Sodium hyaluronate gel microspheres as well as preparation method and application thereof

Abstract

The invention discloses sodium hyaluronate gel microspheres as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The invention requests to protect a sodium hyaluronate gel microsphere, the sodium hyaluronate gel microsphere is formed by hyaluronic acid through self-crosslinking, and a crosslinking bond is an ester bond; the swelling degree range of the sodium hyaluronate gel microspheres is 12.5-70.9, and the particle size range of the sodium hyaluronate gel microspheres is 2.8-2000 [mu] m. The sodium hyaluronate gel microspheres are formed by self-crosslinking of hyaluronic acid, a carbodiimide derivative or an N-hydroxysuccinimide derivative used in a catalytic crosslinking reaction is not connected with sodium hyaluronate through a chemical bond after being catalyzed and can be removed from a product through cleaning, so that the risk of adverse reaction of a crosslinking agent is reduced; and the obtained spherical sodium hyaluronate gel product is easy to inject and use, and is particularly suitable for being used as a short-term hard filling material.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and relates to a sodium hyaluronate gel, in particular to a sodium hyaluronate gel microsphere and a preparation method and application thereof. BACKGROUND

[0002] Hyaluronic acid (HA) is a polymer alternatingly combined by acetylglucosamine and glucuronic acid through glycosidic bond, and often exists in the form of sodium salt. HA is a polysaccharide naturally existing in human connective tissue, and has good biocompatibility, water retention and viscoelasticity, and is widely used in the field of medical cosmetology, such as skin filling, soft tissue repair and joint lubrication.

[0003] Non-crosslinked HA is easily degraded by HA enzyme in vivo, and has low mechanical strength, which limits its application in long-acting filling and shaping. Therefore, a crosslinking agent is often used to form a gel network of HA, so as to improve the degradation resistance and increase the mechanical properties (such as modulus) of the gel. At the same time, among various HA gel forms, spherical particles are more preferred due to their lower tissue irritation and smaller rolling resistance (easy to inject) because of their smooth surface.

[0004] Crosslinked HA has various crosslinking agents to choose from, such as DVS (divinyl sulfone, State Food and Drug Administration Approval 20203130096), BDDE (1,4-butanediol glycidyl ether, State Food and Drug Administration Approval 20193130257), lysine (State Food and Drug Administration Approval 20243131239) and PEG (polyethylene glycol, State Food and Drug Administration Approval 20253130825) used in marketed products. With the progress of crosslinking technology, the safety of crosslinking agents has been continuously improved. However, as a foreign substance, crosslinking agents always have the risk of causing adverse reactions such as allergy.

[0005] In addition, crosslinked HA gel can have higher modulus (achieve hard filling effect) compared to non-crosslinked HA, and the higher the crosslinking degree, the higher the modulus. However, the degradation time of the gel will also increase with the increase of the crosslinking degree, resulting in the fact that HA gel cannot have both short degradation period and high modulus, which limits the application of HA gel in the field of short-term hard filling (such as tissue engineering repair scaffold, postoperative anti-adhesion, joint filling, etc.).

[0006] In order to solve the problems of crosslinking agent allergy risk of HA gel microspheres and the fact that short degradation period and high modulus cannot be achieved simultaneously in the prior art, the present application is proposed. SUMMARY

[0007] The present application aims to overcome the deficiencies of the prior art, the first object is to provide a sodium hyaluronate gel microspheres, the second object is to provide the preparation method of the sodium hyaluronate gel microspheres, the third object is to provide the application of the sodium hyaluronate gel microspheres in the preparation of medical filling materials.

[0008] The above-mentioned purposes of the present application are realized by the following technical solutions: A sodium hyaluronate gel microsphere is formed by self-crosslinking of hyaluronic acid, and the crosslinking bond is an ester bond; the swelling degree of the sodium hyaluronate gel microsphere ranges from 12.5 to 70.9, and the particle size ranges from 2.8 to 2000 μm.

[0009] A preparation method of the sodium hyaluronate gel microsphere as described above, comprising the following steps: Step S1, dissolving hyaluronic acid or its salt and a catalyst in a solvent A to obtain an aqueous phase solution; Step S2, dissolving a tackifier in a solvent B to obtain an oil phase solution; Step S3, mixing the aqueous phase solution and the oil phase solution to perform suspension polymerization, and obtaining the product after the reaction is completed; Wherein: In step S1, the catalyst is composed of carbodiimide derivatives and N-hydroxysuccinimide derivatives; taking a group of glucuronic acid and acetylglucosamine in hyaluronic acid or its salt as a repeating unit, the molar ratio of the repeating unit in hyaluronic acid or its salt to carbodiimide derivatives and N-hydroxysuccinimide derivatives is 1:(0.3-1.5):(0.15-2.25); the solvent A is selected from one or more of water, physiological saline, and phosphate buffer; the molecular weight of the hyaluronic acid or its salt is 40 kDa-1000 kDa, and the concentration thereof in the aqueous phase solution is 5-22 wt.%; In step S2, the tackifier is selected from one or more of cellulose acetate butyrate, cellulose acetate propionate, ethyl cellulose, polymethyl methacrylate, polystyrene, and polyvinyl butyral; the solvent B is selected from a water-immiscible organic solvent.

[0010] Preferably, in step S1, the carbodiimide derivative is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N,N'-dicyclohexyl carbodiimide, N,N'-diisopropyl carbodiimide, and N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide methanesulfonate.

[0011] Preferably, in step S1, the N-hydroxysuccinimide derivative is selected from one or more of N-hydroxysuccinimide, N-hydroxysulfosuccinimide sodium salt, and N-hydroxysulfosuccinimide.

[0012] Preferably, in step S1, the pH of the aqueous solution is in the range of 4.55 ~ 6.95.

[0013] Preferably, in step S2, the solvent B is selected from one or more of butyl acetate, amyl acetate, hexyl acetate, butyl propionate, butyl butyrate, dimethyl carbonate, diethyl carbonate.

[0014] Preferably, in step S2, the concentration of the adhesion promoter in the oil phase solution is 0.5 ~ 15wt.%.

[0015] Preferably, in step S3, the volume ratio of the aqueous solution to the oil phase solution is 1:1 ~ 40.

[0016] Preferably, in step S3, the temperature of the suspension polymerization is 10 ~ 90℃, the reaction time is 0.5 ~ 24 h, and the stirring speed is 200 ~ 1500 rpm.

[0017] The application of the sodium hyaluronate gel microspheres or the sodium hyaluronate gel microspheres prepared by any of the above preparation methods in the preparation of medical filling materials.

[0018] Advantages: 1. The sodium hyaluronate gel microspheres provided by the present application are formed by self-crosslinking of hyaluronic acid, and the carbodiimide derivative or N-hydroxysuccinimide derivative used to catalyze the crosslinking reaction is not chemically linked to the sodium hyaluronate after completing the catalysis, and can be removed from the product by washing, thereby reducing the risk of adverse reactions of the crosslinking agent. 2. The hyaluronic acid in the sodium hyaluronate gel microspheres provided by the present application is crosslinked by an ester bond, and the gel modulus is significantly improved due to the increase in the degree of crosslinking, and the degradation period changes less, so that high modulus and fast degradation can be considered, and it is particularly suitable for use as a short-term hard filling material. 3. Compared with the prior art (such as CN 111647193 A which obtains a sponge-like sodium hyaluronate gel), the preparation method of the present application can obtain spherical sodium hyaluronate gel products by the method of suspension polymerization, and the rolling resistance between spherical particles is small, which is easy to use for injection.

[0019] Mechanism of generating advantages: 1. Mechanism of reducing adverse reaction risk of crosslinking product Carbodiimide derivatives and N-hydroxysuccinimide derivatives are a group of catalysts that can activate carboxyl groups. The glucuronic acid units in HA have carboxyl groups that can be activated. Both glucuronic acid and acetylglucosamine contain hydroxyl groups, and the activated carboxyl groups can react with the hydroxyl groups to form ester bonds, i.e. HA crosslinks with HA without the need to introduce additional crosslinking agents. The specific chemical reaction is shown in the formula (take EDC / NHS as an example).

[0020] ; The ester bond in the HA crosslinked by the ester bond can be broken by hydrolysis, and the glycosidic bond between glucuronic acid and acetylglucosamine in the HA is also not affected by the ester bond and can still be hydrolyzed (or enzymatically hydrolyzed). No crosslinker fragments are released after degradation, so there is no safety risk related to the crosslinker. In contrast, the crosslinker (such as BDDE) used in the prior art technology is directly connected to the HA molecule during crosslinking to form a chemical bond connection structure such as HA-BDDE-HA, which cannot be removed by washing, and thus the crosslinker molecules are inevitably released after the gel degrades in the human body, bringing the risk of adverse reactions related to the crosslinker.

[0021] 2. Mechanism of balancing high modulus and fast degradation The crosslinking bonds of the HA gel crosslinked by the traditional crosslinkers BDDE, DVS, and lysine are ether bonds and amide bonds, which have strong hydrolysis resistance. The network pore size of the crosslinked HA gel is small, which does not allow the penetration of HA enzymes and other macromolecular proteins, and the gel can only start to degrade from the surface. Therefore, considering the hydrolysis of the crosslinking bond and the enzymatic hydrolysis pathway of the HA, the enzymatic hydrolysis pathway is the rate-determining step of the overall degradation of the gel. With the increase of the crosslinking degree, the resistance of the gel to enzymatic hydrolysis increases significantly, and the overall degradation time of the gel in the in vivo environment where hydrolysis and enzymatic hydrolysis occur simultaneously significantly increases with the increase of the crosslinking degree.

[0022] In contrast, the HA gel disclosed in the present application is crosslinked by an ester bond, which is a chemical bond that is easily hydrolyzed. The size of the water molecule is smaller than the network pore size of the gel, and all the chemical bonds in the gel are simultaneously hydrolyzed after the gel is in contact with water. Therefore, the hydrolysis pathway is the rate-determining step of the overall degradation of the gel. Even by increasing the crosslinking degree and increasing the number of ester bonds (while improving the mechanical properties of the gel), since the hydrolysis of the ester bonds in the gel occurs simultaneously (water molecules can penetrate the gel network), rather than starting from the surface of the gel particles, the degradation period of the gel is less related to the crosslinking degree. Further increasing the crosslinking degree can significantly improve the modulus of the gel, but does not significantly increase the degradation time, achieving the balance of fast degradation and high modulus of the gel.

[0023] 3. Mechanism of obtaining spherical sodium hyaluronate gel by suspension polymerization Spherical particles have small rolling resistance between particles and are easy to inject, while irregular particles are difficult to inject due to the existence of protrusions, recesses and the like. The disclosed technology (CN 111647193 A) is prone to produce porous and foaming structures when synthesizing block HA gel. The inventors have found that a small amount of isocyanate (synthetic raw material of carbodiimide derivative) inevitably remains in the production process of carbodiimide derivative, and isocyanate reacts with water to produce carbon dioxide, thereby foaming and crosslinking HA. In the present application, the aqueous solution is dispersed into micrometer-sized droplets, so that carbon dioxide is easy to diffuse and separate from the gel, and dense, round and non-porous crosslinked HA can be formed. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Optical microscope photos of the product of Example 1 after reconstitution with normal saline (A), dry powder of the product of Example 1 (B), and the product of Comparative Example 1 after reconstitution with normal saline (C).

[0025] Figure 2 Storage modulus test results of the products of Examples 1, 4, 6 and 7.

[0026] Figure 3 Infrared spectra of the product prepared in Example 7 and raw materials thereof.

[0027] Figure 4 Particle size distribution graph of the product prepared in Example 7 ~ 10 DETAILED DESCRIPTION

[0028] The following will specifically introduce the substantial content of the present application in combination with examples, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

[0029] I. Preparation of sodium hyaluronate gel microspheres

[0030] Example 1: Step S1, dissolve HA (hyaluronic acid, the same below) with a molecular weight of 100 kDa in deionized water to form a solution with a concentration of 10 wt.%. After adjusting the pH to 4.8±0.05 with 0.1 M HCl / NaOH, add EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, the same below) and NHS (N-hydroxysuccinimide, the same below) in a molar ratio of HA repeat unit (a group of glucuronic acid and acetylglucosamine is recorded as one repeat unit): EDC: NHS = 1:0.5:0.5, stir at room temperature to obtain a uniform solution as the aqueous phase.

[0031] Step S2, dissolve cellulose acetate butyrate in butyl acetate to form a solution with a concentration of 5 wt.% as the oil phase.

[0032] Step S3, 50 mL of the aqueous phase was added to 500 mL of the oil phase under mechanical stirring at 10 ℃ and 1000 rpm for 24 h of suspension polymerization; after the reaction was completed, the microspheres were collected by centrifugation, and then were dialyzed with the oil phase solvent and anhydrous ethanol successively, and the product was obtained after drying.

[0033] Example 2: Step S1, HA with a molecular weight of 200 kDa was dissolved in deionized water to form a solution with a concentration of 8 wt.%. After adjusting the pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added in a molar ratio of HA repeating units: EDC: NHS = 1:0.5:0.5, and a uniform solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0034] The other steps were the same as in Example 1.

[0035] Example 3: Step S1, HA with a molecular weight of 500 kDa was dissolved in deionized water to form a solution with a concentration of 5 wt.%. After adjusting the pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added in a molar ratio of HA repeating units: EDC: NHS = 1:0.5:0.5, and a uniform solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0036] The other steps were the same as in Example 1.

[0037] Example 4: Step S1, HA with a molecular weight of 100 kDa was dissolved in deionized water to form a solution with a concentration of 10 wt.%. After adjusting the pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added in a molar ratio of HA repeating units: EDC: NHS = 1:0.3:0.3, and a uniform solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0038] The other steps were the same as in Example 1.

[0039] Example 5: Step S1, HA with a molecular weight of 1000 kDa was dissolved in deionized water to form a solution with a concentration of 5 wt.%. After adjusting the pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added in a molar ratio of HA repeating units: EDC: NHS = 1:0.5:0.5, and a uniform solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0040] The other steps were the same as in Example 1.

[0041] Example 6: Step S1, HA with molecular weight of 100 kDa was dissolved in deionized water to form a solution with concentration of 10 wt.%. After adjusting pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added according to the molar ratio of HA repeat unit: EDC: NHS = 1: 1.0: 1.0, and a homogeneous solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0042] The other steps were the same as in Example 1.

[0043] Example 7: Step S1, HA with molecular weight of 100 kDa was dissolved in deionized water to form a solution with concentration of 10 wt.%. After adjusting pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added according to the molar ratio of HA repeat unit: EDC: NHS = 1: 1.5: 1.5, and a homogeneous solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0044] The other steps were the same as in Example 1.

[0045] Example 8: Step S1, HA with molecular weight of 40 kDa was dissolved in physiological saline to form a solution with concentration of 6 wt.%. After adjusting pH to 4.6±0.05 with 0.1 M HCl / NaOH, DCC (N, N'-dicyclohexyl carbodiimide, same below) and NHS were added according to the molar ratio of HA repeat unit: DCC: NHS = 1: 0.3: 0.15, and a homogeneous solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0046] Step S2, cellulose acetate propionate was dissolved in amyl acetate to form a solution with concentration of 0.5 wt.%, which was used as the oil phase.

[0047] Step S3, 50 mL of the aqueous phase was added to 50 mL of the oil phase under mechanical stirring at 90 ℃ and 1500 rpm, and suspension polymerization was carried out for 0.5 h; after the reaction was completed, the microspheres were collected by centrifugation, and were successively dialyzed with the oil phase solvent and anhydrous ethanol, and then dried to obtain the product.

[0048] Example 9: Step S1, HA with molecular weight of 150 kDa was dissolved in physiological saline to form a solution with concentration of 22 wt.%. After adjusting pH to 6.5±0.05 with 0.5 M HCl / KOH, DIC (N, N'-diisopropyl carbodiimide, same below) and Sulfo-NHS (N-hydroxysulfosuccinimide sodium salt, same below) were added according to the molar ratio of HA repeat unit: DIC: Sulfo-NHS = 1: 1: 1.5, and a homogeneous solution was obtained by stirring at room temperature, which was used as the aqueous phase.

[0049] Step S2, poly(methyl methacrylate) was dissolved in dimethyl carbonate to form a solution with a concentration of 15 wt.% as the oil phase.

[0050] Step S3, 50 mL of the water phase was added to 2000 mL of the oil phase under mechanical stirring at 40 °C and 200 rpm for 4 h of suspension polymerization; after the reaction was completed, the microspheres were collected by centrifugation, and were dialyzed with the oil phase solvent and anhydrous ethanol in sequence, and the product was obtained after drying.

[0051] Example 10: Step S1, HA with a molecular weight of 150 kDa was dissolved in phosphate buffer to form a solution with a concentration of 5 wt.%. After adjusting the pH to 6.9±0.05 with 0.5 M phosphoric acid / KOH, CMC (N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide methanesulfonate, the same below): NHSS (N-hydroxysulfosuccinimide, the same below) = 1:1.5:2.25 (molar ratio) were added, and a uniform solution was obtained by stirring at room temperature as the water phase.

[0052] Step S2, polyvinyl butyral was dissolved in butyl butyrate to form a solution with a concentration of 12 wt.% as the oil phase.

[0053] Step S3, 50 mL of the water phase was added to 1000 mL of the oil phase under mechanical stirring at 60 °C and 650 rpm for 2 h of suspension polymerization; after the reaction was completed, the microspheres were collected by centrifugation, and were dialyzed with the oil phase solvent and anhydrous ethanol in sequence, and the product was obtained after drying.

[0054] Comparative Example 1 (the difference from Example 1 is that the concentration of HA is different): Step S1, HA with a molecular weight of 100 kDa was dissolved in deionized water to form a solution with a concentration of 4 wt.%. After adjusting the pH to 4.8±0.05 with 0.1 M HCl / NaOH, EDC and NHS were added according to HA repeat unit: EDC: NHS = 1:0.5:0.5 (molar ratio), and a uniform solution was obtained by stirring at room temperature as the water phase.

[0055] The other steps were the same as in Example 1.

[0056] Comparative Example 2 (the difference from Example 2 is that the concentration of HA is different): Step S1: Dissolve HA with a molecular weight of 200 kDa in deionized water to form a solution with a concentration of 4 wt.%. Adjust the pH to 4.8 ± 0.05 with 0.1 M HCl / NaOH, then add EDC and NHS according to the HA repeating unit: EDC: NHS = 1:0.5:0.5 (molar ratio), and stir at room temperature to obtain a homogeneous solution, which is used as the aqueous phase.

[0057] The other steps are the same as in Example 1.

[0058] Comparative Example 3 (different from Example 3 in that the concentration of HA is different): Step S1: Dissolve HA with a molecular weight of 500 kDa in deionized water to form a solution with a concentration of 2 wt.%. Adjust the pH to 4.8 ± 0.05 with 0.1 M HCl / NaOH, then add EDC and NHS according to the HA repeating unit: EDC: NHS = 1:0.5:0.5 (molar ratio), and stir at room temperature to obtain a homogeneous solution, which is used as the aqueous phase.

[0059] The other steps are the same as in Example 1.

[0060] Comparative Example 4 (the difference from Example 4 is the ratio of HA repeating units to EDC and NHS): Step S1: Dissolve HA with a molecular weight of 100 kDa in deionized water to form a solution with a concentration of 10 wt.%. Adjust the pH to 4.8 ± 0.05 with 0.1 M HCl / NaOH, then add EDC and NHS according to the HA repeating unit: EDC: NHS = 1:0.2:0.2 (molar ratio), and stir at room temperature to obtain a homogeneous solution, which is used as the aqueous phase.

[0061] The other steps are the same as in Example 1.

[0062] II. Performance Testing 1. Microsphere Formability (1) Test sample The products prepared in Examples 1 to 3 and Comparative Examples 1 to 3 of this invention.

[0063] (2) Test items Microscopic morphology: physiological saline was added to the sample to be tested and observed under a microscope.

[0064] (3) Test results As shown in Table 1, not all concentrations of the solution can obtain spherical products after suspension polymerization. As shown in Figure 1 (lower left), the micro-morphology of the product of Example 1 can be seen to be fine irregular particles. After re-dissolving in physiological saline, the product of Example 1 swells into spherical gel particles (Figure 1 upper). But the product of Comparative Example 1 is quite different, before swelling in physiological saline, its morphology is not obviously different from that of the product of Example 1; but after adding physiological saline, it cannot maintain the spherical particle morphology, but directly dissolves in the physiological saline to form flocculent floating matter (Figure 1 lower right).

[0065] ; (4) Result analysis The intermediate formed when the carbodiimide derivative catalyzes the activation of the carboxyl group of the N-hydroxysuccinimide derivative is highly reactive and can be consumed by the hydroxyl group in water, i.e. the hydroxyl group in HA and the hydroxyl group in the aqueous solution need to compete to react with the activated carboxyl group in HA. When the concentration of HA in the reaction system is low enough, the number of cross-linking bonds formed in the reaction is insufficient, i.e. the cross-linking degree is insufficient. Therefore, the gel particles obtained after cross-linking cannot resist the swelling effect of water molecules and have broken, and cannot maintain the spherical morphology.

[0066] The products obtained in Examples 2-10 have similar spherification properties as the product obtained in Example 1.

[0067] 2. Cross-linking degree and related properties (1) Test samples The products obtained in Examples 1, 4, 6, 7 and Comparative Example 4 of the present application.

[0068] (2) Test items Swelling degree: the sample to be tested is weighed, and the mass is recorded as m1. The product is added to sufficient physiological saline to swell fully, and the swollen gel is taken out, the surface free water is wiped off and weighed, and the mass is recorded as m2. The swelling degree is (m2-m1) / m1.

[0069] Storage modulus: the sample to be tested is added to sufficient physiological saline to swell fully, and the free water outside the gel microspheres is removed. The gel microspheres are placed on the flat plate rotor of the rheometer, the experimental temperature is 25°C, the constant strain is 1%, the flat thickness is 1 mm, the 25 mm flat plate rotor, and the result under 0.1 Hz is counted.

[0070] Hydrolysis: the sample to be tested is added to sufficient physiological saline to swell fully, and is placed at 70°C to accelerate hydrolysis. The solution is observed once every 0.5 h, and the absence of gel particles in the solution is taken as the end point of hydrolysis.

[0071] Enzymolysis: take 50 mg of the sample to be tested, add 5 mL of 50 U / mL hyaluronic acid sodium enzyme physiological saline solution. After uniform shaking, place it at 37°C for enzyme hydrolysis, and observe it every 1 h. Take the gel-like particles under the microscope as the end point of enzyme hydroysis.

[0072] Pushing force: add enough physiological saline to the sample to be tested, suck the reconstituted gel microspheres into a 1 mL syringe, push out the free water after standing, use a 27G needle, and set the pushing speed to 30 mm / min.

[0073] (3) Test results As shown in Table 2, the performance of the crosslinked HA gel microspheres with different catalyst dosages has significant characteristics.

[0074] (4) Result analysis Crosslinking density and mechanical properties: as the molar ratio of HA repeat unit: EDC: NHS increases from 1:0.3:0.3 (Example 4) to 1:1.5:1.5 (Example 7), the swelling degree decreases from 70.9 to 12.5, and the storage modulus increases from 58 Pa to 3410 Pa (as shown in Table 2), which is about 59 times, proving that the ester bond crosslinking network can realize gradient "high modulus" regulation without external crosslinking agents. The swelling degree of the products obtained in Examples 1-10 is in the range of 12.5-70.9. Figure 2

[0075] Degradation speed-crosslinking degree weak coupling: the hydrolysis time is only extended from 4 h to 11 h (less than 3 times), and the enzyme hydrolysis time is extended from 5 h to 74 h (about 15 times), which is significantly lower than the modulus improvement amplitude (59 times). This "weak coupling" phenomenon verifies the core effect of "high modulus-fast degradation" of the present application, and the mechanism is that the ester bond is an "easy hydrolysis bond", and water molecules can penetrate the entire microsphere network and break at the same time, rather than surface-to-center layer-by-layer degradation. Even if the crosslinking degree is greatly improved, the overall hydrolysis time is still short; the enzyme hydrolysis time is not the rate-determining step of the overall degradation of the material.

[0076] Injectability: the maximum pushing force of the spherical sample is ≤ 5 N, which is much lower than the "comfortable injection" threshold (about 10 N) of the commonly used 27G needle in clinical practice, and the pushing force does not increase significantly with the increase of crosslinking degree, which confirms that the round microspheres still have the advantage of "low rolling resistance" in the "high modulus" state.

[0077] ​​Critical crosslinking degree: the gel formed by Comparative Example 4 (molar ratio 1:0.2:0.2) has too low crosslinking density, still has strong fluidity, and cannot test mechanical and degradation performance, further confirming that sufficient crosslinking level is a necessary condition to ensure the spherical gel particle, consistent with the "microsphere formability" conclusion.

[0078] 3. Infrared spectrum (1) Test sample The product prepared in Example 7.

[0079] (2) Test item According to the Chinese Pharmacopoeia (2020 edition) Four Departments 0402 infrared spectrophotometry.

[0080] (3) Test results The infrared spectrum of Example 7 and the raw materials used therein is shown in Figure 3 .

[0081] (4) Analysis of results Since the infrared characteristics of the products prepared in each example are almost the same, Example 7 (high crosslinking level) is taken as a representative spectrum for analysis.

[0082] Sodium hyaluronate (HA) is a polysaccharide, and its main infrared characteristic peaks include: about 3400 cm -1 : O-H stretching vibration (hydroxyl group); about 2900 cm -1 : C-H stretching vibration (methylene and methine on sugar ring). About 1650 cm -1 and about 1550 cm -1 : Amide I and amide II bands (from N-acetylglucosamine units). These peaks can be observed in both HA raw materials and gel microspheres. At the same time, the infrared peak shape of HA raw materials and gel microspheres is basically the same, indicating that the composition of the gel microspheres is almost HA.

[0083] However, compared with the gel microspheres, some typical characteristic peaks of the HA raw materials have changed. For example, about 1600 cm -1 and about 1410 cm -1 (in the figure, marked as #1 and #2): asymmetric and symmetric stretching vibration of carboxylic acid, the relative peak intensity of the gel microspheres is significantly reduced, indicating that the number of carboxyl groups in the gel microspheres is reduced compared with the HA raw materials; about 1100-1000 cm -1 (in the figure, marked as #3): C-O-C and C-O stretching vibration (glycosidic bond and hydroxyl group), the relative peak intensity of the gel microspheres is reduced, indicating that the number of carboxyl groups in the gel microspheres is reduced compared with the HA raw materials. In addition, compared with the HA raw materials, the gel microspheres have about 1700 cm -1A new characteristic peak has been added at position #4 in the figure. This peak is attributed to the stretching vibration peak of the carbonyl group on the ester bond.

[0084] The changes in the intensity of the infrared peaks and the addition of new peaks indicate that the composition of the gel microspheres remains HA. After cross-linking, the amount of carboxyl and hydroxyl groups decreases, and ester bonds are formed.

[0085] In summary, this indicates that the gel microspheres are formed through self-crosslinking of ester bonds.

[0086] 4. Microsphere particle size (1) Test sample The products prepared in Examples 7 to 10 of this invention.

[0087] (2) Test items Particle size: After swelling in physiological saline to reach steady state, the particle size was determined by wet method according to the third method (light scattering method) of particle size and particle size distribution determination in the Pharmacopoeia of the People's Republic of China (Part IV) (2020 Edition) 0982.

[0088] (3) Test results The particle size distribution diagrams of the products prepared in Examples 7-10 are shown below. Figure 4 As shown in Table 3, the particle size statistics are as follows.

[0089] ; (4) Results Analysis The particle size distribution of gel microspheres can be controlled by parameters such as molecular weight concentration and water-oil ratio, and microspheres with sizes ranging from 2.8 to 2000 μm can be prepared. Sieving the product can also yield products with narrower particle size distributions. The particle sizes of the products obtained in Examples 1-10 are all in the range of 2.8 to 2000 μm.

[0090] In summary: 1. The sodium hyaluronate gel microspheres provided by the present invention are formed by the self-crosslinking of hyaluronic acid, and there will be no carbodiimide derivatives or N-hydroxysuccinimide derivatives connected to sodium hyaluronate by chemical bonds in the product, thus reducing the risk of adverse reactions. 2. The hyaluronic acid in the sodium hyaluronate gel microspheres provided by the present invention is cross-linked through ester bonds. The gel modulus is significantly increased due to the increase in the degree of cross-linking, while the degradation cycle changes little. Thus, it can take into account both high modulus and fast degradation, and is particularly suitable as a short-term rigid filler material. 3. Compared with existing technologies (such as CN 111647193 A for obtaining sponge-like sodium hyaluronate gel), the preparation method of the present invention can obtain spherical sodium hyaluronate gel products through suspension polymerization. The rolling resistance between spherical particles is small, making it easy to inject and use.

[0091] The above embodiments serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific embodiments.

Claims

1. Sodium hyaluronate gel microspheres, characterized in that: The sodium hyaluronate gel microspheres are formed by self-crosslinking of hyaluronic acid, and the crosslinking bonds are ester bonds; the sodium hyaluronate gel microspheres have a swelling degree in the range of 12.5-70.9 and a particle size in the range of 2.8-2000 μm.

2. A method for preparing sodium hyaluronate gel microspheres as described in claim 1, characterized in that, The method comprises the following steps: Step S1, dissolving hyaluronic acid or a salt thereof and a catalyst in a solvent A to obtain an aqueous phase solution; Step S2, dissolving a tackifier in a solvent B to obtain an oil phase solution; Step S3, mixing the aqueous phase solution and the oil phase solution to perform suspension polymerization, and obtaining the sodium hyaluronate gel microspheres after the reaction is completed; In step S1, the catalyst is composed of a carbodiimide derivative and an N-hydroxysuccinimide derivative; taking a group of glucuronic acid and acetylglucosamine in the hyaluronic acid or the salt thereof as one repeating unit, the molar ratio of the repeating unit in the hyaluronic acid or the salt thereof to the carbodiimide derivative and the N-hydroxysuccinimide derivative is 1:(0.3-1.5):(0.15-2.25); the solvent A is selected from one or more of water, physiological saline and phosphate buffer; the molecular weight of the hyaluronic acid or the salt thereof is 40 kDa-1000 kDa, and the concentration of the hyaluronic acid or the salt thereof in the aqueous phase solution is 5-22 wt.%; In step S2, the tackifier is selected from one or more of cellulose acetate butyrate, cellulose acetate propionate, ethyl cellulose, polymethyl methacrylate, polystyrene and polyvinyl butyral; the solvent B is selected from a water-immiscible organic solvent. In step S1, the carbodiimide derivative is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N,N'-dicyclohexyl carbodiimide, N,N'-diisopropyl carbodiimide and N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide methanesulfonate.

3. The method of claim 2, wherein: In step S1, the N-hydroxysuccinimide derivative is selected from one or more of N-hydroxysuccinimide, N-hydroxysulfosuccinimide sodium salt and N-hydroxysulfosuccinimide.

4. The method of claim 2, wherein: In step S1, the pH of the aqueous phase solution is in the range of 4.55-6.

95.

5. The method of claim 2, wherein: In step S2, the solvent B is selected from one or more of butyl acetate, amyl acetate, hexyl acetate, butyl propionate, butyl butyrate, dimethyl carbonate and diethyl carbonate.

6. The method of claim 2, wherein: In step S2, the concentration of the tackifier in the oil phase solution is 0.5-15 wt.%.

7. The method of claim 2, wherein: In step S3, the volume ratio of the aqueous phase solution to the oil phase solution is 1:1-40.

8. The method of claim 2, wherein: In step S3, the temperature of the suspension polymerization is 10-90℃, the reaction time is 0.5-24 h, and the stirring speed is 200-1500 rpm.

9. The method of claim 2, wherein:

10. Use of the sodium hyaluronate gel microspheres of claim 1 or the sodium hyaluronate gel microspheres prepared by the method of any one of claims 2-9 in the preparation of a medical filling material. ​

Images