阅读说明：本技术 一种聚左旋乳酸微粒、可注射软组织填充剂的制备方法 (Preparation method of polylactic acid microparticles and injectable soft tissue filler ) 是由 薛姚 栾婧 于 2019-12-27 设计创作，主要内容包括：本发明提供了一种聚左旋乳酸微粒的制备方法以及可注射软组织填充剂的制备方法。聚左旋乳酸微粒的制备方法包括：步骤一、将聚左旋乳酸溶解在良溶剂中,获得聚左旋乳酸溶液；步骤二、将所述聚左旋乳酸溶液加入反应釜中,在搅拌的条件下施加反溶剂,以从反应溶液中析出聚左旋乳酸微粒；步骤三、过滤悬浮有聚左旋乳酸微粒的所述反应溶液,并提纯干燥以获得聚左旋乳酸微粒。本发明通过无皂良溶剂-反溶剂沉淀法制备出聚左旋乳酸微粒,在微粒的制备过程中不需要加入任何乳化剂和分散剂,工艺过程操作简单,生产成本低廉,生产效率高,适合于工业放大生产。(The invention provides a preparation method of poly-L-lactic acid particles and a preparation method of injectable soft tissue filler. The preparation method of the poly-L-lactic acid particles comprises the following steps: dissolving poly-L-lactic acid in a good solvent to obtain a poly-L-lactic acid solution; adding the poly-L-lactic acid solution into a reaction kettle, and applying an anti-solvent under the stirring condition to separate out poly-L-lactic acid particles from the reaction solution; and step three, filtering the reaction solution in which the poly-L-lactic acid particles are suspended, and purifying and drying to obtain the poly-L-lactic acid particles. The invention prepares the poly-L-lactic acid particles by a soap-free good solvent-anti-solvent precipitation method, does not need to add any emulsifier and dispersant in the preparation process of the particles, has simple technological process operation, low production cost and high production efficiency, and is suitable for industrial scale-up production.)

1. A method for preparing poly-L-lactic acid microparticles, which is characterized by comprising the following steps:

dissolving poly-L-lactic acid in a good solvent to obtain a poly-L-lactic acid solution;

adding the poly-L-lactic acid solution into a reaction kettle, and applying an anti-solvent under the stirring condition to separate out poly-L-lactic acid particles from the reaction solution;

and step three, filtering the reaction solution in which the poly-L-lactic acid particles are suspended, and purifying and drying to obtain the poly-L-lactic acid particles.

2. The method according to claim 1, wherein in the second step, the volume ratio of the anti-solvent to the good solvent is 2:1 to 10: 1.

3. The method according to claim 1, wherein the anti-solvent is applied at a rate of 50 to 200mL/min in the second step.

4. The preparation method according to claim 1, wherein the good solvent is one or a mixture of two of chloroform and dichloromethane; and/or

The anti-solvent is one or the mixture of two of ethanol and methanol.

5. The method according to any one of claims 1 to 3, wherein the poly (L-lactic acid) has a molecular weight of 3 to 200 KDa.

6. A method for preparing an injectable soft tissue filler, comprising the steps of:

adding an auxiliary material to the polylactic acid fine particles obtained by the production method according to any one of claims 1 to 5 to obtain a mixed material;

adding ultrapure water into the mixed material, and uniformly stirring and mixing to obtain a mixed solution;

subpackaging the mixed solution;

freeze-drying the mixed solution after subpackage, packaging and sterilizing to obtain the injectable soft tissue filler.

7. The method according to claim 6, wherein the polylactic acid fine particles are contained in the soft tissue filler in an amount of 30 to 60% by mass.

8. The preparation method of claim 6, wherein the auxiliary material is one or more of sodium hyaluronate, sodium carboxymethylcellulose, collagen and mannitol.

9. The preparation method according to claim 6, wherein in the step of freeze-drying, packaging and sterilizing the subpackaged mixed materials, the sterilization method is irradiation sterilization, and the irradiation dose is 8-15 kGy.

10. The method for preparing the sodium hyaluronate according to any one of claims 6 to 9, wherein the molecular weight of the sodium hyaluronate is 500-1000KDa, and the mass percentage content of the sodium hyaluronate in the injectable soft tissue filler is 10-40%; and/or

The molecular weight of the sodium carboxymethylcellulose is 500-1000KDa, and the mass percentage of the sodium carboxymethylcellulose in the injectable soft tissue filler is 10-40%; and/or

The molecular weight of the collagen is 200-500KDa, and the mass percentage content of the collagen in the injectable soft tissue filler is 10-40%; and/or

The mass percentage of the mannitol is 10-30%.

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of poly-L-lactic acid particles and a preparation method of injectable soft tissue filler.

Background

With the development of the medical beauty industry, injection filling materials are more and more concerned, and the injection filling materials can achieve the beauty effects of lifting eyebrows, filling temples, enlarging cheeks, improving statute lines, folding skin at corners of mouths, filling chin, tightening contour lines of the chin and the like. At present, sodium hyaluronate (hyaluronic acid), which is the most commonly used injection filling material in the beauty industry, has a good filling effect, but because the degradation speed of hyaluronic acid is high, a patient needs to repeatedly inject the hyaluronic acid every 6 months, and the filling effect of the hyaluronic acid injection is not obvious for severe facial depressions and deep wrinkles.

In recent years, polylactic acid has been widely used in the fields of biomedicine, tissue engineering, and cosmetic medicine because of its excellent biocompatibility and biodegradability. In the field of tissue engineering, as a tissue repair material, poly-L-lactic acid has high tissue compatibility, can be slowly degraded into lactic acid and carbon dioxide in biological tissues, and can be completely metabolized by human bodies without any residue. In the field of beauty treatment, the poly-L-lactic acid as a filling material can effectively fill skin depressions, stimulate collagen and fibroblast proliferation in the skin, smooth skin wrinkles and realize skin contour remodeling. Meanwhile, the retention period of the poly-L-lactic acid in the body tissue is about two years generally, and the defects of rapid in-vivo degradation and short retention time of the traditional hyaluronic acid filling material are overcome.

When the poly-L-lactic acid is used as a medical cosmetic filling material, the poly-L-lactic acid is generally required to be prepared into micro-particles or micro-spheres with micron-sized sizes, and then the micro-particles or the micro-spheres are injected into deep tissues of skin through an injector to achieve the effect of filling and beautifying. In the prior art, there are various methods for preparing polylactic acid microspheres, such as preparing polylactic acid microspheres by an ultrasonic emulsification method, preparing polylactic acid microspheres by a supercritical anti-solvent method, and preparing polylactic acid microspheres by taking a porous membrane as an emulsion membrane. Although the polylactic acid microspheres can be prepared by the method, the method has application limitations, such as excessively complex preparation process, high cost and difficult amplification production, and meanwhile, a large amount of emulsifier or dispersant is usually required to be introduced in the preparation process, so that the product purification difficulty is high, the biocompatibility is poor, and the application of the polylactic acid microspheres in the fields of biological medicine and cosmetic medical treatment is limited.

Disclosure of Invention

The invention aims to solve the technical problems of high product purification difficulty and poor biocompatibility caused by the fact that a large amount of emulsifying agent or dispersing agent is usually required to be introduced when polylactic acid particles are prepared in the prior art.

A further purpose of the invention is to solve the technical problems that the process is too complex and the cost is higher when the poly-L-lactic acid particles are prepared in the prior art, and the scale-up production is not facilitated.

The invention also aims to obtain the injectable soft tissue filler which has excellent water phase dispersibility, and the suspension obtained after dispersion is uniform, fine and good in needle penetration.

Particularly, the invention provides a preparation method of poly-L-lactic acid particles, which comprises the following steps:

dissolving poly-L-lactic acid in a good solvent to obtain a poly-L-lactic acid solution;

adding the poly-L-lactic acid solution into a reaction kettle, and applying an anti-solvent under the stirring condition to separate out poly-L-lactic acid particles from the reaction solution;

and step three, filtering the reaction solution in which the poly-L-lactic acid particles are suspended, and purifying and drying to obtain the poly-L-lactic acid particles.

Optionally, in the second step, the volume ratio of the anti-solvent to the good solvent is 2:1-10: 1.

Optionally, in step two, the anti-solvent is applied at a rate of 50-200 mL/min.

Optionally, the good solvent is one or a mixture of two of chloroform and dichloromethane;

optionally, the anti-solvent is one or a mixture of two of ethanol and methanol.

Optionally, the poly-L-lactic acid has a molecular weight of 3-200 KDa.

In particular, the invention also provides a preparation method of the injectable soft tissue filler, which comprises the following steps:

adding auxiliary materials into the poly-L-lactic acid particles obtained by the preparation method to obtain a mixed material;

adding ultrapure water into the mixed material, and uniformly stirring and mixing to obtain a mixed solution;

subpackaging the mixed solution;

freeze-drying the mixed solution after subpackage, packaging and sterilizing to obtain the injectable soft tissue filler.

Optionally, the polylactic acid microparticles account for 30-60% by mass of the soft tissue filler.

Optionally, the auxiliary material is one or more of sodium hyaluronate, sodium carboxymethylcellulose, collagen and mannitol.

Optionally, in the step of freeze-drying, packaging and sterilizing the subpackaged mixed materials, the sterilization method is irradiation sterilization, and the irradiation dose is 8-15 kGy.

Optionally, the molecular weight of the sodium hyaluronate is 500-1000KDa, and the mass percentage content of the sodium hyaluronate in the injectable soft tissue filler is 10-40%;

optionally, the molecular weight of the sodium carboxymethyl cellulose is 500-1000KDa, and the mass percentage of the sodium carboxymethyl cellulose in the injectable soft tissue filler is 10-40%;

optionally, the molecular weight of the collagen is 200-500KDa, and the mass percentage of the collagen in the injectable soft tissue filler is 10-40%;

optionally, the mannitol is 10-30% by mass.

According to the scheme of the embodiment of the invention, the poly-L-lactic acid particles are prepared by a soap-free good solvent-anti-solvent precipitation method, any emulsifier and dispersant are not required to be added in the preparation process of the particles, the technological process is simple to operate, the production cost is low, the production efficiency is high, and the method is suitable for industrial scale-up production.

Furthermore, by controlling the reaction conditions, raw material selection, solvent selection, etc. in the above method, poly (L-lactic acid) fine particles having a small particle diameter (particle diameter of 3 to 80 μm), a narrow distribution, and good stability can be finally obtained.

In addition, the injectable soft tissue filler prepared by the method has the advantages of excellent water phase dispersibility, uniform and fine suspension obtained after dispersion, good needle penetration, long retention period in local tissues, outstanding filling effect, good plasticity, capability of promoting the proliferation of collagen and fiber cells of surrounding tissues, less toxic and side effects and the like when being used as a cosmetic filling material, and is an excellent cosmetic filling material.

The above and other objects, advantages and features of the present invention will become more apparent to those skilled in the art from the following detailed description of specific embodiments thereof, taken in conjunction with the accompanying drawings.

Drawings

Some specific embodiments of the invention will be described in detail hereinafter, by way of illustration and not limitation, with reference to the accompanying drawings. The same reference numbers in the drawings identify the same or similar elements or components. Those skilled in the art will appreciate that the drawings are not necessarily drawn to scale. In the drawings:

FIG. 1 shows a schematic flow diagram of a method of preparing poly-L-lactic acid microparticles according to one embodiment of the present invention;

fig. 2 shows a schematic flow diagram of a method of preparing an injectable soft tissue filler according to one embodiment of the invention;

FIG. 3 shows a scanning electron micrograph of poly (L-lactic acid) microparticles obtained according to a production method of an embodiment of the present invention;

FIG. 4 shows another scanning electron micrograph of poly (L-lactic acid) microparticles obtained according to a production method of an embodiment of the present invention;

FIG. 5 is a graph showing the average aerodynamic particle size of polylactic acid microparticles obtained by the production method according to the example of the present invention;

FIG. 6 shows a scanning electron micrograph of poly (L-lactic acid) microparticles obtained according to a production method of an embodiment of the present invention;

FIG. 7 is another scanning electron micrograph of poly (L-lactic acid) microparticles obtained according to a production method of an embodiment of the present invention;

FIG. 8 is a graph showing the average aerodynamic particle size of poly (L-lactic acid) microparticles obtained by the production method according to the example of the present invention;

FIG. 9 is a graph showing the average aerodynamic particle size of polylactic acid microparticles obtained by the production method according to the example of the present invention;

fig. 10 shows a photograph of an injectable soft tissue filler obtained according to a method of preparing an injectable soft tissue filler according to an embodiment of the invention;

fig. 11 shows a photograph of an injectable soft tissue filler solution according to an embodiment of the invention;

FIG. 12 shows a photograph of an injectable soft tissue filler solution injected through a 26g needle syringe according to an embodiment of the present invention;

figure 13 shows anatomical contrast photographs of injection sites after injection of injectable soft tissue filler solution according to an embodiment of the present invention into rat subcutaneous on days 1, 7, 15 and 30;

fig. 14 shows HE staining photographs of tissue sections of injection sites after injection of injectable soft tissue filler solution according to an embodiment of the present invention subcutaneously into rats on days 1, 7, 15 and 30;

fig. 15 shows Masson stained photographs of tissue sections of injection sites after injection of injectable soft tissue filler solution according to an embodiment of the present invention subcutaneously into rats on days 1, 7, 15 and 30.

Detailed Description

Fig. 1 shows a schematic flow chart of a method for preparing poly-L-lactic acid microparticles according to one embodiment of the present invention. As shown in fig. 1, the preparation method comprises:

dissolving poly-L-lactic acid in a good solvent to obtain a poly-L-lactic acid solution;

adding the poly-L-lactic acid solution into a reaction kettle, and applying an anti-solvent under the stirring condition to separate out poly-L-lactic acid particles from the reaction solution;

and step three, filtering the reaction solution in which the poly-L-lactic acid particles are suspended, and purifying and drying to obtain the poly-L-lactic acid particles.

In the first step, the molecular weight of poly-L-lactic acid can be 3KDa, 10KDa, 20KDa, 50KDa, 100KDa, 150KDa or 200KDa, or any other value of 3-200 KDa. The poly-L-lactic acid with the molecular weight can be directly purchased on the market and also can be prepared by the existing preparation method, so that the poly-L-lactic acid with the molecular weight is selected as a raw material, and the raw material source is wide and convenient. The concentration of poly (L-lactic acid) in the poly (L-lactic acid) solution may be 0.01g/mL, 0.02g/mL, 0.03g/mL, 0.04g/mL, or 0.05g/mL, or any other concentration value from 0.01g/mL to 0.05 g/mL. For a poly-L-lactic acid raw material with a molecular weight, the concentration of the oligomeric L-lactic acid can effectively reduce the agglomeration phenomenon among poly-L-lactic acid particles in the anti-solvent precipitation process, so that the average particle size of the prepared poly-L-lactic acid particles is smaller.

The good solvent may be chloroform or dichloromethane, or a mixed solvent of chloroform and dichloromethane. The poly-L-lactic acid is completely dissolved in the good solvent, and the good solvent is selected as a good foundation for finally preparing and obtaining the poly-L-lactic acid particles meeting the requirements.

In the second step, the anti-solvent may be ethanol or methanol, or a mixed solvent of ethanol and methanol. The volume ratio of the anti-solvent to the good solvent in the first step is 2:1, 3:1, 5:1, 7:1, 8:1, 9: 1 or 10:1, or any other volume ratio of 2-10: 1. The volume ratio of the anti-solvent to the good solvent can only be within the range to effectively separate out the poly-L-lactic acid particles, thereby laying the foundation for subsequently purifying a large amount of poly-L-lactic acid particles.

Wherein, the stirring condition can be that the reaction kettle is provided with magnetic stirring or mechanical stirring, thereby realizing that the anti-solvent is added into the reaction kettle while stirring. When the antisolvent is added, the antisolvent may be added dropwise into the reaction vessel at a constant rate by using a peristaltic pump, and the dropwise addition rate may be 50mL/min, 70mL/min, 90mL/min, 110mL/min, 130mL/min, 150mL/min, 180mL/min, or 200mL/min, or may be any other rate of 50-200 mL/min. Under the same magnetic stirring or mechanical stirring speed, the low anti-solvent dropping speed can prolong the mixing time of the solvent and the anti-solvent, the solvent and the anti-solvent are mixed more uniformly, the concentration of the anti-solvent in the local area of the mixed solution is reduced, and then the precipitation process of the poly-L-lactic acid particles in the solution is more uniform, and the poly-L-lactic acid particles with uniform particle size and narrow distribution are obtained.

In step three, the reaction solution in which the poly (L-lactic acid) microparticles are suspended is poured into a stainless steel screen for filtration. Wherein, the stainless steel screen mesh is 300 meshes, 400 meshes, 500 meshes, 600 meshes, 700 meshes, 800 meshes, 900 meshes or 1000 meshes, and can be any other mesh of 300 meshes and 1000 meshes. The stainless steel screens with different meshes are used for filtering and collecting the poly-L-lactic acid particles with different particle sizes obtained under different reaction conditions.

Wherein the purification step comprises repeatedly washing and filtering the poly (L-lactic acid) microparticles obtained by the filtration to purify the poly (L-lactic acid). The washing solvent used in the washing treatment is ethanol and n-hexane. In the drying step, the purified poly-L-lactic acid particles are dried in vacuum at 30-50 ℃. The average particle diameter of the polylactic acid particles finally obtained is 3-80 μm. The polylactic acid particles finally obtained are in the form of powder.

According to the scheme of the embodiment of the invention, the poly-L-lactic acid particles are prepared by a soap-free good solvent-anti-solvent precipitation method, any emulsifier and dispersant are not required to be added in the preparation process of the particles, the technological process is simple to operate, the production cost is low, the production efficiency is high, and the method is suitable for industrial scale-up production. Namely, the preparation process of the soap-free good solvent-antisolvent precipitation method does not need to use any emulsifier such as polyvinyl alcohol (PVA), methylcellulose and the like, so that the purification process for preparing the poly-L-lactic acid particles is simple and convenient, the emulsifier does not need to be eluted, and the obtained poly-L-lactic acid particles have high raw material purity and no surfactant residue.

The preparation process of the invention does not need to use water phase, so that the separation and drying processes of the prepared poly-L-lactic acid particles are simple and convenient. Compared with water, the organic solvents trichloromethane, dichloromethane, ethanol and methanol used in the soap-free good solvent-anti-solvent precipitation method have small surface tension, so that the organic solvents have good transmittance, high product separation speed and high separation efficiency in the process of sieving and separating products.

Compared with water, the organic solvents trichloromethane, dichloromethane, ethanol and methanol used in the soap-free good solvent-anti-solvent precipitation method have low boiling points and are volatile, so that the drying temperature and the drying time of the poly-L-lactic acid particle product can be greatly reduced, and the poly-L-lactic acid particle product with low solvent residue and high purity can be obtained.

The preparation process of the soap-free good solvent-anti-solvent precipitation method does not need to use a water phase, so that the risk of bacterial contamination of the poly-L-lactic acid particle product in the preparation, purification and drying processes is reduced, because bacteria are easier to breed in the water phase, and the bacteria in the organic solvent can not grow.

Furthermore, by controlling the reaction conditions, raw material selection, solvent selection, etc. in the above method, poly (L-lactic acid) fine particles having a small particle diameter (particle diameter of 3 to 80 μm), a narrow distribution, and good stability can be finally obtained.

Fig. 2 shows a schematic flow diagram of a method of preparing an injectable soft tissue filler according to one embodiment of the present invention. As shown in fig. 2, the preparation method comprises:

s100, adding auxiliary materials into the poly-L-lactic acid particles obtained by the preparation method to obtain a mixed material;

SS200, adding ultrapure water into the mixture, and uniformly stirring and mixing to obtain a mixed solution;

s300, subpackaging the mixed solution;

s400, freeze-drying the split mixed solution, packaging and sterilizing to obtain the injectable soft tissue filler.

In step S100, the content of the poly (L-lactic acid) microparticles in the soft tissue filler may be 30%, 40%, 50%, or 60% by mass, or any other ratio of 30 to 60%. According to the clinical soft tissue filling level requirement, soft tissue fillers of different batches can be prepared by changing the percentage content of the poly-L-lactic acid particles in the filler, and the action time and the lasting effect of the filler in vivo are prolonged.

The auxiliary material can be one or a combination of more of sodium hyaluronate, sodium carboxymethylcellulose, collagen and mannitol. By optimizing the formula of the auxiliary materials, the redissolution time of the filler freeze-dried powder before clinical use is effectively shortened, the dispersity and stability of the poly-L-lactic acid particles in the filler after redissolution are improved, and the uniform, fine and good-needle-penetration poly-L-lactic acid particle suspension is obtained. Meanwhile, after the auxiliary material is injected into organism soft tissues, the auxiliary material has certain effects of nourishing soft tissue cells and repairing tissues, and can achieve a better soft tissue filling effect by matching the auxiliary material with the poly-L-lactic acid.

When the auxiliary material is selected to be sodium hyaluronate, the mass percentage content of the sodium hyaluronate in the injectable soft tissue filler can be 10%, 20%, 30% or 40%, and any other proportion of 10-40% can also be provided. The molecular weight of the sodium hyaluronate is 500KDa, 700KDa, 900KDa or 1000KDa, or any other molecular weight of 500KDa and 1000 KDa. The molecular weight of the sodium hyaluronate is selected within the range, and the sodium hyaluronate can be directly purchased and obtained on the market, so that the sodium hyaluronate with the molecular weight within the range is selected as an auxiliary material, and the auxiliary material is wide and convenient in source.

When the auxiliary material is selected to be sodium carboxymethyl cellulose, the mass percentage content of the sodium carboxymethyl cellulose in the injectable soft tissue filler can be 10%, 20%, 30% or 40%, and any other proportion of the sodium carboxymethyl cellulose in the injectable soft tissue filler can also be 10-40%. The molecular weight of the sodium carboxymethylcellulose can be 500KDa, 700KDa, 900KDa or 1000KDa, and can also be any other molecular weight of 500-1000 KDa. The molecular weight of the sodium carboxymethylcellulose is selected within the range, and the sodium carboxymethylcellulose can be directly purchased and obtained on the market, so that the sodium carboxymethylcellulose with the molecular weight within the range is selected as an auxiliary material, and the auxiliary material is wide and convenient in source.

When the auxiliary material is selected to be collagen, the mass percentage of the collagen in the injectable soft tissue filler can be 10%, 20%, 30% or 40%, and any other proportion of 10-40% can also be used. The molecular weight of the collagen is 200KDa, 300KDa, 400KDa or 500KDa, or any other molecular weight of 200KDa and 500 KDa. The molecular weight of the collagen is selected within the range, and the collagen can be directly purchased and obtained on the market, so that the collagen with the molecular weight within the range is selected as the auxiliary material, and the auxiliary material is wide and convenient in source.

When the auxiliary material is selected to be mannitol, the mass percentage content of the mannitol in the injectable soft tissue filler can be 10%, 20% or 30%, or any other proportion of 10-30%. The mannitol with the molecular weight within the range is selected to be directly purchased and obtained on the market, so that the mannitol with the molecular weight within the range is selected as an auxiliary material, and the auxiliary material is wide and convenient in source.

In step S400, the sterilization method is irradiation sterilization, and the irradiation dose may be 8kGy, 10kGy, 12kGy, 14kGy, or 15kGy, or any other dose from 8 to 15 kGy. The irradiation dose is in the range of 8-15kGy, the poly-L-lactic acid soft tissue filler can be effectively sterilized, and the safety of the filler in clinical use is improved.

According to the scheme of the embodiment of the invention, the injectable soft tissue filler prepared by the method has the advantages of excellent water phase dispersibility, uniform and fine suspension obtained after dispersion, good needle penetration, long retention period in local tissues, outstanding filling effect, good plasticity, capability of promoting the proliferation of collagen and fiber cells of surrounding tissues, less toxic and side effects and the like when being used as a cosmetic filling material, and is an excellent cosmetic filling material.

The following is illustrated in detail by specific examples: