Preparation method of glucan-sodium hyaluronate gel with enzymolysis resistance

Abstract

The invention provides a preparation method of dextran-sodium hyaluronate gel with the performance of resisting enzymolysis of dextranase and hyaluronidase. The preparation method comprises the steps of dissolution, cross-linking reaction, pH adjustment, swelling and dialysis, sieving, filling and sterilization and the like, is simple and convenient, and is beneficial to industrial production. The gel prepared by the method disclosed by the invention is high in safety performance and good in dextranase and hyaluronidase enzymolysis resistance.

Description

Technical Field

[0001] The invention relates to the fields of material science and clinical medicine, and in particular to a method for preparing a dextran-sodium hyaluronate gel with anti-enzymatic performance. Background Art

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

[0003] Sodium hyaluronate is susceptible to degradation by hyaluronidase and free radicals in the human body. Chemical cross-linking can allow long polysaccharide chains to form a hydrogel structure and enhance the stability of the molecule. Common 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 is ring-opened and attacked by the hydroxyl group on the sodium hyaluronate sugar unit to form a stable ether bond. This three-dimensional hydrogel structure can improve the physical and chemical properties of the material and enhance its mechanical strength, thereby extending its durability in the skin to a certain extent, but this improvement is still limited. Therefore, beauty seekers usually need to undergo multiple injections within a year to maintain their ideal cosmetic filling effect.

[0004] Patent CN108250462A discloses a method for preparing enzymatically resistant cross-linked sodium hyaluronate, which 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 enzymatic resistance is still limited.

[0005] Patent CN111012953B enhances the anti-enzymatic performance of the combined material by adding indomethacin-polylactic acid microspheres to the gel and utilizing the inhibitory effect of indomethacin on hyaluronidase. However, dichloromethane is used in the preparation of the microspheres, which introduces the risk of residual toxic organic reagents; in addition, since the combination of gel and polylactic acid microspheres is mainly used for injection deep into the dermis, its application scenarios are relatively limited.

[0006] Glucan is a high molecular weight polysaccharide with a branched structure formed by glucose units connected by 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 bonds and the side chain groups of the polysaccharide skeleton. Glucan can be divided into α-glucan and β-glucan according to the type of glycosidic bonds. α-Glucan, also known as dextran, has long been widely used as a plasma substitute due to its excellent molecular stability and biosafety. In addition, hydrogel materials based on cross-linked dextran have been used as drug delivery carriers for decades due to their excellent drug release properties and biocompatibility. β-glucan is valued for its immunomodulatory and anti-inflammatory effects. A number of β-glucan-based liquid wound dressings have been developed at home and abroad to promote the recovery of skin wounds.

[0007] Patent CN105377898A discloses a method for preparing a binary cross-linked hydrogel, in which dextran and sodium hyaluronate are cross-linked through (poly) phosphodiester bonds, and the formed hydrogel shows stronger resistance to hyaluronidase degradation than the gel cross-linked with sodium hyaluronate alone. Patent CN106661133A discloses a method for grafting dextran into a cross-linked hydrogel, in which small molecule dextran first diffuses freely into the gel and is then grafted to the sodium hyaluronate skeleton through an amide bond. Compared with before grafting, the swelling degree of the gel is reduced and the resistance to hyaluronidase is enhanced. Both methods introduce dextran that resists hyaluronidase degradation through chemical cross-linking, and realize the covalent bond connection between dextran and sodium hyaluronate. The newly formed binary cross-linked product has an additional bonding mode compared to the single network of cross-linked sodium hyaluronate, and the steric hindrance provided by the dextran sugar chain further limits the enzymolysis efficiency of hyaluronidase.

[0008] However, as a biodegradable high molecular weight polysaccharide, dextran is also susceptible to degradation by glycosidases and free radicals in the body. Although the aforementioned binary composite cross-linking method enhances the network structure of the original sodium hyaluronate gel, no cross-linking chemical bonds are formed between the dextran sugar chains to obtain a structural reinforcement similar to that of sodium hyaluronate. Secondly, the aforementioned two composite cross-linking methods have complex process flows, and the intermediate steps involve compounds that are irritating to the skin, so these methods have not been widely used in the fields of biomedicine or medical cosmetology. Summary of the invention

[0009] The object of the present invention is to provide a method for preparing a multi-crosslinked combination of dextran and sodium hyaluronate with optimized anti-enzymatic performance.

[0010] The present invention also aims to provide a hydrogel with a simple process flow and no irritation to the skin.

[0011] In the first aspect of the present invention, a method for preparing a dextran-sodium hyaluronate gel having resistance to dextranase and hyaluronidase enzymatic hydrolysis is provided, comprising the following steps: (1) Dissolution: Add the cross-linking 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; Wherein, the concentration of the cross-linking agent is 0.05-2wt%, and the cross-linking agent is one or more of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, 1,3-diepoxybutane; Wherein, the average molecular weight of the added dextran is: 60~80kDa, and the average molecular weight of sodium hyaluronate is: 1000~1800kDa; or, The average molecular weight of the added dextran is 400-600 kDa, and the average molecular weight of sodium hyaluronate is 300-1800 kDa; And the molar ratio of the dextran to the sodium hyaluronate is 1:4 to 4:1; (2) Cross-linking reaction: Solution A is allowed to cross-link at a temperature of 30-40°C for a time of 6-24 h to obtain a transparent gel B; (3) Adjusting pH: Cut the gel B into 1-2 cm 3 Small pieces, neutralized to a pH between 6.8 and 7.5; (4) Swelling and dialysis: swelling and dialysis in isotonic phosphate buffer at room temperature, with the solution changed every 4 to 6 hours for more than 48 hours; (5) Sieving: The dialyzed gel is passed through a 100-mesh sieve to obtain dextran-sodium hyaluronate gel particles, wherein the particle size of the gel is in the range of 50 to 250 μm; (6) Filling and sterilization: Fill the gel particles obtained in step (5) into a filling syringe and sterilize by wet heat.

[0012] In another preferred embodiment, in step (1), the molar ratio of the dextran to the sodium hyaluronate is 1:1 to 1:4.

[0013] In another preferred embodiment, in step (1), the molar ratio of the dextran to the sodium hyaluronate is 1:4, 1:1 or 4:1.

[0014] 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, The average molecular weight of the added dextran is 450-550 kDa, and the average molecular weight of sodium hyaluronate is 350-1500 kDa.

[0015] In another preferred embodiment, the added dextran and sodium hyaluronate are selected from the following group: (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; (c) Dextran with an average molecular weight of 500 kDa and sodium hyaluronate with an average molecular weight of 1200 kDa.

[0016] In another preferred embodiment, 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 to 1:4.

[0017] In another preferred embodiment, 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 to 4:1.

[0018] In another preferred embodiment, 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.

[0019] In another preferred embodiment, the anti-enzymatic performance refers to the gel's resistance to degradation by two enzymes: dextranase (α-glycosidase) and hyaluronidase (β-glycosidase).

[0020] In another preferred embodiment, the concentration of the dextran is 2-40wt%, preferably 2-20wt%.

[0021] In another preferred embodiment, the concentration of the sodium hyaluronate is 1-25wt%, preferably 2-15wt%.

[0022] In another preferred embodiment, in step (1), the cross-linking agent is 1,4-butanediol diglycidyl ether.

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

[0024] In another preferred embodiment, in step (1), the concentration of the NaOH solution is 0.2-0.3 mol / L.

[0025] In another preferred embodiment, in step (3), dilute hydrochloric acid is used for neutralization.

[0026] 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.

[0027] In another preferred example, in step (4), the phosphate buffer solution has an osmotic pressure of 270-330 mOsmol / L and a pH of 6.8-7.5.

[0028] In another preferred example, in step (s6), the sterilization conditions are as follows: moist heat sterilization at 121 °C for 8-30 min.

[0029] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel is 60% to 90% or 110% to 140% after 2 h.

[0030] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel is 90% to 110% after 2 h.

[0031] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel is 60% to 90% or 110% to 140% after 4 h.

[0032] In another preferred example, after being treated with hyaluronidase, the mass retention rate of the gel is 90% to 110% after 4 h.

[0033] In another preferred example, after being treated with dextranase, the mass retention rate of the gel is 60% to 90% or 110% to 140% after 2 h.

[0034] In another preferred example, after being treated with dextranase, the mass retention rate of the gel is 90% to 110% after 2 h.

[0035] In another preferred example, after being treated with dextranase, the mass retention rate of the gel is 60% to 90% or 110% to 140% after 4 h.

[0036] In another preferred example, after being treated with dextranase, the mass retention rate of the gel is 90% to 110% after 4 h.

[0037] In another preferred example, the treatment process is as follows: (i) According to the swelling degree of each gel, accurately weigh the corresponding mass of gel particles; (ii) Add phosphate buffer solution to the gel particles and incubate in a water bath at 37 °C for 1 hour to ensure sufficient swelling of the gel at 37 °C; (iii) After swelling, remove the excess buffer solution by centrifugation and accurately weigh the mass of the gel, denoted as X 0; (iv) adding an equal amount of hyaluronidase or dextranase; (v) Heat the system in a 37°C water bath, take it out at 2 hours and 4 hours, centrifuge it and weigh the remaining mass of the gel solid, recorded as X 2 and X 4 ; Among them, 2h retention rate = (X 2 / X 0 ) × 100%, 4h retention rate = (X 4 / X 0 ) × 100%.

[0038] In another preferred embodiment, in step (iv), when hyaluronidase is added, the final concentration of the enzyme is 40 U / mL.

[0039] In another preferred embodiment, in step (iv), when dextranase is added, the final concentration of the enzyme is 1 U / mL.

[0040] In the second aspect of the present invention, a dextran-sodium hyaluronate gel having resistance to dextranase and hyaluronidase hydrolysis is provided, wherein the gel is prepared by the method described in the first aspect of the present invention.

[0041] In another preferred embodiment, after the gel is treated with hyaluronidase, the mass retention rate of the gel after 2 hours is 60% to 90% or 110% to 140%.

[0042] In another preferred embodiment, after the gel is treated with hyaluronidase, the mass retention rate of the gel after 2 hours is 90% to 110%.

[0043] In another preferred embodiment, after the gel is treated with hyaluronidase, the mass retention rate of the gel after 4 hours is 60% to 90% or 110% to 140%.

[0044] In another preferred embodiment, after the gel is treated with hyaluronidase, the mass retention rate of the gel after 4 hours is 90% to 110%.

[0045] In another preferred embodiment, after the gel is treated with dextranase, the mass retention rate at 2 h is 60% to 90% or 110% to 140%.

[0046] In another preferred embodiment, after the gel is treated with dextranase, the mass retention rate of the gel after 2 hours is 90% to 110%.

[0047] In another preferred embodiment, after the gel is treated with dextranase, the mass retention rate after 4 hours is 60% to 90% or 110% to 140%.

[0048] In another preferred embodiment, after the gel is treated with dextranase, the mass retention rate of the gel after 4 hours is 90% to 110%.

[0049] In another preferred embodiment, the processing process is as follows: (i) Accurately weigh the corresponding mass of gel particles according to the swelling degree of each gel; (ii) adding phosphate buffer to the gel microparticles and incubating in a 37° C. water bath for 1 hour to ensure that the gel is fully swollen at 37° C.; (iii) After swelling, remove excess buffer by centrifugation and accurately weigh the gel mass, recorded as X 0 ; (iv) adding an equal amount of hyaluronidase or dextranase; (v) Heat the system in a 37°C water bath, take it out at 2 hours and 4 hours, centrifuge it and weigh the remaining mass of the gel solid, recorded as X 2 and X 4 ; Among them, 2h retention rate = (X 2 / X 0 ) × 100%, 4h retention rate = (X 4 / X 0 ) × 100%.

[0050] In another preferred embodiment, in step (iv), when hyaluronidase is added, the final concentration of the enzyme is 40 U / mL.

[0051] In another preferred embodiment, in step (iv), when dextranase is added, the final concentration of the enzyme is 1 U / mL.

[0052] In a third aspect of the present invention, a medical material is provided, wherein the medical material comprises a gel prepared by the method described in the first aspect of the present invention.

[0053] In another preferred embodiment, the medical material is a medical filler.

[0054] 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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0055] The inventors have conducted extensive and in-depth research and, through a large number of screenings, have discovered for the first time a method for preparing a multi-mixed cross-linked combination of dextran and sodium hyaluronate based on optimized anti-enzymatic performance. Specifically, under alkaline conditions, a combination of dextran and sodium hyaluronate with a specific molecular weight and a certain amount of substance is reacted with a commonly used epoxy-containing cross-linking agent. The gel prepared by the method of the present invention has excellent anti-dextranase and hyaluronidase enzymatic performance. On this basis, the present invention has been completed.

[0056] the term In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0057] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one 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.).

[0058] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0059] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0060] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0061] The main advantages of the present invention include: (a) The preparation method of the invention involves multiple cross-linking modes between glucan sugar chains, between sodium hyaluronate sugar chains, and between glucan 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.

[0062] (b) The overall process route of the preparation method of the present invention is simple and easy to operate, the amount of cross-linking agent used is small, and it is more environmentally friendly, green, safe and controllable.

[0063] (c) The gel prepared by the method of the present invention can be used as an innovative tissue filling material. The material can be used as a long-acting skin filler and has broad application prospects in the fields of medical cosmetology.

[0064] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0065] Example 1 This example investigates the effect of mixed cross-linking of medium molecular dextran and low molecular sodium hyaluronate in different ratios on gel properties. The preparation method is as follows: (1) Dissolve 0.5 g BDDE in 100 mL 0.25 M NaOH aqueous solution, mix well, add X g dextran 70 (average molecular weight: 70 kDa) and Y g low molecular weight sodium hyaluronate (average molecular weight: 356 kDa), and stir thoroughly at room temperature until completely dissolved without agglomeration. React at 37 °C for 16 hours to obtain a transparent hydrogel.

[0066] Table 1: Raw material addition amounts and corresponding names in Example 1

[0067] In the three reaction systems, the molar amount of total sugar units was consistent, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units were 2:8, 5:5, and 8:2, respectively.

[0068] (2) The hydrogel obtained by the reaction was roughly cut into pieces with a diameter of 1~2 cm. 3 The gel blocks were neutralized (to a pH between 6.8 and 7.5), swollen, and dialyzed in isotonic phosphate buffer at room temperature for more than 48 hours (the solution was changed every 4 to 6 hours).

[0069] (3) Granulating, filling and sterilizing with a 100-mesh stainless steel sieve to obtain a mixed cross-linked dextran-sodium hyaluronate gel microparticle suspension with a particle size ranging from 50 to 250 μm.

[0070] Example 2 This example investigates the effect of mixed cross-linking of medium molecular weight dextran and medium molecular weight sodium hyaluronate in different ratios on the properties of the inventive gel, and the preparation method is as follows: (1) Dissolve 0.5 g BDDE in 100 mL 0.25 M NaOH aqueous solution, mix well, add X g dextran 70 (average molecular weight: 70 kDa) and Y g medium molecular weight sodium hyaluronate (average molecular weight: 1200 kDa), and stir thoroughly at room temperature until completely dissolved without lumps. React at 37 ° C for 16 hours to obtain a transparent hydrogel.

[0071] Table 2: Raw material addition amounts and corresponding names in Example 2

[0072] In the three reaction systems, the molar amount of total sugar units was consistent, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units were 2:8, 5:5, and 8:2, respectively.

[0073] (2) The hydrogel obtained by the reaction was roughly cut into pieces with a diameter of 1~2 cm. 3 The gel blocks were neutralized (to a pH between 6.8 and 7.5), swollen, and dialyzed in isotonic phosphate buffer at room temperature for more than 48 hours (the solution was changed every 4 to 6 hours).

[0074] (3) Granulating, filling and sterilizing with a 100-mesh stainless steel sieve to obtain a mixed cross-linked dextran-sodium hyaluronate gel microparticle suspension with a particle size ranging from 50 to 250 μm.

[0075] Example 3 This example investigates the effect of mixed cross-linking of high molecular weight dextran and low molecular weight sodium hyaluronate in different ratios on the properties of the inventive gel, and the preparation method is as follows: (1) Dissolve 0.5 g BDDE in 100 mL 0.25 M NaOH aqueous solution, mix well, add X g dextran 500 (average molecular weight: 500 kDa) and Y g low molecular weight sodium hyaluronate (average molecular weight: 356 kDa), and stir thoroughly at room temperature until completely dissolved without agglomerates. React at 37 °C for 16 hours to obtain a transparent hydrogel.

[0076] Table 3: Example 3 Raw material addition amount and corresponding name

[0077] In the three reaction systems, the molar amount of total sugar units was consistent, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units were 2:8, 5:5, and 8:2, respectively.

[0078] (2) The hydrogel obtained by the reaction was roughly cut into pieces with a diameter of 1~2 cm. 3 The gel blocks were neutralized (to a pH between 6.8 and 7.5), swollen, and dialyzed in isotonic phosphate buffer at room temperature for more than 48 hours (the solution was changed every 4 to 6 hours).

[0079] (3) Granulating, filling and sterilizing with a 100-mesh stainless steel sieve to obtain a mixed cross-linked dextran-sodium hyaluronate gel microparticle suspension with a particle size ranging from 50 to 250 μm.

[0080] Example 4 This example investigates the effect of mixed cross-linking of high molecular weight dextran and medium molecular weight sodium hyaluronate in different ratios on the properties of the inventive gel, and the preparation method is as follows: (1) Dissolve 0.5 g BDDE in 100 mL 0.25 M NaOH aqueous solution, mix well, add X g dextran 500 (average molecular weight: 500 kDa) and Y g medium molecular weight sodium hyaluronate (average molecular weight: 1200 kDa), and stir thoroughly at room temperature until completely dissolved without agglomerates. React at 37 °C for 16 hours to obtain a transparent hydrogel.

[0081] Table 4: Raw material addition amounts and corresponding names in Example 4

[0082] In the three reaction systems, the molar amount of total sugar units was consistent, and the molar ratios of dextran sugar units to sodium hyaluronate sugar units were 2:8, 5:5, and 8:2, respectively.

[0083] (2) The hydrogel obtained by the reaction was roughly cut into pieces with a diameter of 1~2 cm. 3 The gel blocks were neutralized (to a pH between 6.8 and 7.5), swollen, and dialyzed in isotonic phosphate buffer at room temperature for more than 48 hours (the solution was changed every 4 to 6 hours).

[0084] (3) Granulate the mixture using a 100-mesh stainless steel screen (particle size range is 50-250 μm), fill, and sterilize to obtain a mixed cross-linked dextran-sodium hyaluronate gel particle suspension.

[0085] Comparison of the swelling degrees of different hybrid cross-linked gels.

[0086] Weigh 0.5 g of each gel and test it according to method 6.6 in YY / T 0962-2021. The results are shown in Table 5.

[0087] Table 5: Comparison of swelling degree of mixed cross-linked gels

[0088] According to the data shown in the above table, the present invention reveals that even under the condition of keeping the sugar unit and the cross-linking agent concentrations consistent, different ratios of the two polysaccharides and the average length of their sugar chains have a significant effect on the water retention performance of the gel. In addition, although the hydrophilicity of sodium hyaluronate is more significant than that of dextran, the gel containing a higher ratio of sodium hyaluronate is not necessarily the highest value in terms of 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 capacity of the gel.

[0089] Compare the resistance of different mixed cross-linked gels to hyaluronidase.

[0090] According to the swelling degree of each gel, the corresponding mass of gel particles was accurately weighed to ensure the consistency of the mass of polysaccharides in each group. Then, phosphate buffer was added to the gel and incubated in a 37°C water bath for 1 hour to ensure that the gel was fully swollen at 37°C. After swelling, the excess buffer was removed by centrifugation and the mass of the gel was accurately weighed, recorded as m 0 Then, an equal amount of hyaluronidase was added to each system, and phosphate buffer was added to make the total reaction volume consistent. The final concentration of the enzyme was 40 U / mL, and a total of 10 tubes were added (Examples 1-3 and 2-3 were excluded). Finally, the system was heated in a 37°C water bath, and the system was taken out at 2 hours and 4 hours respectively. After centrifugation, the remaining mass of the gel solid was weighed and recorded as m 2 and m 4 .

[0091] Calculate the gel mass retention percentage of the reaction system at the 2-hour and 4-hour time points, 2h retention rate = (m 2 / m 0 ) × 100%, 4h retention rate = (m 4 / m 0 ) × 100%.

[0092] The material stability is graded according to the percentage of mass retention rate: Grade A: The mass retention rate is between 90% and 110%, indicating that the material has a low degree of degradation and a stable structure; Grade B: The mass retention rate is between 60% and 90% or 110% and 140%, indicating that the material has undergone moderate degradation; Grade C: The mass retention rate is between 10% and 60%, indicating that the material has undergone significant degradation. Grade D: The mass retention rate is less than 10%, indicating that the material has basically completely lost its hydrogel characteristics. The results are shown in the table below.

[0093] Table 6: Comparison of solid mass change rate of mixed cross-linked gels after hyaluronidase treatment

[0094] No experimental data were collected for Examples 1-3 and 2-3 because the elasticity of the gel after swelling and homogenization was too low and they were not suitable for development as skin filling materials.

[0095] According to the data shown in the above table, after enzyme treatment, the solid mass of most gels decreased, indicating that a large number of β-1,4-glycosidic bonds of hyaluronic acid in the gel were broken and the gel was partially disintegrated. However, the solid mass of some embodiments increased because the enzyme treatment reduced the overall bonding degree of the gel, and the spatial organization of the sugar chains became loose, thereby enhancing the overall hydrophilicity of the gel. Therefore, when the amount of water adsorbed by the gel due to the increase in swelling exceeds the amount of water lost due to the degradation of the sugar chains, the solid mass of the gel will increase. In summary, after hyaluronidase treatment, the embodiments in which the gel quality remains relatively stable have stronger tolerance to the enzyme than the embodiments in which the gel quality is significantly reduced, such as Examples 2-1, 2-2, 3-1 and 3-2.

[0096] Compare the resistance of different mixed cross-linked gels to dextranase.

[0097] According to the swelling degree of each gel, the corresponding mass of gel particles was accurately weighed to ensure that the mass of the polysaccharide in each group was consistent. Next, phosphate buffer was added to the gel and heated in a 37°C water bath for 1 hour to achieve sufficient swelling of the gel. After swelling, excess buffer was removed by centrifugation, and the mass of the gel was accurately weighed and recorded as M0. Then, an equal amount of dextranase was added to each system, and phosphate buffer was added to make the total reaction volume consistent. The final concentration of the enzyme was 1U / mL, for a total of 10 tubes (Examples 1-3 and 2-3 were excluded). Finally, the system was heated in a 37°C water bath, taken out at 2 hours and 4 hours, and the remaining mass of the gel solid was weighed after centrifugation, recorded as M 2 and M 4 .

[0098] Calculate the gel mass retention rate percentage of the reaction system at 2 hours and 4 hours, 2h retention rate = (M 2 / M 0 ) × 100%, 4h retention rate = (M 4 / M 0 ) × 100%. Material stability is graded according to the percentage of mass retention rate: Grade A: The mass retention rate is between 90% and 110%, indicating that the material has a low degree of degradation and a stable structure; Grade B: The mass retention rate is between 60% and 90% or 110% and 140%, indicating that the material has undergone moderate degradation; Grade C: The mass retention rate is between 10% and 60%, indicating that the material has undergone significant degradation. Grade D: The mass retention rate is less than 10%, indicating that the material has basically completely lost its hydrogel characteristics.

[0099] The results are shown in the table below.

[0100] Table 7: Comparison of the solid mass change rate of mixed cross-linked gels after dextranase treatment

[0101] No experimental data were collected for Examples 1-3 and 2-3 because the elasticity of the gel after swelling and homogenization was too low and they were not suitable for development as skin filling materials.

[0102] According to the data shown in the above table, after dextranase treatment, the embodiments in which the gel quality remained relatively stable had stronger tolerance to the enzyme than the embodiments in which the gel quality was significantly reduced, such as embodiments 1-1, 2-2 and 3-1.

[0103] Based on the experimental results of the two enzyme treatments, Examples 2-2 and 3-1 have the best tolerance to degradation by the two enzymes.

[0104] Comparative Example 1 The cross-linked gel using dextran 70 as the sole raw material was prepared for performance comparison with Example 2-2. The preparation method is as follows: (1) Dissolve 0.5 g BDDE in 100 mL 0.25 M NaOH aqueous solution, mix well, add 20 g dextran 70 (average molecular weight: 70 kDa), and stir thoroughly at room temperature until it is completely dissolved without lumps. React at 37 °C for 16 hours to obtain a hydrogel.

[0105] (2) The hydrogel obtained by the reaction was roughly cut into pieces with a diameter of 1~2 cm. 3 The gel blocks were neutralized, swollen, and dialyzed in isotonic phosphate buffer at room temperature for more than 48 hours.

[0106] (3) Granulating, filling and sterilizing with a 100-mesh stainless steel sieve to obtain a cross-linked dextran gel microparticle suspension with a particle size ranging from 50 to 250 μm.

[0107] Comparative Example 2 The cross-linked gel using medium molecular sodium hyaluronate as the sole raw material was prepared for performance comparison with Example 2-2. The preparation method is as follows: (1) Dissolve 0.5 g BDDE in 100 mL 0.25 M NaOH aqueous solution, mix well, add 15 g medium molecular weight sodium hyaluronate (average molecular weight: 1200 kDa), and stir thoroughly at room temperature until completely dissolved without lumps. React at 37 ° C for 16 hours to obtain a hydrogel.

[0108] (2) The hydrogel obtained by the reaction was roughly cut into pieces with a diameter of 1~2 cm. 3 The gel blocks were neutralized, swollen, and dialyzed in isotonic phosphate buffer at room temperature for more than 48 hours.

[0109] (3) Granulating, filling and sterilizing with a 100-mesh stainless steel sieve to obtain a cross-linked sodium hyaluronate gel microparticle suspension with a particle size ranging from 50 to 250 μm.

[0110] Comparing the tolerance of Example 2-2, Comparative Example 1, Comparative Example 2 and commercially available cross-linked sodium hyaluronate gel products to hyaluronidase, dextranase, and co-degradation by the two enzymes, the operation method is consistent with the above.

[0111] Table 8: Comparison of solid mass change rate of cross-linked gel after hyaluronidase and dextranase treatment

[0112] According to the data shown in the above table, Example 2-2, as a mixed cross-linked gel of two polysaccharides, exhibits a stronger comprehensive tolerance to hyaluronidase and dextranase than a single-component cross-linked gel. At the same time, compared with commercially available cross-linked sodium hyaluronate gel products and other mixed cross-linked combinations, Example 2-2 also has more outstanding anti-enzymatic ability.

[0113] According to the embodiments of the present invention, although the above description is a preferred implementation mode, the protection scope of the present invention is not limited thereto, and any changes, modifications, combinations, substitutions or simplifications that do not violate the spirit and principles of the present invention are deemed to be equivalent replacement methods and are included in the protection scope of the present invention.

[0114] discuss The present invention discloses a preparation method of a multi-crosslinked combination of glucan-sodium hyaluronate based on optimized anti-enzymatic performance, wherein a combination of medium-molecular glucan and medium- and low-molecular-weight sodium hyaluronate is crosslinked in a ratio under alkaline conditions to form a mixed polysaccharide hydrogel, and the gel is swollen, dialyzed, homogenized and sterilized to prepare a gel particle suspension. The gel prepared by the method of the present invention has excellent resistance to dextranase and hyaluronidase enzymolysis. Therefore, the inventors infer that the method of the present invention is used to make the topological entanglement and multi-mixed covalent crosslinking between branched glucans, between linear sugar chains of sodium hyaluronate, and between these two high-molecular-weight polysaccharides, thereby significantly improving the complexity of the three-dimensional structure of the hydrogel. However, the scope of protection of the present invention is not limited by the above mechanism.

[0115] Specifically, in the degradation experiments of hyaluronidase and α-glucosidase, the mixed cross-linked gel showed stronger structural stability and stronger resistance to enzymatic degradation than the single polysaccharide component cross-linked gel. The enzymatically resistant mixed cross-linked dextran-sodium hyaluronate gel material prepared by this method can be used as a tissue filler that reduces the amount of cross-linking agent used, is safer and less sensitive, but has long-lasting effects, and can be used in cosmetics, joint cavity injections, tissue fillers, especially in the field of medical cosmetology.

[0116] The method of the present invention adopts a combination of one or several glucans and sodium hyaluronates with a specific molecular weight and a material amount feed ratio, and can prepare a mixed cross-linked glucan-sodium hyaluronate gel with outstanding resistance to enzymolysis. The preparation method of the present invention has simple steps, does not introduce other organic substances except polysaccharide raw materials and commonly used cross-linking agents, and has high safety; multi-component mixed cross-linking further enhances the structural complexity of the gel compared to binary composite cross-linking, and experimental results show a stronger resistance to enzymolysis than a single-component cross-linked gel. The product of the present invention is a new type of tissue filling material, which can be used as a long-lasting skin filler, applied in fields such as medical cosmetology, and has broad application prospects and market potential.

[0117] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims

1. A method for preparing a dextran-sodium hyaluronate gel having resistance to dextranase and hyaluronidase enzymolysis, characterized in that: The following steps are involved: (1) Dissolution: Add the cross-linking 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; Wherein, the concentration of the cross-linking agent is 0.05-2wt%, and the cross-linking agent is one or more of 1,4-butanediol diglycidyl ether, 1,2,7,8-diepoxyoctane, 1,3-diepoxybutane; Wherein, the average molecular weight of the added dextran is: 60~80kDa, and the average molecular weight of sodium hyaluronate is: 1000~1800kDa; or, The average molecular weight of the added dextran is 400-600 kDa, and the average molecular weight of sodium hyaluronate is 300-1800 kDa; Furthermore, the molar ratio of the dextran to the sodium hyaluronate is 1:4 to 4:1; (2) Cross-linking reaction: Solution A is allowed to cross-link at a temperature of 30-40°C for a time of 6-24 h to obtain a transparent gel B; (3) Adjusting pH: Cut the gel B into 1-2 cm 3 Small pieces, neutralized to a pH between 6.8 and 7.5; (4) Swelling and dialysis: swelling and dialysis in isotonic phosphate buffer at room temperature, with the solution changed every 4 to 6 hours for more than 48 hours; (5) Sieving: The dialyzed gel is passed through a 100-mesh sieve to obtain dextran-sodium hyaluronate gel particles, wherein the particle size of the gel is in the range of 50 to 250 μm; (6) Filling and sterilization: Fill the gel particles obtained in step (5) into a filling syringe and sterilize by wet heat.

2. The method according to claim 1, characterized in that The added dextran and sodium hyaluronate are selected from the following group: (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; (c) dextran with an average molecular weight of 500 kDa and sodium hyaluronate with an average molecular weight of 1200 kDa; Furthermore, 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 and the sodium hyaluronate is 1:4 to 1:1; when the molecular weight combination of the added dextran and sodium hyaluronate is (b), the molar ratio of the dextran and the sodium hyaluronate is 1:4 to 4:

1.

3. The method according to claim 1, characterized in that 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 In step (1), the molar ratio of the dextran to the sodium hyaluronate is 1:4 to 1:

1.

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

7. The method according to claim 1, characterized in that 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 refers to an isotonic solution of sodium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate dissolved in water for injection.

9. A dextran-sodium hyaluronate gel having resistance to dextranase and hyaluronidase enzymolysis, characterized in that: The gel is prepared by the method according to claim 1.

10. A medical material, characterized in that: The medical material comprises the gel prepared by the method according to claim 1.

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