Polydopamine-coated heparin sodium-loaded polylactic acid-glycolic acid targeted sustained-release microspheres and preparation method thereof

Abstract

The invention discloses a polydopamine (PDA)-coated polylactic acid-glycolic acid (PLGA) targeted sustained release microsphere (Hep-coated PLGA-PDA MPs) loaded with heparin sodium, which is characterized in that the polylactic acid-glycolic acid microsphere (Hep-coated PLGA-PDA MPs) loaded with the drug heparin sodium is an inner core microsphere, and the outer part of the microsphere is also coated with a PDA layer. The invention also discloses a preparation method of the Hep-coated PLGA MPs, the microspheres have no burst release phenomenon within 2 hours of drug release, the release proportion of heparin sodium is 45.7-57.2% in 24 hours, and the microspheres have the characteristic of slow release. The microsphere disclosed by the invention can be used for developing an intravenous injection sustained-release agent for targeted therapy of thrombus.

Description

Technical Field

[0001] This invention patent relates to the field of microsphere formulation preparation technology, specifically to a targeted sustained-release microsphere loaded with heparin sodium and its preparation method. Background Technology

[0002] Heparin is an important anticoagulant drug, mainly used for the prevention and treatment of thromboembolic diseases. Heparin is a highly sulfonated glycosaminoglycan that binds to antithrombin III through its thiopentasaccharide sequence, causing a conformational change and activation. Activated antithrombin III can inhibit the activity of thrombin, coagulation factor Xa, and other proteases, thereby effectively inhibiting the blood clotting process.

[0003] PLGA is a polymer material formed by the random polymerization of two monomers, lactic acid and glycolic acid, also known as polylactic-co-glycolic acid or poly(glycolic acid-glycolic acid). It is produced through the ring-opening polymerization reaction of glycolide (GA) and lactide (LA).

[0004] Dopamine (DA) is an endogenous catecholamine neurotransmitter synthesized from tyrosine via tyrosine hydroxylase and is an important component of the human neurotransmission system. It plays a variety of important roles in the brain and body, such as regulating mood, motor control, and cognitive function.

[0005] Currently, the market for heparin sodium preparations is limited to a single dosage form, mainly solution-type injections. There is a lack of technology for targeted microsphere injection formulations, which is not conducive to improving the bioavailability of heparin sodium and reducing its toxic side effects. Summary of the Invention

[0006] To address the issues of insufficient targeting function and easy generation of systemic side effects in existing solution-type heparin sodium injections, and to develop new dosage forms of heparin sodium drugs, this invention discloses a heparin sodium sustained-release microsphere that can be used to develop targeted antithrombotic intravenous injections, and its preparation method.

[0007] The content of this invention is as follows: A polylactic acid-glycolic acid (PLGA) targeted sustained-release microsphere (Hep@PLGA-PDA MPs) coated with polydopamine (PDA) and loaded with heparin sodium (Hep) has a core microsphere loaded with heparin sodium and is further coated with a PDA layer.

[0008] The PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium have an average particle size of 1 to 4 µm, preferably 2.06 to 2.24 µm.

[0009] The PDA-coated PLGA targeted sustained-release microspheres loaded with heparin sodium comprise the following components and parts by weight: 0.5 to 2 parts of polylactic acid-glycolic acid; 1.5 to 20 parts of heparin sodium; 0.05 to 0.25 parts of poloxamer; and 0.1 to 0.5 parts of PDA.

[0010] The drug carrier PLGA is one or a combination of two of the following: lactic acid and glycolic acid monomers in a ratio of 50:50 or 75:25. Preferably, PLGA of type 5050 is used, which has the beneficial effects of fast degradation rate and fast drug release.

[0011] The emulsifier poloxamer is one or two of the products with model numbers 188 and 407 that are permitted to be used as excipients for injections in the Chinese Pharmacopoeia. Poloxamer model number 407 is preferred. Microsphere products prepared using this model of emulsifier have the beneficial effects of being easy to form and having good stability.

[0012] The emulsifier poloxamer is an aqueous solution with a mass concentration of 0.5% to 5%.

[0013] The method for preparing PDA-coated PLGA targeted sustained-release microspheres loaded with heparin sodium, wherein the weight-average molecular weight of the heparin sodium is 10,000 to 14,000, preferably 12,000; the weight-average molecular weight of the pharmaceutical excipient-grade polylactic-co-glycolic acid is 38,000 to 54,000, preferably 46,000; and the PDA coating layer is synthesized by self-polymerization of dopamine hydrochloride monomer.

[0014] Furthermore, the PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium are prepared by the following steps: (1) Preparation of Hep@PLGA MPs: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a dichloromethane / ethyl acetate oil phase solution of PLGA. The oil phase solution containing PLGA was added to an aqueous sodium heparin solution and homogenized to obtain a primary emulsion. An emulsifier was added to the primary emulsion, and the mixture was homogenized and emulsified again to obtain a secondary emulsion. The secondary emulsion was centrifuged to precipitate, the supernatant was removed, and the precipitate was washed with pure water in 30 mL increments by vortexing to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze dry it using a freeze dryer to obtain Hep@PLGA MPs lyophilized powder; (2) Preparation of Hep@PLGA-PDA MPs: Dopamine hydrochloride was dissolved in Tirs-HCl buffer and added dropwise to the Hep@PLGA MPs suspension while stirring. The mixture was incubated on a shaker at 37°C and then centrifuged. The precipitate was then freeze-dried to obtain Hep@PLGA-PDA MPs lyophilized powder.

[0015] The oil phase is a PLGA solution with dichloromethane / ethyl acetate (v / v = 1.5:1 ~ 2:1) as a composite solvent, the aqueous phase is an aqueous solution of heparin sodium, the oil / water phase (v / v) ratio is 1:4 ~ 1:29, the solute PLGA / Hep (w / w) ratio is 1:10 ~ 4:10, and the emulsifier during reemulsification is 0.5 ~ 5% (w / w) of poloxamer.

[0016] Furthermore, the oil phase is a 10-20 mg / mL PLGA solution with dichloromethane / ethyl acetate as the composite solvent (v / v = 1.5:1 ~ 2:1), the aqueous phase is an aqueous solution of heparin sodium, the oil / water volume ratio is 1:4 ~ 1:29, the solute PLGA / Hep (w / w) ratio is 1:10 ~ 4:10, and the emulsifier during reemulsification is 2% (w / w) poloxamer.

[0017] Furthermore, the preparation process of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a 10-20 mg / mL PLGA dichloromethane / ethyl acetate oil phase solution. The PLGA-containing oil phase solution was added to an aqueous heparin sodium solution and homogenized at 5000-8000 rpm for 1-5 min to obtain a primary emulsion. The primary emulsion was added to an emulsifier to obtain a system solution, which was homogenized again at 5000-8000 rpm for 6-15 min to form a secondary emulsion. The solution was centrifuged with pure water at 1000-4000 rpm for 6-15 min, the supernatant was removed, the precipitate was added to pure water and vortexed for washing, and the process was repeated 2-4 times to obtain a suspension of Hep@PLGA MPs. Take the Hep@PLGA MPs suspension and freeze-dry it for 16 to 30 hours, preferably 24 hours, to obtain Hep@PLGA MPs freeze-dried powder. The preparation process of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGAMPs suspension. Incubate on a shaker at 37°C for 4-8 hours. Wash the solution by centrifugation at 1000-4000 rpm to remove free drug and emulsifier. Freeze-dry the precipitate at -20 to -30°C for 24 hours to obtain Hep@PLGA-PDA MPs lyophilized powder. In the preparation process of Hep@PLGA MPs in step (1), the oil phase / water phase (v / v) ratio is preferably 1:4 to 1:29, which has a higher encapsulation efficiency (above 57.37%). Furthermore, the preparation method of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a 10-20 mg / mL PLGA dichloromethane / ethyl acetate oil phase solution. The PLGA-containing oil phase solution was added to an aqueous heparin sodium solution and homogenized at 6000-7000 rpm for 1-4 min to obtain a primary emulsion. The primary emulsion was added to an emulsifier to obtain a system solution, which was homogenized again at 6000-7000 rpm for 8-12 min to form a secondary emulsion. The system was washed three times with pure water by centrifugation at 2000-3000 rpm for 8-12 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 20-25 hours using a freeze dryer to obtain Hep@PLGA lyophilized powder; The preparation method of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGAMPs suspension. Incubate on a shaker at 35-38°C for 5-7 hours. Centrifuge at 2000-3000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20-30°C for 20-25 hours to obtain Hep@PLGA-PDA MPs lyophilized powder.

[0018] Furthermore, the preparation method of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a 10 mg / mL PLGA dichloromethane / ethyl acetate oil phase solution. The PLGA-containing oil phase solution was added to an aqueous heparin sodium solution and homogenized at 7000 rpm for 2 min to obtain a primary emulsion. The primary emulsion was added to an emulsifier to obtain a system solution, which was homogenized again at 6000 rpm for 10 min to form a secondary emulsion. The system was centrifuged three times with pure water at 3000 rpm for 10 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 24 h using a freeze dryer to obtain Hep@PLGA lyophilized powder; Based on orthogonal experimental design (using orthogonal array L...), 12 (4×3) 3 The microspheres were prepared using oil-to-water ratios (v / v) of 1:4, 1:9, 1:14, and 1:29; PLGA / Hep (w / w) ratios of 1:10, 2:10, and 4:10; and PLGA solvent-dichloromethane / ethyl acetate mixture (v / v) ratios of 1:1, 1.5:1, and 2:1. The optimal preparation conditions, based on the encapsulation efficiency, are as follows: The oil-to-water ratio (v / v) was 1:4; the solute PLGA / Hep (w / w) ratio was 2:10; the emulsifier for re-emulsification was 2% poloxamer; and the PLGA solvent was a 2:1 mixture of dichloromethane and ethyl acetate (v / v).

[0019] The specific preparation method of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh out the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGAMPs suspension. Incubate on a shaker at 37°C for 6 hours. Centrifuge at 3000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20 to -30°C for 24 hours to obtain Hep@PLGA-PDA lyophilized powder.

[0020] In summary, this invention utilizes dopamine hydrochloride to self-polymerize and form a PDA coating layer, which is then coated onto the surface of experimentally prepared Hep@PLGAMPs to obtain PDA-coated polylactic-coated sodium heparin microspheres (Hep@PLGA-PDA MPs). The specific preparation steps are as follows: according to Figure 1This invention employs a double emulsification method to prepare microspheres. The oil-to-water ratio is designed to be 1:4 to 1:29. PLGA is dissolved in a mixed solution of dichloromethane and ethyl acetate at a ratio of 1.5:1 to 2:1 to obtain a 10-20 mg / mL PLGA solution in dichloromethane and ethyl acetate. The PLGA oil phase is added to a heparin sodium solution in the aqueous phase at a solute / Hep (w / w) ratio of 1:10 to 4:10, and homogenized at 7000 rpm for 2 min to obtain a primary emulsion. The primary emulsion is then added to 2% emulsifier to obtain a 30 mL system solution, which is homogenized again at 9000 rpm for 10 min to form a double emulsion. The system is centrifuged three times with pure water at 3000 rpm for 10 min to wash away residual emulsifier and free heparin sodium. The supernatant and washing solution are transferred for encapsulation efficiency determination. Centrifugation and vortexing with pure water were used to obtain a Hep@PLGA MPs suspension.

[0021] according to Figure 2 Hep@PLGA MPs suspension was prepared. 10.0 mg of dopamine hydrochloride was weighed and dissolved in 10 mL of Tirs-HCl buffer. While stirring, 1 mL of the Hep@PLGA MPs suspension was slowly added dropwise. The mixture was incubated on a shaker at 37°C for 6 h. After centrifugation at 3000 rpm for 10 min, free drug and emulsifier were washed away. Morphological observation was performed to confirm the final product. The precipitate was then freeze-dried at -20 to -30°C for 24 h to obtain Hep@PLGA-PDA lyophilized powder.

[0022] In addition, the targeting principle and innovations of this invention are as follows.

[0023] (1) The core principle of Hep@PLGA-PDA in achieving thrombus targeting through electrostatic adsorption: The specific electrostatic attraction generated by the charge difference between the thrombus site and the microsphere surface is enhanced by combining the microsphere structural characteristics to improve targeting precision. After thrombus formation, the lesion site exposes a large number of negatively charged components, such as collagen fibers, sulfated glycoproteins, and negatively charged groups on the surface of activated platelets, forming a stable microenvironment with a strong negative potential, providing a target for electrostatic adsorption. The PDA coating layer on the microsphere surface is key to achieving electrostatic interaction: the amino and hydroxyl functional groups in the PDA molecule can undergo protonation under physiological pH conditions, giving the microsphere surface a stable positive charge. This positive charge and the negatively charged environment of the thrombus site form a strong electrostatic attraction, becoming the core driving force for the microsphere's active targeting of the thrombus. When Hep@PLGA-PDA is injected intravenously into the bloodstream, the positively charged PDA layer can quickly recognize the negatively charged target at the thrombus site and actively accumulate in the lesion area through electrostatic adsorption, significantly reducing non-specific distribution in normal tissues. Meanwhile, the hydrophilic layer formed by PDA can reduce plasma protein adsorption, avoid clearance by the reticuloendothelial system, prolong the blood circulation time of microspheres, and provide sufficient time for electrostatic adsorption-mediated targeted enrichment, ultimately achieving precise and sustained release of drugs at the thrombus site. This not only enhances the antithrombotic efficacy but also reduces systemic bleeding side effects, solving the problem of poor targeting of traditional solution-type injections.

[0024] (2) The Hep@PLGA-PDA MPs prepared using the technical solution of this invention have the following characteristics: Firstly, the invention is scientific, innovative, and feasible: Thrombus sites have a strong negative charge. This invention uses positively charged PDA groups as target molecules, which enables drugs to better identify and accumulate in the lesion. Furthermore, it has innovatively and successfully prepared PDA-coated Hep@PLGA active targeting microspheres.

[0025] Secondly, the prepared Hep@PLGA-PDA MPs have a significant sustained-release effect: in vitro drug release experiments show that the microspheres of the present invention do not exhibit burst release within 2 hours of drug release, and the heparin sodium release ratio is 45.7-57.2% after 24 hours. The average cumulative release rate of each embodiment is 48.16% after 24 hours.

[0026] Thirdly, a more environmentally friendly preparation process: The commonly used solvent for dissolving PLGA is dichloromethane, which is a hazardous chemical with neurotoxicity and liver damage risks. Therefore, this invention uses ethyl acetate to replace part of the dichloromethane solvent in order to reduce chlorine dioxide residue in the sample and reduce biosafety risks.

[0027] In summary, this microsphere formulation imparts active drug targeting through the electrostatic adsorption principle between the PDA and the thrombus site. Combined with the micro-nano size characteristics of the carrier, it holds promise for providing a precise and safe intravenously injectable drug delivery system for the clinical treatment of cerebral thrombosis, thereby reducing the risk of bleeding and improving efficacy. This invention can be used for targeted intravenous injections, addressing the problems of low drug concentration at the thrombus site and systemic bleeding side effects associated with traditional solution-type heparin sodium injections. It also helps improve the long-term in vivo effect, stability, and biocompatibility of heparin sodium. Attached Figure Description

[0028] Figure 1 Flowchart of the preparation method of Hep@PLGA-PDA MPs; Figure 2 PDA reaction mechanism diagram; Figure 3 Microscopic image of the Hep@PLGA MP microscope specimen prepared in Example 1; Figure 4 Microscopic image of the Hep@PLGA MP microscope specimen prepared in Example 2; Figure 5 Microscopic image of the Hep@PLGA MP microscope specimen prepared in Example 3; Figure 6 Example 1: Hep@PLGA MPs particle size distribution; Figure 7 Example 2: Hep@PLGA MPs particle size distribution; Figure 8 Example 3: Hep@PLGA MPs particle size distribution; Figure 9 Example 1: Cumulative release curve (a) and cumulative release rate curve (b) of Hep@PLGA-PDA MPs heparin sodium; Figure 10 Example 2: Cumulative release curve (a) and cumulative release rate curve (b) of Hep@PLGA-PDA MPs heparin sodium; Figure 11 Example 3: Cumulative release curve (a) and cumulative release rate curve (b) of Hep@PLGA-PDA MPs heparin sodium; Figure 12 (a, b, c) Scanning electron microscope images (1,000x, 2,000x, 20,000x) of the Hep@PLGA-PDA MPs sample prepared in Example 1. Figure 13 (d, e, f) Scanning electron microscope images (1,000x, 2,000x, 20,000x) of the Hep@PLGA-PDA MPs sample prepared in Example 2. Figure 14(g,h,i) Scanning electron microscope images (1,000x, 2,000x, 20,000x) of the Hep@PLGA-PDA MPs sample prepared in Example 3. Figure 15 Particle size distribution of the Hep@PLGA-PDA MPs sample prepared in Example 1; Figure 16 Particle size distribution of the Hep@PLGA-PDA MPs sample prepared in Example 2; Figure 17 Particle size distribution of the Hep@PLGA-PDA MPs sample prepared in Example 3. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1:

[0031] According to Table 1, under the corresponding conditions, the designed oil-to-water ratio (v / v) was 1:9. PLGA was dissolved in a mixed solution of dichloromethane and ethyl acetate at a ratio of 1.5:1 to obtain a 10 mg / mL PLGA solution in dichloromethane and ethyl acetate, which served as the oil phase. The PLGA oil phase was added to a heparin sodium solution in the aqueous phase at a solute / Hep ratio of 1:10 (w / w), and homogenized at 7000 rpm for 2 min to obtain a primary emulsion. This primary emulsion was then added to a 2% poloxamer 407 emulsifier to obtain a 30 mL system solution, which was homogenized again at 9000 rpm for 10 min to form a secondary emulsion. The solution was centrifuged three times with pure water at 3000 rpm for 10 min to wash away residual emulsifier and free heparin sodium. The supernatant and washing solution were transferred for encapsulation efficiency determination. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. The encapsulation efficiency was 57.37%, and the average particle size was 2.13µm.

[0032] according to Figure 1Hep@PLGA MPs suspension was prepared. 10.0 mg of dopamine hydrochloride was dissolved in 10 mL of Tirs-HCl buffer, and 1 mL of the Hep@PLGA MPs suspension was slowly added dropwise while stirring. The mixture was incubated on a shaker at 37°C for 6 h. After centrifugation at 3000 rpm for 10 min, free drug and emulsifier were washed away. Morphological observation was performed to confirm the final product. The precipitate was then freeze-dried at -20 to -30°C for 24 h to obtain Hep@PLGA-PDA lyophilized powder. The drug loading was 14.88%, the average particle size was 2.07 µm, and the cumulative release over 24 h was 0.357 mg.

[0033] Example 2:

[0034] According to Table 1, under corresponding orthogonal conditions, an oil-to-water ratio (v / v) of 1:4 was designed. PLGA was dissolved in a 2:1 mixture of dichloromethane and ethyl acetate to obtain a 10 mg / mL PLGA solution in dichloromethane and ethyl acetate. The oil phase of PLGA was added to a sodium heparin solution in the aqueous phase at a solute / Hep ratio of 2:10. The mixture was homogenized at 7000 rpm for 2 min to obtain a primary emulsion. This primary emulsion was then added to a 2% poloxamer 407 emulsifier to obtain a 30 mL system solution. The system was homogenized again at 9000 rpm for 10 min to form a secondary emulsion. The mixture was centrifuged three times with pure water at 3000 rpm for 10 min to wash away residual emulsifier and free sodium heparin. The supernatant and washing solution were transferred for encapsulation efficiency determination. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. The encapsulation efficiency was 98.72% and the average particle size was 2.45µm.

[0035] according to Figure 1 Hep@PLGA MPs suspension was prepared. 10.0 mg of dopamine hydrochloride was dissolved in 10 mL of Tirs-HCl buffer, and 1 mL of the Hep@PLGA MPs suspension was slowly added dropwise while stirring. The mixture was incubated on a shaker at 37°C for 6 h. After centrifugation at 3000 rpm for 10 min, free drug and emulsifier were washed away. Morphological observation was performed to confirm the final product. The precipitate was then freeze-dried at -20 to -30°C for 24 h to obtain Hep@PLGA-PDA lyophilized powder. The drug loading was 16.49%, the average particle size was 2.06 µm, and the cumulative release over 24 h was 0.355 mg.

[0036] Example 3:

[0037] According to Table 1, under corresponding orthogonal conditions, the oil-to-water ratio (v / v) was designed to be 1:14. PLGA was dissolved in a mixed solution of dichloromethane and ethyl acetate in a 2:1 ratio to obtain a 10 mg / mL PLGA solution in dichloromethane and ethyl acetate. The oil phase of PLGA was added to a sodium heparin solution in the aqueous phase at a solute / Hep ratio of 4:10. The mixture was homogenized at 7000 rpm for 2 min to obtain a primary emulsion. This primary emulsion was then added to a 2% poloxamer 407 emulsifier to obtain a 30 mL system solution. The mixture was homogenized again at 9000 rpm for 10 min to form a secondary emulsion. The mixture was centrifuged three times with pure water at 3000 rpm for 10 min to wash away residual emulsifier and free sodium heparin. The supernatant and washing solution were transferred for encapsulation efficiency determination. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. The encapsulation efficiency was 75.92% and the average particle size was 1.07µm.

[0038] according to Figure 1 Hep@PLGA MPs suspension was prepared. 10.0 mg of dopamine hydrochloride was dissolved in 10 mL of Tirs-HCl buffer, and 1 mL of the Hep@PLGA MPs suspension was slowly added dropwise while stirring. The mixture was incubated on a shaker at 37°C for 6 h. After centrifugation at 3000 rpm for 10 min, free drug and emulsifier were washed away. Morphological observation was performed to confirm the final product. The precipitate was then freeze-dried at -20 to -30°C for 24 h to obtain Hep@PLGA-PDA MPs lyophilized powder. The drug loading was 15.38%, the average particle size was 2.24 µm, and the cumulative release over 24 h was 0.369 mg.

[0039] The polylactic acid-glycolic acid microsphere suspensions loaded with heparin sodium prepared in Examples 1 to 3 were subjected to microscopic observation, microsphere particle size distribution inspection, encapsulation rate test, and release rate test, and were analyzed respectively.

[0040] in, Figure 3 Images 1 through 8 are microscopic images of the Hep@PLGA-PDA MPs prepared in Examples 1 through 3, showing their particle size distribution. Spherical liposome particles can be clearly seen in the images.

[0041] in, Figure 12 Images ~14 are microscopic images of the Hep@PLGA-PDA MPs prepared in Examples 1 to 3, respectively. Spherical microparticles are clearly visible in the images. According to... Figure 15 As shown in the particle size distribution diagram of ~17, the average particle sizes are 2.07µm, 2.06µm, and 2.24µm, respectively.

[0042] See Figure 6 ~8. The particle size distribution of the microspheres was detected using the microscope's built-in analysis software. The specific detection results are as follows: Figure 6 The average particle size of the sample prepared for Example 1 was 2.13 μm. Among the sample microspheres, microspheres with a diameter in the range of 1–1.5 µm accounted for 16% of the total sample, those with a diameter in the range of 1–2 µm accounted for 52%, those with a diameter in the range of 1–2.5 µm accounted for 80%, and those with a diameter in the range of 1–3 µm accounted for 92%. Microspheres with a diameter in the range of 1.5–2 µm accounted for the largest proportion of the total sample, at 36%.

[0043] Figure 7 The average particle size of the sample prepared for Example 2 was 2.45 µm. Among the sample microspheres, microspheres with a diameter in the range of 1–1.5 µm accounted for 3% of the total sample, microspheres with a diameter in the range of 1–2 µm accounted for 33%, microspheres with a diameter in the range of 1–2.5 µm accounted for 63%, and microspheres with a diameter in the range of 1–3 µm accounted for 83%. Microspheres with a diameter in the range of 1.5–2.5 µm accounted for the largest proportion of the total sample, at 60%.

[0044] Figure 8 The average particle size of the sample prepared for Example 3 was 1.07 μm. Among the sample microspheres, microspheres with a diameter in the range of 0–0.5 µm accounted for 2% of the total sample, microspheres with a diameter in the range of 0–1 µm accounted for 52%, microspheres with a diameter in the range of 0–1.5 µm accounted for 82%, and microspheres with a diameter in the range of 0–2 µm accounted for 92%. Microspheres with a diameter in the range of 0.5–1 µm accounted for the largest proportion of the total sample, at 50%.

[0045] Based on the encapsulation results shown in Table 1, the optimal conditions can be determined as follows: oil-to-water ratio (v / v) of 1:4; PLGA / Hep ratio (w / w) of 2:10; 2% (w / w) poloxamer as emulsifier during reemulsification; and PLGA solvent as a mixture of dichloromethane and ethyl acetate (v / v=2:1).

[0046] The encapsulation efficiency test method is as follows: Plotting a standard curve for heparin sodium solution: Prepare standard solutions (concentration range: 0.2 U ~ 4 U / mL, not exceeding 34 μg / mL), and determine A using the toluidine blue method. 632 nm Values ​​are used to plot a standard curve based on the obtained data (the fitting equation is: A). 吸光度 = -0.0098 C肝素钠浓度 + 0.5711, R² = 0.9938).

[0047] Encapsulation efficiency determination: The centrifuged liquid and washing supernatant were combined and diluted 1:10000 with PBS buffer (pH=7.4). The encapsulation efficiency was determined using the toluidine blue method. 632 nm The concentration of heparin sodium in the sample is calculated based on the standard curve, thereby estimating the total amount of heparin sodium in the centrifugation and washing supernatants.

[0048] Calculation formula:

[0049] (Note: In the formula, Cf is the amount of free drug; Ct is the total amount of drug in the heparin sodium sustained-release microparticle suspension.) Table 1. Encapsulation efficiency data of Hep@PLGA MPs prepared in Examples 1 to 3

[0050] Table 1 shows the encapsulation efficiency test results for Examples 1, 2, and 3. It can be seen that the Hep@PLGA MPs prepared by the method in Example 2 have the highest encapsulation efficiency, reaching 98.72%.

[0051] according to Figure 9 , Figure 10 , Figure 11 The release accumulation curve and release curve are for three examples of samples. Each example of lyophilized powder sample was tested in three parallel experiments for in vitro release.

[0052] The specific testing steps are as follows: Take the dried powder, add 1 mL of PBS buffer (pH=7.4), shake thoroughly, and place on a shaker (37℃, 200 rpm). Observation points are 10 min, 30 min, 1 h, 2 h, 3 h, 6 h, 9 h, 12 h, and 24 h. At each observation point, centrifuge the sample at 3000 rpm for 10 min, collect 20 μL of the supernatant, add 20 μL of PBS buffer, and continue shaking. Use each sample as the test specimen, and determine the A632 nm value using the toluidine blue method. Calculate the concentration of heparin sodium in the sample based on the standard curve, thereby calculating the heparin sodium content, and finally plot the release curve.

[0053] Note: In Example 1, the drug release of the sample did not exceed 30% within the first 2 hours, indicating no burst release and high safety. The accumulated amount of the sample increased significantly within the first 2 hours, reaching 0.161 mg, 0.199 mg, and 0.223 mg, with an average accumulation of 0.194 mg. At this time, the release rates of the three groups were 20.08%, 24.85%, and 27.93%, respectively, with an average release rate of 24.29%. Between 2 and 12 hours, the release slowly increased, with accumulated amounts of 0.296 mg, 0.335 mg, and 0.335 mg, respectively, with an average accumulation of 0.322 mg. At this time, the release rates of the three groups were 37.05%, 41.83%, and 41.83%, respectively, with an average release rate of 40.24%. From 12 hours onwards... During the 24-hour period, the three groups showed a small amount of release, with the final cumulative release amounts at 24 hours being 0.351 mg, 0.359 mg, and 0.362 mg, respectively, and the average cumulative release amount being 0.357 mg. The corresponding cumulative release rates were 43.93%, 44.91%, and 45.19%, respectively, with an average cumulative release rate of 44.69%.

[0054] In Example 2, the drug release of the sample did not exceed 30% within the first 2 hours, indicating no burst release and high safety. The drug accumulation in the sample increased slowly within the first 6 hours, with accumulations of 0.186 mg, 0.195 mg, and 0.210 mg, respectively, and an average accumulation of 0.197 mg. At this time, the release rates of the three groups of samples were 26.92%, 28.13%, and 30.33%, respectively, with an average release rate of 28.46%. Between 6 and 9 hours, the release rate increased rapidly, with accumulations of 0.317 mg, 0.336 mg, and 0.346 mg, respectively, and an average accumulation of 0.333 mg. At this time, the release rates of the three groups of samples were 45.93%, 48.56%, and 50.10%, respectively, with an average release rate of 48.20%. Between 9 and 9 hours... During the 24-hour period, the three groups showed a small amount of release, with the final cumulative release amounts at 24 hours being 0.334 mg, 0.386 mg, and 0.392 mg, respectively, and the average cumulative release amount being 0.371 mg. The corresponding cumulative release rates were 48.34%, 55.81%, and 56.69%, respectively, with an average cumulative release rate of 53.61%.

[0055] In Example 3, the drug release of the sample did not exceed 30% within the first 2 hours, indicating no burst release and high safety. The accumulation of the drug significantly increased within the first 3 hours, reaching 0.218 mg, 0.234 mg, and 0.257 mg, with an average accumulation of 0.236 mg. At this point, the release rates of the three groups were 27.23%, 29.20%, and 32.14%, respectively, with an average release rate of 29.53%. Between 3 and 6 hours, the release increased slowly, with accumulations of 0.240 mg, 0.255 mg, and 0.270 mg, respectively, with an average accumulation of 0.255 mg. At this point, the release rates of the three groups were 30.04%, 31.86%, and 33.69%, respectively, with an average release rate of 31.86%. Between 6 and 9 hours, the release increased rapidly, with accumulations of 0.286 mg, 0.312 mg, and 0.318 mg, respectively. The average cumulative release was 0.305 mg, with release rates of 35.79%, 39.02%, and 39.72% for the three groups, respectively, and an average release rate of 38.18%. During the 9-24 hour period, all three groups showed a small amount of release, with final cumulative releases of 0.363 mg, 0.372 mg, and 0.374 mg at 24 hours, respectively, and an average cumulative release of 0.369 mg. The corresponding cumulative release rates were 45.33%, 46.46%, and 46.74%, respectively, with an average cumulative release rate of 46.17%.

[0056] according to Figure 15 , Figure 16 , Figure 17 As can be seen, the prepared microparticles are relatively regular spherical particles with complex textures. The surface of these particles has obvious wrinkles and depressions, which are PDAs that are self-assembled and coated on the Hep@PLGA surface.

[0057] In summary, the characterization data of the Hep@PLGA MPs prepared in this invention show that the Hep@PLGA MPs have a uniform particle size with an average particle size of 2.45 µm, an encapsulation efficiency of 98.72%, and a drug loading of 16.49%. The encapsulation efficiency and drug loading of heparin sodium are significantly improved (compare with reference: Cao Jianjun et al. Preparation and in vitro cell compatibility study of heparin sodium PLGA nanoparticles), and the amount of solvent chlorine dioxide used is reduced.

[0058] The Hep@PLGA-PDA sample accumulated an average of 0.333 mg over 24 hours, with an average cumulative release rate of 48.20%. Hep@PLGA-PDA was successfully prepared using a simple method, and the resulting product exhibited uniform and stable morphology. Hep@PLGA-PDA can enhance thrombus targeting and achieve long-term drug circulation through electrostatic adsorption. Combined with the characteristics of nanocarriers, it holds promise for providing a precise and safe drug delivery system for the clinical treatment of cerebral thrombosis, reducing the risk of bleeding and improving efficacy.

[0059] Example 4: A PDA-coated PLGA-targeted sustained-release microsphere loaded with heparin sodium (Hep@PLGA-PDA MPs), wherein polylactic acid-glycolic acid microspheres loaded with heparin sodium (Hep@PLGA MPs) are the core microspheres, and the microspheres are also coated with a PDA layer.

[0060] Example 5: A PDA-coated PLGA-targeted sustained-release microsphere loaded with heparin sodium (Hep@PLGA-PDA MPs), wherein polylactic acid-glycolic acid microspheres loaded with heparin sodium (Hep@PLGA MPs) are the core microspheres, and the microspheres are also coated with a PDA layer. The average particle size of the entire microsphere is 1~4µm.

[0061] Example 6:

[0062] A PDA-coated PLGA-targeted sustained-release microsphere loaded with heparin sodium (Hep@PLGA-PDA MPs) has a core microsphere of polylactic-coated glycolic acid microspheres loaded with heparin sodium (Hep@PLGA MPs) and an outer PDA layer. The average particle size of the entire microsphere is 2.06 ~ 2.24 µm.

[0063] Example 7:

[0064] A PDA-coated PLGA-based targeted sustained-release microsphere (Hep@PLGA-PDA MPs) loaded with heparin sodium comprises polylactic acid-glycolic acid microspheres (Hep@PLGA MPs) as the core microspheres, with a PDA layer coating the outer layer. The average particle size of the entire microsphere is 2.06~2.24µm. The PDA-coated PLGA-based targeted sustained-release microsphere comprises the following components and parts by weight: 1 part polylactic acid-glycolic acid; 9 parts heparin sodium; 0.10 parts poloxamer; and 0.25 parts PDA. The drug carrier PLGA has a lactic acid to glycolic acid monomer ratio of 50:50. The emulsifier poloxamer is type 188, permitted for use as an excipient in injectable formulations according to the Chinese Pharmacopoeia. The emulsifier poloxamer is an aqueous solution with a mass concentration of 1%.

[0065] Example 8: A PDA-coated PLGA-based targeted sustained-release microspheres loaded with heparin sodium (Hep@PLGA-PDA MPs), wherein the polylactic acid-glycolic acid microspheres loaded with heparin sodium (Hep@PLGA MPs) are the core microspheres, and the microspheres are also coated with a PDA layer. The average particle size of the entire microsphere is 2.06 ~ 2.24 µm, comprising the following components and parts by weight: polylactic acid-glycolic acid: 2 parts, heparin sodium: 20 parts, poloxamer: 0.25 parts, PDA: 0.5 parts; the drug carrier PLGA has a lactic acid to glycolic acid monomer ratio of 75:25, using PLGA of type 5050, which has the beneficial effects of fast degradation rate and fast drug release; the emulsifier poloxamer is type 407, which is permitted for use as an excipient in injections in the Chinese Pharmacopoeia; the emulsifier poloxamer is a 3% aqueous solution.

[0066] Example 9: A method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium: The preparation process of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a 10 mg / mL PLGA dichloromethane / ethyl acetate oil phase solution. The PLGA-containing oil phase solution was added to an aqueous heparin sodium solution and homogenized at 6000 rpm for 4 min to obtain a primary emulsion. The primary emulsion was added to an emulsifier to obtain a system solution, which was homogenized again at 6000 rpm for 7 min to form a secondary emulsion. The system was centrifuged twice with pure water at 2000 rpm for 9 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 20 hours to obtain Hep@PLGA lyophilized powder; The preparation process of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh out the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGAMPs suspension. Incubate on a shaker at 37°C for 7 hours. Centrifuge at 3000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -30°C for 24 hours to obtain Hep@PLGA-PDA lyophilized powder.

[0067] The oil phase is a solution of PLGA in dichloromethane / ethyl acetate, v / v = 1.5:1, the aqueous phase is an aqueous solution of heparin sodium, the oil / water ratio (v / v) is 1:4, the solute PLGA / Hep (w / w) ratio is 1:10, and the emulsifier during reemulsification is 2% (w / w) of poloxamer 188.

[0068] Example 10: A method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium: Step (1) Preparation of Hep@PLGA MPs: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate (dichloromethane / ethyl acetate, v / v = 2:1) to obtain a 15 mg / mL PLGA oil phase solution in dichloromethane / ethyl acetate. The PLGA-containing oil phase solution was added to an aqueous heparin sodium solution (PLGA / Hep (w / w) = 4:10) at a volume ratio of 1:29. The mixture was homogenized at 7000 rpm for 3 min to obtain a primary emulsion. The primary emulsion was added to 1% (w / w) of poloxamer 188 emulsifier to obtain a system solution. The system solution was homogenized again at 7000 rpm for 12 min to form a secondary emulsion. The system was centrifuged three times with pure water at 3000 rpm for 7 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 24 h using a freeze dryer to obtain Hep@PLGA lyophilized powder; the preparation of Hep@PLGA-PDA MPs in step (2): Weigh the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGAMPs suspension. Incubate on a shaker at 37°C for 5 hours. Centrifuge at 4000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20°C for 24 hours to obtain Hep@PLGA-PDA lyophilized powder.

[0069] Example 11: A method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with sodium heparin: Step (1) Preparation of Hep@PLGA MPs: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a 20 mg / mL PLGA dichloromethane / ethyl acetate oil phase solution. The PLGA-containing oil phase solution was added to an aqueous heparin sodium solution at a volume ratio of 1:14 (PLGA / Hep (w / w) was 2:10). The mixture was homogenized at 7000 rpm for 2 min to obtain a primary emulsion. The primary emulsion was added to a 2% poloxamer 407 emulsifier to obtain a system solution. The system solution was homogenized again at 6000 rpm for 10 min to form a secondary emulsion. The system was centrifuged three times with pure water at 3000 rpm for 10 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 24 h using a freeze dryer to obtain Hep@PLGA lyophilized powder; Step (2) Preparation of Hep@PLGA-PDA MPs: Weigh the prescribed amount of DA and dissolve it in Tirs-HCl buffer. While stirring, slowly add the Hep@PLGA MPs suspension. Incubate on a shaker at 37°C for 6 hours. Centrifuge at 3000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20 to -30°C for 24 hours to obtain Hep@PLGA-PDA lyophilized powder. The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polylactic-coated poly(lactic-coated) glycolic acid (PLGA) targeted sustained-release microsphere (Hep@PLGA-PDA MPs) loaded with sodium heparin (Hep), characterized in that... The inner core of the polylactic-coated glycolic acid microspheres (Hep@PLGA MPs) is loaded with sodium heparin, and the outer surface of the microspheres is also coated with a PDA layer.

2. The PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium according to claim 1, characterized in that: The average particle size of the microspheres is 1 to 4 µm, preferably 2.06 to 2.24 µm.

3. The PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium according to claim 1, characterized in that: It includes the following components and parts by weight: polylactic acid-glycolic acid 0.5 to 2 parts; sodium heparin 1.5 to 20 parts; poloxamer 0.05 to 0.25 parts; PDA 0.1 to 0.5 parts; the drug carrier PLGA is one or a combination of two of the following: a lactic acid to glycolic acid monomer ratio of 50:50 or 75:

25.

4. The method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium according to claim 1, characterized in that: The weight-average molecular weight of the heparin sodium drug is 10,000 to 14,000, preferably 12,000; the weight-average molecular weight of the pharmaceutical excipient grade polylactic-co-glycolic acid is 38,000 to 54,000, preferably 46,000; the PDA coating layer is synthesized by self-polymerization of dopamine hydrochloride monomer.

5. The PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium as described in claim 1, wherein the preparation method comprises the following steps: (1) Preparation of Hep@PLGA MPs: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a dichloromethane / ethyl acetate oil phase solution of PLGA. The oil phase solution containing PLGA was added to an aqueous sodium heparin solution and homogenized to obtain a primary emulsion. An emulsifier was added to the primary emulsion, and the mixture was homogenized and emulsified again to obtain a secondary emulsion. The secondary emulsion was centrifuged to precipitate, the supernatant was removed, and the precipitate was washed with pure water by vortexing to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze dry it using a freeze dryer to obtain Hep@PLGA MPs lyophilized powder; (2) Preparation of Hep@PLGA-PDA MPs: Dopamine hydrochloride was dissolved in Tirs-HCl buffer and added dropwise to the Hep@PLGA MPs suspension while stirring. The mixture was incubated on a shaker at 37°C and then centrifuged. The precipitate was then freeze-dried to obtain Hep@PLGA-PDA MPs lyophilized powder.

6. The PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium as described in claim 5, characterized in that: the oil phase is a PLGA solution with dichloromethane / ethyl acetate, v / v = 1.5:1 ~ 2:1 as a composite solvent; the aqueous phase is an aqueous solution of heparin sodium, with an oil / water ratio (v / v) of 1:4 ~ 1:29, a solute PLGA / Hep (w / w) ratio of 1:10 ~ 4:10, and the emulsifier during reemulsification is 0.5 ~ 5% (w / w) of poloxamer.

7. The method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium as described in claim 6, characterized in that: The oil phase is a PLGA solution with dichloromethane / ethyl acetate as the composite solvent, v / v = 1.5:1 ~ 2:1; the aqueous phase is an aqueous solution of sodium heparin; the oil / aqueous phase volume ratio is 1:4 ~ 1:29, and the solute PLGA / Hep (w / w) ratio is 1:10 ~ 4:10; the emulsifier for reemulsification is 2% (w / w) poloxamer.

8. The method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium as described in claim 5, characterized in that: The preparation method of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a dichloromethane / ethyl acetate oil phase solution of PLGA. The oil phase solution containing PLGA was added to an aqueous heparin sodium solution and homogenized at 5000-8000 rpm for 1-5 min to obtain a primary emulsion. An emulsifier was added to the primary emulsion and homogenized again at 5000-8000 rpm for 6-15 min to form a secondary emulsion. The emulsion was centrifuged with pure water at 1000-4000 rpm for 6-15 min, the supernatant was removed, the precipitate was washed with pure water and vortexed, and the cycle was repeated 2-4 times to obtain a suspension of Hep@PLGA MPs. Take the Hep@PLGA MPs suspension and freeze-dry it for 16-30 h, preferably 24 h, to obtain Hep@PLGA freeze-dried powder. The preparation of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGA MPs suspension. Incubate on a shaker at 37°C for 4-8 hours. Centrifuge at 1000-4000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20 to -30°C for 24 hours to obtain Hep@PLGA-PDA lyophilized powder.

9. The method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium as described in claim 5, characterized in that: The preparation method of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a dichloromethane / ethyl acetate oil phase solution of PLGA. The oil phase solution containing PLGA was added to an aqueous heparin solution and homogenized at 6000-7000 rpm for 1-4 min to obtain a primary emulsion. The primary emulsion was added to an emulsifier to obtain a system solution, which was homogenized again at 6000-7000 rpm for 8-12 min to form a secondary emulsion. The system was centrifuged three times with pure water at 2000-3000 rpm for 8-12 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 20-25 hours using a freeze dryer to obtain Hep@PLGA lyophilized powder; The preparation method of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGA MPs suspension. Incubate on a shaker at 35-38°C for 5-7 hours. Centrifuge at 2000-3000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20 to -30°C for 20-25 hours to obtain Hep@PLGA-PDA lyophilized powder.

10. The method for preparing PDA-coated PLGA-targeted sustained-release microspheres loaded with heparin sodium as described in claim 5, characterized in that: The preparation method of Hep@PLGA MPs in step (1) is as follows: PLGA was dissolved in an oil phase solution of dichloromethane and ethyl acetate to obtain a dichloromethane / ethyl acetate oil phase solution of PLGA. The oil phase solution containing PLGA was added to an aqueous heparin sodium solution and homogenized at 7000 rpm for 2 min to obtain a primary emulsion. The primary emulsion was added to an emulsifier to obtain a system solution, which was homogenized again at 6000 rpm for 10 min to form a secondary emulsion. The system was washed three times with pure water by centrifugation at 3000 rpm for 10 min. The centrifuged precipitate was vortexed with pure water to obtain a Hep@PLGA MPs suspension. Take the Hep@PLGA MPs suspension and freeze-dry it for 24 h using a freeze dryer to obtain Hep@PLGA lyophilized powder; The preparation method of Hep@PLGA-PDA MPs in step (2) is as follows: Weigh the prescribed amount of dopamine hydrochloride and dissolve it in Tirs-HCl buffer. While stirring, slowly add the solution dropwise to the Hep@PLGA MPs suspension. Incubate on a shaker at 37°C for 6 hours. Centrifuge at 3000 rpm to wash away free drug and emulsifier. Freeze-dry the precipitate at -20 to -30°C for 24 hours to obtain Hep@PLGA-PDA lyophilized powder.

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