A drug delivery system for treating rheumatoid arthritis and its preparation method and application

Abstract

The present invention relates to the field of targeted drugs, and discloses a drug delivery system for treating rheumatoid arthritis, a preparation method thereof, and an application thereof. The drug delivery system comprises PLGA, halofuginone, a biomimetic hybrid membrane, and phospholipidated hyaluronic acid, wherein the biomimetic hybrid membrane is wrapped on the surface of the PLGA loaded with the halofuginone, and the phospholipidated hyaluronic acid is embedded on the surface of the biomimetic hybrid membrane. The drug delivery system for treating rheumatoid arthritis provided by the present invention not only has a higher encapsulation rate and drug loading amount, but also has a lower preparation cost, and also has excellent biocompatibility and biosafety, and has a more excellent anti-rheumatoid arthritis effect.

Description

Technical Field

[0001] The present invention relates to the field of targeted drugs, and in particular to a drug-carrying system for treating rheumatoid arthritis, and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic autoimmune inflammatory disease characterized by chronic synovial inflammation, cartilage and bone erosion, and ultimately joint structural destruction. Although effective treatments for RA have gradually evolved from nonsteroidal anti-inflammatory drugs to modern biological agents, the clinical remission rate of RA in my country is actually low, and more than one-third of patients discontinue treatment because they cannot tolerate the side effects of Western medicine. There is still a lack of ideal treatment drugs and methods.

[0003] Halofuginone (HF) is a derivative of febrifugine, an extract of Dichroa febrifuga Lour., a traditional Chinese medicine. Existing studies have shown that HF ​​can inhibit the proliferation of rheumatoid arthritis fibroblasts and inflammatory macrophages, and inhibit the expression of inflammatory factors, and has an anti-rheumatoid arthritis effect. However, its short biological half-life, rapid elimination from the body, and non-specific targeting limit its clinical application.

[0004] In the prior art, a drug-carrying system is prepared by using a biomimetic membrane to achieve a sustained release of the drug. CN114306637A discloses a drug-carrying system for treating rheumatoid arthritis, comprising sinomenine hydrochloride, Prussian blue, a biomimetic membrane and phospholipidated hyaluronic acid, wherein the biomimetic membrane is wrapped on the surface of the Prussian blue loaded with sinomenine hydrochloride, and the phospholipidated hyaluronic acid is embedded on the surface of the biomimetic membrane, and the biomimetic membrane comprises a red blood cell membrane and a macrophage membrane. The drug-carrying system has a good ability to target inflamed joints and has a good therapeutic effect.

[0005] CN113041356A discloses a hemoglobin targeted drug delivery system, comprising hemoglobin, Prussian blue nanoparticles, a biomimetic membrane coating layer and a hyaluronic acid modification layer. The hemoglobin targeted drug delivery system can accurately deliver hemoglobin to the lesion site of arthritis, prolong the retention time of the drug in the lesion site of arthritis, and ensure the safety and effectiveness of treatment.

[0006] CN116059186A discloses a bionic PLGA composite material, which is loaded and encapsulated by PLGA for atorvastatin and metformin, and then a bionic membrane is disguised on the outer layer of PLGA, and a phospholipidated hyaluronic acid is inserted on the surface of the bionic membrane to modify it. It utilizes the characteristics of hyaluronic acid interacting with the target cell surface receptor CD44 to achieve effective utilization of the nanocomposite material by the target cells, thereby improving its effect in preventing and treating atherosclerosis. However, the bionic membranes used are all composed of macrophage membranes, which makes the preparation cost of the composite material high.

[0007] However, the above prior art has the problems of low encapsulation efficiency, low drug loading and slow degradation rate in the body. Therefore, it is necessary to provide a drug delivery system suitable for halofuginone with high encapsulation efficiency, high drug loading and rapid degradation in the body. Summary of the invention

[0008] The purpose of the present invention is to overcome the problems of short biological half-life, non-specific targeting, low encapsulation efficiency and low drug loading capacity of halofuginone in the prior art.

[0009] In order to achieve the above object, the present invention provides a drug delivery system for treating rheumatoid arthritis, comprising PLGA, halofuginone, a biomimetic hybrid membrane and phospholipidated hyaluronic acid, wherein the biomimetic hybrid membrane is wrapped on the surface of the PLGA loaded with the halofuginone, and the phospholipidated hyaluronic acid is embedded on the surface of the biomimetic hybrid membrane;

[0010] The relative ability of the drug delivery system to target inflammatory macrophages is 60-100%, and in the drug delivery system, the encapsulation efficiency of halofuginone is 50-80wt%, and the drug loading is 3-30wt%;

[0011] The volume average particle size of the drug delivery system is 160nm-200nm;

[0012] The biomimetic hybrid membrane is a product obtained by mixing red blood cell membrane fragments and macrophage membrane fragments in a mass ratio of 1:0.5-2.

[0013] The second aspect of the present invention provides a method for preparing the drug delivery system for treating rheumatoid arthritis according to the first aspect, the method comprising:

[0014] (1) in the presence of an organic solvent, mixing halofuginone, PLGA and an aqueous phase for reaction I to obtain a PLGA micelle solution;

[0015] (2) performing a mixing reaction II on the PLGA micelle solution and the biomimetic hybrid membrane at a temperature of 30-37° C. and a rotation speed of 300-400 rpm for 1-2 h to obtain a composite material;

[0016] (3) performing a mixing reaction III on the composite material and phospholipidated hyaluronic acid to obtain the drug delivery system for treating rheumatoid arthritis;

[0017] Wherein, the aqueous phase is a 0.5-2 wt % polyvinyl alcohol aqueous solution and / or a 0.1-0.5 wt % poloxamer aqueous solution.

[0018] The third aspect of the present invention provides the use of the drug-carrying system for treating rheumatoid arthritis prepared by the method described in the second aspect in the preparation of anti-rheumatoid arthritis therapeutic drugs.

[0019] Compared with the prior art, the drug delivery system provided by the present invention has at least the following advantages:

[0020] (1) It has good targeting ability for inflamed joints, significantly improving the therapeutic effect of the drug and reducing the drug's non-target organ toxicity;

[0021] (2) The encapsulation efficiency is improved and efficient drug loading can be achieved;

[0022] (3) The preparation process is simple, stable, and low in cost, and has good prospects for mass production and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The SDS-PAGE analysis of the membrane proteins of the preferred erythrocyte membrane fragments, macrophage membrane fragments and biomimetic hybrid membranes provided by the present invention;

[0024] Figure 2 This is a transmission electron microscopy image of a preferred drug delivery system for treating rheumatoid arthritis provided by the present invention;

[0025] Figure 3 The Zeta potential diagram of the preferred PLGA, halofuginone, PLGA micellar solution, composite material and drug delivery system for treating rheumatoid arthritis provided by the present invention;

[0026] Figure 4 The biocompatibility diagram of the preferred halofuginone, PLGA micellar solution and drug delivery system for treating rheumatoid arthritis provided by the present invention;

[0027] Figure 5 This is a graph showing the inhibition rate of RAFLS cells by the preferred halofuginone, PLGA micelle solution and drug delivery system for treating rheumatoid arthritis provided by the present invention;

[0028] Figure 6 The figure is a fluorescence intensity diagram of the preferred PLGA micelle solution and the drug delivery system for treating rheumatoid arthritis provided by the present invention.

[0029] Description of Reference Numerals

[0030] Marker membrane protein internal reference group DETAILED DESCRIPTION

[0031] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0032] As mentioned above, the first aspect of the present invention provides a drug delivery system for treating rheumatoid arthritis, comprising PLGA, halofuginone, a biomimetic hybrid membrane and phospholipidated hyaluronic acid, wherein the biomimetic hybrid membrane is wrapped on the surface of the PLGA loaded with the halofuginone, and the phospholipidated hyaluronic acid is embedded on the surface of the biomimetic hybrid membrane;

[0033] The relative ability of the drug delivery system to target inflammatory macrophages is 60-100%, and in the drug delivery system, the encapsulation rate of halofuginone is 50-80wt%, and the drug loading is 3-30wt%; the volume average particle size of the drug delivery system is 160nm-200nm;

[0034] The biomimetic hybrid membrane is a product obtained by mixing red blood cell membrane fragments and macrophage membrane fragments in a mass ratio of 1:0.5-2.

[0035] It should be noted that, in the present invention, the drug loading (wt%) = the mass of halofuginone loaded on the surface of PLGA / the mass of PLGA in the drug loading system×100wt%.

[0036] It should be noted that, in the present invention, the encapsulation rate (wt%) = the mass of halofuginone loaded on the surface of PLGA / the total mass of halofuginone in the drug-carrying system×100wt%.

[0037] It should be noted that, in the present invention, the relative ability of the drug delivery system to target inflammatory macrophages is calculated as follows: the uptake capacity of the drug delivery system / the uptake capacity of the macrophage membrane drug delivery system. The difference between the macrophage membrane drug delivery system and the drug delivery system of the present invention is that the biomimetic hybrid membranes in the macrophage membrane drug delivery system are all macrophage membranes and do not contain red blood cell membranes.

[0038] Preferably, the content mass ratio of the halofuginone, the PLGA, the biomimetic hybrid membrane and the phospholipidated hyaluronic acid is 0.5-5:10:2.5-10:2-10. The inventors found in the research process that when the mass ratio of the halofuginone to the PLGA is greater than 1:2, for example, 1:1, although the drug loading of PLGA will be increased, the encapsulation rate of the drug will decrease, resulting in drug waste; and when the mass ratio of the halofuginone to the PLGA is less than 1:20, for example, 1:30, it will cause a decrease in the drug loading.

[0039] Preferably, the molar ratio of the monomers provided by polylactic acid to the monomers provided by polyglycolic acid in the PLGA is 50-90:10-50.

[0040] Preferably, the weight average molecular weight of the PLGA is 38000-54000, and the volume average particle size is 150-200 nm. The inventors found that in this preferred case, the drug delivery system has both high targeting ability and good loading capacity.

[0041] As mentioned above, the second aspect of the present invention provides a method for preparing the drug delivery system for treating rheumatoid arthritis described in the first aspect, the method comprising:

[0042] (1) in the presence of an organic solvent, mixing halofuginone, PLGA and an aqueous phase for reaction I to obtain a PLGA micelle solution;

[0043] (2) performing a mixing reaction II on the PLGA micelle solution and the biomimetic hybrid membrane at a temperature of 30-37° C. and a rotation speed of 300-400 rpm for 1-2 h to obtain a composite material;

[0044] (3) performing a mixing reaction III on the composite material and phospholipidated hyaluronic acid to obtain the drug delivery system for treating rheumatoid arthritis;

[0045] Wherein, the aqueous phase is a 0.5-2 wt % polyvinyl alcohol aqueous solution and / or a 0.1-0.5 wt % poloxamer aqueous solution.

[0046] Preferably, the method of the present invention further comprises preparing the biomimetic hybrid membrane using a method comprising the following steps: mixing erythrocyte membrane and macrophage membrane in the presence of a buffer solution, and obtaining the biomimetic hybrid membrane after separation.

[0047] More preferably, the biomimetic hybrid membrane is prepared using a method comprising the following steps:

[0048] At 2-4°C, the red blood cells are hemolyzed, ultrasonically broken, centrifuged I, and the supernatant is further centrifuged II to obtain the red blood cell membrane fragments; the macrophages are extracted with a membrane cell extract, ultrasonically broken, frozen and thawed 4-5 times, centrifuged I, and the supernatant is further centrifuged II to obtain the macrophage membrane fragments; the red blood cell membrane fragments and the macrophage membrane fragments are stirred at a speed of 200-800rpm for 0.5-2h at a dosage mass ratio of 1:0.5-2, and then pressure filtered, and centrifuged again II to obtain the biomimetic hybrid membrane. The inventors found that under this preferred condition, the encapsulation rate of the drug delivery system for treating rheumatoid arthritis provided by the present invention is higher.

[0049] More preferably, the ultrasonic fragmentation conditions at least meet the following requirements: ultrasonic power is 30-100 W, and time is 3-15 min.

[0050] Preferably, the conditions of each centrifugation I independently at least meet the following conditions: a rotation speed of 800-3000 rpm and a time of 5-10 min.

[0051] Preferably, the conditions of each centrifugation II independently at least meet the following conditions: a rotation speed of 10000-13000 rpm and a time of 30-60 min.

[0052] Preferably, the buffer is at least one of phosphate-phosphate buffer, sodium chloride solution and glucose solution.

[0053] More preferably, the buffer is a phosphoric acid-phosphate buffer having a pH of 7.2-7.4 and a concentration of 0.001-0.01 mol / L in terms of phosphate.

[0054] More preferably, the buffer solution is a sodium chloride solution having a pH of 4.5-7.0, and a concentration of the buffer solution of 0.1-0.9 wt % based on sodium chloride.

[0055] More preferably, the buffer solution is a glucose solution with a pH of 3.2-6.5, and a concentration of the buffer solution is 1-5 wt % based on glucose.

[0056] Preferably, in step (1), the operation of the mixing reaction I comprises: first dissolving halofuginone and PLGA in an organic solvent to obtain an oil phase, then adding the oil phase dropwise to the aqueous phase at a rotation speed of 300-1500 rpm to carry out the mixing reaction I for 0.5-2 h, and removing the organic solvent to obtain the PLGA micelle solution; and

[0057] The dripping speed of the oil phase is 50-200 mL / min for every 1 L of the water phase. The inventors found that in this preferred case, the particle size of the drug delivery system for treating rheumatoid arthritis provided by the present invention is more stable.

[0058] Preferably, the organic solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, ethanol, methanol, acetone and dichloromethane.

[0059] According to a preferred embodiment, the method of the present invention further comprises: in step (1), the process of dissolving halofuginone and PLGA in an organic solvent is carried out under ultrasonic conditions, wherein the ultrasonic conditions include: a power of 30-100 W and a time of 2-10 min.

[0060] According to a preferred embodiment, the step of removing the organic solvent in the present invention comprises: after completing the mixing reaction I, dialyzing the obtained product in a dialysis bag with a MWCO (molecular weight cut-off) of 2-2.5 kDa for 12-15 hours to obtain the PLGA micelle solution.

[0061] Preferably, in step (1), the mass ratio of the PLGA to the organic solvent is 1:0.02-0.2.

[0062] Preferably, the volume ratio of the oil phase to the water phase is 1:5-15. The inventors found during the research that when the volume ratio of the oil phase to the water phase is greater than 1:5, such as 1:3, the stability of the drug delivery system is poor and the encapsulation effect is poor; and when the volume ratio of the oil phase to the water phase is less than 1:15, such as 1:25, the average particle size of the drug delivery system is larger.

[0063] Preferably, the mixing reaction III is carried out under stirring conditions and at least meets the following conditions: a rotation speed of 300-800 rpm and a time of 0.5-2 h.

[0064] Preferably, the preparation method of the phospholipidated hyaluronic acid comprises: in the presence of the buffer solution, mixing hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and distearoylphosphatidylethanolamine-polyethylene glycol-amino for reaction IV to obtain the phospholipidated hyaluronic acid.

[0065] More preferably, the preparation method of the phospholipidated hyaluronic acid further comprises: the mass ratio of the hyaluronic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the N-hydroxysuccinimide and the distearoylphosphatidylethanolamine-polyethylene glycol-amino is 1:2-8:5-15:2-8.

[0066] Preferably, the mixing reaction IV is carried out under stirring conditions and at least meets the following conditions: temperature of 25-38° C., rotation speed of 300-1000 rpm, and time of 22-25 h.

[0067] More preferably, the preparation method of the phospholipidated hyaluronic acid comprises: dissolving hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a PBS solution, stirring at 25-30°C for 25-40 minutes, adding distearoyl phosphatidylethanolamine-polyethylene glycol-amino and stirring at 35-38°C for 22-25 hours, dialyzing the resulting mixture in a dialysis bag (MWCO=2.5kDa) for 22-25 hours, and then vacuum freeze-drying at -20 to 4°C for 12-24 hours to obtain the phospholipidated hyaluronic acid.

[0068] As mentioned above, the third aspect of the present invention provides the use of the drug delivery system for treating rheumatoid arthritis prepared by the method described in the second aspect in the preparation of anti-rheumatoid arthritis therapeutic drugs.

[0069] It should be noted that, in various aspects of the present invention, for the same components or terms in various aspects, the present invention is described only once in one aspect without repeated description, which should not be understood by those skilled in the art as a limitation of the present invention.

[0070] The present invention is described in detail below by way of examples. Unless otherwise specified, the raw materials used are common commercial products of analytical grade.

[0071] Halofuginone: provided by Shanghai Taoshu Biotechnology Co., Ltd., product number T001-10008873;

[0072] PLGA: The molar ratio of the monomer provided by polylactic acid to the monomer provided by polyglycolic acid is 50:50, the weight average molecular weight is 40000, the volume average particle size is 125 nm, purchased from Resomer, CAS number is 26780-50-7, brand is 719900-5G;

[0073] Buffer: Phosphoric acid-phosphate buffer, the pH value is 7.4, the concentration of the buffer is 0.01 mol / L based on phosphate.

[0074] Organic solvent: dimethyl sulfoxide;

[0075] Macrophages, normal fibroblast-like synoviocytes, and rheumatoid arthritis fibroblast-like synoviocytes were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0076] Fluorescence spectrophotometer: purchased from Hitachi, Japan, instrument model FL-2500;

[0077] Transmission electron microscope: purchased from the official website of JEOL, the instrument model is JEM-2100F;

[0078] Zeta potential and nanoparticle size analyzer: manufactured by Malvin Instrument Co., Ltd. of the United States, the instrument model is ZetasizerNano;

[0079] Ultrasonic cell disruptor: purchased from Nanjing Saifei Biotechnology Co., Ltd., model Biosafer900-92;

[0080] Laser confocal scanning microscope: purchased from Olympus, Japan, model FV1200; pH of PBS is 7.2-7.4.

[0081] Preparation of phospholipidated hyaluronic acid:

[0082] 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 phosphate-phosphate buffer, stirred at 25°C for 30 min, and then 25 mg of distearoylphosphatidylethanolamine-polyethylene glycol-amino was added and stirred at 37°C for 30 min, and then stirred at 37°C for 24 h. The resulting mixture was dialyzed in a dialysis bag (MWCO = 2.5 kDa) for 24 h and then vacuum freeze-dried at 4°C to obtain phospholipidated hyaluronic acid.

[0083] Preparation Example A-1

[0084] Preparation of biomimetic hybrid membranes:

[0085] At 4°C, 1 mL of red blood cells were hemolyzed, ultrasonically disrupted at 40W for 15 minutes, centrifuged at 1000 rpm for 10 minutes I, the supernatant was further centrifuged at 13000 rpm for 30 minutes II, and the lower precipitate was removed to obtain red blood cell membrane fragments (RBCm); macrophages were extracted with membrane cell extract, ultrasonically disrupted at 40W for 15 minutes, frozen and thawed 4 times, centrifuged at 1000 rpm for 10 minutes I, the supernatant was further centrifuged at 13000 rpm for 30 minutes II, and the lower precipitate was removed to obtain macrophage membrane fragments The red blood cell membrane fragment and the macrophage membrane fragment were stirred at a speed of 300 rpm for 60 minutes in a mass ratio of 1:1, and then pressure filtered, and then centrifuged again at 13000 rpm for 30 minutes to obtain the biomimetic hybrid membrane a1 (M);

[0086] Figure 1 Red blood cell membrane fragments (RBCm), macrophage membrane fragments SDS-PAGE analysis of membrane proteins of biomimetic hybrid membrane a1(M). The figure shows that biomimetic hybrid membrane a1 has both RBCm and of protein expression.

[0087] Preparation Example A-2

[0088] Preparation of biomimetic hybrid membranes:

[0089] This preparation example is carried out using a method similar to that of preparation example A-1, except that the mass ratio of the red blood cell membrane fragment to the macrophage membrane fragment is 1:0.5, to obtain the biomimetic hybrid membrane a2.

[0090] Preparation Example A-3

[0091] Preparation of biomimetic hybrid membranes:

[0092] This preparation example is carried out using a method similar to that of preparation example A-1, except that the mass ratio of the red blood cell membrane fragment to the macrophage membrane fragment is 1:2, and the biomimetic hybrid membrane a3 is obtained.

[0093] Preparation Example A-4

[0094] Preparation of biomimetic hybrid membranes:

[0095] This preparation example is carried out using a method similar to that of preparation example A-1, except that the mass ratio of the red blood cell membrane fragment to the macrophage membrane fragment is 1:0.4, and the biomimetic hybrid membrane a4 is obtained.

[0096] Preparation Example B-1

[0097] This preparation example is used to illustrate that the drug delivery system for treating rheumatoid arthritis provided by the present invention is prepared according to the following method, including:

[0098] (1) PLGA and halofuginone were ultrasonically treated with an organic solvent in a mass ratio of 1:0.2:0.1 (power of 50 W, time of 5 min) to obtain an oil phase, 1 wt% PVA solution (polyvinyl alcohol aqueous solution) was used as the aqueous phase, and the oil phase was added to the aqueous phase at a speed of 100 mL / min for 1 h of mixing reaction I with a rotation speed of 1200 rpm for each 1 L of the aqueous phase, and then dialyzed in a dialysis bag (MWCO = 2.5 kDa) for 12 h to remove the organic solvent to obtain a PLGA micelle solution (P@HF); wherein the volume ratio of the oil phase to the aqueous phase was 1:10;

[0099] (2) The PLGA solution loaded with halofuginone and the biomimetic hybrid membrane (a1) were subjected to a mixing reaction II at 37°C and 300 rpm for 1 h to obtain a composite material (M@P@HF);

[0100] (3) performing a mixing reaction III of the composite material and phospholipidated hyaluronic acid at 600 rpm for 2 h to obtain the drug delivery system for treating rheumatoid arthritis, named P1 (HA-M@P@HF);

[0101] Wherein, the mass ratio of the halofuginone, the PLGA, the biomimetic hybrid membrane and the phospholipidated hyaluronic acid is 2:10:5:2.5;

[0102] The drug delivery system has an encapsulation efficiency of 72 wt %, a drug loading of 14 wt %, a volume average particle size of 178 nm, and a relative ability to target inflammatory macrophages of 98%;

[0103] Figure 2 The transmission electron microscope image of P1 is shown, where Figure 2 It showed that the spherical PLGA loaded with fusinone appeared as a biomimetic hybrid membrane outer layer after being camouflaged as a biomimetic hybrid membrane.

[0104] Preparation Example B-2

[0105] This method is carried out in a similar manner to that of Preparation Example B-1, except that the biomimetic hybrid membrane used is a2, and a drug delivery system for treating rheumatoid arthritis is obtained, which is named P2;

[0106] Among them, the relative ability of the drug delivery system to target inflammatory macrophages is 94%, the encapsulation efficiency is 71wt%, the drug loading is 14wt%, and the volume average particle size is 163nm.

[0107] Preparation Example B-3

[0108] This method is carried out in a similar manner to that of Preparation Example B-1, except that the biomimetic hybrid membrane used is a3, and a drug-carrying system for treating rheumatoid arthritis is obtained, which is named P3;

[0109] Among them, the relative ability of the drug delivery system to target inflammatory macrophages is 99%, the encapsulation efficiency is 70wt%, the drug loading is 14wt%, and the volume average particle size is 171nm.

[0110] Preparation Example B-4

[0111] This method is carried out in a similar manner to that of Preparation Example B-1, except that the volume ratio of the oil phase to the water phase is 1:4, and a drug delivery system for treating rheumatoid arthritis is obtained, which is named P4;

[0112] Among them, the relative ability of the drug delivery system to target inflammatory macrophages is 95%, the encapsulation efficiency is 51wt%, the drug loading is 13wt%, and the volume average particle size is 173nm.

[0113] Preparation Example B-5

[0114] This method is carried out in a similar manner to that of Preparation Example B-1, except that the mass ratio of the halofuginone, the PLGA, the biomimetic hybrid membrane and the phospholipidated hyaluronic acid is 6:10:5:2.5, and a drug delivery system for treating rheumatoid arthritis is obtained, which is named P5;

[0115] Among them, the relative ability of the drug-carrying system to target inflammatory macrophages is 90%, the encapsulation efficiency is 50wt%, the drug loading is 30wt%, and the volume average particle size is 186nm.

[0116] Comparative Example 1

[0117] This comparative example was prepared by a method similar to that of Preparation Example B-1, except that the biomimetic hybrid membrane used was a4, and a drug delivery system for treating rheumatoid arthritis was obtained, which was named DP1;

[0118] Among them, the relative ability of the drug delivery system to target inflammatory macrophages is 59%, the encapsulation efficiency is 72wt%, the drug loading is 14wt%, and the volume average particle size is 163nm.

[0119] Comparative Example 2

[0120] This comparative example is prepared by a method similar to that of Preparation Example B-1, except that the biomimetic hybrid membrane used does not contain macrophage membrane fragments. The specific steps of preparing the biomimetic hybrid membrane include:

[0121] At 4°C, 1 mL of red blood cells were hemolyzed, ultrasonically disrupted at 40W for 15 minutes, and centrifuged at 1000 rpm for 10 minutes (I). The supernatant was further centrifuged at 13000 rpm for 30 minutes (II). The lower precipitate was taken to obtain the red blood cell membrane fragment (RBCm), which was the biomimetic hybrid membrane and was named a5.

[0122] Finally, a drug delivery system for the treatment of rheumatoid arthritis was obtained, named DP4;

[0123] Among them, the relative ability of the drug delivery system to target inflammatory macrophages is 52%, the encapsulation efficiency is 45wt%, the drug loading is 14wt%, and the volume average particle size is 160nm.

[0124] Test Example 1

[0125] The PLGA, halofuginone (HF), PLGA micelle solution (P@HF), composite material (M@P@HF) and drug delivery system for treating rheumatoid arthritis (HA-M@P@HF) in Preparation Example B-1 were placed in a Malvern cuvette, and their Zeta potentials were measured using a Zeta potential analyzer. The results are as follows: Figure 3 As shown in the figure, PLGA, HF, P@HF, M@P@HF, and HA-M@P@HF are all negatively charged. After modification with the biomimetic hybrid membrane (M), the potential of M is reduced because it is negatively charged. When the targeting substance hyaluronic acid (HA) is added, the positively charged distearoylphosphatidylethanolamine-polyethylene glycol-amino is also added, thereby increasing the potential of M@P@HF, further confirming the successful preparation of HA-M@P@HF.

[0126] Test Example 2

[0127] Take fresh blood from SPF adult SD rats, centrifuge at 2400rpm for 10min at 20℃, collect the plasma layer, centrifuge at 3600rpm for 15min, and extract platelet-rich plasma. Mix PLGA, HF, P@HF, HA-M@P@HF in Preparation Example B-1 with plasma, use buffer (PBS) as negative control, and thrombin as positive control, incubate the mixture at room temperature for 1h, and then use a microplate reader to detect the absorbance change at 650nm. The results are as follows: Figure 4 As shown, Figure 4 It was shown that PLGA, HF, P@HF, and HA-M@P@HF did not cause coagulation, thus proving that the drug delivery system for treating rheumatoid arthritis of the present invention has excellent biocompatibility and biosafety.

[0128] Test Example 3

[0129] Take HF, P@HF, and HA-M@P@HF prepared in Example B-1, and then take RAFLS cells at 5×10 3 The cells were inoculated at a density of 10 cells / well into a 96-well plate. After culturing for 24 hours, fresh culture media containing HF (0.5 μM), P@HF, and HA-M@P@HF were added respectively. After culturing for 48 hours, the culture media was removed, the plates were washed with buffer, and serum-free culture media containing 10% MTT was added for 4 hours. After removing the supernatant, 100 μL DMSO (dimethyl sulfoxide) was added to each well, the plates were mixed on a horizontal shaker, and the absorbance OD value was detected at 490 nm on a microplate reader.

[0130] The cell viability was calculated according to the formula: Cell viability (%) = OD (490nm / sample) / OD (490nm / control) × 100%, and the results are shown in Figure 5As can be seen from the figure, compared with the control group, each drug-treated group has an inhibitory effect on RAFLS cells, and the inhibition rate of HA-M@P@HF on RAFLS cells is significantly higher than that of HF alone or P@HF alone. Therefore, the drug delivery system for treating rheumatoid arthritis of the present invention has an excellent anti-rheumatoid arthritis effect.

[0131] Test Example 4

[0132] Take P@HF and HA-M@P@HF in Preparation Example B-1, stack the fluorescent material ce6 on the surface of P@HF and HA-M@P@HF, and measure the content of P@HF and HA-M@P@HF taken by cells by semi-quantitative means of detecting fluorescence intensity. P@HF and HA-M@P@HF were co-cultured with normal macrophages, normal fibroblast synoviocytes (HFLS), activated macrophages induced by lipopolysaccharide (LPS, 100 ng / mL), and rheumatoid arthritis fibroblast synoviocytes (RAFLS) at 37°C and 5% CO2 for 4 hours. The results are as follows. Figure 6 As shown, Figure 6 It was shown that when compared with normal macrophages and normal HFLS, the average fluorescence intensity of activated macrophages and RAFLS cells was significantly enhanced; therefore, the drug delivery system for treating rheumatoid arthritis of the present invention can specifically enhance the uptake of inflammatory macrophages and rheumatoid arthritis synovial fibroblasts, thereby inhibiting the inflammatory response.

[0133] It can be seen from the above that the drug delivery system for treating rheumatoid arthritis provided by the present invention not only has a higher encapsulation rate and drug loading capacity, and a lower preparation cost, but also has excellent biocompatibility and biosafety, and has a more excellent anti-rheumatoid arthritis effect.

[0134] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A drug delivery system for treating rheumatoid arthritis, It is characterized in that It comprises PLGA, halofuginone, a biomimetic hybrid membrane and phospholipidated hyaluronic acid, wherein the biomimetic hybrid membrane is wrapped on the surface of the PLGA loaded with the halofuginone, and the phospholipidated hyaluronic acid is embedded on the surface of the biomimetic hybrid membrane; The relative ability of the drug delivery system to target inflammatory macrophages is 60-100%, and in the drug delivery system, the encapsulation efficiency of halofuginone is 50-80wt%, and the drug loading is 13-30wt%; The volume average particle size of the drug delivery system is 160nm-200nm; The biomimetic hybrid membrane is a product obtained by mixing red blood cell membrane fragments and macrophage membrane fragments in a mass ratio of 1:0.5-2; The mass ratio of the halofuginone, the PLGA, the biomimetic hybrid membrane and the phospholipidated hyaluronic acid is 0.5-5:10:2.5-10:2-10; The molar ratio of the monomers provided by polylactic acid and the monomers provided by polyglycolic acid in the PLGA is 50:50, and the weight average molecular weight is 38000-54000.

2. The drug delivery system according to claim 1, in, The volume average particle size of the PLGA is 150-200 nm.

3. A method for preparing the drug delivery system for treating rheumatoid arthritis according to claim 1 or 2, It is characterized in that The method includes: (1) In the presence of an organic solvent, the halofuginone, PLGA and an aqueous phase are mixed to react I to obtain a PLGA micelle solution; (2) performing a mixing reaction II for 1-2 hours with the PLGA micelle solution and the biomimetic hybrid membrane at a temperature of 30-37° C. and a rotation speed of 300-400 rpm to obtain a composite material; (3) performing a mixing reaction III on the composite material and phospholipidated hyaluronic acid to obtain the drug delivery system for treating rheumatoid arthritis; Wherein, the aqueous phase is a 0.5-2wt% polyvinyl alcohol aqueous solution and / or a 0.1-0.5wt% poloxamer aqueous solution.

4. The method according to claim 3, in, The method further comprises preparing the biomimetic hybrid membrane using a method comprising the following steps: mixing red blood cell membrane and macrophage membrane in the presence of a buffer solution, and obtaining the biomimetic hybrid membrane after separation.

5. The method according to claim 3 or 4, in, In step (1), the operation of the mixing reaction I includes: first dissolving halofuginone and PLGA in an organic solvent to obtain an oil phase, then adding the oil phase dropwise to the aqueous phase at a rotation speed of 300-1500 rpm to carry out the mixing reaction I for 0.5-2 h, and removing the organic solvent to obtain the PLGA micelle solution; and The oil phase is added at a rate of 50-200 mL / min for every 1 L of the water phase.

6. The method according to claim 3 or 4, in, In step (1), the mass ratio of the PLGA to the organic solvent is 1:0.02-0.

2.

7. The method according to claim 5, in, The volume ratio of the oil phase to the water phase is 1:5-15.

8. The method according to claim 3 or 4, in, The mixed reaction II is carried out under stirring conditions and at least meets the following conditions: temperature is 30-37° C. and time is 1-4 h.

9. The method according to claim 3 or 4, in, The preparation method of the phospholipidated hyaluronic acid comprises: in the presence of a buffer solution, mixing hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and distearoylphosphatidylethanolamine-polyethylene glycol-amino for reaction IV to obtain the phospholipidated hyaluronic acid.

10. Use of the drug delivery system for treating rheumatoid arthritis prepared by the method according to any one of claims 3 to 9 in preparing anti-rheumatoid arthritis therapeutic drugs.

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