolysis properties is provided, comprising the following steps:

[0012] (1) Dissolution: Add a crosslinking agent to a 0.1 - 0.5 mol / L NaOH solution, stir evenly, then add dextran and sodium hyaluronate, and continue stirring until completely dissolved to obtain solution A;

[0013] Wherein, the concentration of the crosslinking agent is 0.05 - 2 wt%, and the crosslinking agent is one or more of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, and 1,3-diepoxybutane;

[0014] Wherein, the average molecular weight of the added dextran is: 60 - 80 kDa, and the average molecular weight of sodium hyaluronate is: 1000 - 1800 kDa; or,

[0015] The average molecular weight of the added dextran is: 400 - 600 kDa, and the average molecular weight of sodium hyaluronate is: 300 - 1800 kDa;

[0016] And the molar ratio of the dextran to the sodium hyaluronate is 1:4 - 4:1;

[0017] (2) Crosslinking reaction: Let solution A stand for crosslinking, the crosslinking temperature is 30 - 40 °C, and the crosslinking time is 6 - 24 h to obtain a transparent gel B;

[0018] (3) pH adjustment: Cut the gel B into 1 - 2 cm 3 small pieces, and after neutralization, the pH is between 6.8 and 7.5;

[0019] (4) Swelling and dialysis: Swell and dialyze in an isotonic phosphate buffer at room temperature, change the solution every 4 - 6 hours, and dialyze for more than 48 hours;

[0020] (5) Sieving: Sieve the dialyzed gel through a 100-mesh sieve to obtain dextran-sodium hyaluronate gel particles, and the particle size range of the gel is 50 - 250 μm;

[0021] (6) Filling and sterilization: Fill the gel particles obtained in step (5) into a sealed syringe and sterilize by moist heat.

[0022] In another preferred example, in step (1), the molar ratio of the dextran to the sodium hyaluronate is 1:1 - 1:4.

[0023] In another preferred example, in step (1), the molar ratio of the dextran to the sodium hyaluronate is 1:4, 1:1, or 4:1.

[0024] The average molecular weight of the added dextran is: 65 - 75 kDa, and the average molecular weight of sodium hyaluronate is: 1100 - 1500 kDa; or,

[0025] The average molecular weight of the added dextran is: 450 - 550 kDa, and the average molecular weight of sodium hyaluronate is: 350 - 1500 kDa.

[0026] In another preferred example, the added dextran and sodium hyaluronate are selected from the following group:

[0027] (a) Dextran with an average molecular weight of 70 kDa and sodium hyaluronate with an average molecular weight of 1200 kDa;

[0028] (b) Dextran with an average molecular weight of 500 kDa and sodium hyaluronate with an average molecular weight of 356 kDa;

[0029] (c) Dextran with an average molecular weight of 500 kDa and sodium hyaluronate with an average molecular weight of 1200 kDa.

[0030] In another preferred example, in step (1), when the molecular weight combination of the added dextran and sodium hyaluronate is (a) or (c), the molar ratio of the dextran to the sodium hyaluronate is 1:1 - 1:4.

[0031] In another preferred example, in step (1), when the molecular weight combination of the added dextran and sodium hyaluronate is (b), the molar ratio of the dextran to the sodium hyaluronate is 1:4 - 4:1.

[0032] In another preferred example, in step (1), when the molecular weight combination of the added dextran and sodium hyaluronate is (b), the molar ratio of the dextran to the sodium hyaluronate is 1:4, 1:1 or 4:1.

[0033] In another preferred example, the anti - enzymatic property refers to the anti - degradation property of the gel against two enzymes, dextranase (α - glycosidase) and hyaluronidase (β - glycosidase).

[0034] In another preferred example, the concentration of the dextran is 2 - 40 wt%, preferably 2 - 20 wt%.

[0035] In another preferred example, the concentration of the sodium hyaluronate is 1 - 25 wt%, preferably 2 - 15 wt%.

[0036] In another preferred example, in step (1), the cross - linker is 1,4 - butanediol diglycidyl ether.

[0037] In another preferred example, in step (1), the molar ratio of the dextran to the sodium hyaluronate is 1:1 to 1:4.

[0038] In another preferred example, in step (1), the concentration of the NaOH solution is 0.2 to 0.3 mol / L.

[0039] In another preferred example, in step (3), neutralization is carried out using dilute hydrochloric acid.

[0040] In another preferred example, in step (4), the phosphate buffer solution refers to an isotonic solution of sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate dissolved in water for injection.

[0041] In another preferred example, in step (4), the osmotic pressure of the phosphate buffer solution is 270 to 330 mOsmol / L and the pH is 6.8 to 7.5.

[0042] In another preferred example, in step (s6), the sterilization conditions are as follows: moist heat sterilization at 121 °C for 8 to 30 min.

[0043] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel after 2 h is 60% to 90% or 110% to 140%.

[0044] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel after 2 h is 90% to 110%.

[0045] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel after 4 h is 60% to 90% or 110% to 140%.

[0046] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel after 4 h is 90% to 110%.

[0047] In another preferred example, after being treated with dextranase, the mass retention rate of the gel after 2 h is 60% to 90% or 110% to 140%.

[0048] In another preferred example, after being treated with dextranase, the mass retention rate of the gel after 2 h is 90% to 110%.

[0049] In another preferred example, after being treated with dextranase, the mass retention rate of the gel after 4 h is 60% to 90% or 110% to 140%.

[0050] In another preferred example, after being treated with dextranase, the mass retention rate of the gel after 4 h is 90% to 110%.

[0051] In another preferred example, the process of the treatment is as follows:

[0052] (i) According to the swelling degree of each gel, accurately weigh the corresponding mass of gel particles;

[0053] (ii) Add phosphate buffer solution to the gel particles and incubate in a 37°C water bath for 1 hour to ensure the full swelling of the gel at 37°C;

[0054] (iii) After swelling, remove the excess buffer solution by centrifugation and accurately weigh the mass of the gel, denoted as X0;

[0055] (iv) Add an equal amount of hyaluronidase or dextranase;

[0056] (v) Heat the system in a 37°C water bath, take it out at the 2-hour and 4-hour nodes respectively, centrifuge and weigh the remaining mass of the gel solid, denoted as X2 and X4;

[0057] Among them, the 2-hour retention rate = (X2 / X0) × 100%, and the 4-hour retention rate = (X4 / X0) × 100%.

[0058] In another preferred example, in step (iv), when hyaluronidase is added, the final concentration of the enzyme is: 40 U / mL.

[0059] In another preferred example, in step (iv), when dextranase is added, the final concentration of the enzyme is: 1 U / mL.

[0060] In the second aspect of the present invention, a dextran-sodium hyaluronate gel with anti-dextranase and hyaluronidase enzymatic hydrolysis properties is provided, and the gel is prepared by the method described in the first aspect of the present invention.

[0061] In another preferred example, after being treated with hyaluronidase, the 2-hour mass retention rate of the gel is 60% to 90% or 110% to 140%.

[0062] In another preferred example, after being treated with hyaluronidase, the 2-hour mass retention rate of the gel is 90% to 110%.

[0063] In another preferred example, after being treated with hyaluronidase, the 4-hour mass retention rate of the gel is 60% to 90% or 110% to 140%.

[0064] In another preferred example, after being treated with hyaluronidase, the 4-hour mass retention rate of the gel is 90% to 110%.

[0065] In another preferred example, after being treated with dextranase, the mass retention rate of the gel at 2 h is 60% to 90% or 110% to 140%.

[0066] In another preferred example, after being treated with dextranase, the mass retention rate of the gel at 2 h is 90% to 110%.

[0067] In another preferred example, after being treated with dextranase, the mass retention rate of the gel at 4 h is 60% to 90% or 110% to 140%.

[0068] In another preferred example, after being treated with dextranase, the mass retention rate of the gel at 4 h is 90% to 110%.

[0069] In another preferred example, the process of the treatment is as follows:

[0070] (i) According to the swelling degree of each gel, accurately weigh the corresponding mass of gel particles; z

[0071] (ii) Add phosphate buffer solution to the gel particles and incubate in a 37 °C water bath for 1 hour to ensure the full swelling of the gel at 37 °C;

[0072] (iii) After swelling, remove the excess buffer solution by centrifugation and accurately weigh the mass of the gel, denoted as X0;

[0073] (iv) Add an equal amount of hyaluronidase or dextranase;

[0074] (v) Heat the system in a 37 °C water bath, take it out at the 2-hour and 4-hour nodes respectively, centrifuge and weigh the remaining mass of the gel solid, denoted as X2 and X4;

[0075] Among them, the 2-hour retention rate = (X2 / X0) × 100%, and the 4-hour retention rate = (X4 / X0) × 100%.

[0076] In another preferred example, in step (iv), when hyaluronidase is added, the final concentration of the enzyme is: 40 U / mL.

[0077] In another preferred example, in step (iv), when dextranase is added, the final concentration of the enzyme is: 1 U / mL.

[0078] In the third aspect of the present invention, a medical material is provided, and the medical material includes the gel prepared by the method described in the first aspect of the present invention.

[0079] In another preferred example, the medical material is a medical filler.

[0080] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Detailed Embodiments

[0081] After extensive and in-depth research and a large number of screenings, the inventor of the present invention first discovered a preparation method for a multi-component mixed cross-linking combination of dextran-sodium hyaluronate based on the optimization of anti-enzyme digestion performance. Specifically, under alkaline conditions, a combination of dextran and sodium hyaluronate with a specific molecular weight and molar ratio is reacted with a common epoxy group-containing cross-linking agent. The gel prepared by the method of the present invention has excellent anti-dextranase and hyaluronidase digestion performance. On this basis, the present invention was completed.

[0082] Terms

[0083] To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0084] The term "about" can refer to a value or a composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0085] As used herein, the term "comprising" or "including (containing)" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of..." or "consisting of...".

[0086] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the said range and, where appropriate, fractional values thereof (such as one-tenth and one-hundredth of an integer).

[0087] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0088] The main advantages of the present invention include:

[0089] (a) The preparation method of the invention simultaneously involves multiple cross-linking methods between dextran sugar chains, between sodium hyaluronate sugar chains, and between dextran and sodium hyaluronate sugar chains. On the one hand, this method enriches the bonding forms within the gel and increases the three-dimensional spatial complexity of the hydrogel.

[0090] (b) The overall process route of the preparation method of the present invention is simple and easy to operate, with less amount of cross-linking agent used, more environmentally friendly and green, and safe and controllable.

[0091] (c) The gel prepared by the method of the present invention can be used as an innovative tissue filling material, which can be used as a long-acting skin filler and has broad application prospects in the fields of medical beauty and the like.

[0092] 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, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0093] Example 1

[0094] This example examines the effect of the mixed cross-linking of medium molecular dextran and low molecular sodium hyaluronate with different ratios on the properties of the gel. The preparation method is as follows:

[0095] (1) Dissolve 0.5 g of BDDE in 100 mL of 0.25 M aqueous NaOH solution. After mixing, add X g of dextran 70 (average molecular weight: 70 kDa) and Y g of low molecular sodium hyaluronate (average molecular weight: 356 kDa), and stir well at room temperature until completely dissolved without lumps. React at 37 °C for 16 hours to obtain a transparent hydrogel.

[0096] Table 1: Raw material addition amounts and corresponding names in Example 1

[0097]

[0098] In the three reaction systems, the molar amount of the total sugar units is the same, and the molar ratios of the dextran sugar units to the sodium hyaluronate sugar units are 2:8, 5:5, and 8:2, respectively.

[0099] (2) Roughly cut the obtained hydrogel into gel blocks with a diameter of 1 - 2 cm 3 , neutralize (to a pH between 6.8 and 7.5), swell, and dialyze in an isotonic phosphate buffer solution at room temperature for more than 48 hours (changing the solution every 4 - 6 hours).

[0100] (3) Granulate, fill, and sterilize with a 100-mesh stainless steel sieve to obtain a suspension of mixed cross-linked dextran-sodium hyaluronate gel microparticles with a particle size range of 50 - 250 μm.

[0101] Example 2

[0102] In this example, the effect of the mixed cross - linking of different ratios of medium - molecular - weight dextran and medium - molecular - weight sodium hyaluronate on the properties of the inventive gel was investigated. The preparation method is as follows:

[0103] (1) Dissolve 0.5 g of BDDE in 100 mL of 0.25 M aqueous NaOH solution. After mixing, add X g of dextran 70 (average molecular weight: 70 kDa) and Y g of medium - molecular - weight sodium hyaluronate (average molecular weight: 1200 kDa), and stir well at room temperature until completely dissolved without lumps. React at a constant temperature of 37 °C for 16 hours to obtain a transparent hydrogel.

[0104] Table 2: Raw material addition amounts and corresponding names in Example 2

[0105]

[0106] In the three reaction systems, the molar amount of the total sugar units is the same, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units are 2:8, 5:5, and 8:2, respectively.

[0107] (2) Roughly cut the obtained hydrogel into gel blocks with a diameter of 1 - 2 cm 3 , neutralize (to a pH between 6.8 and 7.5), swell, and dialyze in an isotonic phosphate - buffered solution at room temperature for more than 48 hours (change the solution every 4 - 6 hours).

[0108] (3) Granulate, fill, and sterilize with a 100 - mesh stainless - steel sieve to obtain a suspension of mixed - cross - linked dextran - sodium hyaluronate gel microparticles with a particle size range of 50 - 250 μm.

[0109] Example 3

[0110] In this example, the effect of the mixed cross - linking of different ratios of high - molecular - weight dextran and low - molecular - weight sodium hyaluronate on the properties of the inventive gel was investigated. The preparation method is as follows:

[0111] (1) Dissolve 0.5 g of BDDE in 100 mL of 0.25 M aqueous NaOH solution. After mixing, add X g of dextran 500 (average molecular weight: 500 kDa) and Y g of low - molecular - weight sodium hyaluronate (average molecular weight: 356 kDa), and stir well at room temperature until completely dissolved without lumps. React at a constant temperature of 37 °C for 16 hours to obtain a transparent hydrogel.

[0112] Table 3: Raw material addition amounts and corresponding names in Example 3

[0113]

[0114] In the three reaction systems, the molar amount of the total sugar units is the same, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units are 2:8, 5:5, and 8:2, respectively.

[0115] (2) The obtained hydrogel was roughly cut into gel blocks with a diameter of 1 - 2 cm 3 and then neutralized (to a pH between 6.8 and 7.5), swollen, and dialyzed at room temperature in an isotonic phosphate buffer for more than 48 hours (changing the solution every 4 - 6 hours).

[0116] (3) Granulated with a 100 - mesh stainless - steel sieve, filled, and sterilized to obtain a suspension of mixed cross - linked dextran - sodium hyaluronate gel microparticles with a particle size range of 50 - 250 μm.

[0117] Example 4

[0118] This example investigated the effect of different ratios of high - molecular - weight dextran and medium - molecular - weight sodium hyaluronate on the properties of the inventive gel. The preparation method is as follows:

[0119] (1) Dissolve 0.5 g of BDDE in 100 mL of 0.25 M NaOH aqueous solution, mix well, and then add X g of dextran 500 (average molecular weight: 500 kDa) and Y g of medium - molecular - weight sodium hyaluronate (average molecular weight: 1200 kDa). Stir well at room temperature until completely dissolved without lumps. React at 37 °C for 16 hours to obtain a transparent hydrogel.

[0120] Table 4: Raw material addition amounts and corresponding names in Example 4

[0121]

[0122] In the three reaction systems, the molar amount of the total sugar units is the same, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units are 2:8, 5:5, and 8:2, respectively.

[0123] (2) The obtained hydrogel was roughly cut into gel blocks with a diameter of 1 - 2 cm 3 and then neutralized (to a pH between 6.8 and 7.5), swollen, and dialyzed at room temperature in an isotonic phosphate buffer for more than 48 hours (changing the solution every 4 - 6 hours).

[0124] (3) Granulated with a 100 - mesh stainless - steel sieve (particle size range of 50 - 250 μm), filled, and sterilized to obtain a suspension of mixed cross - linked dextran - sodium hyaluronate gel microparticles.

[0125] Compare the swelling degrees of different mixed cross - linked gels.

[0126] Weigh 0.5 g of each gel and detect it according to the method in 6.6 of YY / T 0962-2021. The results are shown in Table 5.

[0127] Table 5: Comparison of the swelling degrees of the mixed cross-linked gels

[0128]

[0129] Based on the data shown in the above table, the present invention reveals that even under the condition of keeping the sugar unit and cross-linker concentrations consistent, different ratios of two polysaccharides and the average length of their sugar chains have a significant impact on the water retention performance of the gels. In addition, although the hydrophilicity of sodium hyaluronate is more significant than that of dextran, the gel containing a higher proportion of sodium hyaluronate does not necessarily have the highest swelling degree. Therefore, the present invention points out that the hydrophilic properties of sodium hyaluronate and dextran and the topological characteristics of their spatial structures jointly determine the overall water retention ability of the gels.

[0130] Compare the tolerance of different mixed cross-linked gels to hyaluronidase.

[0131] According to the swelling degree of each gel, accurately weigh the corresponding mass of gel microparticles to ensure that the mass of the polysaccharide in each group is the same. Then, add phosphate buffer solution to the gels and incubate them in a water bath at 37 °C for 1 hour to ensure the full swelling of the gels at 37 °C. After swelling, remove the excess buffer solution by centrifugation and accurately weigh the mass of the gels, denoted as m0. Then, add an equal amount of hyaluronidase to each system, and at the same time add phosphate buffer solution to make the total reaction volume consistent. The final concentration of the enzyme is 40 U / mL, with a total of 10 tubes (Examples 1-3 and 2-3 are excluded). Finally, heat the systems in a water bath at 37 °C, take them out at the 2-hour and 4-hour time points respectively, centrifuge and weigh the remaining mass of the gel solids, denoted as m2 and m4.

[0132] Calculate the percentage of the gel mass retention rate at the 2-hour and 4-hour time points of the reaction system. The 2-hour retention rate = (m2 / m0) × 100%, and the 4-hour retention rate = (m4 / m0) × 100%.

[0133] Classify the material stability according to the percentage of the mass retention rate: Grade A: The mass retention rate is in the range of 90% to 110%, indicating a low degree of material degradation and a stable structure; Grade B: The mass retention rate is in the range of 60% to 90% or 110% to 140%, indicating a medium degree of material degradation; Grade C: The mass retention rate is in the range of 10% to 60%, indicating a large degree of material degradation. Grade D: The mass retention rate is less than 10%, indicating that the material has basically completely lost the characteristics of the hydrogel. The results are shown in the following table.

[0134] Table 6: Comparison of the change rate of the solid mass of the mixed cross-linked gels after treatment with hyaluronidase

[0135]

[0136] In Examples 1-3 and 2-3, no experimental data were collected because the gels had too low elasticity after swelling and homogenization and were not suitable for development as skin filling materials.

[0137] According to the data shown in the above table, after enzymatic treatment, the solid mass of most gels decreased, indicating that a large number of β-1,4-glycosidic bonds of hyaluronic acid in the gels were broken and the gels were partially disintegrated. However, the solid mass of some examples increased because the enzymatic treatment reduced the overall bonding degree of the gels, the spatial organization of the sugar chains became looser, and thus the overall hydrophilicity of the gels was enhanced. Therefore, when the amount of water adsorbed by the gels due to the increase in the swelling degree exceeded the amount of water lost due to sugar chain degradation, the solid mass of the gels increased. In summary, after treatment with hyaluronidase, the examples in which the gel mass remained relatively stable had a stronger tolerance to the enzyme than the examples in which the gel mass decreased significantly, such as Examples 2-1, 2-2, 3-1, and 3-2.

[0138] Compare the tolerance of different mixed cross-linked gels to dextranase.

[0139] According to the swelling degree of each gel, accurately weigh the corresponding mass of gel microparticles to ensure that the mass of polysaccharides in each group is the same. Then, add phosphate buffer to the gels and heat them in a water bath at 37 °C for 1 hour to achieve sufficient swelling of the gels. After swelling, remove the excess buffer by centrifugation and accurately weigh the gel mass, denoted as M0. Then, add an equal amount of dextranase to each system, and at the same time add phosphate buffer to make the total reaction volume the same, and the final concentration of the enzyme is 1 U / mL, for a total of 10 tubes (Examples 1-3 and 2-3 were excluded). Finally, heat the system in a water bath at 37 °C, take it out at the 2-hour and 4-hour time points respectively, centrifuge and weigh the remaining mass of the gel solid, denoted as M2 and M4.

[0140] Calculate the percentage of gel mass retention rate at the 2-hour and 4-hour time points of the reaction system. The 2-hour retention rate = (M2 / M0) × 100%, and the 4-hour retention rate = (M4 / M0) × 100%. Classify the material stability according to the percentage of mass retention rate: Grade A: The mass retention rate is in the range of 90% to 110%, indicating low material degradation degree and stable structure; Grade B: The mass retention rate is in the range of 60% to 90% or 110% to 140%, indicating medium degree of material degradation; Grade C: The mass retention rate is in the range of 10% to 60%, indicating large degree of material degradation; Grade D: The mass retention rate is less than 10%, indicating that the material has basically completely lost the characteristics of hydrogel.

[0141] The results are shown in the following table.

[0142] Table 7: Comparison of the solid mass change rate of the mixed cross-linked gel after dextranase treatment

[0143]

[0144] In Examples 1-3 and 2-3, no experimental data were collected because the gels had too low elasticity after swelling and homogenization and were not suitable for development as skin filling materials.

[0145] According to the data shown in the above table, after treatment with dextranase, the examples in which the gel mass remained relatively stable had a stronger tolerance to the enzyme than the examples in which the gel mass decreased significantly, such as Examples 1-1, 2-2, and 3-1.

[0146] Based on the experimental results of the treatment with the two enzymes, Examples 2-2 and 3-1 had the best degradation tolerance to the two enzymes.

[0147] Control Example 1

[0148] A cross-linked gel prepared with dextran 70 as the sole raw material was compared with Example 2-2 in terms of performance. The preparation method was as follows:

[0149] (1) Dissolve 0.5 g of BDDE in 100 mL of 0.25 M aqueous NaOH solution. After mixing, add 20 g of dextran 70 (average molecular weight: 70 kDa). Stir well at room temperature until completely dissolved without lumps. React at 37 °C for 16 hours to obtain a hydrogel.

[0150] (2) Coarsely cut the obtained hydrogel into gel blocks with a diameter of 1-2 cm 3 and neutralize, swell, and dialyze in isotonic phosphate buffer at room temperature for more than 48 hours.

[0151] (3) Granulate, fill, and sterilize with a 100-mesh stainless steel sieve to obtain a suspension of cross-linked dextran gel microparticles with a particle size range of 50-250 μm.

[0152] Control Example 2

[0153] A cross-linked gel prepared with medium molecular weight sodium hyaluronate as the sole raw material was compared with Example 2-2 in terms of performance. The preparation method was as follows:

[0154] (1) Dissolve 0.5 g of BDDE in 100 mL of 0.25 M aqueous NaOH solution. After mixing, add 15 g of medium molecular weight sodium hyaluronate (average molecular weight: 1200 kDa). Stir well at room temperature until completely dissolved without lumps. React at 37 °C for 16 hours to obtain a hydrogel.

[0155] (2) Coarsely cut the obtained hydrogel into gel blocks with a diameter of 1-2 cm 3The gel blocks are neutralized, swollen, and dialyzed at room temperature in isotonic phosphate buffer for more than 48 hours.

[0156] (3) Granulate, fill, and sterilize with a 100-mesh stainless steel sieve to obtain a suspension of cross-linked sodium hyaluronate gel microparticles with a particle size range of 50 - 250 μm.

[0157] Compare the tolerance of Example 2-2, Comparative Example 1, Comparative Example 2, and a commercially available cross-linked sodium hyaluronate gel product to hyaluronidase, dextranase, and the combined degradation of the two enzymes. The operation method is the same as described above.

[0158] Table 8: Comparison of the change rate of the solid mass of cross-linked gels after treatment with hyaluronidase and dextranase

[0159]

[0160] According to the data shown in the above table, as a mixed cross-linked gel of two polysaccharides, Example 2-2 exhibits a stronger comprehensive tolerance to hyaluronidase and dextranase compared to single-component cross-linked gels. At the same time, compared to commercially available cross-linked sodium hyaluronate gel products and other mixed cross-linked combinations, the anti-enzymatic hydrolysis ability of Example 2-2 is also more prominent.

[0161] According to the embodiments of the present invention, although the above description is a preferred embodiment, the protection scope of the present invention is not limited thereto. Any change, modification, combination, substitution, or simplification that does not violate the spirit and principle of the present invention is regarded as an equivalent replacement method and is included in the protection scope of the present invention.

[0162] Discussion

[0163] The present invention discloses a preparation method of a multi-crosslinked combination of dextran-sodium hyaluronate based on the optimization of anti-enzymatic hydrolysis performance. Under alkaline conditions, medium molecular dextran and a combination of medium and low molecular sodium hyaluronate are crosslinked in one step according to a ratio to form a mixed polysaccharide hydrogel, and the gel is swollen, dialyzed, homogenized, and sterilized to prepare a suspension of gel microparticles. Since the gel prepared by the method of the present invention has excellent anti-dextranase and hyaluronidase enzymatic hydrolysis performance. Therefore, the inventor infers that the method of the present invention enables topological entanglement and multi-crosslinked covalent crosslinking between branched dextrans, between linear sugar chains of sodium hyaluronate, and between these two high molecular polysaccharides, thereby significantly enhancing the complexity of the three-dimensional structure of the hydrogel. However, the protection scope of the present invention is not limited by the above mechanism.

[0164] Specifically, in the degradation experiments of hyaluronidase and α-glucosidase, the mixed cross-linked gel exhibited stronger structural stability than the single polysaccharide component cross-linked gel and had stronger anti-enzymatic hydrolysis performance. The anti-enzymatic hydrolysis mixed cross-linked dextran-sodium hyaluronate gel material prepared by this method can be used as a tissue filler with reduced cross-linking agent usage, safer, less allergenic but long-acting maintenance, and can be applied to cosmetics, intra-articular injection solutions, and tissue fillers, especially in the field of medical aesthetics.

[0165] The method of the present invention uses a combination of dextran and sodium hyaluronate with one or several specific molecular weights and molar feed ratios, and can prepare a mixed cross-linked dextran-sodium hyaluronate gel with outstanding anti-enzymatic hydrolysis ability. The preparation method of the present invention has simple steps, does not introduce other organic substances except polysaccharide raw materials and common cross-linking agents, and has high safety; the multi-component mixed cross-linking further enhances the structural complexity of the gel compared with the binary composite cross-linking, and the experimental results show stronger anti-enzymatic hydrolysis ability than the single-component cross-linked gel. The product of the present invention is a novel tissue filling material, can be used as a long-acting skin filler, and is applied in the fields of medical aesthetics, etc., and has broad application prospects and market potential.

[0166] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A method for preparing a dextran-sodium hyaluronate gel with anti-dextranase and anti-hyaluronidase enzymatic hydrolysis properties, characterized in that, It includes the following steps: (1) Dissolution: Add a crosslinking agent to a 0.1 - 0.5 mol / L NaOH solution. After stirring evenly, add dextran and sodium hyaluronate, and continue stirring until completely dissolved to obtain Solution A; Among them, the concentration of the crosslinking agent is 0.05 - 2 wt%, and the crosslinking agent is one or more of 1,4 - butanediol diglycidyl ether, 1,2,7,8 - diepoxyoctane, and 1,3 - diepoxybutane; Among them, the added dextran and sodium hyaluronate are selected from the following groups: (a) Dextran with an average molecular weight of 70 kDa and sodium hyaluronate with an average molecular weight of 1200 kDa; (b) Dextran with an average molecular weight of 500 kDa and sodium hyaluronate with an average molecular weight of 356 kDa; And when the molecular weight combination of the added dextran and sodium hyaluronate is (a), the molar ratio of the dextran to the sodium hyaluronate is 1:4 - 1:1; when the molecular weight combination of the added dextran and sodium hyaluronate is (b), the molar ratio of the dextran to the sodium hyaluronate is 1:4 - 1:1; (2) Cross - linking reaction: Let Solution A stand for cross - linking. The cross - linking temperature is 30 - 40 °C, and the cross - linking time is 6 - 24 h to obtain transparent Gel B; (3) Adjust the pH: Cut the Gel B into small pieces of 1 - 2 cm 3 and neutralize them until the pH is between 6.8 and 7.5; (4) Swelling and dialysis: Swell and dialyze in an isotonic phosphate buffer solution at room temperature, change the solution every 4 - 6 hours, and dialyze for more than 48 hours; (5) Sieving: The dialyzed gel is sieved through a 100 - mesh sieve to obtain dextran - sodium hyaluronate gel particles, and the particle size range of the gel is 50 - 250 μm; (6) Filling and sterilization: Fill the gel particles obtained in step (5) into a sealed syringe and sterilize by moist heat; Among them, the dextran is dextran.

2. The method according to claim 1, wherein When the molecular weight combination of the added dextran and sodium hyaluronate is (a), the molar ratio of the dextran to the sodium hyaluronate is 1:

4.

3. The method according to claim 1, wherein The added dextran and sodium hyaluronate are: dextran with an average molecular weight of 70 kDa and sodium hyaluronate with an average molecular weight of 1200 kDa; and the molar ratio of the dextran to the sodium hyaluronate is 1:

1.

4. The method according to claim 1, wherein In step (1), the cross - linking agent is 1,4 - butanediol diglycidyl ether.

5. The method according to claim 1, characterized in that, When the molecular weight combination of the added dextran and sodium hyaluronate is (b), the molar ratio of the dextran to the sodium hyaluronate is 1:

4.

6. The method according to claim 1, wherein In step (1), the concentration of the NaOH solution is 0.2 - 0.3 mol / L.

7. The method according to claim 1, wherein In step (3), dilute hydrochloric acid is used for neutralization.

8. The method according to claim 1, characterized in that In step (4), the phosphate buffer solution refers to an isotonic solution of sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate dissolved in injection water.

9. A dextran-sodium hyaluronate gel with the performance of resisting enzymatic hydrolysis by dextranase and hyaluronidase, characterized in that, The gel is prepared by the method described in claim 1.

10. A medical material, characterized in that, The medical material includes the gel prepared by the method described in claim 1.

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