Drug-loaded systems for the treatment of rheumatoid arthritis and methods of making and using the same

Abstract

The application relates to the technical field of targeted drugs, and discloses a drug-loaded system for treating rheumatoid arthritis as well as a preparation method and application of the drug-loaded system. The drug-loaded system comprises sinomenine hydrochloride, prussian blue, a biomimetic membrane and phosphatidylated hyaluronic acid, the biomimetic membrane is wrapped on the surface of the prussian blue loaded with the sinomenine hydrochloride, the phosphatidylated hyaluronic acid is embedded on the surface of the biomimetic membrane, and the biomimetic membrane comprises red blood cell membranes and macrophage membranes. The drug-loaded system has good targeting inflammation joint capacity and good treatment effect.

Description

TECHNICAL FIELD

[0001] The present application relates to a targeted drug, in particular to a drug-loaded system for treating rheumatoid arthritis and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis is a common chronic autoimmune inflammatory disease characterized by chronic inflammation of the synovial membrane, erosion of cartilage and bone tissue, and ultimately destruction of the joint structure. Sinomenine is a kind of alkaloid extracted from the dried rhizome of Sinomenium acutum of the family of Stephania. At present, the clinical medicine is mainly hydrochloric acid sinomenine, and the commonly used preparations are Zhengqing Fengtongning sustained-release tablets and Zhengqing Fengtongning injection, which are mainly used for the treatment of rheumatoid arthritis and rheumatic diseases. The anti-inflammatory effect of hydrochloric acid sinomenine has definite curative effect, and there is no cardiovascular system side effect caused by non-steroidal anti-inflammatory drugs, which is a potential world-class anti-rheumatic drug. However, its biological half-life is short, the elimination in vivo is fast, the dosage is large, and the non-specific targeting limits its wide clinical application.

[0003] The prior art (CN113041356A) also prepares a targeted drug-loaded system by combining prussian blue, a biomimetic membrane and hyaluronic acid with heparin, but the therapeutic effect of the targeted drug-loaded system still needs to be further improved. SUMMARY

[0004] The purpose of the present application is to overcome the problems of large dosage of hydrochloric acid sinomenine, non-specific targeting and the like in the prior art and the need for further improvement of the therapeutic effect of the existing targeted drug-loaded system, and to provide a drug-loaded system for treating rheumatoid arthritis and a preparation method and application thereof. The drug-loaded system has good targeting ability for inflammatory joints and good therapeutic effect.

[0005] In order to achieve the above-mentioned purpose, the present application provides a drug-loaded system for treating rheumatoid arthritis, which comprises hydrochloric acid sinomenine, prussian blue, a biomimetic membrane and phosphatidylated hyaluronic acid. The biomimetic membrane is wrapped on the surface of prussian blue loaded with hydrochloric acid sinomenine, the phosphatidylated hyaluronic acid is embedded on the surface of the biomimetic membrane, and the biomimetic membrane comprises red blood cell membrane and macrophage membrane.

[0006] Preferably, the mass ratio of the prussian blue, the hydrochloric acid sinomenine, the biomimetic membrane and the phosphatidylated hyaluronic acid is 1:1-10:0.5-1:0.5-1.

[0007] Further preferably, the mass ratio of the prussian blue, the hydrochloric acid sinomenine, the biomimetic membrane and the phosphatidylated hyaluronic acid is 1:4-8:0.5-1:0.5-1.

[0008] Preferably, the mass ratio of the red blood cell membrane and the macrophage membrane is 1:0.5-1.

[0009] Preferably, the Prussian blue is hollow mesoporous Prussian blue.

[0010] Further preferably, the preparation method of the hollow mesoporous Prussian blue comprises: dissolving Prussian blue and polyvinylpyrrolidone II in hydrochloric acid II, heating reaction II, and then solid-liquid separation II, washing, and drying II.

[0011] More preferably, the mass ratio of the Prussian blue and the polyvinylpyrrolidone II is 1:2-8.

[0012] Preferably, the concentration of the hydrochloric acid II is 0.5-2 mol / L.

[0013] Preferably, the heating reaction II is performed at a temperature of 140-160℃ for 4-6 hr.

[0014] Preferably, the drying II is vacuum freeze-drying.

[0015] Preferably, the vacuum freeze-drying is performed at a temperature of -20-4℃ for 12-24 hr.

[0016] The second aspect of the present application provides a preparation method of the drug delivery system for treating rheumatoid arthritis according to the first aspect, comprising the following steps:

[0017] (1) preparing a Prussian blue dispersion;

[0018] (2) mixing sinomenine hydrochloride and the Prussian blue dispersion to perform a mixing reaction I, and then removing impurities to obtain a Prussian blue dispersion loaded with sinomenine hydrochloride;

[0019] (3) mixing the Prussian blue dispersion loaded with sinomenine hydrochloride and a biomimetic membrane dispersion to perform a mixing reaction II, and then performing solid-liquid separation III to obtain a biomimetic membrane-coated Prussian blue composite material loaded with sinomenine hydrochloride;

[0020] (4) mixing the composite material and phosphatidylated hyaluronic acid to perform a mixing reaction III to obtain the drug delivery system.

[0021] Preferably, the preparation method of the biomimetic membrane dispersion comprises: mixing and crushing red blood cell membranes and macrophage membranes in a buffer.

[0022] Preferably, in step (1), the preparation method of the Prussian blue dispersion comprises: ultrasonically mixing Prussian blue with water to obtain the Prussian blue dispersion.

[0023] Preferably, the mass ratio of the Prussian blue and the water is 1:0.05-1.

[0024] Preferably, the condition of the ultrasonic mixing comprises: ultrasonic power of 30-100 W, time of 3-15 min.

[0025] Preferably, the Prussian blue is hollow mesoporous Prussian blue.

[0026] Further preferably, the preparation method of the hollow mesoporous Prussian blue comprises: dissolving Prussian blue and polyvinylpyrrolidone II in hydrochloric acid II, heating reaction II, and then solid-liquid separation II, washing, and drying II.

[0027] More preferably, the mass ratio of the Prussian blue and the polyvinylpyrrolidone II is 1:2-8.

[0028] Preferably, the concentration of the hydrochloric acid II is 0.5-2 mol / L.

[0029] Preferably, the condition of the heating reaction II comprises: temperature of 140-160℃, time of 4-6 hr.

[0030] Preferably, the drying II is vacuum freeze-drying.

[0031] Preferably, the condition of the vacuum freeze-drying comprises: temperature of -20-4℃, time of 12-24 hr.

[0032] Preferably, in step (2), the mixing reaction I comprises: stirring the mixed solution of the sinomenine hydrochloride and the Prussian blue dispersion.

[0033] Further preferably, the condition of the stirring comprises: rotation speed of 300-800 rpm, time of 10-12 hr.

[0034] Preferably, in step (2), the method for removing impurities is dialysis.

[0035] Preferably, the dialysis time is 12-24 hr.

[0036] Preferably, in step (3), the mixing reaction II comprises: stirring the mixed solution of the Prussian blue dispersion loaded with sinomenine hydrochloride and the biomimetic membrane dispersion.

[0037] Further preferably, the condition of the stirring comprises: rotation speed of 300-800 rpm, temperature of 30-37℃, time of 2-4 hr.

[0038] Preferably, in step (3), the method for solid-liquid separation I is centrifugal separation.

[0039] Preferably, the centrifugal separation condition comprises a rotation speed of 10,000-13,000 rpm for 10-20 min.

[0040] Preferably, in step (4), the preparation method of the phosphatidylated hyaluronic acid comprises dissolving hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and distearoylphosphatidyl ethanolamine-polyethylene glycol-amino in a phosphate buffer and stirring reaction, freeze-drying.

[0041] Preferably, in the preparation method of the phosphatidylated hyaluronic acid, the mass ratio of the hyaluronic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the N-hydroxysuccinimide and the distearoylphosphatidyl ethanolamine-polyethylene glycol-amino is 1:2-8:5-15:2-8.

[0042] Preferably, in the preparation method of the phosphatidylated hyaluronic acid, the stirring reaction condition comprises a rotation speed of 300-800 rpm for 2-24 hr.

[0043] Preferably, in the preparation method of the phosphatidylated hyaluronic acid, the freeze-drying is vacuum freeze-drying.

[0044] Preferably, the vacuum freeze-drying condition comprises a temperature of -20-4℃ for 12-24 hr.

[0045] Preferably, in step (4), the mixing reaction III comprises stirring the composite material and the phosphatidylated hyaluronic acid.

[0046] Further preferably, the stirring condition comprises a rotation speed of 300-800 rpm for 2-4 hr.

[0047] Preferably, the mass ratio of the Prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidylated hyaluronic acid is 1:1-10:0.5-1:0.5-1.

[0048] Further preferably, the mass ratio of the Prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidylated hyaluronic acid is 1:4-8:0.5-1:0.5-1.

[0049] Preferably, in the preparation method of the biomimetic membrane dispersion, the mass ratio of the red blood cell membrane and the macrophage membrane is 1:0.5-1.

[0050] Preferably, in the preparation method of the biomimetic membrane dispersion, the mixed pulverization is ultrasonic pulverization.

[0051] Further preferably, the ultrasonic pulverization conditions include a power of 50-120 W and a time of 1-3 min.

[0052] Preferably, the method for preparing the biomimetic membrane dispersion liquid further comprises stirring the mixed and pulverized solid mixture.

[0053] Preferably, the stirring conditions include a rotation speed of 300-800 rpm and a time of 2-3 hr.

[0054] The third aspect of the present application provides a use of the drug-loaded system for treating rheumatoid arthritis according to the first aspect or the drug-loaded system for treating rheumatoid arthritis prepared by the method according to the second aspect in the preparation of an anti-rheumatoid arthritis therapeutic drug.

[0055] The drug-loaded system of the present application can effectively improve the targeting effect of the drug-loaded system by wrapping the biomimetic membrane including the red blood cell membrane and the macrophage membrane on the surface of the Prussian blue loaded with sinomenine hydrochloride and wrapping the phosphatidyl hyaluronic acid on the surface of the biomimetic membrane, thereby effectively reducing the use amount (3 mg / kg) of sinomenine hydrochloride, effectively improving the treatment effect of rheumatoid arthritis, and more efficiently blocking the inflammatory response and the bone destruction process of rheumatoid arthritis, which provides a new theoretical support for the development of anti-rheumatoid arthritis drugs and related clinical detection and treatment, and has important scientific significance, practical value and economic value. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a scanning transmission electron microscope image of the drug-loaded system in Example 1 of the present application;

[0057] Figure 2 is a resonance energy transfer diagram of the fusion of the red blood cell membrane (RBCm) and the macrophage membrane in the present application;

[0058] Figure 3 is an ultraviolet-visible spectrum diagram of free sinomenine hydrochloride, Prussian blue dispersion liquid, Prussian blue nanocomposite loaded with sinomenine hydrochloride, and the drug-loaded system in Example 1 of the present application;

[0059] Figure 4 is a diagram of the specific targeting activation of the biomimetic membrane wrapped Prussian blue composite material loaded with sinomenine hydrochloride on macrophages and rheumatoid arthritis fibroblast-like synoviocytes;

[0060] Figure 5 is a diagram of the uptake of the activated macrophages and rheumatoid arthritis fibroblast-like synoviocytes on the biomimetic membrane wrapped Prussian blue composite material loaded with sinomenine hydrochloride;

[0061] Figure 6 ​The effect diagram of the sinomenine hydrochloride biomimetic nano-targeting drug delivery system for treating rheumatoid arthritis model rats. DETAILED DESCRIPTION

[0062] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Any numerical value, however, can be expressed as a range by either adding to or subtracting from the stated value. In certain instances, a range can be expressed as from about one particular value, to about another particular value, when said range is meant to include one or both of the values.

[0063] As described above, the first aspect of the present application provides a drug delivery system for treating rheumatoid arthritis, comprising sinomenine hydrochloride, Prussian blue, a biomimetic membrane and phosphatidyl hyaluronic acid, the biomimetic membrane being wrapped on the surface of the Prussian blue loaded with the sinomenine hydrochloride, the phosphatidyl hyaluronic acid being embedded on the surface of the biomimetic membrane, the biomimetic membrane comprising red blood cell membrane and macrophage membrane.

[0064] According to the present application, the biomimetic membrane comprises red blood cell membrane and macrophage membrane, that is, the biomimetic membrane is a fusion membrane of red blood cell membrane and macrophage membrane. The phosphatidyl hyaluronic acid is hyaluronic acid treated by phosphatidyl.

[0065] The inventors found in the research process that in the drug delivery system, the biomimetic membrane comprising red blood cell membrane and macrophage membrane is wrapped on the surface of the Prussian blue loaded with sinomenine hydrochloride, and the phosphatidyl hyaluronic acid is embedded on the outside, which can effectively improve the targeting effect of the drug delivery system, further reduce the dosage of sinomenine hydrochloride, and improve the treatment effect on rheumatoid arthritis.

[0066] In order to further improve the treatment effect on rheumatoid arthritis, preferably, the mass ratio of the Prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidyl hyaluronic acid is 1:1-10:0.5-1:0.5-1.

[0067] In order to further improve the treatment effect on rheumatoid arthritis, preferably, the mass ratio of the Prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidyl hyaluronic acid is 1:4-8:0.5-1:0.5-1.

[0068] In order to further improve the treatment effect on rheumatoid arthritis, preferably, the mass ratio of the red blood cell membrane and the macrophage membrane is 1:0.5-1.

[0069] In order to further improve the treatment effect on rheumatoid arthritis, preferably, the Prussian blue is hollow mesoporous Prussian blue.

[0070] Preferably, the preparation method of the hollow mesoporous Prussian blue comprises: dissolving Prussian blue and polyvinylpyrrolidone II in hydrochloric acid II, heating reaction II, then solid-liquid separation II, washing, and drying II. The hollow mesoporous Prussian blue prepared by this method has high porosity, which can increase the loading capacity of sinomenine hydrochloride and the embedding amount of the biological biomimetic membrane, and effectively improve the treatment effect on rheumatoid arthritis.

[0071] Preferably, the mass ratio of the Prussian blue and the polyvinylpyrrolidone II is 1:2-8. Studies have shown that the hollow mesoporous Prussian blue prepared under this condition has a higher loading rate of sinomenine hydrochloride.

[0072] Preferably, the concentration of the hydrochloric acid II is 0.5-2 mol / L.

[0073] Preferably, the heating reaction II is carried out under the following conditions: temperature 140-160℃, time 4-6 hr. Under this reaction condition, the porosity of Prussian blue can be further improved, and the treatment effect on rheumatoid arthritis can be effectively improved.

[0074] In order to further improve the drying effect, preferably, the drying II is vacuum freeze drying. Preferably, the vacuum freeze drying is carried out under the following conditions: temperature -20-4℃, time 12-24 hr.

[0075] As described above, the second aspect of the present application provides a preparation method of a drug-loaded system for treating rheumatoid arthritis, comprising the following steps:

[0076] (1) preparing a Prussian blue dispersion;

[0077] (2) mixing sinomenine hydrochloride and the Prussian blue dispersion to carry out a mixing reaction I, removing impurities, and obtaining a Prussian blue dispersion loaded with sinomenine hydrochloride;

[0078] (3) mixing the Prussian blue dispersion loaded with sinomenine hydrochloride and a biological biomimetic membrane dispersion to carry out a mixing reaction II, solid-liquid separation III, and obtaining a biological biomimetic membrane-wrapped Prussian blue composite material loaded with sinomenine hydrochloride;

[0079] (4) mixing the composite material and phosphatidyl hyaluronic acid to carry out a mixing reaction III, and obtaining the drug-loaded system;

[0080] The preparation method of the biological biomimetic membrane dispersion is: mixing and crushing red blood cell membranes and macrophage membranes in a buffer.

[0081] According to the application, in step (2), the impurity removal method can be any method disclosed in the prior art. Preferably, the impurity removal method is dialysis. As a specific embodiment of the application, the dialysis time is 12-24 hours. In step (3), the solid-liquid separation III can be achieved by centrifugation, filtration or suction filtration. In the preparation method of the biomimetic membrane dispersion, the mixing and crushing can be performed by crushing the red blood cell membrane and the macrophage membrane in a buffer solution respectively, and then mixing the crushed solutions; or by mixing the red blood cell membrane and the macrophage membrane in a buffer solution, and then crushing. The buffer solution can be any buffer solution with high biocompatibility, and specifically can be a phosphate-phosphate buffer solution or a carbonate-hydrochloride buffer solution, with a pH of 7.2-7.4 and a concentration of 0.001-0.01 mol / L of phosphate or carbonate.

[0082] The inventors have found that the drug delivery system prepared by the above method has better targeting effect in the treatment of rheumatoid arthritis, can further reduce the dosage of sinomenine hydrochloride, and improve the treatment effect on rheumatoid arthritis.

[0083] Preferably, in step (1), the preparation method of the Prussian blue dispersion includes: ultrasonic mixing Prussian blue and water to obtain the Prussian blue dispersion.

[0084] In order to further improve the dispersion effect of Prussian blue and improve the loading effect of Prussian blue on sinomenine hydrochloride, preferably, the mass ratio of the Prussian blue to the water is 1:0.05-1.

[0085] In order to further improve the dispersion effect of Prussian blue and improve the loading effect of Prussian blue on sinomenine hydrochloride, preferably, the ultrasonic mixing conditions include: ultrasonic power of 30-100 W and time of 3-15 min.

[0086] In order to further improve the treatment effect on rheumatoid arthritis, preferably, the Prussian blue is hollow mesoporous Prussian blue.

[0087] Preferably, the preparation method of the hollow mesoporous Prussian blue includes: dissolving Prussian blue and polyvinylpyrrolidone II in hydrochloric acid II, heating reaction II, and then performing solid-liquid separation II, washing, and drying II. The hollow mesoporous Prussian blue prepared by the method has high porosity, can improve the loading amount of sinomenine hydrochloride and the embedding amount of the biomimetic membrane, and effectively improves the treatment effect on rheumatoid arthritis.

[0088] Preferably, the mass ratio of the Prussian blue and the polyvinylpyrrolidone II is 1:2-8. Studies have shown that the hollow mesoporous Prussian blue prepared under this condition has a higher loading rate of sinomenine hydrochloride.

[0089] Preferably, the concentration of the hydrochloric acid II is 0.5-2 mol / L.

[0090] Preferably, the conditions of the heating reaction II include a temperature of 140-160℃ and a time of 4-6 hr. Under this reaction condition, the porosity of the Prussian blue can be further improved, and the treatment effect on rheumatoid arthritis can be effectively improved.

[0091] In order to further improve the drying effect, preferably, the drying II is vacuum freeze drying. Preferably, the conditions of the vacuum freeze drying include a temperature of -20-4℃ and a time of 12-24 hr.

[0092] The Prussian blue can be commercially available or prepared. Preferably, the preparation method of the Prussian blue includes: dissolving potassium ferricyanide and polyvinylpyrrolidone I in hydrochloric acid I, heating reaction I, and then solid-liquid separation I, washing, and drying I.

[0093] In order to further improve the reaction effect, preferably, the mass ratio of the potassium ferricyanide and the polyvinylpyrrolidone I is 1:5-18.

[0094] Preferably, the concentration of the hydrochloric acid I is 0.005-0.02 mol / L.

[0095] Preferably, the conditions of the heating reaction I include a temperature of 60-100℃ and a time of 18-22 hr.

[0096] Preferably, the drying I and the drying II are vacuum freeze drying, and the conditions of the vacuum freeze drying include a temperature of -20-4℃ and a time of 12-24 hr.

[0097] Under the above reaction conditions, the potassium ferricyanide and the polyvinylpyrrolidone I have a good reaction effect, and the generation rate of the Prussian blue is higher.

[0098] Preferably, the solid-liquid separation I and the solid-liquid separation II are centrifugal separation, and the conditions of the centrifugal separation include a rotation speed of 10000-13000 rpm and a time of 10-20 min. The structure can be ensured while having a good separation effect, so as to ensure the loading effect.

[0099] Preferably, in step (2), the mixing reaction I comprises stirring the mixture of the sinomenine hydrochloride and the prussian blue dispersion. The stirring can effectively increase the loading capacity of sinomenine hydrochloride in the prussian blue.

[0100] Preferably, in the mixing reaction I, the stirring condition comprises a rotation speed of 300-800 rpm and a time of 10-12 hours.

[0101] In order to further reduce the use of sinomenine hydrochloride and improve the therapeutic effect on rheumatoid arthritis, preferably, in step (3), the mixing reaction II comprises stirring the mixture of the sinomenine hydrochloride-loaded prussian blue dispersion and the biomimetic membrane dispersion.

[0102] Preferably, in the mixing reaction II, the stirring condition comprises a rotation speed of 300-800 rpm and a time of 2-4 hours. Studies have shown that better reaction results can be achieved under this stirring condition.

[0103] Preferably, in step (3), the solid-liquid separation III is performed by centrifugal separation.

[0104] Preferably, the centrifugal separation condition comprises a rotation speed of 10,000-13,000 rpm and a time of 10-20 minutes.

[0105] Preferably, in step (4), the preparation method of the phosphatidylated hyaluronic acid comprises dissolving hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and distearoylphosphatidyl ethanolamine-polyethylene glycol-amino in a phosphate buffer and performing a stirring reaction to phosphatidylate the hyaluronic acid, and freeze-drying. Studies have shown that the drug delivery system prepared from the phosphatidylated hyaluronic acid prepared by this method has better targeting effect, which can further improve the therapeutic effect on rheumatoid arthritis.

[0106] In order to further improve the therapeutic effect on rheumatoid arthritis, preferably, in the preparation method of the phosphatidylated hyaluronic acid, the mass ratio of the hyaluronic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the N-hydroxysuccinimide, and the distearoylphosphatidyl ethanolamine-polyethylene glycol-amino is 1:2-8:5-15:2-8.

[0107] The phosphate buffer is a phosphate-phosphate buffer. In order to further improve the targeting effect of the prepared drug delivery system, preferably, in the preparation method of the phosphatidylated hyaluronic acid, the pH of the phosphate buffer is 7.2-7.4.

[0108] Preferably, in the preparation method of the phosphatidylated hyaluronic acid, the stirring reaction conditions include: rotation speed of 300-800 rpm, and reaction time of 2-24 hr. Studies have shown that the phosphatidylated hyaluronic acid prepared under the conditions can improve the targeting effect of the drug delivery system.

[0109] Preferably, in the preparation method of the phosphatidylated hyaluronic acid, the freeze-drying conditions include: temperature of -20-4 ℃, and time of 12-24 hr.

[0110] Preferably, in step (4), the mixing reaction III includes: stirring the composite material and the phosphatidylated hyaluronic acid. The reaction efficiency of the composite material and the phosphatidylated hyaluronic acid can be improved, thereby improving the targeting effect of the drug delivery system.

[0111] Preferably, in the mixing reaction III, the stirring conditions include: rotation speed of 300-800 rpm, temperature of 30-37 ℃, and time of 2-4 hr.

[0112] In order to further improve the therapeutic effect of the prepared system on rheumatoid arthritis, preferably, the mass ratio of the Prussian blue, the sinomenine hydrochloride, the biomimetic membrane, and the phosphatidylated hyaluronic acid is 1:1-10:0.5-1:0.5-1.

[0113] In order to further improve the therapeutic effect of the prepared system on rheumatoid arthritis, preferably, the mass ratio of the Prussian blue, the sinomenine hydrochloride, the biomimetic membrane, and the phosphatidylated hyaluronic acid is 1:4-8:0.5-1:0.5-1.

[0114] In order to further improve the therapeutic effect of the prepared system on rheumatoid arthritis, preferably, in the preparation method of the biomimetic membrane dispersion, the mass ratio of the red blood cell membrane and the macrophage membrane is 1:0.5-1.

[0115] In order to further improve the fusion effect of the red blood cell membrane and the macrophage, preferably, in the preparation method of the biomimetic membrane dispersion, the mixed crushing is ultrasonic crushing.

[0116] Preferably, the ultrasonic crushing conditions include: power of 50-120 W, and time of 1-3 min.

[0117] In order to further improve the fusion effect of the red blood cell membrane and the macrophage, preferably, in the preparation method of the biomimetic membrane dispersion, the mixed crushed solid mixture is further stirred.

[0118] Preferably, the stirring conditions include a rotation speed of 300-800 rpm, a temperature of 30-37℃, and a time of 2-3 hr.

[0119] As described above, the third aspect of the present application provides a use of the drug-loaded system for treating rheumatoid arthritis according to the first aspect or the drug-loaded system for treating rheumatoid arthritis prepared by the preparation method according to the second aspect in the preparation of an anti-rheumatoid arthritis therapeutic drug. The drug-loaded system has good therapeutic effect on rheumatoid arthritis and can effectively reduce the use amount of sinomenine hydrochloride.

[0120] According to a particularly preferred embodiment of the present application, a preparation method of a drug-loaded system for treating rheumatoid arthritis is provided, which comprises the following steps:

[0121] (1) Dissolve potassium ferricyanide and polyvinylpyrrolidone in hydrochloric acid with a concentration of 0.005-0.02 mol / L at a mass ratio of 1:5-18, heat and react at 60-100℃ for 18-22 hr, centrifuge, wash, freeze and vacuum dry to obtain Prussian blue; dissolve the obtained Prussian blue and polyvinylpyrrolidone in hydrochloric acid with a concentration of 0.5-2 mol / L at a mass ratio of 1:2-8, high-heat react in a reaction kettle at 140-160℃ for 4-6 hr, centrifuge, wash, freeze and vacuum dry to obtain hollow mesoporous Prussian blue, and disperse with water (the mass ratio of hollow mesoporous Prussian blue to water is 1:0.05-1) to obtain a Prussian blue dispersion;

[0122] (2) Mix sinomenine hydrochloride with the Prussian blue dispersion to obtain a mixed solution, stir the mixed solution at 300-800 rpm for 10-12 hr, remove impurities by dialysis, and obtain a Prussian blue dispersion loaded with sinomenine hydrochloride;

[0123] Mix red blood cell membranes and macrophage membranes in a phosphate-phosphate buffer solution with a pH of 7.2-7.4 and ultrasonically pulverize (power: 50-120 W, time: 1-2 min), and stir the pulverized solid mixture (rotation speed: 300-800 rpm, time: 2-3 hr) to obtain a biomimetic membrane dispersion;

[0124] (3) Mix the Prussian blue dispersion loaded with sinomenine hydrochloride and the biomimetic membrane dispersion to obtain a mixed solution, stir the mixed solution at 300-800 rpm for 2-4 hr, centrifuge, and obtain a biomimetic membrane-wrapped Prussian blue composite material loaded with sinomenine hydrochloride;

[0125] (4) dissolving hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide in a phosphate-phosphate buffer solution with pH of 7.2-7.4, stirring for 20-40 min, then dissolving distearoylphosphatidyl ethanolamine-polyethylene glycol-amino in the above solution and continuing to stir for 2-24 hr, dialysis, to obtain phosphatidylated hyaluronic acid, the mass ratio of the hyaluronic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the N-hydroxysuccinimide and the distearoylphosphatidyl ethanolamine-polyethylene glycol-amino being 1:2-8:5-15:2-8;

[0126] mixing the composite material and the phosphatidylated hyaluronic acid to obtain a mixed solution, stirring the mixed solution at 300-800 rpm for 2-4 hr to obtain the drug-loaded system;

[0127] wherein the mass ratio of the sinomenine hydrochloride, the Prussian blue, the biomimetic membrane and the phosphatidylated hyaluronic acid is 1:4-8:0.5-1:0.5-1, and the mass ratio of the red blood cell membrane and the macrophage membrane is 1:0.5-1.

[0128] The application will be described in detail below through examples. In the following examples, the sinomenine hydrochloride is provided by Hunan Zhengqing Pharmaceutical Group, with product number YK-0509; the heat-inactivated Mycobacterium tuberculosis H37Ra is purchased from the United States BD Company, with product number 8138819; the mineral oil is purchased from the United States Sigma Aldric Company, with product number M8410; the macrophages, normal fibroblast-like synoviocytes and rheumatoid arthritis fibroblast-like synoviocytes are purchased from the China Academy of Sciences Cell Bank. The fluorescence spectrophotometer is purchased from the Japan Hitachi Company, with instrument model number FL-2500; the transmission electron microscope is purchased from the Japan Electronic (JEOL) official website, with instrument model number JEM-2100F; the ultraviolet-visible spectrophotometer is purchased from the United States Beckman Coulter Company, with instrument model number DU800. The ultrasonic cell disrupter is purchased from Nanjing Saifei Biological Technology Co., Ltd., with model number Biosafer 900-92; the laser confocal scanning microscope is purchased from the Japan Olympus, with model number FV1200; the small animal live imaging system is purchased from the United States PerkinElmer, with model number Lumina xr; the small animal gas anesthesia machine is purchased from the United States Surgivet, with model number SurgiVet CDS9000; the paw swelling tester is purchased from the Italy UGO Basile, with model number 37140; the microsyringe is purchased from the United States Hamilton Company, with model number 250 μL; the PBS has a pH of 7.2-7.4. Others are all common commercially available products.

[0129] Example 1

[0130] (1) Potassium ferricyanide and polyvinylpyrrolidone were dissolved in hydrochloric acid with a concentration of 0.01 mol / L at a mass ratio of 1:11, and the reaction was carried out at 80°C under oil bath heating for 20 hours. The product solution obtained by the reaction was centrifuged at a speed of 12000 rpm for 15 minutes, washed with water three times, and freeze-dried under vacuum to obtain Prussian blue. The obtained Prussian blue and polyvinylpyrrolidone were dissolved in hydrochloric acid with a concentration of 1 mol / L at a mass ratio of 1:5, and the reaction was carried out in a reaction kettle at 140°C for 4 hours. Then, the product solution obtained by the reaction was centrifuged at a speed of 12000 rpm for 15 minutes, washed with water three times, and freeze-dried under vacuum to obtain hollow mesoporous Prussian blue. Water was added to disperse the hollow mesoporous Prussian blue (the mass ratio of hollow mesoporous Prussian blue to water was 1:0.1) to obtain a Prussian blue dispersion (PB);

[0131] (2) A mixture solution was obtained by mixing the sinomenine hydrochloride aqueous solution and the Prussian blue dispersion, wherein the mass ratio of sinomenine hydrochloride to hollow mesoporous Prussian blue was 6:1. The mixture solution was stirred at a speed of 600 rpm for 12 hours, and then dialyzed in a dialysis bag (MWCO = 1 kDa) for 24 hours to obtain a Prussian blue dispersion loaded with sinomenine hydrochloride (PB@SIN);

[0132] Fresh blood of SD rats was centrifuged at 4°C and a speed of 2000 rpm for 10 minutes, and the precipitate was washed with PBS multiple times. Then, 0.25*PBS was mixed with the precipitate and placed on ice for 2 hours. The second layer solution was obtained by centrifuging at 12000 rpm and 4°C for 5 minutes to obtain red blood cell membrane RBCm. The macrophage membrane was prepared by using a membrane protein extraction kit The macrophage membrane was resuspended in membrane extraction reagent A (containing 1% PMSF by mass); after being placed on ice for 1 hour, it was repeatedly frozen and thawed 5 times at -80°C and 37°C, each for 30 minutes. The macrophage membrane was obtained by centrifuging at 12000 rpm and 4°C for 30 minutes. The two were mixed at a weight ratio of 1:1, ultrasonicated on ice for 2 minutes (power 80W), and stirred at a speed of 600 rpm for 2 hours to obtain a biomimetic membrane dispersion (M);

[0133] (3) A mixture solution was obtained by mixing the Prussian blue dispersion loaded with sinomenine hydrochloride and the biomimetic membrane dispersion, wherein the mass ratio of the biomimetic membrane to the hollow mesoporous Prussian blue was 1:1. The mixture solution was stirred at a speed of 600 rpm for 2 hours, and then centrifuged to obtain a biomimetic membrane-wrapped Prussian blue composite material loaded with sinomenine hydrochloride (M@PB@SIN);

[0134] (4) 5 mg of hyaluronic acid, 25 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 50 mg of N-hydroxysuccinimide were dissolved in 1 ml of PBS solution, stirred at room temperature for 30 min, 25 mg of distearoylphosphatidyl ethanolamine-polyethylene glycol-amino was added and stirred at 37°C for 30 min, and then stirred at room temperature for 24 hr, the obtained solution was dialyzed in a dialysis bag (MWCO = 2.5 kDa) for 24 hr, and then freeze-dried to obtain phosphatidylated hyaluronic acid (HA);

[0135] The composite material and the phosphatidylated hyaluronic acid were mixed to obtain a mixed solution, wherein the mass ratio of the composite material to the phosphatidylated hyaluronic acid was 1:0.5 based on the hollow mesoporous Prussian blue, the mixed solution was stirred at 600 rpm for 2 hr to obtain the drug-loaded system (HA@M@PB@SIN);

[0136] The mass ratio of the hollow mesoporous Prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidylated hyaluronic acid was 1:6:1:0.5.

[0137] Example 2

[0138] (1) Potassium ferricyanide and polyvinylpyrrolidone were dissolved in hydrochloric acid with a concentration of 0.005 mol / L at a mass ratio of 1:5, and the reaction was carried out in an oil bath at 60°C for 22 hr, then the product solution obtained by the reaction was centrifuged at a speed of 10000 rpm for 20 min, washed with water for three times, and freeze-dried under vacuum to obtain Prussian blue; the obtained Prussian blue and polyvinylpyrrolidone were dissolved in hydrochloric acid with a concentration of 0.5 mol / L at a mass ratio of 1:2, and the reaction was carried out in a reaction kettle at 140°C for 4 hr, then the product solution obtained by the reaction was centrifuged at a speed of 10000 rpm for 20 min, washed with water for three times, and freeze-dried under vacuum to obtain hollow mesoporous Prussian blue, which was dispersed in water by ultrasonic dispersion (wherein the mass ratio of the hollow mesoporous Prussian blue to water was 1:0.05) to obtain a Prussian blue dispersion (PB);

[0139] (2) A sinomenine hydrochloride aqueous solution and the Prussian blue dispersion were mixed to obtain a mixed solution, wherein the mass ratio of the sinomenine hydrochloride to the hollow mesoporous Prussian blue was 1:1, the mixed solution was stirred at 300 rpm for 12 hr, and then dialyzed in a dialysis bag (MWCO = 1 kDa) for 18 hr to obtain a Prussian blue dispersion loaded with sinomenine hydrochloride (PB@SIN);

[0140] Fresh blood of SD rats was centrifuged at 4℃ and 2000 rpm for 10 min, and the precipitate was washed with PBS for multiple times; then, 0.25* PBS was mixed with the precipitate and placed on ice for 2 hr; the second layer solution was obtained by centrifuging at 12000 rpm and 4℃ for 5 min to obtain red blood cell membrane RBCm; the membrane protein extraction kit was used to prepare macrophage membrane The macrophage cells were resuspended in the membrane extraction reagent A (containing 1 mass% PMSF); after being placed on ice for 1 hr, the repeated freezing and thawing was performed for 5 times at-80℃ and 37℃ for 30 min each time; the macrophage membrane was obtained by centrifuging at 12000 rpm and 4℃ for 30 min; the mixture of the two was ultrasonicated on ice for 2 min (power 50 W) at a weight ratio of 1:0.5, and mixed and stirred at 600 rpm for 2 hr to obtain the biomimetic membrane dispersion (M);

[0141] (3) The biomimetic membrane dispersion loaded with sinomenine hydrochloride was obtained by mixing the biomimetic membrane dispersion and the prussian blue dispersion loaded with sinomenine hydrochloride, wherein the mass ratio of the hollow mesoporous prussian blue and the biomimetic membrane was 1:0.5; the mixture was stirred at 300 rpm for 4 hr, and then centrifuged to obtain the biomimetic membrane coated prussian blue composite material loaded with sinomenine hydrochloride (M@PB@SIN);

[0142] (4) 5 mg of hyaluronic acid, 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 25 mg of N-hydroxysuccinimide were dissolved in 1 ml of PBS solution, and stirred at room temperature for 30 min; 10 mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol-amino was added and stirred at 37℃ for 30 min, and then stirred at room temperature for 12 hr; the obtained solution was dialyzed in a dialysis bag (MWCO=2.5 kDa) for 24 hr, and then freeze-dried to obtain phosphatidyl hyaluronic acid (HA);

[0143] The composite material and the phosphatidyl hyaluronic acid were mixed to obtain a mixture, wherein the mass ratio of the composite material and the phosphatidyl hyaluronic acid was 1:0.5 in terms of the hollow mesoporous prussian blue; the mixture was stirred at 300 rpm for 4 hr to obtain the drug-loaded system (HA@M@PB@SIN);

[0144] The mass ratio of the hollow mesoporous prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidyl hyaluronic acid was 1:1:0.5:0.5.

[0145] Example 3

[0146] (1) Potassium ferricyanide and polyvinylpyrrolidone were dissolved in 0.02 mol / L hydrochloric acid at a mass ratio of 1:18. The mixture was heated in an oil bath at 100°C for 18 hours. The resulting product solution was centrifuged at 12,000 rpm for 15 minutes, washed three times with water, and then freeze-dried under vacuum to obtain Prussian blue. The obtained Prussian blue and polyvinylpyrrolidone were dissolved in 1 mol / L concentrated hydrochloric acid at a mass ratio of 1:8. The mixture was heated in a reaction vessel at 160°C for 6 hours. The resulting product solution was centrifuged at 12,000 rpm for 15 minutes, washed three times with water, and then freeze-dried under vacuum to obtain hollow mesoporous Prussian blue. Water was added and ultrasonically dispersed (the mass ratio of hollow mesoporous Prussian blue to water was 1:1) to obtain Prussian blue dispersion (PB).

[0147] (2) The aqueous solution of sinomenine hydrochloride and the Prussian blue dispersion were mixed to obtain a mixture, wherein the mass ratio of hollow mesoporous Prussian blue and sinomenine hydrochloride was 1:10. The mixture was stirred at 600 rpm for 12 hours and dialyzed in a dialysis bag (MWCO = 1 kDa) for 24 hours to obtain a Prussian blue dispersion loaded with sinomenine hydrochloride (PB@SIN).

[0148] Fresh blood from SD rats was centrifuged at 2000 rpm for 10 min at 4 °C, and the precipitate was washed repeatedly with PBS. Then, 0.25×PBS was mixed with the precipitate and placed on ice for 2 h. The mixture was centrifuged at 12000 rpm at 4 °C for 5 min, and the second layer was collected to obtain erythrocyte membranes (RBCs). Macrophage membranes were prepared using a membrane protein extraction kit. Macrophages were resuspended in membrane extraction reagent A (containing 1% PMSF by mass); after being placed on ice for 1 hour, they were repeatedly frozen and thawed 5 times at -80°C and 37°C for 30 minutes each time; macrophage membranes were obtained by centrifugation at 12000 rpm and 4°C for 30 minutes; the mixture of the two at a weight ratio of 1:1 was sonicated on ice for 2 minutes (power of 120W) and stirred at 600 rpm for 2 hours to obtain biomimetic membrane dispersion (M).

[0149] (3) The Prussian blue dispersion loaded with sinomenine hydrochloride and the biomimetic membrane dispersion were mixed to obtain a mixture, wherein the mass ratio of the biomimetic membrane and the hollow mesoporous Prussian blue was 1:1. The mixture was stirred at 600 rpm for 2 hours and centrifuged to obtain the Prussian blue composite material loaded with sinomenine hydrochloride encapsulated by the biomimetic membrane (M@PB@SIN).

[0150] (4) 5 mg of hyaluronic acid, 40 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 75 mg of N-hydroxysuccinimide were dissolved in 1 ml of PBS solution, stirred at room temperature for 30 min, 40 mg of distearoylphosphatidyl ethanolamine-polyethylene glycol-amino was added and stirred at 37°C for 30 min, then stirred at room temperature for 12 hr, the obtained solution was dialyzed in a dialysis bag (MWCO = 2.5 kDa) for 24 hr, then freeze-dried to obtain phosphatidylated hyaluronic acid (HA);

[0151] The composite material and the phosphatidylated hyaluronic acid were mixed to obtain a mixed solution, wherein the mass ratio of the composite material to the phosphatidylated hyaluronic acid was 1:1 based on the hollow mesoporous prussian blue, the mixed solution was stirred at 300 rpm for 4 hr to obtain the drug-loaded system (HA@M@PB@SIN);

[0152] The mass ratio of the hollow mesoporous prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidylated hyaluronic acid was 1:10:1:1.

[0153] Example 4

[0154] The drug-loaded system was prepared according to the method of Example 1, except that step (1) comprised:

[0155] Potassium ferricyanide and polyvinylpyrrolidone were dissolved in hydrochloric acid with a concentration of 0.005 mol / L at a mass ratio of 1:5, and the reaction was carried out at 60°C under oil bath heating for 22 hr, then the product solution obtained by the reaction was centrifuged at a speed of 10000 rpm for 20 min, washed with water for three times, and freeze-dried under vacuum to obtain prussian blue, which was dispersed in water (the mass ratio of prussian blue to water was 1:0.5) to obtain a prussian blue dispersion (PB).

[0156] Comparative Example 1

[0157] The drug-loaded system was prepared according to the method of Example 1, except that step (2) comprised:

[0158] The sinomenine hydrochloride aqueous solution (6 mg / mL) and the prussian blue dispersion were mixed to obtain a mixed solution, which was stirred at 600 rpm for 12 hr, and then dialyzed in a dialysis bag (MWCO = 1 kDa) for 24 hr to obtain a prussian blue dispersion loaded with sinomenine hydrochloride (PB@SIN);

[0159] Fresh blood of SD rats was centrifuged at 4℃ and 2000 rpm for 10 min, and the precipitate was washed with PBS for several times; then, 0.25x PBS was mixed with the precipitate and placed on ice for 2 hr; centrifugation was performed at 12000 rpm and 4℃ for 5 min, and the second layer solution was taken to obtain red blood cell membranes (RBCm); a membrane protein extraction kit was used to prepare rheumatoid arthritis fibroblast-like synoviocyte membranes. The rheumatoid arthritis fibroblast-like synoviocyte membranes were resuspended in membrane extraction reagent A (containing 1% PMSF by mass); after being placed on ice for 1 hr, the membranes were repeatedly frozen and thawed at-80℃ and 37℃ for 5 times, each for 30 min; centrifugation was performed at 12000 rpm and 4℃ for 30 min to obtain rheumatoid arthritis fibroblast-like synoviocyte membranes; the two were mixed in a weight ratio of 1:1, and the mixture was ultrasonicated on ice for 2 min (power 80 W) and mixed and stirred at 600 rpm for 2 hr to obtain a cell biomimetic membrane dispersion (1 mg / mL) of red blood cell membranes and rheumatoid arthritis fibroblast-like synoviocyte membranes fused with each other (RFM).

[0160] Step (3) comprises mixing the PB@SIN dispersion and the RFM dispersion to obtain a mixed solution, wherein the mass ratio of the RFM biomimetic membrane and the hollow mesoporous Prussian blue is 1:1, the mixed solution is stirred at 600 rpm for 2 hr, and centrifugation is performed to obtain a RFM biomimetic membrane-wrapped Prussian blue composite material loaded with sinomenine hydrochloride (RFM@PB@SIN).

[0161] Step (4) comprises dissolving 5 mg of hyaluronic acid, 40 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 75 mg of N-hydroxysuccinimide in 1 ml of a PBS solution, stirring at room temperature for 30 min, adding 40 mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol-amino, stirring at 37℃ for 30 min, and then stirring at room temperature for 12 hr; the obtained solution is dialyzed in a dialysis bag (MWCO = 2.5 kDa) for 24 hr, and then freeze-dried to obtain phosphatidylated hyaluronic acid (HA).

[0162] The composite material and the phosphatidylated hyaluronic acid are mixed to obtain a mixed solution, wherein the mass ratio of the composite material and the phosphatidylated hyaluronic acid, in terms of the hollow mesoporous Prussian blue, is 1:1, the mixed solution is stirred at 300 rpm for 4 hr, and the drug-loaded system (HA@RFM@PB@SIN) is obtained.

[0163] Test Example

[0164] According to the method of Example 1, the drug-loaded system prepared in Example 1 is subjected to transmission electron microscope imaging analysis, and the results are shown in FIG. 2, which shows that the uniformly dispersed square hollow mesoporous Prussian blue is successfully prepared, and a biomimetic membrane outer layer of about 5 nm appears after the biomimetic membrane is disguised. Figure 1 ​

[0165] According to the method of Example 1, the mass ratio of red blood cell membrane and macrophage membrane was adjusted, and then the fusion was performed Energy resonance transfer analysis showed that the red blood cell membrane and macrophage membrane were successfully fused, as shown in Figure 2

[0166] The prussian blue dispersion liquid, the prussian blue nanomaterial loaded with sinomenine hydrochloride and the drug-loaded system prepared in Example 1 were analyzed by ultraviolet-visible spectrophotometry, and the ultraviolet absorption spectrum curves were obtained as shown in Figure 3 From the figure, it can be seen that the characteristic absorption peak of prussian blue is located near 710 nm. After prussian blue loaded sinomenine hydrochloride, the ultraviolet absorption appeared at 262 nm, which is the characteristic absorption peak of sinomenine hydrochloride. Finally, the drug-loaded system appeared the ultraviolet characteristic absorption peak of biological biomimetic membrane at 405 nm.

[0167] In step (3) of Example 1 and Comparative Example 1, the biological biomimetic membrane wrapped prussian blue composite material loaded with sinomenine hydrochloride was collected, and then it was stirred with fluorescent molecules rhodamine according to the mass ratio of 1:4, so that the rhodamine was stacked on the surface of the composite material. The content of the composite material loaded with sinomenine hydrochloride taken up by cells was determined by semi-quantitative means of detecting fluorescence intensity. The biological biomimetic membrane wrapped prussian blue composite material loaded with sinomenine hydrochloride (10 μg / ml) was co-cultured with normal macrophages, normal fibroblast synoviocytes (HFLS), activated macrophages induced by lipopolysaccharide (LPS, 100 ng / ml) and rheumatoid arthritis fibroblast synoviocytes (RA-HFLS) at 37°C, 5% CO2 for 4 hr. The results are shown in Figure 4 Compared with normal macrophages and normal HFLS respectively, the average fluorescence intensity of activated macrophages and RA-HFLS cells was significantly enhanced; the results showed that the drug-loaded system could specifically enhance the uptake of inflammatory macrophages and rheumatoid arthritis synoviocyte fibroblasts, thereby inhibiting the inflammatory response.

[0168] ​In Example 1 and Comparative Example 1, the biomimetic membrane (M) or the biomimetic membrane (RFM) fused with rheumatoid arthritis fibroblast-like synoviocytes was collected, and then the biomimetic membrane (M) or the biomimetic membrane (RFM) loaded with sinomenine hydrochloride and prussian blue composite material was stirred uniformly with fluorescent molecules rhodamine at a mass ratio of 1:4, so that the rhodamine was stacked on the surface of the composite material, and the content of the composite material loaded with sinomenine hydrochloride was determined by detecting the fluorescence intensity in a semi-quantitative manner. The M loaded with sinomenine hydrochloride and prussian blue composite material (M@PB@SIN) or the RFM loaded with sinomenine hydrochloride and prussian blue composite material (RFM@PB@SIN) (10 μg / ml) was co-cultured with LPS (100 ng / ml) induced activated macrophages and RA-HFLS at 37°C in 5% CO2 for 4 hours. The results are shown in Figure 5 Compared with the RFM@PB@SIN nanosystem, the M@PB@SIN nanosystem significantly enhanced the average fluorescence intensity of activated macrophages and RA-HFLS cells. The results showed that the sinomenine hydrochloride biomimetic nanotargeted drug delivery system could simultaneously target inflammatory macrophages and rheumatoid arthritis synoviocyte fibroblasts, thereby inhibiting inflammation.

[0169] A rheumatoid arthritis animal model was established, and the prussian blue dispersion liquid (2 mg / kg), the sinomenine hydrochloride loaded prussian blue dispersion liquid (containing SIN 3 mg / kg, PB 2 mg / kg), the sinomenine hydrochloride biomimetic nanotargeted drug delivery system (containing SIN 3 mg / kg, PB 2 mg / kg), and the single SIN (3 mg / kg) and methotrexate (MIX 1 mg / kg) were used to treat the rheumatoid arthritis model rats. As shown in Figure 6 Compared with the model group (Model) rats, the single SIN group rats showed no significant improvement in the volume of the hind paw and the arthritis score, while the sinomenine hydrochloride biomimetic nanotargeted drug delivery system group (HA@M@PB@SIN) rats showed significant reduction in the volume of the hind paw and the arthritis score, and tended to be normal with time. The results showed that the sinomenine hydrochloride biomimetic nanotargeted drug delivery system could significantly improve the effectiveness of rheumatoid arthritis treatment.

[0170] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A drug delivery system for the treatment of rheumatoid arthritis, characterized in that The application relates to a drug delivery system, which comprises sinomenine hydrochloride, prussian blue, a biomimetic membrane and phosphatidyl hyaluronic acid, wherein the biomimetic membrane is wrapped around the prussian blue loaded with sinomenine hydrochloride, the phosphatidyl hyaluronic acid is embedded on the surface of the biomimetic membrane, the biomimetic membrane comprises red blood cell membranes and macrophage membranes, and the mass ratio of the prussian blue, the sinomenine hydrochloride, the biomimetic membrane and the phosphatidyl hyaluronic acid is 1:1-10:0.5-1:0.5-1, and the mass ratio of the red blood cell membranes and the macrophage membranes is 1:0.5-1.

2. The drug-loaded system for the treatment of rheumatoid arthritis according to claim 1, characterized in that, The prussian blue is hollow mesoporous prussian blue.

3. The drug-loaded system for use in the treatment of rheumatoid arthritis according to claim 2, characterized in that, The preparation method of the hollow mesoporous prussian blue comprises the following steps: dissolving prussian blue and polyvinylpyrrolidone II in hydrochloric acid II, heating reaction II, and then performing solid-liquid separation II, washing and drying II.

4. The drug-loaded system for use in the treatment of rheumatoid arthritis according to claim 3, characterized in that, The mass ratio of the prussian blue and the polyvinylpyrrolidone II is 1:2-8. The concentration of the hydrochloric acid II is 0.5-2 mol / L. The heating reaction II is performed under the following conditions: a temperature of 140-160 DEG C and a time of 4-6 hours. The drying II is vacuum freeze drying, and the vacuum freeze drying is performed under the following conditions: a temperature of -20-4 DEG C and a time of 12-24 hours.

5. A method for preparing the drug-loaded system for treating rheumatoid arthritis according to any one of claims 1 to 4, characterized by, The application further discloses a preparation method of the drug delivery system. The preparation method comprises the following steps: (1) preparing a prussian blue dispersion liquid; (2) mixing sinomenine hydrochloride and the prussian blue dispersion liquid to perform a mixing reaction I, removing impurities, and obtaining a prussian blue dispersion liquid loaded with sinomenine hydrochloride; (3) mixing the prussian blue dispersion liquid loaded with sinomenine hydrochloride and a biomimetic membrane dispersion liquid to perform a mixing reaction II, performing solid-liquid separation III, and obtaining a biomimetic membrane wrapped prussian blue composite material loaded with sinomenine hydrochloride; (4) mixing the composite material and phosphatidyl hyaluronic acid to perform a mixing reaction III, and obtaining the drug delivery system.

6. The preparation method according to claim 5, characterized in that, The preparation method of the biomimetic membrane dispersion liquid comprises the following steps: mixing and crushing red blood cell membranes and macrophage membranes in a buffer solution. In step (1), the preparation method of the prussian blue dispersion liquid comprises the following steps: ultrasonically mixing prussian blue and water to obtain the prussian blue dispersion liquid. The mass ratio of the prussian blue and the water is 1:0.05-1.

7. The production method according to claim 5 or 6, characterized by, The ultrasonic mixing is performed under the following conditions: an ultrasonic power of 30-100 W and a time of 3-15 minutes. In step (2), the mixing reaction I comprises the following steps: stirring the mixed liquid of the sinomenine hydrochloride and the prussian blue dispersion liquid. The stirring is performed under the following conditions: a rotating speed of 300-800 rpm and a time of 10-12 hours. The method for removing impurities is dialysis, and the dialysis time is 12-24 hours. In step (3), the mixing reaction II comprises the following steps: stirring the mixed liquid of the prussian blue dispersion liquid loaded with sinomenine hydrochloride and the biomimetic membrane dispersion liquid. The stirring is performed under the following conditions: a rotating speed of 300-800 rpm, a temperature of 30-37 DEG C and a time of 2-4 hours. The solid-liquid separation III is performed by centrifugal separation, and the centrifugal separation is performed under the following conditions: a rotating speed of 10000-13000 rpm and a time of 10-20 minutes.

8. The production method according to claim 5 or 6, characterized by, In step (4), the preparation method of the phospholipidized hyaluronic acid comprises: dissolving hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and distearoylphosphatidyl ethanolamine-polyethylene glycol-amino in a phosphate buffer and stirring to react, and freeze-drying.

9. The production method according to claim 8, characterized by, The mass ratio of the hyaluronic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the N-hydroxysuccinimide and the distearoylphosphatidyl ethanolamine-polyethylene glycol-amino is 1:2-8:5-15:2-8; The stirring reaction condition comprises: a rotation speed of 300-800 rpm and a time of 2-24 hr; In step (4), the mixing reaction III comprises: stirring the composite material and the phospholipidized hyaluronic acid; The stirring condition comprises: a rotation speed of 300-800 rpm and a time of 2-4 hr.

10. The production method according to claim 5 or 6, characterized by, In the preparation method of the biomimetic membrane dispersion liquid, the mixed crushing is ultrasonic crushing; The ultrasonic crushing condition comprises: a power of 50-120 W and a time of 1-3 min; In the preparation method of the biomimetic membrane dispersion liquid, the mixed crushing further comprises: stirring the solid mixture after the mixed crushing; The stirring condition comprises: a rotation speed of 300-800 rpm and a time of 2-3 hr.

11. Use of the drug-loaded system for treating rheumatoid arthritis according to any one of claims 1 to 4 or the drug-loaded system for treating rheumatoid arthritis prepared by the preparation method according to any one of claims 5 to 10 in the preparation of an anti-rheumatoid arthritis therapeutic drug.

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