Composition comprising microspheres and crosslinked hyaluronic acid or salt thereof, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a composition comprising microspheres and crosslinked hyaluronic acid or a salt thereof, and a preparation method therefor and a use thereof. The composition comprises crosslinked hyaluronic acid or a salt thereof, microspheres, a phosphate buffer, and non-crosslinked hyaluronic acid or a salt thereof. In the present invention, under a condition of a large amount of an alkali solution, a mixture of high- and low-molecular-weight hyaluronic acid or a salt thereof is used to form a loose structure; under a condition of a small amount of an alkali solution, low-molecular-weight hyaluronic acid or a salt thereof is used to form a compact network structure; the loose structure and the compact network structure are mixed to continue a crosslinking reaction to form a gel having a soft elastic mosaic structure in which loose pores wrap elastic hard spheres; then the gel is mixed with the microspheres, the entry of rigid microspheres in the gel enables the composition to have both a soft elastic function and rigid support performance, and the soft elastic function and the rigid support performance have a synergistic effect; the gel can also effectively protect the microspheres during moist heat sterilization, reducing the degradation of the microspheres, prolonging the effect of the microspheres in stimulating collagen regeneration, making applicable skin layers and sites to be filled more extensive, and improving the safety of the composition in use.

Description

A composition containing microspheres and cross-linked hyaluronic acid or its salt, a method for preparing the composition and its application. Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a composition containing microspheres and cross-linked hyaluronic acid or its salts, its preparation method, and its application. Background Technology

[0002] Hyaluronic acid is a natural mucopolysaccharide composed of repeating (1-β-4)D-glucuronic acid and (1-β-3)N-acetyl-D-glucosamine disaccharide units. It is an important component of human and animal skin, vitreous humor, joint lubricating fluid, and cartilage tissue. Sodium hyaluronate plays multiple roles in the human body, including space filling, hydration, joint lubrication, and providing a matrix through which cells can migrate. Despite its numerous advantages, the weak mechanical properties and easy degradation in vivo of natural hyaluronic acid limit its application in biomaterials. Cross-linked sodium hyaluronate, a polymeric gel obtained by chemically cross-linking sodium hyaluronate, overcomes the short retention time of natural sodium hyaluronate.

[0003] Patent document CN106279726B discloses a cross-linked sodium hyaluronate gel and its preparation method. Under ultrasonic conditions, a cross-linking agent is added dropwise to an alkaline aqueous solution of sodium hyaluronate to initiate a cross-linking reaction. After the reaction, the pH of the system is adjusted to 7.1-7.5 with acid, and the mixture is stirred for 1-2 hours. Then, it is precipitated with anhydrous ethanol to obtain the cross-linked sodium hyaluronate gel. However, this method does not include a step to remove the cross-linking agent, which can easily lead to residues of the cross-linking agent.

[0004] Patent document CN112940300B discloses a method for preparing cross-linked hyaluronic acid gel, comprising the following steps: (1) mixing an aqueous solution containing a cross-linking agent and hyaluronic acid, its metal salt, or a mixture thereof in a non-neutral environment to form a mixed solution; (2) placing the mixed solution at a temperature below 0°C and above the eutectic point of the mixed solution for a sufficient time to form a solid-liquid heterogeneous system; (3) subjecting the heterogeneous system to a cross-linking reaction at a temperature below 0°C and above the eutectic point of the mixed solution; and (4) melting the solid phase in the heterogeneous system after the cross-linking reaction, optionally neutralizing, purifying, and homogenizing it. However, the neutralization step described in this method does not guarantee a more gradual decrease in the pH of the system, which can easily damage the three-dimensional cross-linked structure of the sodium hyaluronate gel system.

[0005] Polyester microspheres, collagen microspheres, silk fibroin microspheres, hydroxyapatite microspheres, etc., are often used as novel filler materials for medical aesthetics due to their biocompatibility, biodegradability, and non-toxicity. However, when microspheres are mixed with carriers such as matrices and sterilized, the carriers cannot effectively protect the microspheres. As a result, after the product is injected, problems such as rapid degradation of microspheres, incompatibility with the injection site tissue, and inflammation may still occur.

[0006] In summary, existing technologies cannot simultaneously meet the industrialization requirements of hyaluronic acid gel, such as simple preparation process, good safety, effective removal of cross-linking agents, strong viscosity, high cohesion, and low water absorption. They also cannot effectively guarantee the degradation effect of microspheres and achieve collagen regeneration. Summary of the Invention

[0007] This invention overcomes the deficiencies of existing technologies and provides a composition containing microspheres and cross-linked hyaluronic acid or its salts, its preparation method, and its application. Using Semo cross-linking technology (Soft Elastic Mosaic Cross-Linking Technology), a cross-linked hyaluronic acid or its salt gel is first prepared, and then mixed with microspheres. The inclusion of rigid microspheres in the gel further enhances the supporting performance of the soft elastic interlocking structure, giving the composition both the soft elasticity of a gel and rigid support properties, resulting in a synergistic effect. Simultaneously, the gel effectively protects the microspheres during moist heat sterilization, reducing microsphere degradation, prolonging the effect of microspheres stimulating collagen regeneration, and broadening the applicable skin layers and filling sites, while also improving the safety of the composition.

[0008] In a first aspect, the present invention provides a composition comprising microspheres and cross-linked hyaluronic acid or a salt thereof, wherein the method for preparing the cross-linked hyaluronic acid or a salt thereof in the composition comprises the following steps:

[0009] (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;

[0010] (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;

[0011] (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.

[0012] Furthermore, the mass concentration of the first alkaline solution is higher than that of the second alkaline solution.

[0013] Furthermore, the molecular weight of hyaluronic acid I or its salt is higher than that of hyaluronic acid II, III, IV or their salts.

[0014] 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, 4...). 7mg / mL, 48mg / mL, 49mg / mL, 50mg / mL, 51mg / mL, 52mg / mL, 53mg / mL, 54mg / mL, 55mg / mL, 56mg / mL, 57mg / mL , 58 mg / mL, 59 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL), preferably 12 to 90 mg / mL, more preferably 15 to 80 mg / mL.

[0015] Further, the concentration of the second alkaline solution in step (2) is 0.1 to 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 to 40 mg / mL, more preferably 5 to 30 mg / mL.

[0016] Further, the molecular weight of the hyaluronic acid I or its salt is 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, and more preferably 1200-1600 kDa.

[0017] In one embodiment of the present invention, the hyaluronic acid I or its salt has a molecular weight of 1500 kDa.

[0018] Further, the molecular weight of the hyaluronic acid II, III, IV or its salts is 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.

[0019] Furthermore, the molecular weights of the hyaluronic acids II, III, IV, or their salts may be the same or different.

[0020] In one embodiment of the present invention, the molecular weight of the hyaluronic acid II, III, IV or its salt is 600 kDa.

[0021] Further, the mass ratio of hyaluronic acid I or its salt to hyaluronic acid II or its salt in step (1) is 2-10:1, preferably 3-9:1.

[0022] In one embodiment of the present invention, the mass ratio of hyaluronic acid I or its salt to hyaluronic acid II or its salt in step (1) is 3:1.

[0023] Further, the mass ratio of the cross-linked hyaluronic acid I′ or its salt to the cross-linked hyaluronic acid II′ or its salt is 1-10:1; preferably 2-6:1.

[0024] Furthermore, the amount of hyaluronic acid IV or its salt added is 5-30% of that of hyaluronic acid I or its salt.

[0025] Furthermore, in step (3), the concentration of the alkaline solution diluted with water is equal to the concentration of the second alkaline solution in step (2).

[0026] Furthermore, the reaction temperature described in steps (1), (2), and (3) is 20–30 °C.

[0027] Furthermore, the reaction time described in steps (1), (2), and (3) is 4 to 30 hours.

[0028] Furthermore, the temperature and time of the reaction in steps (1), (2), and (3) can be the same or different.

[0029] In one embodiment of the present invention, the reaction temperature in step (1) is 25°C and the reaction time is 20h.

[0030] Furthermore, the hyaluronic acid I or its salt, hyaluronic acid II or its salt, hyaluronic acid III or its salt, and hyaluronic acid IV or its salt may be derived from animals or obtained through bacterial fermentation.

[0031] Furthermore, 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; sodium hyaluronate is preferred.

[0032] Further, the alkaline solution is selected from one or more solutions prepared from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium sulfite, and potassium sulfite, preferably a sodium hydroxide solution. The first alkaline solution and the second alkaline solution may be the same or different; preferably, the first alkaline solution and the second alkaline solution are the same.

[0033] Furthermore, the crosslinking agent is selected from one or more combinations of 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone, polyethylene glycol, genipin, and carbodiimide, preferably 1,4-butanediol diglycidyl ether.

[0034] Furthermore, the degree of crosslinking of the crosslinked hyaluronic acid or its salt is 1% to 6%, preferably 1.5% to 5%.

[0035] Furthermore, the microspheres are selected from one or more of polyester microspheres, collagen microspheres, silk fibroin microspheres, and hydroxyapatite microspheres.

[0036] Furthermore, the microspheres are polyester microspheres.

[0037] Further, the polyester is selected from one or more of polylactic acid (PLLA), polylactic acid (PDLA), racemic polylactic acid (PDLLA), polylactic acid coglycolic acid (PLGA), polymethyl methacrylate (PMMA), polycaprolactone (PCL), PEG-PLLA (levolactic acid-ethylene glycol copolymer), or hydroxyapatite-HAP-PLLA.

[0038] Further, the content of microspheres in the composition is 1% to 35% (e.g., 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%.

[0039] Further, the mass concentration of the cross-linked hyaluronic acid or its salt is 1–30 mg / mL (e.g., 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.

[0040] This invention first involves compounding high- and low-molecular-weight hyaluronic acid or its salts. Under conditions of a large alkaline solution, 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. The low-molecular-weight hyaluronic acid or its salts can freely undergo random translational or rotational movements while stretching, enabling them to interpenetrate or stand upright between the high-molecular-weight hyaluronic acid or its salts, achieving cross-linking between the two molecules. Simultaneously, the large alkaline solution volume degrades the molecular chains of the hyaluronic acid or its salts during cross-linking, ultimately forming a three-dimensional loose network structure with large pore spacing. Subsequently, under conditions of a small alkaline solution volume, the low-molecular-weight hyaluronic acid or its salts is cross-linked. The low alkaline solution volume results in stronger hydrogen bonds between the hyaluronic acid or its salts, more widespread molecular chain entanglement, and a rapid and easy reaction between the hyaluronic acid or its salts and the cross-linking agent to form a compact network structure, yielding micron-sized gel particles. However, due to the incomplete cross-linking reaction under conditions of high alkaline solution volume, some cross-linking agent remains. By adding water to dilute the solution, the conditions of high alkaline solution volume are changed to low alkaline solution volume. Micron-sized gel particles, hyaluronic acid, or its salts are then added to continue the reaction, consuming the incompletely reacted cross-linking agent. This allows the compact structure to penetrate into a more porous 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 porous region has good extensibility, the gel exhibits excellent support, extensibility, and deformation resistance, as well as better water absorption and retention, better tissue compatibility, and is more suitable for flat injection filling of the inner and outer surfaces of cavities.

[0041] Furthermore, this invention mixes microspheres with the aforementioned cross-linked hyaluronic acid or its salt gel. Because the gel has many loose pores, most of the rigid microspheres can enter these pores during the mixing process, thus preventing microsphere aggregation and allowing the microspheres to be more uniformly dispersed within the gel's soft, elastic interlocking structure. This also achieves a certain degree of encapsulation of the microspheres, enabling the gel to effectively protect them during moist heat sterilization, better preserving their filling and dispersing properties, reducing degradation, and extending the degradation time and safety performance after implantation. The entry of rigid microspheres further enhances the support performance of the soft, elastic interlocking structure, giving the composition both the soft elasticity of a gel and rigid support properties. These two properties have a synergistic effect, thereby prolonging the effect of microspheres stimulating collagen regeneration. This results in a wider range of applicable skin layers and filling sites, improving the safety of the composition.

[0042] Furthermore, the composition further includes non-crosslinked hyaluronic acid or its salts, phosphate buffer, optional local anesthetic, and / or water-soluble cellulose.

[0043] Furthermore, the non-crosslinked hyaluronic acid salt is selected from any one of the following: non-crosslinked sodium hyaluronate, non-crosslinked potassium hyaluronate, non-crosslinked calcium hyaluronate, non-crosslinked magnesium hyaluronate, non-crosslinked ammonium hyaluronate, non-crosslinked tetrabutylammonium hyaluronate, non-crosslinked bismuth hyaluronate, and non-hyaluronic acid zinc, preferably non-crosslinked sodium hyaluronate.

[0044] Further, the concentration of the non-crosslinked hyaluronic acid or its salt is 0.2–10 mg / mL (e.g., 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 mg / mL, 3.5 mg / mL, 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.

[0045] Further, the molecular weight of the non-crosslinked hyaluronic acid or its salt is 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, and more preferably 400-700 kDa.

[0046] Furthermore, the phosphate buffer solution comprises: disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium chloride.

[0047] 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). The concentrations are 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, and 10 mg / mL. Preferably, the concentration of disodium hydrogen phosphate is 3 to 10 mg / mL, and more preferably, the concentration of disodium hydrogen phosphate is 4 to 8 mg / mL.

[0048] In one embodiment of the present invention, the concentration of the disodium hydrogen phosphate is 10 mg / mL.

[0049] Further, the concentration of sodium dihydrogen phosphate is 0.01–9 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, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 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 sodium dihydrogen phosphate is 1–7 mg / mL, and more preferably, the concentration of sodium dihydrogen phosphate is 2–6 mg / mL.

[0050] In one embodiment of the present invention, the concentration of sodium dihydrogen phosphate is 4 mg / mL.

[0051] Further, the concentration of potassium chloride is 0.01–7 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). The concentrations of potassium chloride are 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, and 7 mg / mL. Preferably, the concentration of potassium chloride is 0.05–5 mg / mL, and more preferably, the concentration of potassium chloride is 0.1–3 mg / mL.

[0052] In one embodiment of the present invention, the concentration of potassium chloride is 1 mg / mL.

[0053] Furthermore, the local anesthetic is selected from one or a combination of amide and ester types.

[0054] Further, the local anesthetic is selected from: lidocaine or its salt, bupivacaine or its salt, butancaine or its salt, carticaine or its salt, cincocaine or its salt, chlorotetracaine or its salt, ethyl p-piperidinylacetaminobenzoate or its salt, eticaine or its salt, mepivacaine or its salt, oxicaine or its salt, prilocaine or its salt, ropivacaine or its salt, tolicaine or its salt, trimethaine or its salt, vadocaine or its salt, articaine or its salt, levobupivacaine or its salt, amicaine or its salt, cocaine or its salt. The group consisting of one or more of the following: propylpacaine or its salt, chloromecaine or its salt, cyclomecaine or its salt, propylpacaine or its salt, dicaine or its salt, benzocaine or its salt, butacaine or its salt, tintosicaine or its salt, butyl aminobenzoate or its salt, chloroprocaine or its salt, dimethylcaine or its salt, oxybubucaine or its salt, piperocaine or its salt, paraethoxycaine or its salt, procaine or its salt, propyloxycaine or its salt, tricaine or its salt, preferably lidocaine or its salt, and more preferably lidocaine hydrochloride.

[0055] Further, the content of the local anesthetic ranges 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, the content of the local anesthetic ranges from 0.1 to 8 mg / mL, more preferably, the content of the local anesthetic ranges from 0.5 to 8 mg / mL, and even more preferably, the content of the local anesthetic ranges from 0.5 to 5 mg / mL.

[0056] In one embodiment of the present invention, the content of the local anesthetic is in the range of 3 mg / mL.

[0057] Furthermore, the water-soluble cellulose is selected from one or more of hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, ethyl methyl cellulose and carboxymethyl cellulose, preferably hydroxypropyl methyl cellulose.

[0058] Furthermore, the water-soluble cellulose content ranges from 0.01 to 40 mg / mL, preferably from 0.2 to 25 mg / mL, and more preferably from 0.5 to 10 mg / mL.

[0059] A second aspect of the present invention provides a method for preparing a composition containing microspheres and cross-linked hyaluronic acid or a salt thereof, comprising the following steps:

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Preferably, the tissue engineering materials include soft tissue filler materials, cartilage repair materials, and tissue engineering scaffolds.

[0064] 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.

[0065] 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:

[0066] 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

[0067] 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.

[0068] 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.

[0069] The beneficial effects of this invention are:

[0070] (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.

[0071] (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

[0072] Figure 1 is an electron microscope schematic diagram of the composition of Example 1.

[0073] Figure 2 shows schematic diagrams of 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

[0074] 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.

[0075] All raw materials and reagents used in the examples and comparative examples are commercially available.

[0076] Example 1

[0077] (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′.

[0078] (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′.

[0079] (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.

[0080] (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.

[0081] Example 2

[0082] 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.

[0083] Example 3

[0084] 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.

[0085] Example 4

[0086] 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.

[0087] Example 5

[0088] 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.

[0089] Example 6

[0090] 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.

[0091] Comparative Example 1

[0092] 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.

[0093] Comparative Example 2

[0094] 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.

[0095] Comparative Example 3

[0096] 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.

[0097] Comparative Example 4

[0098] 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.

[0099] Performance testing

[0100] Performance example 1

[0101] The composition of Example 1 was lyophilized and observed using a scanning electron microscope, and its structure is shown in Figure 1. As can be seen from Figure 1, 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.

[0102] Performance example 2

[0103] 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.

[0104] As shown in Figure 2, 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 from the microspheres was observed in the composition of Example 1, with only a small portion of the 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, allowing 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 from the microspheres, with a large number of microspheres settling at the bottom of the test tube. This suggests that the gel failed to effectively encapsulate the microspheres and ensure their stable suspension and dispersion in the gel, also indicating that microspheres mixed with conventionally cross-linked sodium hyaluronate are prone to poor long-term storage stability.

[0105] Performance Example 3

[0106] 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:

[0107] 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.

[0108] 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.

[0109] The characterization results are shown in Table 1.

[0110] Table 1. Elastic modulus and crosslinking degree results of different samples

[0111] 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.

[0112] 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.

[0113] Performance example 4

[0114] 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:

[0115] 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.

[0116] 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.

[0117] 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.

[0118] The characterization results are shown in Table 2.

[0119] Table 2. pH and degradation performance results of different samples

[0120] 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.

[0121] 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.

[0122] 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; (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; (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 and hyaluronic acid IV or its salt obtained in step (2), mix and react to obtain the cross-linked hyaluronic acid or its salt. 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 10-100 mg / mL, preferably 12-90 mg / mL, more preferably 15-80 mg / mL; the concentration of the second alkaline solution in step (2) is 0.1-50 mg / mL, preferably 1-40 mg / mL, more preferably 5-30 mg / mL.

3. The composition according to claim 1, characterized in that, The molecular weight of hyaluronic acid I or its salt is 900-3000 kDa, preferably 1000-2000 kDa, and more preferably 1200-1600 kDa; the molecular weight of hyaluronic acid II, III, IV or its salt is 100-1000 kDa, preferably 300-800 kDa, and more preferably 400-700 kDa.

4. The composition according to any one of claims 1-3, 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, preferably 3-9:1; Preferably, the ratio of cross-linked hyaluronic acid I′ or its salt to cross-linked hyaluronic acid II′ or its salt is 1-10:1, more preferably 2-6:

1.

5. The composition according to any one of claims 1-3, 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.

6. The composition according to any one of claims 1-3, characterized in that, The reaction temperature in steps (1), (2), and (3) is 20–30°C; preferably, the reaction time in steps (1), (2), and (3) is 4–30 h.

7. The composition according to any one of claims 1-3, characterized in that, The hyaluronic acid salt is selected from any one of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate, bismuth hyaluronate, and zinc hyaluronate, preferably sodium hyaluronate; Preferably, the degree of crosslinking of the obtained crosslinked hyaluronic acid or its salt is 1% to 6%, more preferably 1.5% to 5%; Preferably, the alkaline solution is selected from one or more solutions prepared from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium sulfite, and potassium sulfite, and more preferably a sodium hydroxide solution. Preferably, the crosslinking agent is selected from one or more combinations of 1,4-butanediol diglycidyl ether, divinyl sulfone, polyethylene glycol, genipin and carbodiimide, more preferably 1,4-butanediol diglycidyl ether.

8. The composition according to any one of claims 1-3, characterized in that, The microspheres are selected from one or more of polyester microspheres, collagen microspheres, silk fibroin microspheres, and hydroxyapatite microspheres; preferably polyester microspheres; more preferably, the polyester microspheres are selected from one or more of PLLA, PDLA, PDLLA, PLGA, PMMA, PCL, PEG-PLLA, and HA-PLLA.

9. The composition according to any one of claims 1-3, characterized in that, The composition contains 1% to 35% microspheres, preferably 10% to 32%, and more preferably 15% to 30%. Preferably, the mass concentration of the cross-linked hyaluronic acid or its salt is 1 to 30 mg / mL.

10. The composition according to any one of claims 1-3, characterized in that, The composition further includes non-crosslinked hyaluronic acid or its salt, phosphate buffer, and optionally a local anesthetic and / or water-soluble cellulose; Preferably, the phosphate buffer solution comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium chloride; Preferably, the local anesthetic is selected from one or a combination of amide and ester types; Preferably, the water-soluble cellulose is selected from one or more of hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, ethyl methyl cellulose, and carboxymethyl cellulose.

11. A method for preparing the composition according to any one of claims 1-10, 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.

12. The preparation method according to claim 11, 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.

13. The use of a composition according to any one of claims 1-10 or a composition prepared by any one of claims 11-12 in the preparation of pharmaceuticals, tissue engineering materials or cosmetics; Preferably, the tissue engineering material includes soft tissue filler, cartilage repair material, or tissue engineering scaffold.

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