CN110859828B - Slow-release hydrogen sulfide donor microsphere and preparation method and application thereof - Google Patents

Slow-release hydrogen sulfide donor microsphere and preparation method and application thereof Download PDF

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CN110859828B
CN110859828B CN201911171402.7A CN201911171402A CN110859828B CN 110859828 B CN110859828 B CN 110859828B CN 201911171402 A CN201911171402 A CN 201911171402A CN 110859828 B CN110859828 B CN 110859828B
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张绘莉
陈爱政
高祺
张卉
郝浏智
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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    • A—HUMAN NECESSITIES
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    • A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
    • A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P9/12—Antihypertensives

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Abstract

The invention relates to the field of high polymer materials, in particular to a sustained-release hydrogen sulfide donor microsphere and a preparation method and application thereof. The preparation method of the sustained-release hydrogen sulfide donor microsphere provided by the invention comprises the following steps: providing a dispersed phase which is an emulsion comprising an aqueous phase and an oil phase selected from an organic solution comprising donor microsphere material and ACS 14; providing a continuous phase; distributing the dispersed phase in the continuous phase to provide microspheroidal droplets of the dispersed phase; and solidifying, washing and drying the provided microsphere liquid drops. The invention constructs a sustained-release H2The biodegradable high-molecular polymer microspheres prepared from the S drug-loaded porous microspheres have good particle size monodispersity, uniform size and good aerodynamic performance, can be inhaled through respiratory tracts for administration, not only improve the lung targeting property and biological tolerance of the drug, but also greatly enhance the effect of treating pulmonary hypertension by hydrogen sulfide.

Description

Slow-release hydrogen sulfide donor microsphere and preparation method and application thereof
Technical Field
The invention relates to the field of high polymer materials, in particular to a sustained-release hydrogen sulfide donor microsphere and a preparation method and application thereof.
Background
Pulmonary hypertension is a complex disease of the cardio-pulmonary vascular system that is mainly manifested by increased vascular pressure and pulmonary vascular remodeling that severely limits the function of the right ventricle and leads to right heart failure and even death. The currently used therapeutic drugs include endothelin receptor antagonists, prostacyclin and its analogs, phosphatase inhibitors, and the like. The main problems with these drugs are that they usually require a combination and have low targeting,the side effect of the medicine is great. Endogenous hydrogen sulfide gas is a gas medium participating in various physiological activities of human bodies, and plays roles in dilating blood vessels and reducing blood pressure in a cardiovascular system. However, H2S is a transient gaseous molecule and NaHS, the most common donor, is unable to release H2S at a controlled rate, which often results in extremely high local concentrations and causes cytotoxicity. S-aspirin (ACS-14) is a synthetic hydrogen sulfide donor drug that connects aspirin and dithioethione (5- (4-hydroxyphenyl) -3H-1, 2-dithio-3-thione, ADTOH) via ester bonds. ACS-14, however, is poorly water soluble and needs to be dissolved in dimethyl sulfoxide, which increases drug toxicity. In addition, intraperitoneal injection of ACS-14 does not target the pulmonary vasculature and the systemic response of the drug is increased (e.g., decreased systolic blood pressure, liver injury, etc.). These disadvantages of ACS-14 clearly affect the utility and efficacy of the drug in PAH.
The conventional method for successfully playing the function in the lung is to use a high molecular material as a carrier to prepare drug-loaded porous microspheres, and the drug-loaded porous microspheres are delivered to the lung organ. The method for preparing the drug-loaded porous microspheres is an emulsion volatilization method, and the biggest problem of the method is that the prepared porous microspheres have large particle size distribution span and poor particle size uniformity, so that the delivery effect is poor.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a sustained-release hydrogen sulfide donor microsphere, a preparation method and a use thereof, which are used for solving the problems in the prior art.
In order to achieve the above and other related objects, the present invention provides a method for preparing sustained-release hydrogen sulfide donor microspheres, comprising:
a) providing a dispersed phase which is an emulsion comprising an aqueous phase and an oil phase selected from an organic solution comprising donor microsphere material and ACS 14;
b) providing a continuous phase;
c) distributing the dispersed phase provided in step a) into the continuous phase provided in step b) to provide microspheroidal droplets of the dispersed phase;
d) solidifying, washing and drying the microsphere liquid drops provided by the step c).
In some embodiments of the invention, the aqueous phase is selected from an aqueous ammonium bicarbonate solution, and the aqueous phase comprises 4.5-5.5 g of solute per 100ml of solution by volume mass concentration, preferably 4.9-5.1 g of solute per 100ml of solution.
In some embodiments of the present invention, the donor microsphere material is selected from polylactic-co-glycolic acid, which includes the donor microsphere material and ACS14, and the organic solution includes 2.5-3.5 g of the donor microsphere material per 100ml of the solution, preferably, 2.9-3.1 g of the donor microsphere material per 100ml of the solution.
In some embodiments of the present invention, the organic solution comprising the donor microsphere material and ACS14 comprises 1-2 g ACS14 per 100ml solution, preferably 1.4-1.6 g ACS14 per 100ml solution.
In some embodiments of the present invention, the organic solution comprising the donor microsphere material and ACS14 is a solvent selected from haloalkane solvents, preferably from dichloromethane.
In some embodiments of the present invention, the continuous phase is selected from an aqueous solution of polyvinyl alcohol, wherein the aqueous solution of polyvinyl alcohol comprises 4.5 to 5.5g of polyvinyl alcohol per 100ml of solution, preferably, 4.9 to 5.1g of polyvinyl alcohol per 100ml of solution.
In some embodiments of the present invention, the volume ratio of the water phase to the oil phase in the emulsion is 1: 2.5-3.5, preferably 1: 2.9-3.1, and the emulsion is prepared by fully mixing the water phase and the oil phase, preferably by ultrasonication.
In some embodiments of the present invention, the dispersed phase provided in step a) is distributed in the continuous phase provided in step b) by a coaxial microfluidic method.
In some embodiments of the present invention, in the coaxial micro-fluidic control method, the flow rate ratio of the continuous phase to the dispersed phase is 2000-4000: 1, preferably 2700-3300: 1.
In some embodiments of the invention, the slow release hydrogen sulfide donor microspheres are for pulmonary administration.
In some embodiments of the invention, the slow release hydrogen sulfide donor microspheres are inhalants.
The invention also provides the slow-release hydrogen sulfide donor microsphere prepared by the preparation method.
The invention also provides application of the sustained-release hydrogen sulfide donor microsphere in preparation of medicines.
In some embodiments of the invention, the drug is selected from a drug for treating an endogenous H2S-associated disease;
and/or, the drug is a slow release drug;
and/or, the medicament is for pulmonary administration;
and/or, the medicament is an inhalant.
The invention also provides a pharmaceutical composition comprising the slow-release hydrogen sulfide donor microsphere.
Drawings
FIG. 1 shows a schematic diagram of the microfluidic preparation of the sustained-release hydrogen sulfide donor microsphere of the invention.
FIG. 2 is a schematic micrograph showing the microspheres of the slow-release hydrogen sulfide donor prepared in example 1 of the present invention.
FIG. 3 is a schematic micrograph showing the microspheres of the slow-release hydrogen sulfide donor prepared in example 1 of the present invention.
FIG. 4 is a schematic diagram showing the results of biosafety experiments of the sustained-release hydrogen sulfide donor microsphere of the present invention.
FIG. 5 is a schematic diagram showing the lung tissue targeting experimental results of the slow-release hydrogen sulfide donor microsphere of the present invention.
FIG. 6 is a schematic diagram showing the effect of the sustained-release hydrogen sulfide donor microsphere in pulmonary hypertension.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.
The inventor provides the sustained-release hydrogen sulfide donor microsphere and the preparation method and the application thereof through a large amount of practical researches, the sustained-release hydrogen sulfide donor microsphere has better particle size uniformity and good monodispersity, and can slowly release endogenous H2S, and thus can be used as a variety of therapeutics for the treatment of endogenous H2S-related diseases, and the present invention has been completed based on these.
The invention provides a preparation method of a slow-release hydrogen sulfide donor microsphere, which comprises the following steps:
a) providing a dispersed phase which is an emulsion comprising an aqueous phase and an oil phase selected from an organic solution comprising donor microsphere material and ACS 14;
b) providing a continuous phase;
c) distributing the dispersed phase provided in step a) into the continuous phase provided in step b) to provide microspheroidal droplets of the dispersed phase;
d) solidifying, washing and drying the microsphere liquid drops provided by the step c).
The preparation method of the sustained-release hydrogen sulfide donor microsphere provided by the invention can comprise the following steps: providing a dispersed phase that is an emulsion comprising an aqueous phase and an oil phase selected from an organic solution comprising donor microsphere material and ACS 14. The dispersed phase is generally an emulsion, which can be prepared by thoroughly mixing an oil phase and an aqueous phase, and a suitable method of thoroughly mixing an oil phase and an aqueous phase and preparing an emulsion should be known to those skilled in the art, and in a specific embodiment of the present invention, the dispersed phase can be prepared by ultrasonication or the like. In the process of preparing the dispersed phase, the water phase and the oil phase generally need to have a proper ratio, for example, the volume ratio of the used water phase and the used oil phase can be 1: 2.5-3.5, 1: 2.5-2.7, 1: 2.7-2.9, 1: 2.9-3.1, 1: 3.1-3.3, or 1: 3.3-3.5, in a specific embodiment of the present invention, the volume ratio of the used water phase and the used oil phase is 1:3 ± 0.01, and too much volume ratio, i.e. too much oil phase, generally causes too high liquid viscosity to increase shearing difficulty, too large microsphere particle size, too low volume ratio, i.e. too much water phase, generally causes too low concentration to break during the curing process, thereby affecting the yield. The aqueous phase used generally requires solutes of the appropriate type and concentration, for example, the aqueous phase can be selected from aqueous ammonium bicarbonate solutions, which can allow faster porogenic rates and generally no harmful substances remain; for another example, each 100ml of the solution may include 4.5-5.5 g, 4.5-4.7 g, 4.7-4.9 g, 4.9-5.1 g, 5.1-5.3 g, or 5.3-5.5 g of solute by volume mass concentration, and in a specific embodiment of the present invention, each 100ml of the solution may include 5 ± 0.1g of solute, generally speaking, an excessively high concentration may cause an increase in the size of the microspheres during pore-forming process to affect aerodynamic performance, an excessively low concentration may cause poor pore-forming performance, and an excessively small number of pores may affect aerodynamic performance. In the organic solution including the donor microsphere material and ACS14, which is used as the oil phase, the solvent used is generally easy to volatilize at room temperature without leaving organic solvent, and specifically may be a haloalkane solvent or the like, and in one embodiment of the present invention, the solvent used may be dichloromethane or the like. The organic solution used as the oil phase and including the donor microsphere material and ACS14 generally includes the donor microsphere material of a suitable type and concentration, for example, the donor microsphere material may be selected from polylactic acid-glycolic acid, etc., and further, for example, the donor microsphere material may include 2.5-3.5 g, 2.5-2.7 g, 2.7-2.9 g, 2.9-3.1 g, 3.1-3.3 g, 3.3-3.5 g per 100ml of the solution by volume mass concentration, and in one embodiment of the present invention, 3 + -0.1 g of the donor microsphere material may be included per 100ml of the solution, and too high concentration of the donor microsphere material generally results in too large viscosity to form too large microsphere diameter, and too low concentration generally results in easy breakage of liquid droplets during curing. The organic solution used as the oil phase and including the donor microsphere material and ACS14 generally includes ACS14 with a suitable concentration, for example, 1-2 g, 1-1.2 g, 1.2-1.4 g, 1.4-1.6 g, 1.6-1.8 g, and 1.8-2 g ACS14 by volume mass concentration per 100ml of the solution, in one embodiment of the present invention, 1.5 + -0.1 g ACS14 per 100ml of the solution, and an excessively high concentration of ACS14 generally causes excessive loss and waste, and an excessively low concentration generally causes a decrease in therapeutic effect.
The preparation method of the sustained-release hydrogen sulfide donor microsphere provided by the invention can comprise the following steps: providing a continuous phase that is generally not significantly toxic and is capable of protecting the droplets from stabilization prior to curing, which may be specifically an aqueous solution of polyvinyl alcohol or the like. The aqueous solution of polyvinyl alcohol used as the continuous phase generally needs to have a suitable concentration, for example, the concentration by volume and mass per 100ml of the solution may include 4.5-5.5 g, 4.5-4.7 g, 4.7-4.9 g, 4.9-5.1 g, 5.1-5.3 g, or 5.3-5.5 g of polyvinyl alcohol, and in one embodiment of the present invention, 5g ± 0.1g of polyvinyl alcohol may be included per 100ml of the solution, and a high concentration of polyvinyl alcohol generally causes too much adhesion to the surface of the microspheres to be difficult to remove, and a low concentration generally causes the microspheres to be easily aggregated and fused during the curing process.
The preparation method of the sustained-release hydrogen sulfide donor microsphere provided by the invention can comprise the following steps: distributing the dispersed phase provided in step a) in the continuous phase provided in step b) to provide microspheroidal droplets of the dispersed phase. In one embodiment of the present invention, the dispersed phase provided in step a) may be distributed in the continuous phase provided in step b) by a coaxial micro-fluidic method. The coaxial microfluidic method is known to those skilled in the art and is a technique for micro-scale manipulation of droplets in which a dispersed phase and a mobile phase generally have a defined flow direction, and the dispersed phase is introduced into the mobile phase at a flow rate such that microspheroidal droplets of the dispersed phase may be formed. In the coaxial micro-fluidic control method, the flow rate ratio of the continuous phase to the dispersed phase is usually required to be appropriate, for example, in the coaxial micro-fluidic control method, the flow rate ratio of the continuous phase to the dispersed phase may be 2000-4000: 1, 2000-2200: 1, 2200-2400: 1, 2400-2600: 1, 2600-2800: 1, 2800-3000: 1, 3000-3200: 1, 3200-3400: 1, 3400-3600: 1, 3600-3800: 1, or 3800-4000: 1.
In the preparation method of the sustained-release hydrogen sulfide donor microsphere provided by the invention, a person skilled in the art can select a suitable method to treat the liquid drop provided in the step c) to provide the sustained-release hydrogen sulfide donor microsphere. For example, it may include: solidifying, washing and drying the microsphere liquid drops provided by the step c). The solidification generally refers to standing the microsphere liquid drop for a certain time under a proper condition, and the specific time can be 21-27 hours, so that the organic solvent and the pore-forming agent can be volatilized from the liquid drop to form the solid microsphere with pores. The washing generally refers to washing the microspheres in a solid form with a suitable solvent (e.g., distilled water, etc.), so as to remove residual polyvinyl alcohol, etc. in one embodiment of the present invention, the microspheres may be soaked in distilled water and slowly stirred, the microspheres may be washed several times, and the distilled water may be replaced during the washing process. The drying typically removes excess solvent, etc., remaining in the microsphere product to provide slow release hydrogen sulfide donor microspheres, and suitable drying methods will be known to those skilled in the art, e.g., lyophilization, etc.
The second aspect of the present invention provides a sustained-release hydrogen sulfide donor microsphere, which generally comprises a porous microsphere, wherein ACS-14 is loaded on the porous microsphere, and the sustained-release hydrogen sulfide donor microsphere can be prepared by the preparation method of the sustained-release hydrogen sulfide donor microsphere provided in the first aspect of the present invention. The slow-release hydrogen sulfide donor microsphere has good particle size uniformity and monodispersity, the geometric diameter of the microsphere is 16-32 μm, 16-20 μm, 20-24 μm, 24-28 μm or 28-32 μm, so that the microsphere has good aerodynamic performance and can be used for pulmonary administration and/or used as an inhalant. The slow-release hydrogen sulfide donor microsphere is loaded with ACS-14, because the microsphere has an obvious through-hole structure, the aperture of a hole is usually 1-2 μm, 1-1.2 μm, 1.2-1.4 μm, 1.4-1.6 μm, 1.6-1.8 μm or 1.8-2 μm, the ACS-14 can be distributed on the surface and/or inside of the microsphere, and the slow-release hydrogen sulfide donor microsphere can slowly release endogenous H2S, controllable H2The S release rate can greatly improve the biological tolerance and the targeting property of the medicineAre used as various medicines for treating endogenous H2S-related diseases.
In a third aspect, the invention provides the use of the sustained-release hydrogen sulfide donor microspheres provided in the second aspect of the invention in the preparation of a medicament. As mentioned above, the sustained-release hydrogen sulfide donor microsphere provided by the invention can slowly release endogenous H2S, can thus be used for the preparation of a medicament for the treatment of endogenous H2S-related diseases, and is typically a slow-release drug. As a donor microsphere preparation, the microsphere has better particle size uniformity, good monodispersity and good aerodynamic performance, and can be generally used for pulmonary administration and can be generally used as an inhalant during administration.
In a fourth aspect, the invention provides a pharmaceutical composition comprising the sustained-release hydrogen sulfide donor microspheres provided in the second aspect of the invention. In the pharmaceutical composition provided by the invention, the slow-release hydrogen sulfide donor microspheres can be a single effective component, and can also be combined with other active components to form a combined preparation.
The invention constructs a sustained-release H2The biodegradable high-molecular polymer microspheres prepared from the S drug-loaded porous microspheres have good particle size monodispersity, uniform size and good aerodynamic performance, can be inhaled through respiratory tracts to deliver drugs, not only improves the lung targeting property and biological tolerance of the drugs, but also greatly enhances the effect of treating pulmonary hypertension by hydrogen sulfide, has simple, convenient and quick preparation method, saves raw materials, has stable and safe preparation process, and shows good application prospect and practical value in the field of pulmonary hypertension treatment.
The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
Example 1
Preparing microspheres:
100ml of polyvinyl alcohol aqueous solution with the mass concentration of 5% (w/v) is prepared, 50 ml of the polyvinyl alcohol aqueous solution is sucked into a 50 ml syringe, the head of the syringe is connected with one end of a continuous phase capillary, and the other 50 ml of polyvinyl alcohol aqueous solution is filled into a crystallization dish with the capacity of 100ml for collection.
Preparing 1 ml of ammonium bicarbonate aqueous solution with the mass concentration of 5% (w/v), preparing 3 ml of dichloromethane solution with the mass ratio of polylactic acid-glycolic acid to hydrogen sulfide releasing aspirin of 2:1, mixing the two solutions, ultrasonically crushing the two solutions into uniform emulsion, collecting the emulsion by using a 5 ml syringe, and connecting the head of the syringe with one end of a disperse phase capillary tube.
The device for preparing the ACS-14-loaded polylactic acid-glycolic acid for the preparation method of the porous microspheres for pulmonary administration by microfluidics comprises a dispersed phase capillary tube, a continuous phase capillary tube and a silica gel hose for connecting the dispersed phase capillary tube and the continuous phase capillary tube. One end of the continuous phase capillary tube is connected with the head of the continuous phase injector, the other end of the continuous phase capillary tube is connected with the silica gel hose, the disperse phase capillary tube is inserted into the silica gel hose from a position 1 cm away from the other end of the silica gel hose (the end of the silica gel hose which is not connected with the continuous phase capillary tube), one end of the disperse phase capillary tube is connected with the head of the disperse phase injector, and the other end of the disperse phase capillary tube extends (the end of the silica gel hose which is far away from the capillary tube) to a position below the liquid level of a collector filled with 5% of polyvinyl alcohol water solution. The inner diameter of the silica gel hose is 1 mm, the outer diameter is 2 mm, the outer diameter of the continuous phase capillary is 0.9 mm, the inner diameter is 0.8 mm, the outer diameter of the disperse phase capillary is 0.3 mm, and the inner diameter is 0.15 mm.
The two syringes were placed on two microinjection pumps, respectively, the continuous phase flow rate was adjusted to 3 ml/min, the dispersed phase flow rate was adjusted to 0.001 ml/min, and the two microinjection pumps were started simultaneously to start the preparation of microspheres.
And standing the collected microspheres in a crystallizing dish for 24 hours, washing the microspheres with distilled water for three times, and putting the microspheres into a vacuum freeze dryer to obtain a final product. The structural photographs of the product are shown in fig. 2 and fig. 3, and from the microphotographs, the geometric diameter of the drug-loaded porous microspheres is 25 micrometers, and the drug-loaded porous microspheres have a through hole structure.
Further, the nano measure 1.2 software is used to measure the particle size of 100 microspheres and statistical analysis is performed, and it can be seen that particles with a particle size of 25 ± 3 microns account for 80% of all particles.
Example 2
Biological safety of ACS-14 drug-loaded microspheres:
and (3) detecting the cell viability of the pulmonary artery endothelial cells (HPAECs) of the ACS-14 and ACS-14 drug-loaded microspheres under the incubation of the same concentration and time by using a CCK-8 kit.
The experimental procedure was as follows:
1. cultured HPAECs were added to a 96-well plate at a cell density of 5000 cells/well in an amount of 100. mu.l.
2. Incubated overnight in an incubator.
3. MSs (75.4mg/L), ACS-14 (50. mu.M) and ACS-14MSs (50. mu.M, ACS-14 molecular weight 388.99g/mol, microsphere drug loading 25.8% were added to 96-well plates, respectively, so that the microspheres were incubated at a concentration of (50. mu. 388.99. mu.10-3)/25.8% 75.4mg/L for 0h,6h,12h, and 24h, respectively.
4. To each well, 10. mu.l of CCK-8 solution was added and incubated in an incubator for 2 hours.
5. Absorbance at 450nm was measured with a microplate reader.
The results of a specific experiment on the biosafety of ACS-14 drug-loaded microspheres are shown in fig. 4, wherein,*p is less than 0.05ACS-14MSs and is compared with the MSs group;#p < 0.05ACS-14MSs compared with ACS-14 group.
Example 3
Lung tissue targeting of ACS-14 drug-loaded microspheres:
and (3) performing intratracheal administration on SD rats by using fluorescent microspheres for 0h,6h,12h and 24h respectively, and detecting the microsphere residual quantity in each tissue.
After rats are anesthetized by isoflurane gas, near-infrared fluorochrome-labeled ACS-14 drug-loaded microspheres (45mg/kg) are administrated through a trachea, after the administration is carried out for 0h,6h,12h and 24h, the rats are killed, hearts, lungs, livers and kidneys of the rats are separated, the fluorescence intensity in each tissue is detected by a Maestro in-vivo imaging system, and the excitation/emission wavelength is 750nm/780 nm.
The specific experimental results of lung tissue targeting of ACS-14 drug-loaded microspheres are shown in FIG. 5.
Example 4
The function of the ACS-14 drug-loaded microspheres in pulmonary hypertension is as follows:
200-240g of 8-week-old SD rats were randomly divided into five groups of 10 rats, and the pulmonary hypertension was induced by intraperitoneal injection of monocrotaline 60mg/kg to each group except the control group. 7 days after the monocrotaline injection, rats were administered Sildenafil at 25mg/kg per day (MCT + Sildenafil), ACS-1412 mg/kg per day (MCT + ACS-14), and non-drug-loaded microspheres at 45mg/kg per day (MCT + MSs) or ACS-14 microspheres (prepared in example 1) at 45mg/kg per day (MCT + ACS-14MSs), respectively. The specific experimental results of the detection of pulmonary artery pressure of rats in each group when monocrotaline is injected for 21 days are shown in figure 6, wherein,*p < 0.05 is the comparison of each group with the control group;#p < 0.05 is (MCT + Sildenafil) group, (MCT + ACS-14MSs) group is compared with MCT + MSs group respectively;@p < 0.05 is (MCT + ACS-14MSs) compared to MCT + ACS-14.
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (12)

1. A preparation method of the sustained-release hydrogen sulfide donor microsphere comprises the following steps:
a) providing a dispersed phase which is an emulsion comprising an aqueous phase and an oil phase selected from an organic solution comprising a donor microsphere material and S-aspirin; the water phase is selected from ammonium bicarbonate water solution, the donor microsphere material is selected from polylactic acid-glycolic acid, and the organic solvent is selected from dichloromethane;
b) providing a continuous phase; the continuous phase is selected from aqueous polyvinyl alcohol solution;
c) distributing the dispersed phase provided in step a) in the continuous phase provided in step b) by a coaxial micro-fluidic method to provide microsphere droplets of the dispersed phase;
d) solidifying, washing and drying the microsphere liquid drops provided by the step c).
2. The method according to claim 1, wherein the aqueous phase comprises 4.5 to 5.5g of solute per 100ml of solution by volume mass concentration;
and/or in the organic solution containing the donor microsphere material and the S-aspirin, each 100ml of the solution contains 2.5-3.5 g of the donor microsphere material according to the volume mass concentration;
and/or in the organic solution containing the donor microsphere material and the S-aspirin, 1-2 g of S-aspirin is contained in each 100ml of solution according to the volume mass concentration;
and/or in the polyvinyl alcohol aqueous solution, each 100ml of the solution contains 4.5-5.5 g of polyvinyl alcohol according to volume mass concentration.
3. The preparation method according to claim 1, wherein the aqueous phase comprises 4.9 to 5.1g of solute per 100ml of solution by volume mass concentration;
and/or in the organic solution containing the donor microsphere material and the S-aspirin, each 100ml of the solution can contain 2.9-3.1 g of the donor microsphere material according to the volume mass concentration;
and/or in the organic solution containing the donor microsphere material and the S-aspirin, each 100ml of the solution can contain 1.4-1.6 g S-aspirin according to the volume mass concentration;
and/or the polyvinyl alcohol aqueous solution can contain 4.9-5.1 g of polyvinyl alcohol per 100ml of solution according to volume mass concentration.
4. The preparation method according to claim 1, wherein the volume ratio of the water phase to the oil phase in the emulsion is 1: 2.5-3.5, and the emulsion is prepared by fully mixing the water phase and the oil phase.
5. The preparation method according to claim 1, wherein the volume ratio of the water phase to the oil phase in the emulsion is 1: 2.9-3.1, and the emulsion is prepared from the water phase and the oil phase by ultrasonic crushing.
6. The method of claim 1, wherein the ratio of the flow rates of the continuous phase and the dispersed phase in the coaxial microfluidic method is 2000-4000: 1.
7. The method of claim 1, wherein the ratio of flow rates of the continuous phase and the dispersed phase in the coaxial microfluidic method is 2700-3300: 1.
8. The method of claim 1, wherein the sustained-release hydrogen sulfide donor microspheres are for pulmonary administration;
and/or the slow-release hydrogen sulfide donor microspheres are inhalants.
9. The sustained-release hydrogen sulfide donor microspheres prepared by the preparation method according to any one of claims 1 to 8.
10. The use of the sustained-release hydrogen sulfide donor microspheres of claim 9 in the preparation of a medicament for preventing and treating pulmonary hypertension diseases.
11. The use of claim 10, wherein the medicament is selected from the group consisting of for treating endogenous H2(ii) S-related disease agents;
and/or, the drug is a slow release drug;
and/or, the medicament is for pulmonary administration;
and/or, the medicament is an inhalant.
12. A pharmaceutical composition comprising the sustained-release hydrogen sulfide donor microspheres of claim 9.
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