阅读说明：本技术 一种含左旋聚乳酸的透明质酸复合微球及其制备方法 (Hyaluronic acid composite microsphere containing levorotatory polylactic acid and preparation method thereof ) 是由 王丽 解荡 于 2021-09-16 设计创作，主要内容包括：本发明涉及A61L,更具体地,本发明涉及一种含左旋聚乳酸的透明质酸复合微球及其制备方法。所述复合微球包括透明质酸微球和左旋聚乳酸粉体,所述左旋聚乳酸粉体位于透明质酸微球内部。本发明提供一种含有左旋聚乳酸的透明质酸钠交联微球反应条件,且均使用高度生物相容性、生物降解性的原料,得到的微球具有高的热稳定性和生物相容性。通过将左旋聚乳酸包裹在透明质酸微球的内部,在注射后能激发细胞二次活力并刺激肌体自身胶原增加来达到抗衰和预防衰老的作用,可用于化妆品、组织填充与修复、生物支架、眼科、药物缓释与靶向给药等化妆品、医药、医美等多个领域,具有广泛的应用前景。(The invention relates to A61L, in particular to hyaluronic acid composite microspheres containing L-polylactic acid and a preparation method thereof. The composite microspheres comprise hyaluronic acid microspheres and levorotatory polylactic acid powder, and the levorotatory polylactic acid powder is positioned inside the hyaluronic acid microspheres. The invention provides a reaction condition of sodium hyaluronate cross-linked microspheres containing levorotatory polylactic acid, and the microspheres all use raw materials with high biocompatibility and biodegradability, and have high thermal stability and biocompatibility. The levorotatory polylactic acid is coated in the hyaluronic acid microspheres, so that the secondary activity of cells can be stimulated and the collagen of a body can be stimulated to increase after injection to achieve the effects of resisting aging and preventing aging, and the levorotatory polylactic acid can be used in a plurality of fields of cosmetics, medicines, cosmetology and the like, such as cosmetics, tissue filling and repairing, biological scaffolds, ophthalmology, medicine slow release, targeted drug delivery and the like, and has wide application prospects.)

1. The hyaluronic acid composite microsphere containing the levorotatory polylactic acid is characterized by comprising a hyaluronic acid microsphere and levorotatory polylactic acid powder, wherein the levorotatory polylactic acid powder is positioned in the hyaluronic acid microsphere.

2. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 1, wherein the raw materials for preparing the L-polylactic acid powder comprise L-polylactic acid and a hydrophilic modifier, and the L-polylactic acid accounts for 30-80 wt% of the total weight of the L-polylactic acid and the hydrophilic modifier.

3. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 2, wherein the raw materials for preparing the hydrophilic modifier comprise 1-15 wt% of hyaluronic acid aqueous solution, 1-9 wt% of degradable polymer aqueous solution and a cross-linking agent.

4. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 3, wherein the degradable polymer in the aqueous solution of degradable polymer accounts for 3-10 wt% of hyaluronic acid in the aqueous solution of hyaluronic acid.

5. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 3, wherein the molar amount of the cross-linking agent per gram of hyaluronic acid is 0.005-0.02 mol.

6. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 3, wherein the degradable polymer is selected from one or more of chitosan, chitin, sodium alginate and glucan.

7. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 3, wherein the preparation method of the L-polylactic acid powder comprises the following steps: mixing a hyaluronic acid aqueous solution, a cross-linking agent and a degradable high polymer aqueous solution, adding the levorotatory polylactic acid, standing, solidifying, drying and crushing to obtain the levorotatory polylactic acid.

8. The hyaluronic acid composite microsphere containing the L-polylactic acid according to any one of claims 1 to 7, wherein the hyaluronic acid microsphere is a hyaluronic acid-hydrophilic polymer microsphere, and the hydrophilic polymer is one or more selected from chitosan, chitin, sodium alginate, dextran, polyvinyl alcohol, polyethylene glycol and poly (glycolide-lactide).

9. The hyaluronic acid composite microsphere containing L-polylactic acid according to claim 8, wherein the raw materials for preparing the hyaluronic acid microsphere comprise 1-15 wt% of hyaluronic acid aqueous solution, 1-9 wt% of hydrophilic polymer aqueous solution, and the L-polylactic acid powder accounts for 10-40 wt% of the raw materials for preparing the hyaluronic acid microsphere.

10. The method for preparing hyaluronic acid composite microspheres containing L-polylactic acid according to claim 9, comprising:

preparation of an aqueous phase: mixing the raw materials for preparing the hyaluronic acid microspheres to obtain a water phase;

reverse suspension polymerization: and adding the levorotatory polylactic acid powder into the water phase, adding the oil phase, reacting for 4-7 h at 50-75 ℃, filtering, washing and drying to obtain the composite microsphere.

Technical Field

The invention relates to A61L, in particular to hyaluronic acid composite microspheres containing L-polylactic acid and a preparation method thereof.

Background

Levorotatory polylactic acid (PLLA) has good biocompatibility and degradability, is used in the medical field for nearly 40 years, is used as a medical and American injection product, and is approved as a legal filling injection by the FDA in the united states in 2009. Because of good compatibility and absorbability, the safety is extremely high.

Hyaluronic Acid (HA), as a natural anionic polysaccharide, HAs high water retention, skin nutritional metabolism improvement, aging prevention and other effects, and HAs great application potential in the medical field because HA is non-immunogenic. The hyaluronic acid gel is mainly applied to shaping, face filling or dent filling and the like in the cosmetic industry, and the main component of many commercially available products is hyaluronic acid at present.

Materials such as levorotatory polylactic acid and hyaluronic acid with high biocompatibility and degradability can fully play respective roles when being prepared into microspheres and the like together to obtain a more efficient product, but compared with hydrophilic hyaluronic acid, the levorotatory polylactic acid is difficult to dissolve in water and cannot be fused with water-soluble microspheres, and generally adopts a chemical bond reaction mode, for example, CN111375124A is subjected to emulsion polymerization through an oil phase and a water phase containing the levorotatory polylactic acid, so that a hydrophilic microsphere system is difficult to form with the hyaluronic acid, and the smoothness degree and the compression elasticity of the microspheres are difficult to keep stable.

Disclosure of Invention

In order to solve the above problems, a first aspect of the present invention provides hyaluronic acid composite microspheres containing levorotatory polylactic acid, the composite microspheres including hyaluronic acid microspheres and levorotatory polylactic acid powder, the levorotatory polylactic acid powder being located inside the hyaluronic acid microspheres.

Levorotatory polylactic acid powder

In order to promote the formation of the composite microspheres of polylactic acid and hyaluronic acid, the hydrophilic modifier and the levorotatory polylactic acid are added and blended to form gel, and the gel is dried to obtain the polylactic acid powder with the hydrophilic modifier, so that the obtained powder can enter a water phase containing hyaluronic acid to form the composite microspheres in the microsphere formation process. In one embodiment, the raw material for preparing the levorotatory polylactic acid powder comprises levorotatory polylactic acid and a hydrophilic modifier, wherein the levorotatory polylactic acid accounts for 30-80 wt% of the total weight of the levorotatory polylactic acid and the hydrophilic modifier.

In one embodiment, the hydrophilic modifier is prepared from raw materials comprising:

1 to 15% by mass of an aqueous hyaluronic acid solution, and 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt% may be mentioned; the pH of the hyaluronic acid aqueous solution is 3-8, and can be 3, 4, 5, 6, 7 and 8.

The preparation method of the hyaluronic acid aqueous solution in the hydrophilic modifier is not particularly limited, and for example, the hyaluronic acid aqueous solution is obtained by adding hyaluronic acid into 1-3 mol/L inorganic salt aqueous solution. As examples of inorganic salts, there are included, but not limited to, sodium chloride, potassium chloride, sodium bromide, sodium sulfate, and the like.

A crosslinking agent; the crosslinking agent of the present invention may be an aldehyde crosslinking agent, an epoxy crosslinking agent, a carbodiimide crosslinking agent, such as glutaraldehyde, butanediol diglycidyl ether, carbodiimide, etc., which are well known in the art, or a carboxylic acid activated crosslinking agent, such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, N-hydroxysulfosuccinimide, and a complex thereof, and preferably a carboxylic acid activated crosslinking agent, in order to reduce irritation and improve biosafety.

The degradable polymer aqueous solution having a mass concentration of 1 to 9 wt% may include 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, and 9 wt%. The degradable polymer is selected from one or more of chitosan, chitin, sodium alginate and glucan, preferably chitosan, and a small amount of polycation and hyaluronic acid are added to be used as a hydrophilic modifier, so that gel formation can be promoted, and meanwhile, the density and adhesiveness of the modifier on the surface of polylactic acid can be improved, and further, the subsequent entering of the hyaluronic acid microsphere water phase is promoted and dispersed in the microsphere, and powder aggregation is reduced.

The invention does not specifically limit the preparation method of the degradable high polymer aqueous solution in the hydrophilic modifier, for example, the degradable high polymer is added into an organic acid aqueous solution with the mass concentration of 0.5-2 wt% to obtain the degradable high polymer aqueous solution. As examples of the organic acid solution, formic acid, acetic acid, butyric acid, and the like are included, but not limited thereto.

In one embodiment, the degradable polymer in the aqueous solution of degradable polymer accounts for 3-10 wt% of the hyaluronic acid in the aqueous solution of hyaluronic acid, and may be, for example, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%.

In one embodiment, the amount of the crosslinking agent is 0.005 to 0.02mol per gram of hyaluronic acid, and examples thereof include 0.005mol, 0.006mol, 0.008mol, 0.01mol, 0.012mol, 0.015mol, 0.017mol, and 0.02 mol. The inventor finds that activating hyaluronic acid by adding a small amount of cross-linking agent can further promote standing and solidification into a gel shape, promote the adhesion of degradable polymers such as hyaluronic acid and chitosan on the surface of polylactic acid after drying, improve the compatibility at the interface, promote the physical cross-linking density, play a certain role in fixation and promote the compression elasticity when acting with hyaluronic acid microspheres. The cross-linking agent of the present invention may be one or a mixture of two or more, and when two or more, such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide, the molar ratio may be (1-5): (1 to 5), there may be mentioned, 1: 5. 3: 5. 1: 1. 3: 1. 5: 1.

in one embodiment, the method for preparing the levorotatory polylactic acid powder comprises the following steps: mixing a hyaluronic acid aqueous solution, a cross-linking agent and a degradable high polymer aqueous solution, adding the levorotatory polylactic acid, standing, solidifying, drying and crushing to obtain the levorotatory polylactic acid.

In one embodiment, the method for preparing the levorotatory polylactic acid powder comprises the following steps:

mixing hyaluronic acid aqueous solution and a cross-linking agent at-2-5 ℃ (such as ice bath), standing for 6-12 h (such as standing overnight, about 6-9 h), adding degradable high polymer aqueous solution, mixing, adding levorotatory polylactic acid, standing for solidification, drying, and crushing to obtain the levorotatory polylactic acid. The crushing can be carried out by grinding and crushing, and the levorotatory polylactic acid powder with the particle size of 20-40 mu m is screened out. In the mixing process of the hyaluronic acid aqueous solution and the cross-linking agent, the cross-linking agent can be dissolved by adding a small amount of water and then mixed, and the cross-linking agent is less in dosage and less in water for dissolving, so that the method is not particularly limited.

Hyaluronic acid microspheres

The hyaluronic acid microsphere can refer to the preparation method of the microsphere with the application number of 2021109412967, and other active agents or hydrophilic polymers can be used as the material of the hyaluronic acid microsphere. In one embodiment, the hyaluronic acid microspheres are hyaluronic acid-hydrophilic polymer microspheres, and the hydrophilic polymer is one or more selected from chitosan, chitin, sodium alginate, dextran, polyvinyl alcohol, polyethylene glycol and poly (glycolide), and is preferably a hydrophilic polymer with high biocompatibility. Preferably chitosan. According to the invention, the chitosan is used as a component of the composite microsphere and also can be used as a cross-linking agent to compositely cross-link hyaluronic acid and derivatives thereof, and the levorotatory polylactic acid is wrapped in the microsphere to prepare the microsphere, the reaction condition is simple, the cross-linking agent is made of a natural polymer material, and the obtained gel microsphere has high thermal stability and biocompatibility.

In one embodiment, the raw materials for preparing the hyaluronic acid microspheres comprise:

1 to 15% by mass of an aqueous hyaluronic acid solution, and 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt% may be mentioned; the pH value of the hyaluronic acid aqueous solution is 3-8, and can be 3, 4, 5, 6, 7 and 8; the mass concentrations of the hyaluronic acid aqueous solution in the hyaluronic acid microsphere and the hydrophilic modifier can be the same or different, and are not particularly limited.

An active agent; the active agent is not particularly limited, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, N-hydroxysulfosuccinimide and a compound thereof are listed.

The hydrophilic polymer aqueous solution having a mass concentration of 1 to 9 wt% may be, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, and the present invention does not specifically limit the preparation method of the hydrophilic polymer aqueous solution, and for example, the hydrophilic polymer aqueous solution is obtained by adding an organic acid aqueous solution having a mass concentration of 0.5 to 2 wt% to the hydrophilic polymer. As examples of the organic acid solution, formic acid, acetic acid, butyric acid, and the like are included, but not limited thereto.

The levorotatory polylactic acid powder accounts for 10-40 wt% of the raw materials for preparing the hyaluronic acid microspheres, and 10 wt%, 12 wt%, 15 wt%, 17 wt%, 20 wt%, 22 wt%, 25 wt%, 30 wt%, 32 wt%, 35 wt%, 37 wt% and 40 wt% can be enumerated.

The second aspect of the present invention provides a preparation method of the hyaluronic acid composite microsphere containing l-polylactic acid, comprising:

preparation of an aqueous phase: mixing the raw materials for preparing the hyaluronic acid microspheres to obtain a water phase;

reverse suspension polymerization: and adding the levorotatory polylactic acid powder into the water phase, adding the oil phase, reacting for 4-7 h at 50-75 ℃, filtering, washing and drying to obtain the composite microsphere.

In one embodiment, in the preparation of the aqueous phase, the aqueous hyaluronic acid solution and the activating agent in the raw material for preparing the hyaluronic acid microspheres are mixed at-2 to 5 ℃ (such as ice bath), and are allowed to stand for 6 to 12 hours (such as overnight, about 6 to 9 hours), and the aqueous hydrophilic polymer solution is added and mixed to obtain the aqueous phase.

The oil phase can be an oil phase solvent, such as liquid paraffin, ethyl acetate, petroleum ether, butyl acetate and the like, or a mixed solution of an emulsifier and the oil phase solvent, wherein when the emulsifier is included, the mass concentration of the emulsifier in the oil phase is 1-6 wt%, and the emulsifier is not specifically limited, such as cellulose acetate butyrate, cellulose acetate, polyoxyethylene castor oil, sorbitan ester, polyoxyethylene sorbitan ester and the like.

In one embodiment, the weight ratio of the oil phase to the aqueous phase is greater than 1, preferably (5 to 12): 1, is not particularly limited.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a reaction condition of sodium hyaluronate cross-linked microspheres containing levorotatory polylactic acid, and the microspheres all use raw materials with high biocompatibility and biodegradability, and have high thermal stability and biocompatibility.

(2) The levorotatory polylactic acid is coated in the hyaluronic acid microspheres, so that the secondary activity of cells can be stimulated and the collagen of a body can be stimulated to increase after injection to achieve the effects of resisting aging and preventing aging, and the levorotatory polylactic acid can be used in a plurality of fields of cosmetics, medicines, cosmetology and the like, such as cosmetics, tissue filling and repairing, biological scaffolds, ophthalmology, medicine slow release, targeted drug delivery and the like, and has wide application prospects.

(3) According to the invention, after hydrogel is obtained by activating and crosslinking in a system containing hyaluronic acid and hydrophilic macromolecules such as chitosan as main raw materials, powder obtained by treating levorotatory polylactic acid with a hydrophilic modifier is added into an oil-water mixed system, and the levorotatory polylactic acid powder can be coated inside microspheres by a polymerization mode of reversed-phase suspension polymerization to obtain the hyaluronic acid micron-sized microspherical hydrogel, so that the preparation is simple.

(4) The composite microspheres prepared by the method are round and full, have uniform particle size, and the levorotatory polylactic acid powder is dispersed in the microspheres to form a polylactic acid-hyaluronic acid two-phase system without influencing the compression elasticity of the microspheres.

Drawings

FIG. 1 is an SEM image of microspheres provided in example 4.

Fig. 2 is a partially enlarged view of fig. 1.

FIG. 3 is an SEM image of microspheres provided in example 5.

Fig. 4 is a partially enlarged view of fig. 3.

FIG. 5 is an SEM image of microspheres provided in example 6.

Fig. 6 is a partially enlarged view of fig. 5.

Detailed Description

Examples

Embodiments 1 to 3 provide a levorotatory polylactic acid powder and a preparation method thereof

Example 1

The present embodiment provides a levorotatory polylactic acid powder and a method for preparing the same, including:

dissolving hyaluronic acid into 2mol/L NaCl solution to prepare a hyaluronic acid aqueous solution with the concentration of 10 wt%, adjusting the pH value to 6, and carrying out ice bath for later use.

Weighing EDC (0.007 mol per gram of hyaluronic acid) and dissolving in a small amount of deionized water, slowly adding into the solution of the first step in an ice bath, and uniformly stirring. NHS (0.007 mol per gram of hyaluronic acid) is weighed and dissolved in a small amount of deionized water, the solution is slowly added into the solution of the first step, after the mixture is uniformly stirred, the preservative film is sealed and transferred to a refrigerated cabinet with the temperature of 4 ℃ for activation overnight, and the solution of the second step is obtained.

Dissolving chitosan in 1 wt% acetic acid water solution to prepare 3 wt% chitosan water solution, and mixing the solution and the chitosan water solution uniformly to obtain solution. The amount of chitosan was 5 wt% based on the weight of the solid hyaluronic acid.

Adding the solution of the third step into the levorotatory polylactic acid powder, wherein the dosage of the levorotatory polylactic acid is 50 wt% of the levorotatory polylactic acid powder and the solution of the third step, standing, solidifying into a gel shape, drying at 70 ℃, drying, and grinding into powder, thus obtaining the levorotatory polylactic acid powder.

Example 2

The present embodiment provides a levorotatory polylactic acid powder and a method for preparing the same, including:

dissolving hyaluronic acid into 2mol/L NaCl solution to prepare a hyaluronic acid aqueous solution with the concentration of 10 wt%, adjusting the pH value to 5, and carrying out ice bath for later use.

Weighing EDC (0.005 mol per gram of hyaluronic acid) and dissolving in a small amount of deionized water, slowly adding into the solution of the first step in an ice bath, and uniformly stirring. Adding 0.005mol of NHS per gram of hyaluronic acid into a small amount of deionized water, slowly adding into the solution I, uniformly stirring, sealing by a preservative film, transferring into a refrigerated cabinet at 4 ℃ for activation overnight, and obtaining the solution II.

Dissolving chitosan in 1 wt% acetic acid water solution to prepare 3 wt% chitosan water solution, and mixing the solution and the chitosan water solution uniformly to obtain solution. The amount of chitosan was 5 wt% based on the weight of the solid hyaluronic acid.

Adding the solution of the third step into the levorotatory polylactic acid powder, wherein the dosage of the levorotatory polylactic acid is 50 wt% of the levorotatory polylactic acid powder and the solution of the third step, standing, solidifying into a gel shape, drying at 70 ℃, drying, and grinding into powder, thus obtaining the levorotatory polylactic acid powder.

Example 3

The present embodiment provides a levorotatory polylactic acid powder and a method for preparing the same, including:

dissolving hyaluronic acid into 2mol/L NaCl solution to prepare a hyaluronic acid aqueous solution with the concentration of 8 wt%, adjusting the pH value to 6, and carrying out ice bath for later use.

Weighing EDC (0.007 mol per gram of hyaluronic acid) and dissolving in a small amount of deionized water, slowly adding into the solution of the first step in an ice bath, and uniformly stirring. Adding 0.007mol of NHS into each gram of hyaluronic acid, dissolving the mixture in a small amount of deionized water, slowly adding the mixture into the solution I, uniformly stirring, sealing the preservative film, transferring the preservative film to a refrigerated cabinet at 4 ℃ for activation overnight, and obtaining the solution II.

Dissolving chitosan in 1 wt% acetic acid water solution to prepare 3 wt% chitosan water solution, and mixing the solution and the chitosan water solution uniformly to obtain solution. The amount of chitosan was 5 wt% based on the weight of the solid hyaluronic acid.

Adding the solution of the third step into the levorotatory polylactic acid powder, wherein the dosage of the levorotatory polylactic acid is 60 wt% of the levorotatory polylactic acid powder and the solution of the third step, standing, solidifying into a gel shape, drying at 70 ℃, drying, and grinding into powder, thus obtaining the levorotatory polylactic acid powder.

Example 4 to 6 provide a composite microsphere and a method for preparing the same

Example 4

The embodiment provides a composite microsphere and a preparation method thereof, and the preparation method comprises the following steps:

dissolving hyaluronic acid in 2mol/L NaCl, stirring uniformly until the concentration of hyaluronic acid is 8 wt%, adjusting the pH value to about 4, weighing EDC (0.005 mol per gram of hyaluronic acid) and dissolving in a small amount of deionized water, slowly adding into hyaluronic acid water solution in ice bath, and stirring uniformly. Weighing N HS (0.005 mol per gram of hyaluronic acid), dissolving in a small amount of deionized water, slowly adding into hyaluronic acid aqueous solution, uniformly stirring, sealing by a preservative film, transferring to a refrigerated cabinet at 4 ℃ for activation overnight; dissolving chitosan by using 1 wt% of acetic acid, mixing and stirring activated hyaluronic acid and chitosan (5 wt% of hyaluronic acid solid) uniformly to obtain a water phase, adding pre-ground powder containing L-polylactic acid, namely the powder in the embodiment 1, wherein the powder in the embodiment 1 is 20 wt% of the weight of the water phase, taking butyl acetate, adding cellulose acetate (with the concentration of 7 wt%) to form a reaction oil phase, mixing the water phase and the oil phase uniformly, stirring the oil-water mixed reaction system at 50 ℃ continuously for reaction for 7 hours, filtering the reaction mixture after the reaction is finished, collecting microspheres, washing the microspheres by using butyl acetate and ethanol sequentially, and drying in vacuum to obtain the embolism microspheres.

Example 5

The embodiment provides a composite microsphere and a preparation method thereof, and the preparation method comprises the following steps:

dissolving hyaluronic acid in 2mol/L NaCl, stirring uniformly until the concentration of hyaluronic acid is 8 wt%, adjusting the pH value to about 6, weighing EDC (0.005 mol per gram of hyaluronic acid) and dissolving in a small amount of deionized water, slowly adding into hyaluronic acid water solution in ice bath, and stirring uniformly. Weighing N HS (0.001 mol per gram of hyaluronic acid), dissolving in a small amount of deionized water, slowly adding into a hyaluronic acid aqueous solution, uniformly stirring, sealing by a preservative film, transferring to a refrigerated cabinet at 4 ℃ for activation overnight; dissolving chitosan by using 1 wt% of acetic acid, mixing and stirring activated hyaluronic acid and chitosan (the dosage is 5 wt% of hyaluronic acid solid dosage) uniformly to obtain a water phase, adding pre-ground powder containing L-polylactic acid, the example 2, the powder dosage is 25 wt% of the weight of the water phase, taking butyl acetate, adding cellulose acetate (the concentration is 7 wt%) to form a reaction oil phase, after uniformly mixing the water phase and the oil phase, continuously stirring the oil-water mixed reaction system at 70 ℃ for reaction for 7 hours, filtering the reaction mixture after the reaction is finished, collecting microspheres, washing with butyl acetate and ethanol sequentially, and drying in vacuum to obtain the embolism microspheres.

Example 6

The embodiment provides a composite microsphere and a preparation method thereof, and the preparation method comprises the following steps:

dissolving hyaluronic acid in 2mol/L NaCl, stirring uniformly until the concentration of hyaluronic acid is 10 wt%, adjusting the pH value to about 4, weighing EDC (0.005 mol per gram of hyaluronic acid) and dissolving in a small amount of deionized water, slowly adding into hyaluronic acid water solution in ice bath, and stirring uniformly. Weighing N HS (0.005 mol per gram of hyaluronic acid), dissolving in a small amount of deionized water, slowly adding into hyaluronic acid aqueous solution, uniformly stirring, sealing by a preservative film, transferring to a refrigerated cabinet at 4 ℃ for activation overnight; dissolving chitosan by using 1 wt% of acetic acid, mixing and stirring activated hyaluronic acid and chitosan (the dosage is 7 wt% of hyaluronic acid solid dosage) uniformly to obtain a water phase, adding pre-ground powder containing L-polylactic acid, the example 3, the powder dosage is 20 wt% of the weight of the water phase, taking butyl acetate, adding cellulose acetate (the concentration is 7 wt%) to form a reaction oil phase, after uniformly mixing the water phase and the oil phase, continuously stirring and reacting the oil-water mixed reaction system at 50 ℃ for 5 hours, filtering the reaction mixture after the reaction is finished, collecting microspheres, washing with butyl acetate and ethanol sequentially, and drying in vacuum to obtain the embolism microspheres. The compression elasticity of the microspheres prepared under the above conditions was 45 wt%.

Evaluation of Performance

1. Compression elasticity: the microspheres provided in the examples were subjected to a compression elasticity test, test apparatus: a physical property analyzer; setting parameters: compression mode, compressive induction force 10g, induction mode: automatically, the lowering speed of the compression bar is 1mm/s, the duration is 10 seconds, the displacement of the downward moving compression head is measured, the compression elasticity of the microspheres is calculated according to the displacement/microsphere diameter of 100%, and the compression elasticity is generally qualified when 40% or more, and the results are shown in table 1.

Table 1 performance characterization test

Examples	Elasticity under compression
		4	55％
5	60％
		6	45％

2. Microspherical shape: SEM observation is carried out on the microspheres provided in the examples 4 to 6, and the microspheres are respectively enlarged as shown in figures 1, 3 and 5, and as shown in figures 2, 4 and 6, the microspheres provided in the examples 4 to 6 are found to be round and full, have uniform particle size, comprise polylactic acid powder inside (the substance like bubbles inside the microspheres is polylactic acid powder), and are mutually dispersed and not aggregated.

3. Biological safety: the microspheres provided by the embodiments 4-6 are subjected to muscle implantation inflammatory reaction, and the inflammatory reaction is found to be 0 grade, which shows that the microspheres provided by the invention have good biocompatibility and safety. The PVA microspheres on the market at present have grade 1 muscle implantation inflammatory response.

According to the test results, the hyaluronic acid composite microspheres containing the L-polylactic acid have good particle size uniformity and compression elasticity, form a micron-sized gel microsphere system with L-polylactic acid coated by the microspheres, and can be used in the fields of various medicines, cosmetics, medicines, cosmetology and the like.