A composition containing microspheres and cross-linked hyaluronic acid or a salt thereof, and a method for preparing and using the same

Abstract

The present application discloses a composition containing microspheres and cross-linked hyaluronic acid or its salt, and a preparation method and application thereof. The composition comprises cross-linked hyaluronic acid or its salt, microspheres, phosphate buffer, and non-cross-linked hyaluronic acid or its salt. The present application uses mixed hyaluronic acid or its salt with high and low molecular weights under the condition of a large amount of alkali solution to form a loose structure, and uses hyaluronic acid or its salt with a small molecular weight under the condition of a small amount of alkali solution to form a compact network structure. The two are mixed to continue the cross-linking reaction to form a gel with a soft elastic hybrid structure of loose pores wrapping elastic hard spheres. The gel is mixed with microspheres, and the entry of rigid microspheres in the gel makes the composition have both soft elastic function and rigid support performance, and the two have a synergistic effect. The gel can also effectively protect the microspheres during moist heat sterilization, reduce the degradation of the microspheres, prolong the effect of stimulating collagen regeneration of the microspheres, and is suitable for more extensive skin levels and filling sites, and improves the safety of the composition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterials, and particularly relates to a composition containing microspheres and cross-linked hyaluronic acid or a salt thereof, and a preparation method and application thereof. BACKGROUND

[0002] Hyaluronic acid is a natural mucopolysaccharide, which is connected by (1-β-4) D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine disaccharide units in repetition, and is an important component of human and animal skin, vitreous body, joint lubricating fluid and cartilage tissue. Sodium hyaluronate has multiple effects in the human body, including space filling, hydration, joint lubrication, and providing a matrix through which cells can migrate. Although hyaluronic acid has many advantages, its mechanical properties are weak, and it is easily degraded in the body, which limits its application in biomaterials. Cross-linked sodium hyaluronate is a high molecular gel obtained by chemical cross-linking modification of sodium hyaluronate, which makes up for the shortcoming of short retention time of natural sodium hyaluronate.

[0003] Patent document CN106279726B discloses a cross-linked sodium hyaluronate gel and a preparation method thereof. Under ultrasonic conditions, a cross-linking agent is added dropwise to an alkaline aqueous solution of sodium hyaluronate to perform a cross-linking reaction. After the reaction is completed, the pH value of the system is adjusted to 7.1-7.5 with acid, and then stirred for 1-2 h. Then, anhydrous ethanol is used for precipitation to obtain the cross-linked sodium hyaluronate gel. However, there is no step for removing the cross-linking agent in this method, which may cause residual cross-linking agent.

[0004] Patent document CN112940300B discloses a preparation method of a cross-linked hyaluronic acid gel, which comprises the following steps:

[0005] (1) uniformly mixing an aqueous solution containing a cross-linking agent and hyaluronic acid, a metal salt thereof, or a mixture thereof in a non-neutral environment to form a mixed solution; (2) placing the mixed solution at a temperature lower than 0℃ and higher than the eutectic point of the mixed solution and keeping it for a time sufficient to form a solid-liquid heterogeneous system to form a solid-liquid heterogeneous system; (3) placing the heterogeneous system at a temperature lower than 0℃ and higher than the eutectic point of the mixed solution to perform a cross-linking reaction; (4) melting the solid phase in the heterogeneous system after the cross-linking reaction, and optionally neutralizing, purifying and homogenizing. However, the neutralization step in this method cannot ensure that the pH of the system decreases more gently, thereby easily destroying the three-dimensional cross-linked structure of the sodium hyaluronate gel system.

[0006] Polyester microspheres, collagen microspheres, silk fibroin microspheres, hydroxyapatite microspheres, etc. are usually used as a new type of medical and aesthetic filling material due to their biocompatibility, biodegradability and non-toxicity, but the microspheres mixed with the carrier such as the matrix after sterilization cannot be well protected by the carrier, so that the product is prone to exist after injection and filling, and the microspheres degrade too fast, and are not compatible with the tissue of the injection site, and inflammation occurs.

[0007] In summary, the prior art cannot meet the industrialization needs of simple preparation process, good safety, effective removal of crosslinking agent, strong viscosity, high cohesion and low water absorption of hyaluronic acid gel, and it is also difficult to effectively ensure the degradation effect of the microspheres and realize the regeneration of collagen. SUMMARY

[0008] The present application overcomes the defects in the prior art and provides a composition containing microspheres and cross-linked hyaluronic acid or salt thereof and a preparation method and application thereof. The cross-linked hyaluronic acid or salt thereof is prepared by Semo cross-linking technology (Soft Elastic Mosaic Cross-Linking Technology, abbreviated as Semo cross-linking technology), and then mixed with the microspheres. The entry of rigid microspheres in the gel can further enhance the support performance of the soft elastic mosaic structure, so that the composition has both the soft elastic function of the gel and the rigid support performance, and the two have a synergistic effect. At the same time, the gel can effectively protect the microspheres during moist heat sterilization, reduce the degradation of the microspheres, prolong the effect of stimulating collagen regeneration of the microspheres, and is suitable for a wider range of skin levels and filling sites, and improves the safety of the composition.

[0009] In a first aspect of the present application, a composition containing microspheres and cross-linked hyaluronic acid or salt thereof is provided, and the preparation method of the cross-linked hyaluronic acid or salt thereof in the composition comprises the following steps:

[0010] (1) mixing, reacting hyaluronic acid I or salt thereof, hyaluronic acid II or salt thereof, a first alkaline solution and a cross-linking agent to obtain cross-linked hyaluronic acid I' or salt thereof;

[0011] (2) mixing, reacting hyaluronic acid III or salt thereof, a second alkaline solution and a cross-linking agent to obtain cross-linked hyaluronic acid II' or salt thereof;

[0012] (3) diluting the cross-linked hyaluronic acid I' or salt thereof obtained in step (1) with water, adding the cross-linked hyaluronic acid II' or salt thereof obtained in step (2) and hyaluronic acid IV or salt thereof, and mixing and reacting to obtain the cross-linked hyaluronic acid or salt thereof.

[0013] Further, the mass concentration of the first alkaline solution is higher than that of the second alkaline solution.

[0014] Further, the hyaluronic acid I or salt thereof has a molecular weight higher than that of the hyaluronic acid II, III, IV or salt thereof.

[0015] Further, the concentration of the first alkaline solution in step (1) is 10-100 mg / mL (e.g., 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL), preferably 12-90 mg / mL, more preferably 15-80 mg / mL.

[0016] Further, the concentration of the second alkaline solution in step (2) is 0.1-50 mg / mL (e.g., 0.1 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL), preferably 1-40 mg / mL, more preferably 5-30 mg / mL.

[0017] Further, the hyaluronic acid I or salt thereof has a molecular weight of 900-3000 kDa (e.g., 900 kDa, 950 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, 3000 kDa), preferably 1000-2000 kDa, more preferably 1200-1600 kDa.

[0018] In one embodiment of the present application, the hyaluronic acid I or salt thereof has a molecular weight of 1500 kDa.

[0019] Further, the hyaluronic acid II, III, IV or salt thereof has a molecular weight of 100-1000 kDa (e.g. 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 1000 kDa), preferably 300-800 kDa, more preferably 400-700 kDa.

[0020] Further, the hyaluronic acid II, III, IV or salt thereof has a molecular weight of 100-1000 kDa (e.g. 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 1000 kDa), preferably 300-800 kDa, more preferably 400-700 kDa.

[0021] In one embodiment of the present application, the hyaluronic acid II, III, IV or salt thereof has a molecular weight of 600 kDa.

[0022] Further, the mass ratio of the hyaluronic acid I or salt thereof to the hyaluronic acid II or salt thereof in step (1) is 2-10:1, preferably 3-9:1.

[0023] In one embodiment of the present application, the mass ratio of the hyaluronic acid I or salt thereof to the hyaluronic acid II or salt thereof in step (1) is 3:1.

[0024] Further, the mass ratio of the cross-linked hyaluronic acid I' or salt thereof to the cross-linked hyaluronic acid II' or salt thereof is 1-10:1; preferably 2-6:1.

[0025] Further, the amount of the hyaluronic acid IV or salt thereof added is 5-30% of the amount of the hyaluronic acid I or salt thereof.

[0026] Further, the concentration of the alkaline solution diluted with water in step (3) is equal to the concentration of the second alkaline solution in step (2).

[0027] Further, the temperature of the reaction in steps (1), (2), (3) is 20-30°C.

[0028] Further, the time of the reaction in steps (1), (2), (3) is 4-30 h.

[0029] Further, the temperature and time of the reaction in steps (1), (2), (3) can be the same or different.

[0030] In one embodiment of the present application, the temperature of the reaction in step (1) is 25°C and the time of the reaction is 20 h.

[0031] Further, the hyaluronic acid I or its salt, the hyaluronic acid II or its salt, the hyaluronic acid III or its salt, and the hyaluronic acid IV or its salt can be derived from animals or obtained by bacterial fermentation.

[0032] Further, the hyaluronic acid salt is selected from any one of sodium hyaluronate (HA), potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate, bismuth hyaluronate, and zinc hyaluronate; preferably sodium hyaluronate.

[0033] Further, the alkaline solution is selected from one or more of the following: sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium sulfite, potassium sulfite, preferably sodium hydroxide solution. The first alkaline solution and the second alkaline solution can be the same or different; preferably, the first alkaline solution and the second alkaline solution are the same.

[0034] Further, the crosslinking agent is selected from one or more of the following: 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone, polyethylene glycol, genipin, and carbodiimide, preferably 1,4-butanediol diglycidyl ether.

[0035] Further, the crosslinked hyaluronic acid or its salt has a crosslinking degree of 1% to 6%, preferably 1.5% to 5%.

[0036] Further, the microspheres are selected from one or more of the following: polyester microspheres, collagen microspheres, silk fibroin microspheres, and hydroxyapatite microspheres.

[0037] Further, the microspheres are polyester microspheres.

[0038] Further, the polyester is selected from one or more of the following: poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), poly-DL-lactic acid (PDLLA), poly-lactic-co-glycolic acid (PLGA), polymethyl methacrylate (PMMA), polycaprolactone (PCL), polyethylene glycol-poly-L-lactic acid (PEG-PLLA), and hydroxyapatite-poly-L-lactic acid (HAP-PLLA).

[0039] Further, the content of the microspheres in the composition is 1% to 35% (such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%), preferably 10% to 32%, more preferably 15% to 30%.

[0040] Further, the mass concentration of the cross-linked hyaluronic acid or salt thereof is 1-30 mg / mL (such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL, 13 mg / mL, 13.5 mg / mL, 14 mg / mL, 14.5 mg / mL, 15 mg / mL, 15.5 mg / mL, 16 mg / mL, 16.5 mg / mL, 17 mg / mL, 17.5 mg / mL, 18 mg / mL, 18.5 mg / mL, 19 mg / mL, 19.5 mg / mL, 20 mg / mL), preferably 5-25 mg / mL, more preferably 10-20 mg / mL.

[0041] The present application firstly realizes the cross-linking of high and low molecular weight hyaluronic acid or salt thereof by compounding high and low molecular weight hyaluronic acid or salt thereof, breaking the hydrogen bond between hyaluronic acid or salt thereof under the condition of a large amount of alkali solution, and fully extending the molecular chain of hyaluronic acid or salt thereof. At the same time, the molecular chain of low molecular weight hyaluronic acid or salt thereof can freely perform random translation or rotation movement, so that the low molecular weight hyaluronic acid or salt thereof can be inserted or erected between the high molecular weight hyaluronic acid or salt thereof, realizing the cross-linking of high and low molecular weight hyaluronic acid or salt thereof. At the same time, the large amount of alkali solution has a certain degradation effect on the molecular chain of hyaluronic acid or salt thereof during cross-linking, and finally forms a three-dimensional loose network structure with a large network spacing. Subsequently, the low molecular weight hyaluronic acid or salt thereof is cross-linked under the condition of a small amount of alkali solution. The amount of alkali solution is low, the hydrogen bond between hyaluronic acid or salt thereof is strong, the molecular chain is generally entangled, and hyaluronic acid or salt thereof is easy to quickly react with the cross-linking agent to form a compact network structure, obtaining micrometer gel particles. Due to the condition of a large amount of alkali solution, the cross-linking reaction is not complete, and there is still a part of cross-linking agent. By adding water to adjust and dilute, the condition of a large amount of alkali solution is changed to the condition of a small amount of alkali solution, and the micrometer gel particles and hyaluronic acid or salt thereof are added to continue the reaction to consume the unreacted cross-linking agent, so that the compact structure can be inserted into the relatively loose structure to form a cross-linked hyaluronic acid or salt thereof gel with a soft elastic hybrid structure of loose porosity wrapping elastic hard ball. Due to the good ductility of the loose porosity region, the gel has good supporting performance, ductility and deformation resistance, and has better water absorption and water retention, better tissue compatibility, and is more suitable for surface paving injection and filling in the cavity.

[0042] In addition, the present application mixes the microspheres with the cross-linked hyaluronic acid or salt gel described above. Since the gel has many loose pores, most of the rigid microspheres can enter the loose pores during the mixing process, thereby avoiding the agglomeration of the microspheres, making the microspheres more uniformly dispersed in the soft and elastic hybrid structure of the gel, and to some extent achieving the encapsulation of the microspheres, so that the gel can effectively protect the microspheres during moist heat sterilization, better retain the filling performance and dispersion performance of the microspheres, reduce the degradation of the microspheres, and prolong the degradation time and safety performance of the microspheres after implantation in the body; the entry of the rigid microspheres can further enhance the support performance of the soft and elastic hybrid structure, making the composition have both the soft and elastic function of the gel and the rigid support performance, and the two have a synergistic effect, thereby prolonging the effect of the microspheres in stimulating collagen regeneration, making the skin layers and filling sites more extensive, and improving the safety of the composition in use.

[0043] Further, the composition further comprises non-cross-linked hyaluronic acid or a salt thereof, a phosphate buffer, optionally a local anesthetic, and / or a water-soluble cellulose.

[0044] Further, the non-cross-linked hyaluronic acid salt is selected from any one of non-cross-linked sodium hyaluronate, non-cross-linked potassium hyaluronate, non-cross-linked calcium hyaluronate, non-cross-linked magnesium hyaluronate, non-cross-linked ammonium hyaluronate, non-cross-linked tetrabutylammonium hyaluronate, non-cross-linked bismuth hyaluronate, and non-cross-linked zinc hyaluronate, preferably non-cross-linked sodium hyaluronate.

[0045] Further, the concentration of the non-cross-linked hyaluronic acid or salt thereof is 0.2-10 mg / mL (such as 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.5, 3 mg / mL, 3.5, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL), preferably 0.5-8 mg / mL, more preferably 1-6 mg / mL.

[0046] Further, the non-crosslinked hyaluronic acid or salt thereof has a molecular weight of 100-1000 kDa (e.g., 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 1000 kDa), preferably 300-800 kDa, more preferably 400-700 kDa.

[0047] Further, the phosphate buffer comprises: disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium chloride.

[0048] Further, the concentration of the disodium hydrogen phosphate is 0.01-10 mg / mL (e.g., 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL), preferably, the concentration of the disodium hydrogen phosphate is 3-10 mg / mL, further preferably, the concentration of the disodium hydrogen phosphate is 4-8 mg / mL.

[0049] In one embodiment of the present application, the concentration of the disodium hydrogen phosphate is 10 mg / mL.

[0050] Further, the concentration of the sodium phosphate monobasic is 0.01-9 mg / mL (such as 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 1 mg / mL, 1.5, 2 mg / mL, 2.5, 3 mg / mL, 3.5, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL), preferably, the concentration of the sodium phosphate monobasic is 1-7 mg / mL, further preferably, the concentration of the sodium phosphate monobasic is 2-6 mg / mL.

[0051] In one embodiment of the application, the concentration of the sodium phosphate monobasic is 4 mg / mL.

[0052] Further, the concentration of the potassium chloride is 0.01-7 mg / mL (such as 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.5, 3 mg / mL, 3.5, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL), preferably, the concentration of the potassium chloride is 0.05-5 mg / mL, further preferably, the concentration of the potassium chloride is 0.1-3 mg / mL.

[0053] In one embodiment of the application, the concentration of the potassium chloride is 1 mg / mL.

[0054] Further, the local anesthetic is selected from the group consisting of one or a combination of amide type and ester type.

[0055] Further, the local anesthetic is selected from the group consisting of one or more of lidocaine or a salt thereof, bupivacaine or a salt thereof, butanilicaine or a salt thereof, carticaine or a salt thereof, cinchocaine or a salt thereof, clibucaine or a salt thereof, dimethisoquin or a salt thereof, etidocaine or a salt thereof, mepivacaine or a salt thereof, oxetacaine or a salt thereof, propiram or a salt thereof, ropivacaine or a salt thereof, tolycaine or a salt thereof, trimecaine or a salt thereof, vascain or a salt thereof, articaine or a salt thereof, levobupivacaine or a salt thereof, amylocaine or a salt thereof, cocaine or a salt thereof, prilocaine or a salt thereof, chloromycocaine or a salt thereof, cyclomycocaine or a salt thereof, propymycocaine or a salt thereof, decocaine or a salt thereof, benzocaine or a salt thereof, butacaine or a salt thereof, butoxycaine or a salt thereof, butamben or a salt thereof, chloroprocaine or a salt thereof, dimethocaine or a salt thereof, orbiprocaine or a salt thereof, piperocaine or a salt thereof, paraoxycaine or a salt thereof, procaine or a salt thereof, propoxycaine or a salt thereof, tricaine or a salt thereof, preferably lidocaine or a salt thereof, further preferably lidocaine hydrochloride.

[0056] Further, the local anesthetic is present in an amount ranging from 0.1 to 10 mg / mL (e.g. 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL), preferably in an amount ranging from 0.1 to 8 mg / mL, further preferably in an amount ranging from 0.5 to 8 mg / mL, more further preferably in an amount ranging from 0.5 to 5 mg / mL.

[0057] In one embodiment of the application, the local anesthetic is present in an amount of 3 mg / mL.

[0058] Further, the water-soluble cellulose is selected from the group consisting of one or more of hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, ethyl methylcellulose and carboxymethyl cellulose, preferably hydroxypropyl methylcellulose.

[0059] Further, the water-soluble cellulose is present in an amount ranging from 0.01 to 40 mg / mL, preferably from 0.2 to 25 mg / mL, more preferably from 0.5 to 10 mg / mL.

[0060] The second aspect of the application provides a method for preparing a composition comprising microspheres and cross-linked hyaluronic acid or a salt thereof, comprising the steps of:

[0061] After dialysis, the cross-linked hyaluronic acid or its salt is removed. The pH of the system is first adjusted to 10-11 with hydrochloric acid solution, and then the pH of the system is slowly adjusted to neutral with lactic acid solution. Microspheres are then added, mixed evenly, and sterilized by moist heat to obtain the composition.

[0062] Furthermore, after adjusting the pH of the system to neutral, phosphate buffer, non-crosslinked hyaluronic acid or its salt may be added, and optionally, local anesthetics and / or water-soluble cellulose may be added.

[0063] A third aspect of the present invention provides the use of a composition containing microspheres and cross-linked hyaluronic acid or its salt as described in the first aspect or a composition containing microspheres and cross-linked hyaluronic acid or its salt prepared in the second aspect in the preparation of pharmaceuticals, tissue engineering materials, and cosmetics.

[0064] Preferably, the tissue engineering materials include soft tissue filler materials, cartilage repair materials, and tissue engineering scaffolds.

[0065] The fourth aspect of the present invention provides a cosmetic non-medical use of a composition comprising microspheres and cross-linked hyaluronic acid or a salt thereof as described in the first aspect above, or a composition comprising microspheres and cross-linked hyaluronic acid or a salt thereof prepared according to the second aspect, for improving the appearance of the skin, smoothing wrinkles, or reshaping the face or body of a subject.

[0066] The fifth aspect of this invention provides a non-medical method for cosmetic purposes, including improving the appearance of skin, smoothing wrinkles, or reshaping the face or body of a subject, comprising:

[0067] I) Provide a composition containing microspheres and cross-linked hyaluronic acid or its salt as described in the first aspect above, or a composition containing microspheres and cross-linked hyaluronic acid or its salt prepared according to the second aspect, and

[0068] II) Inject the composition containing microspheres and cross-linked hyaluronic acid or its salt as described in the first aspect above, or the composition containing microspheres and cross-linked hyaluronic acid or its salt prepared in the second aspect, into the skin of the subject.

[0069] Furthermore, the composition containing microspheres and cross-linked hyaluronic acid or its salts from step II is injected into the dermis and / or subcutaneous tissue.

[0070] The beneficial effects of this invention are:

[0071] (1) This invention involves compounding high- and low-molecular-weight hyaluronic acid or its salts. Under conditions of a large alkaline solution volume, the hydrogen bonds between the hyaluronic acid or its salts are broken, allowing the molecular chains of the hyaluronic acid or its salts to fully extend and stretch. Simultaneously, the low-molecular-weight hyaluronic acid or its salts can interpenetrate or stand upright between the high-molecular-weight hyaluronic acid or its salts, achieving cross-linking of the high- and low-molecular-weight hyaluronic acid or its salts. At the same time, the large alkaline solution volume has a certain degradation effect on the molecular chains of the hyaluronic acid or its salts during cross-linking, ultimately forming a three-dimensional loose network structure with a large interpore spacing. Subsequently, under conditions of a small alkaline solution volume, the low-molecular-weight hyaluronic acid or its salts is cross-linked. With a low alkaline solution volume, the hydrogen bonds between the hyaluronic acid or its salts are stronger, and the molecular chains are more widely entangled. The hyaluronic acid or its salts and the cross-linking agent react quickly to form a compact network structure, resulting in micron-sized gel particles. However, due to the large amount of alkaline solution, the cross-linking reaction is not complete, and some cross-linking agent remains. By adding water to adjust the dilution, the condition of large alkaline solution is changed to that of small alkaline solution. Micron-sized gel particles, hyaluronic acid or its salt are added to continue the reaction and consume the unreacted cross-linking agent. This allows the compact structure to penetrate into the more loose structure, forming a soft elastic interlocking structure of cross-linked hyaluronic acid or its salt gel with loose pores encapsulating elastic hard spheres. Because the loose pore area has good extensibility, the gel has good support, extensibility and deformation resistance, as well as better water absorption and retention.

[0072] (2) In this invention, microspheres are mixed with the above-mentioned cross-linked hyaluronic acid or its salt gel. Since the above-mentioned gel has many loose pores, most of the rigid microspheres can enter the loose pores during the mixing process, thereby avoiding the aggregation of microspheres and making the microspheres more uniformly dispersed in the soft elastic interlocking structure of the gel. It also achieves a certain degree of encapsulation of the microspheres, so that the gel can effectively protect the microspheres during moist heat sterilization, better retain the filling and dispersing properties of the microspheres, reduce the degradation of microspheres, prolong the degradation time and safety performance after the microspheres are implanted in the body, thereby prolonging the effect of microspheres stimulating collagen regeneration. The entry of rigid microspheres can also further enhance the supporting performance of the soft elastic interlocking structure, so that the composition has both the soft elastic function of the gel and the rigid supporting performance. The two have a synergistic effect, and the applicable skin layers and filling sites are more extensive, improving the safety of the composition. Attached Figure Description

[0073] Figure 1 This is an electron microscope schematic diagram of the composition of Example 1.

[0074] Figure 2The diagram shows the compositions of Example 1 and Comparative Example 4 before and after centrifugation (left 1: composition of Comparative Example 4 before centrifugation, left 2: composition of Example 1 before centrifugation, right 1: composition of Comparative Example 4 after centrifugation, right 2: composition of Example 1 after centrifugation). Detailed Implementation

[0075] In order to better understand the technical content of the present invention, the following embodiments are provided in detail. The purpose of these embodiments is only to better understand the content of the present invention and not to limit the scope of protection of the present invention.

[0076] All raw materials and reagents used in the examples and comparative examples are commercially available.

[0077] Example 1

[0078] (1) Preparation of cross-linked sodium hyaluronate I′: Take 1.5g of 1500kDa sodium hyaluronate raw material and 0.5g of 600kDa sodium hyaluronate raw material, add 20mL of water, add 5mL of 250mg / mL sodium hydroxide solution, add 0.06mL of BDDE solution, mix well, and react at 25℃ for 20h to obtain cross-linked sodium hyaluronate I′.

[0079] (2) Preparation of cross-linked sodium hyaluronate II′: Take 0.6g of 600kDa sodium hyaluronate raw material, add 6mL of water, add 1.5mL of 100mg / mL sodium hydroxide solution to dissolve, add 0.03mL of BDDE solution, mix evenly, react at 25℃ for 20h, and form the gel into micron particles to obtain cross-linked sodium hyaluronate II′.

[0080] (3) Preparation of cross-linked sodium hyaluronate: Add the cross-linked sodium hyaluronate I′ from step (1) to 37.5 mL of water and stir evenly. Add the micron-sized particles of cross-linked sodium hyaluronate II′ from step (2) and 0.3 g of 600 kDa sodium hyaluronate raw material to the cross-linked sodium hyaluronate I′, mix evenly, and continue to react at 25 °C for 6 h. After the reaction is completed, cross-linked sodium hyaluronate is obtained.

[0081] (4) The cross-linked sodium hyaluronate obtained in step (3) was removed by dialysis. The pH of the system was first adjusted to about 10-11 with 8% hydrochloric acid solution, and then the pH of the system was slowly adjusted to neutral with 30% lactic acid solution. Then, 100 mL of phosphate buffer (10 g of disodium hydrogen phosphate, 4 g of sodium dihydrogen phosphate, and 1 g of potassium chloride were weighed and dissolved in 1000 mL of water), 0.58 g of lidocaine hydrochloride, 0.4 g of 600 kDa sodium hyaluronate, and 34.9 g of L-lactic acid-ethylene glycol copolymer microspheres were added. Water was added to 194 mL, mixed evenly, and sterilized by moist heat to obtain the cross-linked sodium hyaluronate gel composition (the theoretical molar cross-linking degree of the composition is 7.5%), which is defined as composition 1.

[0082] Example 2

[0083] In Example 1, the molecular weight of sodium hyaluronate was changed from 1500kDa to 900kDa and 600kDa to 300kDa in step (1). In steps (2)-(3), the molecular weight of sodium hyaluronate was changed from 600kDa to 300kDa. The remaining steps were the same as in Example 1 (the theoretical molar crosslinking degree of the combination group was 7.5%), resulting in Composition 2.

[0084] Example 3

[0085] The molecular weight of sodium hyaluronate in step (1) of Example 1 was replaced with 2800kDa and 600kDa was replaced with 1000kDa. The molecular weight of sodium hyaluronate in steps (2)-(3) was replaced with 1000kDa. All other steps were the same as in Example 1 (the theoretical molar crosslinking degree of the combination group was 7.5%), resulting in composition 3.

[0086] Example 4

[0087] The only difference is that the volume of sodium hydroxide in step (1) of Example 1 was replaced with 2 mL instead of 5 mL, and the volume of sodium hydroxide in step (2) was replaced with 0.6 mL instead of 1.5 mL. All other steps were the same as in Example 1 (the theoretical molar crosslinking degree of the combination group was 7.5%), resulting in composition 4.

[0088] Example 5

[0089] The only difference between the 34.9g L-lactic acid-ethylene glycol copolymer microspheres in step (4) of Example 1 and the 9.7g hydroxyapatite microspheres, and the absence of lidocaine hydrochloride, is that the remaining steps are the same as in Example 1 (the theoretical molar crosslinking degree of the combination group is 7.5%), resulting in composition 5.

[0090] Example 6

[0091] In Example 1, step (4) was performed by replacing 34.9g of L-lactic acid-ethylene glycol copolymer microspheres with 58.2g of L-polylactic acid microspheres and adding 0.15g of hydroxypropyl methylcellulose. All other steps were the same as in Example 1 (the theoretical molar crosslinking degree of the combination group was 7.5%), resulting in composition 6.

[0092] Comparative Example 1

[0093] The only difference between step (1) of Example 1, which is "adding 5 mL of 250 mg / mL sodium hydroxide solution", and step (2) is "adding 5 mL of 100 mg / mL sodium hydroxide solution", is that the other steps are the same as in Example 1 (the theoretical molar crosslinking degree of the combination group is 7.5%), and comparative composition 1 is obtained.

[0094] Comparative Example 2

[0095] The only difference between step (1) of Example 1, which is "take 1.5g of 1500kDa sodium hyaluronate raw material and 0.5g of 600kDa sodium hyaluronate raw material", and step (2g of 1500kDa sodium hyaluronate raw material), is that the other steps are the same as in Example 1 (the theoretical molar crosslinking degree of the combination group is 7.5%), is that comparative composition 2 is obtained.

[0096] Comparative Example 3

[0097] The only difference between step (1) of Example 1, which is "take 1.5g of 1500kDa sodium hyaluronate raw material and 0.5g of 600kDa sodium hyaluronate raw material", and step (2g of 600kDa sodium hyaluronate raw material), is that the other steps are the same as in Example 1 (the theoretical molar crosslinking degree of the combination group is 7.5%), is that the comparative composition 3 is obtained.

[0098] Comparative Example 4

[0099] Preparation of cross-linked sodium hyaluronate gel: 2.9 g of 1500 kDa sodium hyaluronate raw material was added to 20 mL of water, followed by 12.5 mL of 20 mg / mL sodium hydroxide solution to dissolve it. 0.1 mL of BDDE solution was added, and the mixture was stirred thoroughly. The mixture was reacted at 25°C for 30 h to obtain cross-linked sodium hyaluronate gel. Then, the gel composition was prepared using the same procedure as in Example 1 (the theoretical molar degree of cross-linking for the composition group was 7.5%), resulting in comparative composition 4.

[0100] Performance testing

[0101] Performance example 1

[0102] The composition of Example 1 was lyophilized and observed using a scanning electron microscope. Its structure is as follows: Figure 1 As shown. From Figure 1 As can be seen, the microspheres are uniformly dispersed in the network interlocking structure of the composition, and the loose network structure provides good encapsulation for most of the microspheres.

[0103] Performance example 2

[0104] Take the composition samples of Example 1 and Comparative Example 4, centrifuge at high speed, let stand for 10 min, observe the layering phenomenon of the composition, and evaluate the stability of the composition.

[0105] from Figure 2As can be seen, before centrifugation, the compositions of Example 1 and Comparative Example 4 were generally milky white gels without stratification, indicating that the microspheres were uniformly dispersed in the gel. After centrifugation, only slight separation of the gel and microspheres was observed in the composition of Example 1, with only a small portion of microspheres settling at the bottom of the test tube, while most microspheres remained dispersed in the gel. This indicates that using the cross-linked sodium hyaluronate of Example 1 provided better encapsulation of the L-lactic acid-ethylene glycol copolymer microspheres in the composition, thus enabling the microspheres to be better suspended and dispersed in the gel, resulting in better stability. In contrast, after centrifugation, the composition of Comparative Example 4 showed significant separation of the gel and microspheres, with a large number of microspheres settling at the bottom of the test tube. This indicates that the gel failed to effectively encapsulate the microspheres and ensure their stable suspension and dispersion in the gel, also suggesting that microspheres mixed with conventionally cross-linked sodium hyaluronate are prone to poor long-term storage stability.

[0106] Performance Example 3

[0107] The composition samples of the examples and comparative examples were subjected to performance testing, including elastic modulus and degree of crosslinking. The specific testing methods are as follows:

[0108] Elastic modulus: 2g of the composition sample was taken and the modulus of the composition sample before and after moist heat sterilization was measured using a rheometer (TA) at 25℃ and 1Hz.

[0109] Crosslinking degree: Take 12g of the crosslinked sodium hyaluronate samples prepared in step (3) of Examples 1-6 and Comparative Examples 1-3, and 12g of the crosslinked sodium hyaluronate sample in Comparative Example 4, add 200g of ethanol, stir and mix, and let stand to precipitate. Collect the precipitate, dry it at 80℃, and degrade it with 2mL of 0.5mol / L sulfuric acid solution at 100℃ until the solution is transparent. Neutralize with 1mol / L sodium hydroxide and freeze dry. Using deuterated DMSO as solvent, the molar crosslinking degree is determined by hydrogen nuclear magnetic resonance according to the area normalization method. Crosslinking degree = [δ = 1.5 peak area / 4] / [δ = 1.9 peak area / 3], where the peak at 1.5ppm represents the (-methylene-) group of BDDE molecule, and the peak at 1.9ppm represents n-acetylglucosamine in crosslinked sodium hyaluronate gel.

[0110] The characterization results are shown in Table 1.

[0111] Table 1. Elastic modulus and crosslinking degree results of different samples

[0112]

[0113] As shown in Table 1, the crosslinking efficiency in Examples 1-6 is relatively high, all exceeding 65%, and the modulus loss rate is low. This allows for better resistance to moist heat sterilization and better maintenance of stability. This is mainly because the present invention uses high and low molecular weight HA to form a loose network structure under conditions of a large alkaline solution volume. Then, under conditions of a small alkaline solution volume, low molecular weight HA is used as a raw material. Due to the strong hydrogen bonding between HA molecules, the molecular chains are more entangled, facilitating a rapid reaction to form a compact network structure, resulting in micron-sized gel particles. The first crosslinking product with a large alkaline solution volume is diluted with water to change the alkaline solution volume to a smaller volume. Micron-sized gel particles and HA are then added to continue the reaction, allowing the compact structure to penetrate the relatively loose structure, forming a soft-elastic interlocking structure of crosslinked HA gel with loose pores encapsulating elastic hard spheres. By controlling the amount of alkali and the molecular weight, the crosslinking reaction efficiency can be improved while controlling the network structure, resulting in a high final crosslinking efficiency. Subsequently, the cross-linked HA gel was mixed with microspheres. Since the gel has many loose pores, the microspheres can enter the loose pores during the mixing process, achieving a certain degree of encapsulation of the microspheres. The entry of rigid spheres further enhances the supporting performance of the soft elastic interlocking structure, so that the composition has both the soft elastic function of the gel and the rigid supporting performance. The two can play a synergistic effect, further improving the overall structural stability of the gel, so that it can withstand moist heat sterilization, and the loss rate of elastic modulus after sterilization is low.

[0114] The crosslinking efficiency and modulus loss rate of Comparative Examples 1-4 were significantly worse than those of Example 1. This is because the alkali content was the same in Comparative Example 1, making it difficult to form an interpenetrating chimeric structure, which affected the synergistic effect between the microspheres and the chimeric structure, resulting in a high modulus loss rate. In Comparative Examples 2-3, the molecular weight of the HA raw material was a single molecular weight during the first crosslinking, making it difficult to form a loose network structure. The microspheres could not easily enter the network structure, resulting in poor dispersibility and easy aggregation of the microspheres, thus leading to a high modulus loss rate. In Comparative Example 4, under conventional single molecular weight conditions, crosslinking mostly occurred between parallel chains, and the crosslinked network was relatively dense with small pore spacing. The microspheres could not enter the interior of the gel structure, so the composition had poor protection for the microspheres during moist heat sterilization, resulting in a high overall elastic modulus loss rate.

[0115] Performance example 4

[0116] The pH value and degradation performance of the composition samples from the examples and comparative examples were tested. The specific testing methods are as follows:

[0117] pH value: Take 3.0g of the composition, dilute it with water in an equal mass ratio, and use a pH meter to measure the pH value before and after moist heat sterilization.

[0118] Degradation performance: Take 10g of the compositions of Examples 1-4, 6 and Comparative Examples 1-4, add 20mL of 1mol / L hydrochloric acid solution, and degrade at 70℃. Replace the hydrochloric acid solution every 2 days and observe the time for complete degradation of the composition.

[0119] Take 10g of the composition from Example 5, add 20mL of phosphate buffer solution at pH 4.0, and degrade it at 80°C. Observe the time for complete degradation of the composition.

[0120] The characterization results are shown in Table 2.

[0121] Table 2. pH and degradation performance results of different samples

[0122] Item name pH value before sterilization pH value after sterilization pH value change rate Degradation performance (d) Example 1 7.25 7.18 -0.07 42 Example 2 7.26 7.12 -0.14 38 Example 3 7.25 7.11 -0.14 39 Example 4 7.24 7.11 -0.13 38 Example 5 7.27 7.31 +0.04 37 Example 6 7.25 7.07 -0.18 46 Comparative Example 1 7.24 7.05 -0.19 37 Comparative Example 2 7.25 7.04 -0.21 37 Comparative Example 3 7.27 7.06 -0.21 36 Comparative Example 4 7.26 7.05 -0.21 36

[0123] As shown in Table 2, the samples from Examples 1-4 and 6 of this invention exhibited higher crosslinking efficiency, smaller pH changes, and longer microsphere degradation times. Specifically, the microsphere degradation time in Example 1 was 42 days, higher than that in Comparative Examples 1-4. This is because the crosslinked sodium hyaluronate in Example 1 possesses a soft, elastic interlocking structure. When this crosslinked sodium hyaluronate is mixed with the microspheres, due to the numerous loose pores in the gel, some rigid microspheres can enter these pores during the mixing process, thus preventing microsphere aggregation and allowing for more uniform dispersion of the microspheres within the soft, elastic interlocking structure of the gel. This also achieves a certain degree of encapsulation of the microspheres, effectively protecting them during moist heat sterilization, better preserving their filling and dispersion properties, and reducing microsphere degradation. Therefore, sterilization... The pH change was small and the degradation time was long. In contrast, in Comparative Example 1, the alkali content was the same, making it difficult to form an interpenetrating chimeric structure, which affected the synergistic effect between the microspheres and the chimeric structure. In Comparative Examples 2-3, because the molecular weight of the HA raw material was a single molecular weight during the first crosslinking, it was difficult to form a loose network structure. The microspheres could not enter the loose network structure, resulting in poor dispersibility and easy aggregation of the microspheres. Therefore, the protective effect on the microspheres was reduced. In Comparative Example 4, under the conventional single molecular weight conditions, crosslinking mostly occurred between parallel chains, and the crosslinked network was relatively dense with small pore spacing. The microspheres could not enter the interior of the gel structure. Therefore, the composition had poor protection for the microspheres during moist heat sterilization. Thus, the overall pH change rate of the compositions in Comparative Examples 1-4 was high and the degradation time was short.

[0124] Furthermore, the microspheres added in Example 5 were hydroxyapatite. As those skilled in the art know, hydroxyapatite contains a large number of hydroxyl groups, making the microspheres alkaline. If degradation occurs during moist heat sterilization, the degradation products will increase the pH of the system. Example 5 shows that the composition of the gel mixed with hydroxyapatite microspheres prepared according to the present invention only increased the pH by 0.04 after moist heat sterilization, demonstrating a small pH change rate. This further proves that the composition of the present invention can effectively protect the microspheres and reduce their degradation. Simultaneously, the degradation time of the composition in Example 5 was 37 days, indicating that the composition prepared by the process of the present invention effectively prolongs the degradation time and filling effect of the microspheres after implantation.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition comprising microspheres and cross-linked hyaluronic acid or a salt thereof, characterized in that, The composition comprises cross-linked hyaluronic acid or its salt, and microspheres; the preparation method of the cross-linked hyaluronic acid or its salt includes the following steps: (1) Hyaluronic acid I or its salt, hyaluronic acid II or its salt, a first alkaline solution and a crosslinking agent are mixed and reacted to obtain crosslinked hyaluronic acid I′ or its salt. The concentration of the first alkaline solution is 10~100mg / mL. (2) Hyaluronic acid III or its salt, a second alkaline solution and a crosslinking agent are mixed and reacted to obtain crosslinked hyaluronic acid II′ or its salt. The concentration of the second alkaline solution is 0.1~50 mg / mL. (3) After diluting the cross-linked hyaluronic acid I′ or its salt obtained in step (1) with water, add the cross-linked hyaluronic acid II′ or its salt obtained in step (2) and hyaluronic acid IV or its salt, mix and react to obtain the cross-linked hyaluronic acid or its salt. The molecular weight of hyaluronic acid I or its salt is 900~3000kDa, and the molecular weight of hyaluronic acid II, III, IV or its salt is 100~1000kDa; The mass concentration of the first alkaline solution is higher than that of the second alkaline solution; the molecular weight of hyaluronic acid I or its salt is higher than that of hyaluronic acid II, III, IV or their salts.

2. The composition according to claim 1, characterized in that, The concentration of the first alkaline solution in step (1) is 12~90 mg / mL, and the concentration of the second alkaline solution in step (2) is 1~40 mg / mL.

3. The composition according to claim 2, characterized in that, The concentration of the first alkaline solution in step (1) is 15~80 mg / mL; the concentration of the second alkaline solution in step (2) is 5~30 mg / mL.

4. The composition according to claim 1, characterized in that, The molecular weight of hyaluronic acid I or its salt is 1000~2000kDa, and the molecular weight of hyaluronic acid II, III, IV or its salt is 300~800kDa.

5. The composition according to claim 4, characterized in that, The molecular weight of hyaluronic acid I or its salt is 1200~1600kDa; the molecular weight of hyaluronic acid II, III, IV or its salt is 400~700kDa.

6. The composition according to any one of claims 1-5, characterized in that, The mass ratio of hyaluronic acid I or its salt to hyaluronic acid II or its salt in step (1) is 2-10:

1.

7. The composition according to claim 6, characterized in that, The mass ratio of hyaluronic acid I or its salt to hyaluronic acid II or its salt in step (1) is 3-9:

1.

8. The composition according to any one of claims 1-5, characterized in that, The ratio of cross-linked hyaluronic acid I′ or its salt to cross-linked hyaluronic acid II′ or its salt is 1-10:

1.

9. The composition according to claim 8, characterized in that, The ratio of cross-linked hyaluronic acid I′ or its salt to cross-linked hyaluronic acid II′ or its salt is 2-6:

1.

10. The composition according to any one of claims 1-5, characterized in that, The amount of hyaluronic acid IV or its salt added is 5-30% of that of hyaluronic acid I or its salt.

11. The composition according to any one of claims 1-5, characterized in that, The reaction temperature described in steps (1), (2), and (3) is 20~30℃.

12. The composition according to claim 11, characterized in that, The reaction time described in steps (1), (2), and (3) is 4 to 30 hours.

13. The composition according to any one of claims 1-5, characterized in that, The hyaluronic acid salt is selected from any one of the following: sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate, bismuth hyaluronate, and zinc hyaluronate.

14. The composition according to claim 13, characterized in that, Hyaluronic acid salt is sodium hyaluronate.

15. The composition according to any one of claims 1-5, characterized in that, The degree of cross-linking of the obtained cross-linked hyaluronic acid or its salt is 1% to 6%.

16. The composition according to claim 15, characterized in that, The degree of cross-linking of the obtained cross-linked hyaluronic acid or its salt is 1.5% to 5%.

17. The composition according to any one of claims 1-5, characterized in that, The alkaline solution is selected from one or more of the following: sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium sulfite, and potassium sulfite.

18. The composition according to claim 17, characterized in that, The alkaline solution is a sodium hydroxide solution.

19. The composition according to any one of claims 1-5, characterized in that, The crosslinking agent is selected from one or more of the following: 1,4-butanediol diglycidyl ether, divinyl sulfone, polyethylene glycol, genipin, and carbodiimide.

20. The composition according to claim 19, characterized in that, The crosslinking agent is 1,4-butanediol diglycidyl ether.

21. The composition according to any one of claims 1-5, characterized in that, The microspheres are selected from one or more of polyester microspheres, collagen microspheres, silk fibroin microspheres, and hydroxyapatite microspheres.

22. The composition according to claim 21, characterized in that, The microspheres are polyester microspheres.

23. The composition according to claim 22, characterized in that, The polyester microspheres are selected from one or more of PLLA, PDLA, PDLLA, PLGA, PMMA, PCL, PEG-PLLA, and HA-PLLA.

24. The composition according to any one of claims 1-5, characterized in that, The composition contains 1% to 35% microspheres.

25. The composition according to claim 24, characterized in that, The composition contains 10% to 32% microspheres.

26. The composition according to claim 25, characterized in that, The composition contains 15% to 30% microspheres.

27. The composition according to any one of claims 1-5, characterized in that, The mass concentration of the cross-linked hyaluronic acid or its salt is 1~30 mg / mL.

28. The composition according to any one of claims 1-5, characterized in that, The composition further includes non-crosslinked hyaluronic acid or its salts, phosphate buffer, and optionally a local anesthetic and / or water-soluble cellulose.

29. The composition according to claim 28, characterized in that, The phosphate buffer solution comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium chloride.

30. The composition according to claim 28, characterized in that, The local anesthetic is selected from one or a combination of amide and ester types.

31. The composition according to claim 28, characterized in that, The water-soluble cellulose is selected from one or more of hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, ethyl methylcellulose, and carboxymethylcellulose.

32. A method for preparing the composition according to any one of claims 1-31, comprising the following steps: After dialysis, the cross-linked hyaluronic acid or its salt is removed. The pH of the system is first adjusted to 10-11 with hydrochloric acid solution, and then the pH of the system is adjusted to neutral with lactic acid solution. Microspheres are then added, mixed, and sterilized by moist heat to obtain a composition containing microspheres and cross-linked hyaluronic acid or its salt.

33. The preparation method according to claim 32, characterized in that, After adjusting the pH of the system to neutral, phosphate buffer, non-crosslinked hyaluronic acid or its salts may be added, and optionally, local anesthetics and / or water-soluble cellulose may be added.

34. The use of a composition according to any one of claims 1-31 or a composition prepared by any one of claims 32-33 in the preparation of pharmaceuticals, tissue engineering materials or cosmetics.

35. The application according to claim 34, characterized in that, The tissue engineering materials include soft tissue fillers, cartilage repair materials, or tissue engineering scaffolds.

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