CN114983945B

CN114983945B - Microsphere loaded with ammonium glycyrrhetate and medical application thereof

Info

Publication number: CN114983945B
Application number: CN202210519400.8A
Authority: CN
Inventors: 黄晓颖; 金佳佳; 陈钧; 施可庆; 姚丹
Original assignee: First Affiliated Hospital of Wenzhou Medical University
Current assignee: First Affiliated Hospital of Wenzhou Medical University
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2024-03-26
Anticipated expiration: 2042-05-12
Also published as: CN114983945A

Abstract

The invention relates to an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases, wherein the microspheres comprise the following components in percentage by mass based on the total weight of the microspheres: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; and (2) a surfactant: 0.1% -5%; stabilizing agent: 30% -60%. The microsphere prepared by the invention can be administered by aerosol inhalation, and can avoid the blocking of a respiratory system in the respiratory process, directly enter and stay in the lung, and can more quickly and directly exert the pharmacodynamic action of ammonium glycyrrhetate compared with the traditional intravenous administration or oral administration, thereby effectively reducing the injury degree of lung tissues caused by acute lung injury.

Description

Microsphere loaded with ammonium glycyrrhetate and medical application thereof

Technical Field

The invention relates to the field of medicines, in particular to application of ammonium glycyrrhetate-loaded microspheres in preparation of a medicine for treating acute lung injury diseases.

Background

Acute lung injury is a syndrome which is caused by various reasons, is based on diffuse lung cell injury, has characteristic pathological changes of lung tissue structure, has pathological characteristics of pulmonary edema and pulmonary micro-atelectasis, rapidly influences gas exchange function and has clinical characteristics and increased lung inflammation and permeability. The pathological features of acute lung injury are alveolar capillary endothelial cell and alveolar epithelial cell injury, manifested as extensive pulmonary edema and tiny atelectasis. Its pathophysiological changes are mainly increased intra-pulmonary flow and decreased lung compliance. It clinically manifests as hypoxia, respiratory rate and diffuse infiltration of the two lungs in the X-ray chest film.

The cause of the acute lung injury is complex, the death rate is high, and severe infection, shock, wound, smoking, drug poisoning, radioactive radiation, high oxygen and the like can lead to the acute lung injury, but the pathological mechanism of the acute lung injury is not completely elucidated. Although lung protective ventilation has been promoted to some extent in recent years, the mortality rate of acute lung injury has been controlled, but the mortality rate is as high as 30% -40%. At present, no specific medicine aiming at acute lung injury exists clinically.

The traditional Chinese medicine is one of the most important components of the traditional Chinese medicine, and the immunoregulation function of the traditional Chinese medicine is widely applied to clinical treatment. Ammonium glycyrrhizate (Monoammonium glycyrrhizinate, MAG), also known as monoammonium glycyrrhizate, is a bioactive plant component isolated from Glycyrrhiza uralensis Fisch. Ammonium glycyrrhetate can be converted into 18 beta-glycyrrhetinic acid, and the 18 beta-glycyrrhetinic acid has various pharmacological effects of anti-inflammation, anti-allergy, anti-gastric ulcer, anti-hepatitis and the like. Ammonium glycyrrhetate has also been found to reduce cytokine production and increase mortality in LPS-induced endotoxin shock in mice, and has also been reported to exert anti-inflammatory effects by modulating TNF- α/IL-10 balance.

Although a large number of documents report that ammonium glycyrrhizate has anti-inflammatory and antiallergic effects, because of strong immunosuppressive effects of ammonium glycyrrhizate, adverse reactions can be caused by long-term massive use, and clinical use of the ammonium glycyrrhizate is limited. Particularly for the treatment of acute lung injury, the traditional modes of intravenous administration, oral administration and the like are difficult to reach the lesion site rapidly, so that development of a new pharmaceutical preparation and an improved administration mode are needed to reduce the toxic and side effects.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases.

Ammonium glycyrrhizate is widely present in various plants, including certain traditional Chinese medicines, vegetable fruits and the like, and pure ammonium glycyrrhizate can be obtained after optimizing an extraction process, and many published documents report how to extract and purify ammonium glycyrrhizate from plants. Ammonium glycyrrhizate raw materials with higher purity are already available in the market. Ammonium glycyrrhetate (Monoammonium Glycyrrhizinate) has strong affinity to sterol metabolizing enzyme in liver, thereby preventing inactivation of cortisol and aldosterone, and has obvious corticosteroid-like effects, such as anti-inflammatory effect, antiallergic effect, protective film structure and the like, without obvious corticosteroid-like side effects. However, the preparation of ammonium glycyrrhetate has the challenges of low solubility and low bioavailability, and the prior art is to increase the solubility by solubilization or encapsulation, and the content of active substances is low. At present, various methods for preparing the compound ammonium glycyrrhizate preparation are disclosed at home and abroad. However, the compound ammonium glycyrrhetate preparation prepared by the method has the problems of large and uneven particles, so that the solubility of the compound ammonium glycyrrhetate preparation is poor when the compound ammonium glycyrrhetate preparation is prepared into injection, and the compound ammonium glycyrrhetate preparation is unfavorable for drug absorption.

The inventors of the present application have unexpectedly found through many years of research that ammonium glycyrrhizate has a therapeutic effect on Lipopolysaccharide (LPS) -induced Acute Lung Injury (ALI) in mice, and elucidated possible mechanisms of action thereof. Ammonium glycyrrhetate pretreatment reduces LPS-induced lung histopathological damage, reduces lung wet/dry weight ratio and protein concentration in bronchoalveolar lavage fluid (BALF). Meanwhile, ammonium glycyrrhetate reduces the number of inflammatory cells in the lung and inhibits IL-1 beta in TNF-alpha and BALF. In addition, it was also demonstrated that ammonium glycyrrhetate inhibited LPS-induced activation of NF- κB signaling pathways in the lung. This study showed that the therapeutic mechanism of ammonium glycyrrhizate for Acute Lung Injury (ALI) in mice may be due to inhibition of NF- κb signaling pathway. However, it is difficult for conventional administration means such as intravenous injection, oral administration, etc. to allow the drug to rapidly reach the site of lung tissue injury caused by acute lung injury and exert pharmacological effects.

Microencapsulation technology (microencapsulation) is a technology for preparing a drug library Microcapsule (Microcapsule) by wrapping solid or liquid drugs (capsule cores) by using natural or synthetic polymer materials (capsule materials) as capsule wall shells. If the drug is dissolved and/or dispersed in a matrix of polymeric material, the tiny spherical entities forming a matrix (matrix) are called microspheres (microspheres). If the medicine is dissolved and/or dispersed in the polymer matrix, the microsphere is called microsphere, the microsphere with the diameter smaller than 1-5 microns can avoid the blocking of respiratory system in the process of breathing, directly enter and stay in the lung (the particles with the particle diameter larger than 5 microns are blocked in nasal mucosa and bronchus cilia and are discharged out of the body, the particles with the particle diameter smaller than 0.3 micron are lighter and can be discharged out of the body along with breathing).

According to the principle, the inventor prepares a microsphere loaded with ammonium glycyrrhetate and applies the microsphere to treat diseases related to acute lung injury.

Therefore, in a first aspect, the present invention provides an application of an ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases, wherein the microsphere comprises the following components in percentage by mass based on the total weight of the microsphere: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; and (2) a surfactant: 0.1% -5%; stabilizing agent: 30% -60%.

In another preferred embodiment, the PLGA content of the microsphere according to the present invention is between 35% and 60% by mass, more preferably between 40% and 50% by mass, based on the total weight of the microsphere. In the above PLGA microspheres, the PLGA is a polylactic Acid-glycolic Acid copolymer (PLGA) formed by randomly polymerizing two monomers, i.e., lactic Acid (LA) and Glycolic Acid (GA), and is a degradable functional polymer organic compound. By adjusting the proportion, relative molecular mass, viscosity and other physical and chemical properties of 2 monomers of PLGA, the degradation rate of PLGA in vivo can be influenced, and further important quality parameters such as the encapsulation rate, drug loading rate, drug release period and the like of the sustained and controlled release injection are influenced, so that the purpose of continuously and long-acting release of the loaded drug in a controllable manner is finally realized. Two monomers L in PLGA used in the invention: the ratio of G may be 50:50. 65: 35. 75:25 or 85:15, preferably 50:50. The molecular weight of PLGA used may be 1000-100000, preferably 2000-50000, more preferably 5000-20000.

In another preferred embodiment, the ammonium glycyrrhizate content of the microsphere is 2% -10%, more preferably 4% -7%.

In another preferred embodiment, the microsphere according to the present invention, wherein the surfactant is selected from one or any combination of tween-80, tween-20, glyceryl monostearate, polyvinyl alcohol, polyglyceryl fatty acid ester, span80, span60, sodium dodecyl sulfonate, more preferably tween 80. In another preferred embodiment, the surfactant content of the microsphere is 0.5% -4%, more preferably 1% -3%. The role of the surfactant during the microsphere preparation is to emulsify and solubilize.

In another preferred embodiment, the microsphere according to the present invention, wherein the stabilizer is selected from one or any combination of mannitol, polyvinyl alcohol, glycerol, propylene glycol, ethylene glycol, more preferably propylene glycol. In another preferred embodiment, the stabilizer is present in the microsphere in an amount of 35% to 55%, more preferably 40% to 50%. The stabilizer has the functions of delaying the hydrolysis of the medicine and increasing the stability of the medicine, preventing unnecessary medicine precipitation and stabilizing the microsphere in the microsphere preparation process.

In another preferred embodiment, the microspheres according to the invention further comprise moisture in an amount of 0.1% to 3%, more preferably 0.5% to 2%.

In another preferred embodiment, the microspheres of the invention have a particle size in the range of 1 to 10 microns, more preferably 1 to 5 microns.

In another preferred embodiment, the invention provides an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases, wherein the microspheres comprise the following components in percentage by mass based on the total weight of the microspheres: 35% -60% of PLGA; ammonium glycyrrhizate: 2% -10%; and (2) a surfactant: 0.5% -4%; stabilizing agent: 35% -55%; the particle size of the microsphere ranges from 1 to 10 microns;

wherein the surfactant is one or any combination of Tween-80, tween-20, glyceryl monostearate, polyvinyl alcohol, polyglyceryl fatty acid ester, span80, span60 and sodium dodecyl sulfonate;

wherein the stabilizer is selected from one or any combination of mannitol, polyvinyl alcohol, glycerol, propylene glycol and ethylene glycol.

In another preferred embodiment, the invention provides an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases, wherein the microspheres comprise the following components in percentage by mass based on the total weight of the microspheres: 35% -60% of PLGA; ammonium glycyrrhizate: 2% -10%; tween 80:0.5% -4%; propylene glycol: 35% -55%; the microsphere particle size is 1-5 microns.

In another preferred embodiment, the invention provides an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases, wherein the microspheres comprise the following components in percentage by mass based on the total weight of the microspheres: 40% -50% of PLGA; ammonium glycyrrhizate: 4% -7%; tween 80:1% -3%; propylene glycol: 40% -50%; 0.1% -3% of water; the microsphere particle size is 1-5 microns.

In another preferred embodiment, the invention provides the use of an ammonium glycyrrhizinate-loaded microsphere for the preparation of a medicament for treating acute lung injury diseases, wherein the ammonium glycyrrhizinate-loaded microsphere can be used for treating acute lung injury caused by bacterial infection, novel coronavirus, shock, smoking, trauma, toxicosis, inhalation of irritant gas, radiation, high oxygen and low oxygen.

Further, the bacterial infection includes, but is not limited to, acute lung injury caused by bacterial lipopolysaccharide, and also includes acute lung injury caused by lipopolysaccharide secreted by the infected bacteria after the human or animal is infected with the bacteria.

Further, the inhalation stimulating gas is selected from the group consisting of phosgene, diphosgene, triphosgene, chlorine, nitrogen oxides, formaldehyde, dimethyl sulfate, hydrogen chloride, hydrogen bromide, hydrogen fluoride, ammonia, ozone, and sulfur dioxide.

In another preferred embodiment, the present invention provides the use of an ammonium glycyrrhizinate-loaded microsphere, preferably having a particle size in the range of 1-10 μm, more preferably 1-5 μm, for the manufacture of a medicament for the treatment of acute lung injury diseases, said ammonium glycyrrhizinate-loaded microsphere being administered by aerosol inhalation. When the aerodynamic diameter of the microsphere is 1-5 mu m, the microsphere can avoid the blocking of a respiratory system in the respiratory process, directly enter and stay in the lung, better plays the pharmacodynamic action of ammonium glycyrrhetate, and can reduce the degree of lung tissue injury caused by acute lung injury.

In another preferred embodiment, the invention provides the use of the ammonium glycyrrhetate-loaded microspheres in the preparation of a medicament for treating acute lung injury diseases, wherein the ammonium glycyrrhetate-loaded microspheres can reduce the increase degree of the lung dry weight to wet weight ratio.

In another preferred embodiment, the invention provides the use of an ammonium glycyrrhizinate-loaded microsphere for the preparation of a medicament for treating acute lung injury diseases, said ammonium glycyrrhizinate-loaded microsphere being capable of reducing the level of inflammatory factors of alveolar lavage fluid, said inflammatory factors being TNF-alpha and IL-1 beta.

The second aspect of the invention provides an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases, wherein the microspheres comprise the following components in percentage by mass based on the total weight of the microspheres: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; and (2) a surfactant: 0.1% -5%; stabilizing agent: 30% -60%; wherein, the microsphere loaded with ammonium glycyrrhizate is prepared by an electrostatic spraying method, and comprises the following steps:

(1) Fully dissolving PLGA, ammonium glycyrrhizate, a surfactant and a stabilizer in an organic solvent, and performing ultrasonic emulsification to form a uniform mixed solution;

(2) Placing the mixed solution under electrostatic spraying equipment for electrostatic spraying, and breaking the sprayed charged fog drops under the action of an electric field to form monodisperse emulsion drops;

(3) And collecting the monodisperse emulsion droplets in a receiving device provided with a collecting liquid, collecting PLGA microspheres after the organic solvent volatilizes, and drying to obtain the microspheres coated with ammonium glycyrrhizate.

In another preferred embodiment, in step (1) of the method, PLGA, surfactant and stabilizer are as defined above.

In another preferred embodiment, in step (1) of the process, the organic solvent is one or more of dichloromethane, chloroform, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, chloroform, trifluoroethanol, hexafluoroisopropanol or acetone, preferably dichloromethane or ethyl acetate, most preferably dichloromethane.

In another preferred embodiment, in step (1) of the method, PLGA, ammonium glycyrrhizate, surfactant and stabilizer are fully dissolved in methylene chloride and are phacoemulsified for 5-60 minutes to form a uniform mixed solution, more preferably for 20-40 minutes to form a uniform mixed solution.

In another preferred embodiment, in step (1) of the method, PLGA, ammonium glycyrrhizate, tween 80 and propylene glycol are fully dissolved in methylene chloride and are phacoemulsified for 5 to 60 minutes to form a uniform mixed solution, more preferably for 20 to 40 minutes.

In another preferred embodiment, in the method step (2), the electrostatic spraying device comprises a direct current high voltage power supply, a microfluidic drive pump, a syringe, and a stainless steel needle. By applying a high potential to the stainless steel needle and controlling the microfluidic pump to cause the mixed solution in the syringe to flow at a constant rate, the solvent is reduced to micron-sized droplets, and a sufficient electric field between the liquid and the counter electrode can cause the accumulation of surface charges on the liquid. The curved surface becomes unstable and the meniscus forms a cone called taylor cone. The charged droplets are separated from the cone and fly toward the counter electrode. Due to the combined action of the viscous force and interfacial tension, the fluid is stretched and eventually breaks down to form monodisperse emulsion droplets. In another preferred embodiment, the monodisperse emulsion droplets and the resulting ammonium glycyrrhetate-entrapped PLGA microspheres are adjustable in particle size by varying the flow rate or applied electric field force. In a preferred embodiment, the microspheres are formed in a uniform size and high degree of formation at a voltage of between 7 and 14KV and a flow rate of between 0.3 and 1.5ml/h, and the particle size of the microspheres can be controlled in the range of between 1 and 10 microns. When the flow rate is 0.3-0.7ml/h, the particle size of the microspheres can be controlled to be 1-5 microns.

In another preferred embodiment, in step (3) of the method, the collection liquid is water, ethanol or a mixed solution thereof, preferably 60% -80% (V/V) ethanol solution. To prevent aggregation of the microspheres, tween-80 is preferably added to the ethanol solution in an amount of 0.01% -0.05% (V/V) to better maintain the stability of the resulting PLGA microspheres. Further, the receiving device in the step (3) may be placed in a fume hood, and the PLGA microspheres encapsulating ammonium glycyrrhizate are formed by diffusion and volatilization of the organic solvent in the air. In another preferred embodiment, a heating magnetic stirrer is placed at the bottom of the receiving device, a magnetic stirrer is used, the evaporation of the organic solvent is accelerated by stirring, and the PLGA microspheres are collected and dried, so that the PLGA microspheres coated with ammonium glycyrrhetate are obtained.

In another preferred embodiment, the PLGA microspheres prepared according to the above method comprise the following components in percentage by mass based on the total weight of the microspheres: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; tween 80:0.1% -5%; propylene glycol: 30% -60%; the microsphere particle size is 1-5 microns.

In another preferred embodiment, the invention provides an application of ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases, wherein the microsphere comprises the following components in percentage by mass based on the total weight of the microsphere: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; tween 80:0.1% -5%; propylene glycol: 30% -60%; the particle size of the microsphere ranges from 1 to 10 microns; wherein, the microsphere loaded with ammonium glycyrrhizate is prepared by an electrostatic spraying method, and comprises the following steps:

(1) Fully dissolving PLGA, ammonium glycyrrhizate, tween 80 and propylene glycol stabilizer in dichloromethane, and performing ultrasonic emulsification for 5-60 minutes to form a uniform mixed solution;

(2) Placing the mixed solution under electrostatic spraying equipment for electrostatic spraying, wherein the electrostatic spraying equipment comprises a direct-current high-voltage power supply, a microfluidic driving pump, an injector and a stainless steel needle, controlling the voltage of the stainless steel needle to be 7-14KV, controlling the microfluidic pump to enable the mixed solution in the injector to flow at the speed of 0.3-1.5ml/h, and breaking the sprayed charged fog drops under the action of an electric field to form monodisperse emulsion drops;

(3) Collecting the monodisperse emulsion droplets in a receiving device provided with a collecting liquid, wherein the collecting liquid is 60% -80% (V/V) ethanol solution, and adding 0.01% -0.05% (V/V) Tween-80; and after the dichloromethane volatilizes, collecting PLGA microspheres and drying to obtain the PLGA microspheres coated with ammonium glycyrrhizate.

The invention adopts an electrostatic spraying method to prepare PLGA microspheres coated with ammonium glycyrrhetate, and uses the prepared microspheres for treating diseases related to acute lung injury, and has the advantages that:

1) Compared with the traditional complex preparation method of the drug microsphere, the preparation method of the invention is prepared by an electrostatic spraying method, the size of the microsphere is further reduced by volatilizing an organic solvent, the particle size of the microsphere produced by the method is uniform, the average particle size is 1-10 mu m, more preferably can be controlled to be 1-5 mu m, the preparation process is simple and easy to implement, and the reaction condition requirement is low;

2) According to the invention, the injector and the stainless steel needle are adopted for preparing the liquid drops, the channel is simple, the construction steps are few, the complex machining process is not needed, the process is simple, the liquid drop size of the monodisperse emulsion and the corresponding size of the solidified polymer microsphere can be adjusted through the flow rate or the voltage, and the operation is convenient;

3) The PLGA microsphere coated with ammonium glycyrrhetate prepared by the invention has smooth scanning result, uniform particle size, high encapsulation efficiency up to 62% -75%, drug loading rate of 5.3% -6.8%, release time of 5 days, mild slow release and low burst release rate. The preparation method is simple and easy to implement, and the reaction condition requirement is low.

4) The microsphere prepared by the method can be used for loading hydrophilic drugs well, and compared with the traditional microsphere, the loaded microsphere has higher encapsulation rate and drug loading rate, and the method can be used for greatly improving the drug application efficiency and saving manpower and material resources.

(5) The microsphere prepared by the invention can be administered by aerosol inhalation, and can avoid the blocking of a respiratory system in the respiratory process, directly enter and stay in the lung, and can more quickly and directly exert the pharmacodynamic action of ammonium glycyrrhetate compared with the traditional intravenous administration or oral administration mode, thereby reducing the injury degree of lung tissues caused by acute lung injury.

In summary, the invention provides an application of ammonium glycyrrhetate-loaded microspheres in preparing a medicament for treating acute lung injury diseases. The technical scheme of the invention is summarized as follows:

1. the application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases comprises the following components in percentage by mass: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; and (2) a surfactant: 0.1% -5%; stabilizing agent: 30% -60%.

2. The use according to claim 1, characterized in that: the PLGA content in the microsphere accounts for 35-60% of the total weight of the microsphere.

3. The use according to claim 2, characterized in that: the PLGA content in the microsphere accounts for 40-50% of the total weight of the microsphere.

4. The use according to claim 1, characterized in that: PLGA two monomers L used in the microsphere: the ratio of G is 50: 50. 65: 35. 75:25 or 85:15.

5. the use according to claim 1, characterized in that: the molecular weight of PLGA used in the microsphere is 1000-100000, 2000-50000 or 5000-20000.

6. The use according to claim 1, characterized in that: the ammonium glycyrrhizate content of the microsphere for the use is 2% -10%, more preferably 4% -7%.

7. The use according to claim 1, characterized in that: the surfactant in the microsphere is one or any combination of Tween-80, tween-20, glyceryl monostearate, polyvinyl alcohol, polyglyceryl fatty acid ester, span80, span60 and sodium dodecyl sulfonate.

8. The use according to claim 7, characterized in that: the surfactant in the microsphere is Tween 80.

9. The use according to claim 1, characterized in that: the content of the surfactant in the microsphere is 0.5% -4%.

10. Use according to claim 9, characterized in that: the content of the surfactant in the microsphere is 1% -3%.

11. The use according to claim 1, characterized in that: the stabilizer in the microsphere is selected from one or any combination of mannitol, polyvinyl alcohol, glycerol, propylene glycol and ethylene glycol.

12. Use according to claim 11, characterized in that: the stabilizer in the microsphere is propylene glycol.

13. The use according to claim 1, characterized in that: the content of the stabilizer in the microsphere is 35% -55%.

14. Use according to claim 13, characterized in that: the content of the stabilizer in the microsphere is 40% -50%.

15. Use according to any one of claims 1-14, characterized in that: the microsphere further comprises water, and the content of the water is 0.1% -3%.

16. The use according to claim 15, characterized in that: the content of moisture in the microsphere is 0.5% -2%.

17. Use according to any one of claims 1-14, characterized in that: the particle size of the microsphere ranges from 1 to 10 microns.

18. The use according to claim 17, characterized in that: the particle size of the microsphere ranges from 1 to 5 microns.

19. The application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases comprises the following components in percentage by mass: 35% -60% of PLGA; ammonium glycyrrhizate: 2% -10%; and (2) a surfactant: 0.5% -4%; stabilizing agent: 35% -55%; the particle size of the microsphere ranges from 1 to 10 microns;

20. The application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases comprises the following components in percentage by mass: 35% -60% of PLGA; ammonium glycyrrhizate: 2% -10%; tween 80:0.5% -4%; propylene glycol: 35% -55%; the microsphere particle size is 1-5 microns.

21. The application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases comprises the following components in percentage by mass: 40% -50% of PLGA; ammonium glycyrrhizate: 4% -7%; tween 80:1% -3%; propylene glycol: 40% -50%; 0.1% -3% of water; the microsphere particle size is 1-5 microns.

22. Use according to any one of claims 1-21, characterized in that: the ammonium glycyrrhetate-loaded microsphere can be used for treating acute lung injury caused by bacterial infection, new coronavirus, shock, smoking, trauma, poison poisoning, inhalation of irritant gas, radiation, high oxygen and hypoxia.

23. The use according to claim 22, characterized in that: the bacterial infection includes but is not limited to acute lung injury caused by bacterial lipopolysaccharide, and also includes acute lung injury caused by lipopolysaccharide secreted by the infected bacteria after the human or animal is infected by the bacteria.

24. The use according to claim 22, characterized in that: the inhalation stimulating gas is selected from phosgene, diphosgene, triphosgene, chlorine, nitrogen oxides, formaldehyde, dimethyl sulfate, hydrogen chloride, hydrogen bromide, hydrogen fluoride, ammonia, ozone and sulfur dioxide.

25. Use according to any one of claims 1-21, characterized in that: the microsphere loaded with ammonium glycyrrhetate is administrated by atomization inhalation, and the particle size range of the microsphere is preferably 1-10 mu m.

26. The use according to claim 25, characterized in that: the microsphere loaded with ammonium glycyrrhetate is administrated by atomization inhalation, and the particle size range of the microsphere is preferably 1-5 mu m.

27. Use according to any one of claims 1-21, characterized in that: the ammonium glycyrrhetate-loaded microspheres can reduce the increase degree of the lung dry weight ratio.

28. Use according to any one of claims 1-21, characterized in that: the ammonium glycyrrhetate-loaded microspheres can reduce the level of inflammatory factors of alveolar lavage fluid, wherein the inflammatory factors are TNF-alpha and IL-1 beta.

29. The application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases comprises the following components in percentage by mass: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; and (2) a surfactant: 0.1% -5%; stabilizing agent: 30% -60%; wherein, the microsphere loaded with ammonium glycyrrhizate is prepared by an electrostatic spraying method, and comprises the following steps:

30. The use according to claim 29, characterized in that: in the method step (1), the organic solvent is one or more of dichloromethane, chloroform, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, chloroform, trifluoroethanol, hexafluoroisopropanol or acetone.

31. The use according to claim 30, characterized in that: the organic solvent is dichloromethane or ethyl acetate.

32. The use according to claim 31, characterized in that: the organic solvent is dichloromethane.

33. The use according to claim 29, characterized in that: in the step (1) of the method, PLGA, ammonium glycyrrhizate, a surfactant and a stabilizer are fully dissolved in methylene dichloride, and are subjected to ultrasonic emulsification for 5-60 minutes to form a uniform mixed solution.

34. The use according to claim 33, characterized in that: in the step (1) of the method, PLGA, ammonium glycyrrhizate, a surfactant and a stabilizer are fully dissolved in methylene dichloride, and are subjected to ultrasonic emulsification for 20-40 minutes to form a uniform mixed solution.

35. The use according to claim 33, characterized in that: in the step (1) of the method, PLGA, ammonium glycyrrhizate, tween 80 and propylene glycol are fully dissolved in methylene dichloride, and are subjected to ultrasonic emulsification for 5-60 minutes to form a uniform mixed solution.

36. The use according to claim 35, characterized in that: in the step (1) of the method, PLGA, ammonium glycyrrhizate, tween 80 and propylene glycol are fully dissolved in methylene dichloride, and the mixture is subjected to ultrasonic emulsification for 20-40 minutes to form a uniform mixed solution.

37. The use according to claim 29, characterized in that: in the method step (2), the electrostatic spraying equipment comprises a direct-current high-voltage power supply, a microfluidic driving pump, an injector and a stainless steel needle; by applying a high potential to the stainless steel needle and controlling the microfluidic pump to cause the mixed solution in the syringe to flow at a constant rate, the fluid is stretched and eventually broken to form monodisperse emulsion droplets due to the combined action of the viscous force and interfacial tension.

38. The use according to claim 37, characterized in that: in the step (2) of the method, the particle size of the monodisperse emulsion droplets and the particle size of the PLGA microspheres which are formed after the monodisperse emulsion droplets and are coated with ammonium glycyrrhetate are adjustable by changing the flow rate or the applied electric field force.

39. The use according to claim 38, characterized in that: in the step (2) of the method, when the voltage is between 7 and 14KV and the flow rate is between 0.3 and 1.5ml/h, the particle size range of the microspheres can be controlled between 1 and 10 microns.

40. The use according to claim 39, characterized in that: in the method step (2), the particle size of the microspheres can be controlled to be 1-5 microns when the flow rate is 0.3-0.7 ml/h.

41. The use according to claim 29, characterized in that: in the step (3) of the method, the collecting liquid is water, ethanol or a mixed solution thereof.

42. The use according to claim 41, characterized in that: in the method step (3), the collected liquid is 60% -80% (V/V) ethanol solution.

43. The use according to claim 42, characterized in that: in the method step (3), the collected liquid is 60% -80% (V/V) ethanol solution, and 0.01% -0.05% (V/V) Tween-80 is added.

44. The use according to claim 29, characterized in that: the receiving device in the step (3) of the method can be placed in a fume hood, and the PLGA microspheres coated with ammonium glycyrrhizate are formed through the diffusion and volatilization of an organic solvent in the air.

45. The use according to any one of claims 29-44, characterized in that: the heating magnetic stirrer can be arranged at the bottom of the receiving device, the magnetic stirrer is used for accelerating the volatilization of the organic solvent through stirring, and the PLGA microspheres are collected and dried to obtain the PLGA microspheres coated with ammonium glycyrrhizate.

46. The use according to any one of claims 29-44, characterized in that: the PLGA microspheres prepared according to the method have a particle size range of 1-5 microns.

47. The application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases comprises the following components in percentage by mass: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; tween 80:0.1% -5%; propylene glycol: 30% -60%; the particle size of the microsphere ranges from 1 to 10 microns; wherein, the microsphere loaded with ammonium glycyrrhizate is prepared by an electrostatic spraying method, and comprises the following steps:

Drawings

FIG. 1 is a schematic diagram of an electrostatic spraying apparatus for preparing PLGA microspheres coated with ammonium glycyrrhizate according to the present invention.

FIG. 2 is a real-time generated image of PLGA microspheres coated with ammonium glycyrrhizate prepared by electrostatic spraying. Wherein fig. 2 (a) is a real-time generated image of a droplet observed by a high-speed camera, the taylor cone forming a jet and generating a droplet; FIGS. 2 (b) - (c) are electron microscopy images of corresponding PLGA polymer microspheres; FIG. 2 (d) shows the particle size distribution of the corresponding PLGA polymer microspheres.

FIG. 3 is a graph showing the relationship between flow rate and microsphere particle size.

FIG. 4 is a graph showing the drug release profile of PLGA microspheres coated with ammonium glycyrrhetate prepared by electrostatic spraying.

FIG. 5 fluorescence intensity plot of FITC over time in microspheres.

FIG. 6 alveolar lavage fluid viable cell count.

FIG. 7 lung dry to wet weight ratio.

FIG. 8 lung tissue HE staining.

FIG. 9 alveolar inflammatory factor (TNF- α) detection. Compared with the model group, the expression level of TNF-alpha in mice treated by the atomized inhaled ammonium glycyrrhizate microsphere is obviously reduced

FIG. 10 alveolar inflammatory factor (IL-1. Beta.) detection. Compared with the model group, the IL-1 beta expression level of the mice in the atomized inhalation ammonium glycyrrhizate microsphere treatment group is obviously reduced.

Detailed Description

In order to better understand the technical solutions of the present invention for those skilled in the art, the following embodiments of the present invention are described in further detail with reference to the accompanying drawings. The experimental methods used in the examples below, unless otherwise indicated, are conventional methods, and the reagents, methods and apparatus used, unless otherwise indicated, are conventional in the art.

EXAMPLE 1 preparation of PLGA microspheres coated with ammonium Glycyrrhizate

An electrostatic spraying device for preparing PLGA microspheres coated with ammonium glycyrrhizate is provided, and is shown in figure 1. The preparation process comprises the following steps:

(1) Preparing spray mixed solution

Hydrophilic ammonium glycyrrhetate is selected as a loading drug of 0.03g, a hydrophobic polymer is selected from polylactic acid-glycolic acid copolymer (molecular weight 8000, 50:50) of 0.3g, a cosurfactant is selected from Tween-80. Mu.l, and a stabilizer is selected from propylene glycol 330. Mu.l. Firstly, 0.3g of polylactic acid-glycolic acid copolymer (molecular weight 8000) is dissolved in 4500 mu l of volatile organic solvent dichloromethane to be fully dissolved, then 200 mu l of stabilizer propylene glycol is added, then 8 mu l of cosurfactant Tween-80 is added, then 150 mu l of water is added to be ultrasonically emulsified for 30min, then 0.03g of hydrophilic ammonium glycyrrhetate is added as a loading drug, and then ultrasonic emulsification is carried out again for 15-30min until the stabilizer propylene glycol is fully dissolved.

(2) Preparing monodisperse emulsion droplets:

the spray liquid is pumped into 1ml syringes and placed on peristaltic pumps respectively, the syringes are fitted with 1mm stainless steel needles, the flow rates are set, and the peristaltic pumps are started to operate. Due to the combined action of the viscous force and interfacial tension, the fluid is stretched and eventually breaks down to form monodisperse emulsion droplets. Fig. 2 (a) is a real-time generated image of a droplet observed by a high-speed camera, the taylor cone being a jet and generating a droplet.

When the voltage is between 7 KV and 14KV and the flow rate is between 0.3ml/h and 1.5ml/h, the formed microspheres are uniform in size and high in forming degree. When the flow rates are consistent, the microspheres change along with the voltage, and the size change is weak; the control voltage is 9KV, other factors are unchanged, the flow rates are respectively set to be 0.3mL/h, 0.5mL/h, 0.7mL/h, 0.9mL/h, 1.1mL/h, 1.3mL/h and 1.5mL/h, and as can be seen from FIG. 3, the particle size of monodisperse emulsion droplets increases with the increase of the flow rate within the range of 0.3-1.5 mL/h; the above results indicate that by varying the flow rate, the regulation of the microsphere particle size can be achieved.

(3) Preparation of PLGA microspheres

Preparing a collection liquid: the collection solution is 70% ethanol solution to prevent aggregation of microsphere, and 0.01% tween-80 is added into ethanol solution to maintain stability of the produced microsphere. The collection liquid was placed in a 100ml petri dish as a receiving device and placed in a fume hood. The bottom of the culture dish is provided with a heating magnetic stirrer, a tetrafluoro magnetic stirrer is used for controlling the rotation speed of the collection liquid to be between 200rpm, and PLGA microspheres coated with ammonium glycyrrhizate are formed through the diffusion and volatilization of methylene dichloride in the air. Collecting PLGA microspheres and drying to obtain PLGA microspheres coated with ammonium glycyrrhetate, wherein fig. 2 (b) - (c) are electron microscope pictures of corresponding PLGA polymer microspheres; the electron micrograph shows that the microspheres loaded with ammonium glycyrrhetate are regular spheres, the size of the microspheres is uniform, and the particle size distribution range is 1-10 microns, as shown in figure 2 d. In particular, when the voltage is between 7 KV and 14KV and the flow rate is between 0.3ml/h and 1.5ml/h, the formed microspheres are uniform in size and high in forming degree, and the particle size range of the microspheres can be controlled to be between 1 and 10 microns. In actual preparation, when the flow rate is controlled to be 0.3-0.7ml/h, the particle size of the microsphere can be controlled to be 1-5 microns, and the content of each component of the formed PLGA microsphere accounts for the total weight of the microsphere, and the mass percentages are respectively as follows: PLGA 44.4%; 4.44% of ammonium glycyrrhizate; tween 80.36%; 49.8% of propylene glycol.

Example 2 determination of drug release Curve of ammonium Glycyrrhizate-entrapped PLGA microspheres

The microspheres prepared in example 1, which were uniform in size, were placed in 5ml of pure water, absorbance of the drug in water was measured at intervals using an ultraviolet spectrophotometer, the amount of the released drug was counted, and a drug release profile was made, as shown in fig. 4. Compared with the common ammonium glycyrrhizate preparation, the PLGA microsphere has slower drug release rate, can prolong the time in vivo, ensures that the drug always maintains the blood concentration required by effective treatment in vivo, avoids frequent and multiple dosing, improves the compliance of patients and improves the curative effect.

Example 3

Fluorescein Isothiocyanate (FITC) is coated in the microsphere, and the fluorescence intensity in the FITC in the microsphere is photographed at intervals by using an inverted fluorescence microscope, so that the overall fluorescence condition of the microsphere can be conveniently observed, and the microsphere with larger particle size (the flow rate in the preparation process is 3 ml/h) is prepared. As shown in fig. 5, by observing the change in fluorescence intensity over time to simulate the release of ammonium glycyrrhetate, it can be seen that the change in fluorescence intensity over time was decaying, but remained for a while.

Example 4 Experimental grouping and administration

15 male C57BL/6J mice were purchased from Zhejiang Violet Lihua laboratory animal technologies Co., ltd., license number: SYXK (Zhe) 2021-0017; animal feeding is performed at the university of wenzhou medical science laboratory animal center. Animal experiment facilities continue to maintain barrier environmental standards. Control range of main environmental index: the temperature of the room temperature is 20-26 ℃ and the daily temperature difference is less than or equal to 4 ℃. The relative humidity is 40-70 ℃. Minimum ventilation times 15 times/hour, light illumination: dark=12 h, light=12 h. Animals are fed into standard boxes, and 5 animals are fed into each box, wherein the feeding space accords with the regulation of the minimum space required by experimental animals in national standard GB 14125-2010 of the people's republic of China. All animals are fed and managed by trained personnel, and the diet activity of the animals is kept free in the whole feeding process.

Ammonium glycyrrhetate (Monoammonium Glycyrrhizinate) was purchased from aladine, and PLGA microspheres coated with ammonium glycyrrhetate were prepared by the method of reference example 1.

Experimental animals were randomly divided into 3 groups of 5 animals each according to body weight, and the groups were as follows: (1) and (3) atomizing and inhaling an ammonium glycyrrhizate microsphere group in a control group (2) model group. Four hours after LPS (10 mg/kg) was injected into the trachea, corresponding treatments were given. Taking lung tissue after 24 hours, and storing in a refrigerator at-80 ℃.

Example 5 alveolar lavage assay

After killing mice, the right lung was clamped with forceps and immediately air-filled into PBS to lavage the first leaf of the left lung 4 times, 200 μl of each time was blown 3 times, sucked into the EP tube, centrifuged at 3000 rpm, the supernatant was sucked, and the lavage fluid of alveoli after lavage was stored in a-80℃low temperature refrigerator.

EXAMPLE 6 viable cell count detection

Phosphate buffer (PBS, pH 7.4) was injected into the first leaf of left lung through the trachea for lavage 4 times, 200. Mu.l of each time was blown 3 times, the sucked BALF was sucked into the centrifuge tube, centrifuged at 3000r/min for 10min, the supernatant was removed, after sucking the supernatant, 1ml of erythrocyte lysate was added to the pellet for resuspension, centrifuged at 3000r/min for 10min, the supernatant was removed, 50. Mu.l of PBS was added to the pellet for resuspension of cells, and cell counting was performed with a fully automatic cell counter.

As shown in fig. 6, the total number of viable cells in the alveolar lavage fluid was significantly increased in the model group compared to the control group, indicating that the model was successfully established. Compared with the model group, the atomized inhaled ammonium glycyrrhetate microsphere can obviously reduce the total number of living cells. The experimental result shows that the atomized inhalation of the ammonium glycyrrhetate microspheres can significantly improve the alveolar inflammatory reaction.

Example 7 detection of the Dry-to-wet ratio

After the mice were sacrificed, the right lung first leaf and left lung second leaf were removed without lavage, and the lung wet weight was weighed, dried in an oven for 48 hours, and the dry weight was measured.

As shown in fig. 7, the dry-wet weight ratio of mice in the model group was increased as compared with the control group, and the dry-wet weight ratio of the atomized inhalation ammonium glycyrrhizate microsphere group was decreased after the treatment.

EXAMPLE 8 HE staining of Lung tissue

And (3) placing the third leaf of the left lung into a proper amount of paraformaldehyde fixing solution, making paraffin sections, HE dyeing, and photographing the dyed pieces under a microscope.

As shown in fig. 8, the model group mice showed more inflammatory cell exudation in alveoli, with occasional alveolar edema, compared to the control group; compared with the mice in the model group, the atomized inhalation microsphere group can significantly improve the above changes, and the results show that the atomized inhalation ammonium glycyrrhizinate microsphere can significantly improve the acute lung injury induced by LPS.

Example 9 alveolar inflammatory factor detection

-taking out alveolar lavage fluid at a low temperature of 80 ℃ by using a refrigerator, and detecting the expression levels of inflammatory factors TNF-alpha and IL-1 beta of the alveolar lavage fluid according to the instruction of a kit.

As shown in fig. 9 and 10, the expression levels of TNF- α and IL-1β in the alveolar lavage fluid of the model group mice were significantly increased compared to the control group, indicating that the inflammatory injury of alveoli was promoted after LPS stimulation. Compared with a model group, the expression level of TNF-alpha and IL-1 beta of mice in the atomized inhalation ammonium glycyrrhizate microsphere treatment group is obviously reduced; the results show that TNF-alpha and IL-1 beta are sensitive indicators for detecting alveolar lavage fluid of atomized inhaled ammonium glycyrrhizate microspheres.

Claims

1. The application of the ammonium glycyrrhetate-loaded microsphere in preparing a medicament for treating acute lung injury diseases caused by bacterial infection comprises the following components in percentage by mass: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; and (2) a surfactant: 0.1% -5%; stabilizing agent: 30% -60%, the grain diameter of the microsphere is 1-10 μm;

wherein, the microsphere is prepared by an electrostatic spraying method comprising the following steps:

(3) Collecting the monodisperse emulsion droplets in a receiving device provided with a collecting liquid, collecting PLGA microspheres after the organic solvent volatilizes, and drying to obtain microspheres coated with ammonium glycyrrhizate;

wherein the surfactant in the microsphere is selected from one or any combination of Tween-80, tween-20, glyceryl monostearate, polyvinyl alcohol, polyglyceryl fatty acid ester, span80, span60 and sodium dodecyl sulfonate;

wherein the stabilizer in the microsphere is selected from one or any combination of mannitol, polyvinyl alcohol, glycerol, propylene glycol and ethylene glycol.

2. Use according to claim 1, characterized in that: the PLGA content in the microsphere accounts for 35-60% of the total weight of the microsphere.

3. Use according to claim 2, characterized in that: the PLGA content in the microsphere accounts for 40-50% of the total weight of the microsphere.

4. Use according to claim 1, characterized in that: PLGA two monomers L used in the microsphere: the ratio of G is 50: 50. 65: 35. 75:25 or 85:15.

5. Use according to claim 1, characterized in that: the molecular weight of PLGA used in the microsphere is 1000-100000, 2000-50000 or 5000-20000.

6. Use according to claim 1, characterized in that: the content of ammonium glycyrrhizate in the microsphere for the application is 2-10%.

7. Use according to claim 6, characterized in that: the ammonium glycyrrhizate content in the microsphere for the application is 4-7%.

8. Use according to claim 7, characterized in that: the surfactant in the microsphere is Tween 80.

9. Use according to claim 1, characterized in that: the content of the surfactant in the microsphere is 0.5% -4%.

11. Use according to claim 1, characterized in that: the stabilizer in the microsphere is propylene glycol.

12. Use according to claim 1, characterized in that: the content of the stabilizer in the microsphere is 35% -55%.

13. Use according to claim 12, characterized in that: the content of the stabilizer in the microsphere is 40% -50%.

14. Use according to claim 1, characterized in that: the microsphere further comprises water, and the content of the water is 0.1% -3%.

15. Use according to claim 14, characterized in that: the content of moisture in the microsphere is 0.5% -2%.

16. Use according to claim 1, characterized in that: the particle size of the microsphere ranges from 1 to 5 microns.

17. The use according to claim 1, wherein the microsphere comprises the following components in percentage by mass based on the total weight of the microsphere: 35% -60% of PLGA; ammonium glycyrrhizate: 2% -10%; and (2) a surfactant: 0.5% -4%; stabilizing agent: 35% -55%; the microsphere particle size is 1-10 microns.

18. The use according to claim 1, wherein the microsphere comprises the following components in percentage by mass based on the total weight of the microsphere: 35% -60% of PLGA; ammonium glycyrrhizate: 2% -10%; tween 80:0.5% -4%; propylene glycol: 35% -55%; the microsphere particle size is 1-5 microns.

19. The use according to claim 1, wherein the microsphere comprises the following components in percentage by mass based on the total weight of the microsphere: 40% -50% of PLGA; ammonium glycyrrhizate: 4% -7%; tween 80:1% -3%; propylene glycol: 40% -50%; 0.1% -3% of water; the microsphere particle size is 1-5 microns.

20. Use according to claim 1, characterized in that: the acute lung injury caused by bacterial infection comprises acute lung injury caused by lipopolysaccharide secreted by infected bacteria after the human or animal is infected by the bacteria.

21. Use according to any one of claims 1-20, characterized in that: the microsphere loaded with ammonium glycyrrhetate is administered by atomization inhalation, and the particle size of the microsphere is 1-10 mu m.

22. Use according to claim 21, characterized in that: the microsphere loaded with ammonium glycyrrhetate is administered by atomization inhalation, and the particle size of the microsphere is 1-5 mu m.

23. Use according to any one of claims 1-20, characterized in that: the ammonium glycyrrhetate-loaded microspheres can reduce the increase degree of the lung dry weight ratio.

24. Use according to any one of claims 1-20, characterized in that: the ammonium glycyrrhetate-loaded microspheres can reduce the level of inflammatory factors of alveolar lavage fluid, wherein the inflammatory factors are TNF-alpha and IL-1 beta.

25. Use according to claim 1, characterized in that: in the preparation method step (1) of the application, the organic solvent is one or more of dichloromethane, chloroform, ethyl acetate, methyl acetate, ethyl propionate, propyl acetate, chloroform, trifluoroethanol, hexafluoroisopropanol or acetone.

26. Use according to claim 25, characterized in that: the organic solvent in the step (1) of the preparation method in the application is dichloromethane or ethyl acetate.

27. Use according to claim 26, characterized in that: the organic solvent in step (1) of the preparation method in the application is methylene dichloride.

28. Use according to claim 1, characterized in that: in the step (1) of the preparation method in the application, PLGA, ammonium glycyrrhizate, a surfactant and a stabilizer are fully dissolved in methylene dichloride, and are subjected to ultrasonic emulsification for 5-60 minutes to form a uniform mixed solution.

29. Use according to claim 28, characterized in that: in the step (1) of the preparation method in the application, PLGA, ammonium glycyrrhizate, a surfactant and a stabilizer are fully dissolved in methylene dichloride, and the mixture is subjected to ultrasonic emulsification for 20-40 minutes to form a uniform mixed solution.

30. Use according to claim 28, characterized in that: in the step (1) of the preparation method in the application, PLGA, ammonium glycyrrhizate, tween 80 and propylene glycol are fully dissolved in methylene dichloride, and are subjected to ultrasonic emulsification for 5-60 minutes to form a uniform mixed solution.

31. Use according to claim 30, characterized in that: in the step (1) of the preparation method in the application, PLGA, ammonium glycyrrhizate, tween 80 and propylene glycol are fully dissolved in methylene dichloride, and the mixture is subjected to ultrasonic emulsification for 20-40 minutes to form a uniform mixed solution.

32. Use according to claim 1, characterized in that: in the preparation method step (2) in the application, the electrostatic spraying equipment comprises a direct-current high-voltage power supply, a microfluidic driving pump, a syringe and a stainless steel needle; by applying a high potential to the stainless steel needle and controlling the microfluidic pump to cause the mixed solution in the syringe to flow at a constant rate, the fluid is stretched and eventually broken to form monodisperse emulsion droplets due to the combined action of the viscous force and interfacial tension.

33. Use according to claim 32, characterized in that: in the step (2) of the preparation method in the application, the particle size of the monodisperse emulsion droplets and the PLGA microspheres which are formed after the monodisperse emulsion droplets and are coated with ammonium glycyrrhetate can be adjusted by changing the flow rate or the applied electric field force.

34. Use according to claim 33, characterized in that: in the step (2) of the preparation method in the application, when the voltage is between 7 and 14KV and the flow rate is between 0.3 and 1.5ml/h, the particle size of the microspheres is controlled to be between 1 and 10 microns.

35. Use according to claim 34, characterized in that: in the step (2) of the production method for the use, the particle diameter of the microspheres is controlled to be 1 to 5 μm when the flow rate is 0.3 to 0.7 ml/h.

36. Use according to claim 1, characterized in that: in the step (3) of the preparation method in the application, the collecting liquid is water, ethanol or a mixed solution thereof.

37. Use according to claim 36, characterized in that: in the preparation method step (3) of the application, the collection liquid is 60% -80% (V/V) ethanol solution.

38. Use according to claim 37, characterized in that: in the preparation method step (3) of the application, the collection liquid is 60-80% (V/V) ethanol solution, and 0.01-0.05% (V/V) Tween-80 is added.

39. Use according to claim 1, characterized in that: the receiving device in the step (3) of the preparation method in the application can be placed in a fume hood, and the PLGA microspheres coated with ammonium glycyrrhizate are formed by the diffusion and volatilization of an organic solvent in the air.

40. The use according to any one of claims 25-39, wherein: the heating magnetic stirrer can be arranged at the bottom of the receiving device, the magnetic stirrer is used for accelerating the volatilization of the organic solvent through stirring, and the PLGA microspheres are collected and dried to obtain the PLGA microspheres coated with ammonium glycyrrhizate.

41. The use according to any one of claims 25-39, wherein: the PLGA microspheres prepared according to the method have a particle size range of 1-5 microns.

42. The use according to claim 1, wherein the microsphere comprises the following components in percentage by mass based on the total weight of the microsphere: 30% -65% of PLGA; ammonium glycyrrhizate: 1% -15%; tween 80:0.1% -5%; propylene glycol: 30% -60%; the particle size of the microsphere ranges from 1 to 10 microns; wherein, the microsphere loaded with ammonium glycyrrhizate is prepared by an electrostatic spraying method, and comprises the following steps:

(2) Placing the mixed solution under electrostatic spraying equipment for electrostatic spraying, wherein the electrostatic spraying equipment comprises a direct-current high-voltage power supply, a microfluidic driving pump, an injector and a stainless steel needle, controlling the voltage of the stainless steel needle to be 7-14V, controlling the microfluidic pump to enable the mixed solution in the injector to flow at the speed of 0.3-1.5ml/h, and breaking the sprayed charged fog drops under the action of an electric field to form monodisperse emulsion drops;

(3) Collecting the droplets of the monodisperse emulsion in a receiving device provided with a collection liquid, wherein

Collecting 60% -80% (V/V) ethanol solution, and adding 0.01% -0.05% (V/V) Tween-80; and after the dichloromethane volatilizes, collecting PLGA microspheres and drying to obtain the PLGA microspheres coated with ammonium glycyrrhizate.