Methotrexate-baijiapolysaccharide nanoparticles, preparation method and application thereof

Abstract

The present application relates to the technical field of rheumatoid arthritis drugs, and particularly relates to methotrexate-baiji polysaccharide nanoparticles and a preparation method and application thereof.The present application provides methotrexate-baiji polysaccharide nanoparticles, which have a structure shown in formula 1.The nanoparticles are formed by connecting methotrexate and baiji polysaccharide through a disulfide bond, and in theory, can realize drug release in response to high active oxygen stimulation in the microenvironment of rheumatoid arthritis.In addition, the present application researches and proves that the nanoparticles can effectively inhibit the proliferation and migration of fibroblast-like synoviocytes, regulate the polarization of M1 macrophages to M2 macrophages, and reduce synovial inflammation.The methotrexate-baiji polysaccharide nanoparticles of the present application have simple preparation process operation, are convenient to use, and have a good development and utilization prospect.Formula 1

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of rheumatoid arthritis drugs, and in particular to a methotrexate-baijiu polysaccharide nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting about 0.5% to 1.0% of the global population, especially women, the risk of which is 2 to 3 times that of men. The main feature of RA is severe inflammation at the synovial joint, which leads to unbearable joint pain, swelling and stiffness, and even disability and premature death. Methotrexate (MTX) is the first choice for treating RA, and plays a therapeutic role mainly through oral and injection administration. There are many challenges in these two administration methods. First, the bioavailability of MTX oral administration has individual differences, and the absorption is easily affected by food and gastrointestinal environment, and also causes systemic adverse reactions such as gastrointestinal reactions, abnormal liver and kidney functions, etc. Second, MTX intravenous injection can cause mucositis (such as oral ulcer), liver and kidney toxicity, and other systemic toxicity risks. Third, oral or intravenous administration lacks targeting, and the enrichment of drugs in the diseased joint is insufficient, and the dosage needs to be increased to achieve the therapeutic effect, which further aggravates the side effects. Fourth, the traditional administration method cannot adjust the release rate according to the degree of inflammation. Studies have found that the occurrence of RA inflammation is closely related to M1 / M2 macrophage imbalance, abnormal proliferation of fibroblast-like synoviocytes, and high reactive oxygen species (ROS) levels. Therefore, based on the particularity of the microenvironment of RA, it is reasonable to develop a multifunctional nanoparticle for intra-articular injection, which can maintain the balance of M1 / M2 macrophages, inhibit the abnormal proliferation of fibroblast-like synoviocytes in RA patients, and respond to ROS stimulation to achieve on-demand drug release to effectively treat rheumatoid arthritis. The nanoparticle can also be applied to local drug delivery for diseases with high pathological microenvironment of ROS. SUMMARY

[0003] In view of this, the present application aims to provide a methotrexate-baijiu polysaccharide nanoparticle and a preparation method and application thereof. The methotrexate-baijiu polysaccharide nanoparticle has microenvironment ROS responsiveness.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a methotrexate-baijiu polysaccharide nanoparticle, which has the structure shown in formula 1:

[0006] Formula 1.

[0007] The present application also provides a preparation method of the methotrexate-baijiu polysaccharide nanoparticle according to the above technical solutions, which comprises the following steps:

[0008] mixing methotrexate, the first activating agent, cystamine dihydrochloride and the first solvent, and performing a light-avoiding reaction to obtain a methotrexate-cystamine dihydrochloride intermediate;

[0009] mixing bletilla striata polysaccharide, sodium periodate and water, and performing a light-avoiding oxidation, then adding ethylene glycol to terminate the oxidation to obtain oxidized bletilla striata polysaccharide;

[0010] mixing the oxidized bletilla striata polysaccharide, the second activating agent and the second solvent, and performing activation to obtain an activated oxidized bletilla striata polysaccharide solution;

[0011] mixing the methotrexate-cystamine dihydrochloride intermediate and the third solvent, and mixing with the activated oxidized bletilla striata polysaccharide solution to perform a Schiff base reaction to obtain the methotrexate-bletilla striata polysaccharide nanoparticle.

[0012] Preferably, the first activating agent and the second activating agent are both 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide;

[0013] The mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in the first activating agent is (0.04~0.08):(0.02~0.05);

[0014] The mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in the second activating agent is (0.01~0.02):(0.006~0.012).

[0015] Preferably, the mass ratio of methotrexate and 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the first activating agent is (0.1~0.2):(0.04~0.08);

[0016] The mass ratio of methotrexate and cystamine dihydrochloride is (0.1~0.2):(0.05~0.1).

[0017] Preferably, the mass ratio of bletilla striata polysaccharide and sodium periodate is (1~2):(0.2~0.6);

[0018] The dosage ratio of bletilla striata polysaccharide and ethylene glycol is (1.0~2.0)g:(5~10)mL.

[0019] Preferably, the temperature of the light-avoiding oxidation is 20℃~25℃, and the time is 2~10h.

[0020] Preferably, the mass ratio of the oxidized Bletilla striata polysaccharide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the second activating agent and N-hydroxysuccinimide in the second activating agent is (0.01-0.02):(0.01-0.02):(0.006-0.012).

[0021] The activation is carried out in the dark, and the temperature of the activation is 20-25 DEG C, and the time is 1-3h.

[0022] Preferably, the mass ratio of the methotrexate-cystamine dihydrochloride intermediate and the oxidized Bletilla striata polysaccharide is (0.03-0.06):(0.01-0.02).

[0023] Preferably, the Schiff base reaction is carried out in the dark, and the temperature of the Schiff base reaction is 20-25 DEG C, and the time is 18-36h.

[0024] The application also provides application of the methotrexate-Bletilla striata polysaccharide nanoparticle in the preparation of a drug for treating a disease with a high active oxygen pathological microenvironment.

[0025] The application provides a methotrexate-Bletilla striata polysaccharide nanoparticle (MTX-ss-OBSP NPs) having the structure shown in formula 1.

[0026] Formula 1.

[0027] The methotrexate-Bletilla striata polysaccharide nanoparticle has a microenvironment ROS responsiveness, and the nanoparticle is formed by connecting methotrexate and Bletilla striata polysaccharide through a disulfide bond, and can realize drug release in response to high active oxygen stimulation in a rheumatoid arthritis microenvironment. In addition, researches of the application prove that the nanoparticle can effectively inhibit fibroblast-like synoviocyte proliferation and migration, regulate M1 macrophage polarization to M2 macrophage, and reduce synovial inflammation. The methotrexate-Bletilla striata polysaccharide nanoparticle has simple preparation process, is convenient to use, and has a good development and utilization prospect.

[0028] Compared with methotrexate or Bletilla striata polysaccharide, the methotrexate-Bletilla striata polysaccharide nanoparticle has the following advantages: on the one hand, the particle size of the methotrexate-Bletilla striata polysaccharide nanoparticle is less than 300 nm, which can significantly increase the specific surface area of the drug and improve the water solubility of methotrexate; on the other hand, Bletilla striata polysaccharide belongs to a high molecular compound, and the solution thereof is difficult to enter cells to play a pharmacological role; the preparation of the nanoparticle can more easily enter cells to remove high-level ROS in the cells, improve synovial inflammation, and regulate M2 macrophage polarization, thereby slowing down the progress of rheumatoid arthritis.

[0029] The application further provides a preparation method of the methotrexate-baiji polysaccharide nanoparticle, and the preparation method comprises the following steps: mixing methotrexate, a first activating agent, cystamine dihydrochloride and a first solvent, and performing a light-proof reaction to obtain a methotrexate-cystamine dihydrochloride intermediate; mixing baiji polysaccharide, sodium periodate and water, and performing a light-proof oxidation, and then adding ethylene glycol to terminate the oxidation to obtain oxidized baiji polysaccharide; mixing the oxidized baiji polysaccharide, a second activating agent and a second solvent, and performing activation to obtain an activated oxidized baiji polysaccharide solution; mixing the methotrexate-cystamine dihydrochloride intermediate and a third solvent, and then mixing the methotrexate-cystamine dihydrochloride intermediate and the third solvent with the activated oxidized baiji polysaccharide solution to perform a Schiff base reaction to obtain the methotrexate-baiji polysaccharide nanoparticle. The preparation method has simple preparation process and easily obtained raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Infrared spectrograms of the baiji polysaccharide (BSP), oxidized baiji polysaccharide (OBSP), methotrexate (MTX), methotrexate-cystamine dihydrochloride intermediate (MTX-ss-NH2) and methotrexate-baiji polysaccharide nanoparticle (MTX-ss-OBSP NPs) described in Example 1;

[0031] Figure 2 Transmission electron microscope images and particle size distribution diagrams of the methotrexate-baiji polysaccharide nanoparticle described in Example 1;

[0032] Figure 3 ROS response diagram (particle size and PDI change) of the methotrexate-baiji polysaccharide nanoparticle described in Example 1;

[0033] Figure 4 In vitro release diagram of methotrexate in the methotrexate-baiji polysaccharide nanoparticle described in Example 1;

[0034] Figure 5 Results diagrams of the effects of the methotrexate, oxidized baiji polysaccharide and methotrexate-baiji polysaccharide nanoparticle on FLS cell viability described in Example 1;

[0035] Figure 6 Results diagrams of the effects of the methotrexate, oxidized baiji polysaccharide and methotrexate-baiji polysaccharide nanoparticle on FLS cell viability described in Example 1;

[0036] Figure 7 Results diagrams of the effects of the methotrexate, oxidized baiji polysaccharide and methotrexate-baiji polysaccharide nanoparticle on FLS cell migration described in Example 1;

[0037] Figure 8 Results diagrams of the effects of the methotrexate, oxidized baiji polysaccharide and methotrexate-baiji polysaccharide nanoparticle on FLS cell migration described in Example 1;

[0038] Figure 9 The results of the flow cytometry test of the effects of methotrexate, oxidized radix bistortae polysaccharide and methotrexate-radix bistortae polysaccharide nanoparticles on the intracellular ROS of FLS cells are shown in the result graph of Example 1;

[0039] Figure 10 The result graph of the promotion of methotrexate, oxidized radix bistortae polysaccharide and methotrexate-radix bistortae polysaccharide nanoparticles to M1 macrophages to M2 macrophages is shown in Example 1. DETAILED DESCRIPTION

[0040] The present application provides a methotrexate-radix bistortae polysaccharide nanoparticle having a structure shown in Formula 1:

[0041] Formula 1.

[0042] The present application also provides a preparation method of the methotrexate-radix bistortae polysaccharide nanoparticle described in the above technical solution, comprising the following steps:

[0043] Mixing methotrexate, a first activating agent, cystamine dihydrochloride and a first solvent to perform a light-shielded reaction to obtain a methotrexate-cystamine dihydrochloride intermediate;

[0044] Mixing radix bistortae polysaccharide, sodium periodate and water to perform a light-shielded oxidation, and then adding ethylene glycol to terminate the oxidation to obtain oxidized radix bistortae polysaccharide;

[0045] Mixing the oxidized radix bistortae polysaccharide, a second activating agent and a second solvent to perform activation to obtain an activated oxidized radix bistortae polysaccharide solution;

[0046] Mixing the methotrexate-cystamine dihydrochloride intermediate and a third solvent, and then mixing with the activated oxidized radix bistortae polysaccharide solution to perform a Schiff base reaction to obtain the methotrexate-radix bistortae polysaccharide nanoparticle.

[0047] In the present application, the preparation process of the methotrexate-radix bistortae polysaccharide nanoparticle is shown in Formula 2:

[0048] Formula 2.

[0049] In the present application, all the preparation raw materials are commercially available products well known to those skilled in the art without special instructions.

[0050] The present application mixes methotrexate, a first activating agent, cystamine dihydrochloride and a first solvent to perform a light-shielded reaction to obtain a methotrexate-cystamine dihydrochloride intermediate.

[0051] In the present application, the first activating agent preferably comprises 1-ethyl-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of the 1-ethyl-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is preferably (0.04~0.08):(0.02~0.05), more preferably (0.05~0.07):(0.03~0.04). In the embodiments of the present application, the mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N- hydroxysuccinimide can be 0.04:0.02. In the present application, the first activating agent functions to activate the carboxyl group.

[0052] In the present application, the first solvent preferably comprises dimethyl sulfoxide (DMSO), an acetic acid solution with a mass concentration of 3%~5%, or a sodium bicarbonate solution with a mass concentration of 6%~8%. In the embodiments of the present application, the first solvent can be DMSO.

[0053] In the present application, the mixing preferably comprises mixing methotrexate and part of the first solvent to obtain a methotrexate solution; mixing the first activating agent and the rest of the first solvent to obtain a first activating agent solution; adding cystamine dihydrochloride after mixing the methotrexate solution and the first activating agent solution; or the mixing preferably comprises mixing methotrexate, the first activating agent, and the first solvent, and then adding cystamine dihydrochloride.

[0054] When the mixing comprises mixing methotrexate and part of the first solvent to obtain a methotrexate solution; mixing the first activating agent and the rest of the first solvent to obtain a first activating agent solution; and adding cystamine dihydrochloride after mixing the methotrexate solution and the first activating agent solution, the present application does not have any special limitation on the preparation process of the methotrexate solution and the first activating agent solution, which can be performed using processes well known to those skilled in the art. In the present application, the mixing of the methotrexate solution and the first activating agent solution is preferably light-shielded stirring after adding the first activating agent solution into the methotrexate solution, the temperature of the light-shielded stirring is preferably room temperature (the specific temperature range is 20~25℃), and the time is preferably 1~3h. In the embodiments of the present application, the temperature of the light-shielded stirring can be 25℃, and the time can be 2h. The present application does not have any special limitation on the adding process of the cystamine dihydrochloride, which can be performed using processes well known to those skilled in the art. The present application does not have any special limitation on the ratio of the part of the first solvent and the rest of the first solvent, which can be mixed at any ratio.

[0055] When the mixing includes adding cystamine dihydrochloride after mixing methotrexate, the first activating agent and the first solvent, the present application does not have any special limitation on the process of the mixing, which can be performed by using the process well known to those skilled in the art. After the mixing is completed, the present application also preferably includes light-protected stirring, the temperature of the light-protected stirring is preferably room temperature (the specific temperature range is 20-25℃), and the time is preferably 18-24h. In the embodiment of the present application, the temperature of the light-protected stirring can be 25℃, and the time can be 24h. The present application does not have any special limitation on the process of adding the cystamine dihydrochloride, which can be performed by using the process well known to those skilled in the art.

[0056] In the present application, the mass ratio of methotrexate to 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the first activating agent is preferably (0.1-0.2):(0.04-0.08), and more preferably 0.1:0.04.

[0057] In the present application, the mass ratio of methotrexate to cystamine dihydrochloride is preferably (0.1-0.2):(0.05-0.1), and more preferably (0.12-0.18):(0.06-0.09). In the embodiment of the present application, the mass ratio of methotrexate to cystamine dihydrochloride can be 0.1:0.05.

[0058] In the present application, the usage ratio of methotrexate to the first solvent is preferably 0.1g:(5mL-10mL), and preferably 0.1g:5mL. In the embodiment of the present application, the usage ratio of methotrexate to the first solvent can be 0.1g:5mL.

[0059] In the present application, the temperature of the light-protected reaction is preferably 20-25℃, and more preferably 25℃; and the time is preferably 18-24h, and more preferably 24h. In the embodiment of the present application, the temperature of the light-protected reaction can be 25℃, and the time can be 24h. In the present application, the light-protected reaction is preferably performed in a protective atmosphere, which is preferably one of a nitrogen atmosphere, an argon atmosphere and a helium atmosphere, and more preferably a nitrogen atmosphere. In the embodiment of the present application, the protective atmosphere can be a nitrogen atmosphere.

[0060] In the present application, the light-protected reaction is an amidation reaction in which the carboxyl group in the methotrexate and the amino group in the cystamine dihydrochloride form an amide bond.

[0061] After the light-protected reaction is completed, the present invention preferably further includes pouring the obtained reaction solution into a buffer solution, stirring until a yellow flocculent precipitate is formed, and then sequentially centrifuging, washing, and freeze-drying. In the present invention, the pH value of the buffer solution is preferably 9.4-9.8, more preferably 9.6. In an embodiment of the present invention, the pH value of the buffer solution can be 9.6. The present invention does not impose any special limitations on the stirring process, and any process well known to those skilled in the art can be used. The present invention does not impose any special limitations on the centrifugation process, and any process well known to those skilled in the art can be used to ensure that the solvent is completely removed. In an embodiment of the present invention, centrifugation is repeated three times to remove the solvent. In the present invention, the washing agent is preferably ultrapure water, and the washing method is preferably centrifugation; the drying is preferably freeze-drying, and the present invention does not impose any special limitations on the freeze-drying process, and any process well known to those skilled in the art can be used.

[0062] After obtaining the methotrexate-cystamine dihydrochloride intermediate, the present invention mixes Bletilla striata polysaccharide, sodium periodate and water, oxidizes it in the dark, and then adds ethylene glycol to terminate the oxidation to obtain oxidized Bletilla striata polysaccharide.

[0063] In this invention, the water is preferably ultrapure water.

[0064] In this invention, the mixing process is preferably carried out by mixing Bletilla striata polysaccharide and water, followed by the addition of sodium periodate. In this invention, the mixing of Bletilla striata polysaccharide and water is preferably carried out under stirring conditions. This invention does not impose any special limitations on the stirring process; any process well-known to those skilled in the art can be used.

[0065] In this invention, the preferred mass ratio of Bletilla striata polysaccharide to sodium periodate is (1~2):(0.2~0.6), more preferably (1.0~1.5):(0.3~0.5). In an embodiment of this invention, the mass ratio of Bletilla striata polysaccharide to sodium periodate can be 1:0.4.

[0066] In this invention, the preferred mass ratio of Bletilla striata polysaccharide to water is (1~2) g:(20~60) mL, more preferably (1~1.5) g:(25~35) mL. In an embodiment of this invention, the mass ratio of Bletilla striata polysaccharide to water can be 1 g:25 mL.

[0067] In this invention, the preferred temperature for the light-protected oxidation is 20-25°C, more preferably 25°C; the preferred time for the light-protected oxidation is 2-10 hours, more preferably 6 hours. In an embodiment of this invention, the temperature for the light-protected oxidation can be 25°C, and the time can be 6 hours.

[0068] In the present application, the mass and volume ratio of the bletilla striata polysaccharide and ethylene glycol is preferably 1.0-2.0 g: 5-10 mL, and more preferably 1.0-1.5 g: 5-7.5 mL. In the embodiment of the present application, the mass and volume ratio of the bletilla striata polysaccharide and ethylene glycol can be 1 g: 5 mL.

[0069] In the present application, the condition for terminating the oxidation is preferably stirring for 0.5-1 h.

[0070] In the present application, after the termination of the oxidation, the hydroxyl groups in the bletilla striata polysaccharide are converted into aldehyde groups.

[0071] After the termination of the oxidation, the present application further preferably comprises dialysis and drying performed in sequence; the dialysis is preferably dialysis for 3 days using a dialysis bag; the drying is preferably freeze-drying; the present application does not have any special limitation on the process of the freeze-drying, which can be performed using a process well known to those skilled in the art.

[0072] After obtaining the oxidized bletilla striata polysaccharide, the present application mixes the oxidized bletilla striata polysaccharide, a second activating agent and a second solvent to perform activation, thereby obtaining an activated oxidized bletilla striata polysaccharide solution.

[0073] In the present application, the second activating agent is preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is preferably (0.01-0.02): (0.006-0.012). In the embodiment of the present application, the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide can be 0.01: 0.006.

[0074] In the present application, the second solvent preferably comprises ultrapure water or DMSO, and in the embodiment of the present application, the second solvent can be ultrapure water.

[0075] In the present application, the mixing preferably comprises mixing the oxidized bletilla striata polysaccharide and the second solvent, and then adding the second activating agent; the present application does not have any special limitation on the mixing process of the oxidized bletilla striata polysaccharide and the second solvent and the adding process of the second activating agent, which can be performed using a process well known to those skilled in the art.

[0076] In the present application, the activation is preferably carried out under light-avoiding and stirring conditions, and the present application does not have any special limitation on the light-avoiding and stirring conditions, which can be carried out by using the conditions well known to those skilled in the art. In the present application, the temperature of the activation is preferably 20-25℃, and more preferably 25℃; and the time of the activation is preferably 1-3h, and more preferably 2h. In the embodiments of the present application, the temperature of the activation can be 25℃, and the time can be 2h.

[0077] After obtaining the activated oxidized BFP solution, the methotrexate-cystamine dihydrochloride intermediate and the third solvent are mixed, and then mixed with the activated oxidized BFP solution to carry out a Schiff base reaction, so as to obtain the methotrexate-BFP nanoparticle.

[0078] In the present application, the third solvent preferably includes DMSO, an acetic acid solution with a mass concentration of 3-5%, or a sodium bicarbonate solution with a mass concentration of 6-8%. In the embodiments of the present application, the third solvent can be DMSO.

[0079] In the present application, the mass of the methotrexate-cystamine dihydrochloride intermediate and the volume of the third solvent are preferably (0.03-0.06) g:(3-6) mL, and more preferably 0.03 g:3 mL.

[0080] The present application does not have any special limitation on the mixing process, which can be carried out by using the process well known to those skilled in the art.

[0081] In the present application, the mass ratio of the methotrexate-cystamine dihydrochloride intermediate to the oxidized BFP is preferably (0.03-0.06):(0.01-0.02), and more preferably (0.04-0.05):(0.012-0.017). In the embodiments of the present application, the mass ratio of the methotrexate-cystamine dihydrochloride intermediate to the oxidized BFP can be 0.03:0.01.

[0082] In the present application, the Schiff base reaction is preferably carried out under light-avoiding and stirring conditions, and the present application does not have any special limitation on the light-avoiding and stirring conditions, which can be carried out by using the process well known to those skilled in the art. In the present application, the temperature of the Schiff base reaction is preferably 20-25℃, and more preferably 25℃; and the time of the Schiff base reaction is preferably 18-36h, and more preferably 20-30h. In the embodiments of the present application, the temperature of the Schiff base reaction can be 25℃, and the time can be 24h.

[0083] The application also provides application of the methotrexate-baiji polysaccharide nanoparticle or the methotrexate-baiji polysaccharide nanoparticle prepared by the preparation method in the preparation of a drug for treating a disease with a high active oxygen pathological microenvironment. In the application, the disease with a high active oxygen pathological microenvironment preferably includes rheumatoid arthritis. The application does not have any special limitation on the method of the application, and the method known to those skilled in the art can be used.

[0084] The technical solutions in the application will be clearly and completely described below in combination with the embodiments in the application. Obviously, the described embodiments are only some of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0085] Embodiment 1

[0086] 0.1 g of methotrexate was dissolved in 5 mL of DMSO to obtain a methotrexate solution;

[0087] 0.04 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.02 g of N-hydroxysuccinimide were dissolved in 5 mL of DMSO to obtain an activator solution;

[0088] The activator solution was added to the methotrexate solution, and after stirring at room temperature (25 DEG C) in the dark for 2 h, 0.05 g of cystamine dihydrochloride was added, and the reaction was carried out at room temperature (25 DEG C) in the dark for 24 h. Under stirring, the obtained reaction solution was slowly poured into a buffer solution with pH=9.6 to generate yellow flocculent precipitate. The solvent was removed by centrifugation for 3 times, and then the buffer solution was cleaned by centrifugation with ultrapure water. After freeze-drying, a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark) was obtained;

[0089] 1.0 g of baiji polysaccharide and 25 mL of ultrapure water were stirred and mixed until completely dissolved. 0.4 g of sodium periodate was added, and oxidation was carried out at 25 DEG C in the dark for 6 h. 5 mL of ethylene glycol was added, and stirring (in the dark, at 25 DEG C) was continued for 30 min. Then, the reaction was terminated. The obtained reaction solution was loaded into a dialysis bag, and dialysis was carried out for 3 days. After dialysis, freeze-drying was carried out to obtain oxidized baiji polysaccharide;

[0090] After 0.01 g of the oxidized baiji polysaccharide and 1 mL of ultrapure water were mixed and dissolved, 0.01 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.006 g of N-hydroxysuccinimide were added. Stirring was carried out at room temperature in the dark for 2 h to obtain an activated oxidized baiji polysaccharide solution;

[0091] After 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO, it was added to the activated oxidized bletilla striata polysaccharide solution, and after stirring in the dark for 24 h, dialysis and freeze-drying were performed to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0092] Example 2

[0093] After 0.2 g of methotrexate, 0.08 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and 0.04 g of N-hydroxysuccinimide were dissolved in 10 mL of DMSO, stirring was performed at room temperature (25°C) in the dark for 1 h, 0.10 g of cystamine dihydrochloride was added, and reaction was performed at room temperature (25°C) in the dark for 24 h. The obtained reaction solution was slowly poured into a buffer solution with pH=9.6 under stirring until a yellow flocculent precipitate was generated. The solvent was removed by centrifugation for 3 times, and the buffer solution was cleaned by centrifugation with ultrapure water. Freeze-drying was performed to obtain a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark);

[0094] After 1.0 g of bletilla striata polysaccharide and 25 mL of ultrapure water were stirred and mixed until completely dissolved, 0.4 g of sodium periodate was added, and oxidation was performed at a temperature of 25°C in the dark for 2 h. Oxidation was terminated by adding 5 mL of ethylene glycol, and after stirring for 30 min (in the dark, at a temperature of 25°C), the obtained reaction solution was loaded into a dialysis bag, dialysis was performed for 3 days, and freeze-drying was performed after dialysis was completed to obtain oxidized bletilla striata polysaccharide;

[0095] After 0.01 g of the oxidized bletilla striata polysaccharide and 1 mL of ultrapure water were mixed and dissolved, 0.01 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.006 g of N-hydroxysuccinimide were added, and stirring was performed in the dark at room temperature for 2 h to obtain an activated oxidized bletilla striata polysaccharide solution;

[0096] After 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO, it was added to the activated oxidized bletilla striata polysaccharide solution, and after stirring in the dark for 24 h, dialysis and freeze-drying were performed to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0097] Example 3

[0098] 0.1 g methotrexate, 0.04 g 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.02 g N-hydroxysuccinimide were dissolved in 10 mL DMSO, after stirring at room temperature (25°C) for 2 h in the dark, 0.05 g cystamine dihydrochloride was added, and the reaction was carried out at room temperature (25°C) for 24 h in the dark. The resulting reaction solution was slowly poured into a buffer solution with pH = 9.6 under stirring until a yellow flocculent precipitate was produced. The solvent was removed by centrifugation for 3 times, and the buffer solution was cleaned by centrifugation with ultrapure water. The methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark) was obtained by freeze-drying.

[0099] 1.0 g Bletilla striata polysaccharide and 25 mL ultrapure water were stirred and mixed until completely dissolved. 0.4 g sodium periodate was added, and oxidation was carried out at 25°C in the dark for 4 h. 5 mL ethylene glycol was added, and stirring (in the dark at 25°C) was continued for 30 min. The oxidation was terminated, and the resulting reaction solution was loaded into a dialysis bag. Dialysis was carried out for 3 days. After dialysis, freeze-drying was carried out to obtain oxidized Bletilla striata polysaccharide.

[0100] 0.01 g of the oxidized Bletilla striata polysaccharide and 1 mL DMSO were mixed and dissolved. 0.01 g 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.006 g N-hydroxysuccinimide were added. The resulting solution was stirred at room temperature in the dark for 3 h to obtain an activated oxidized Bletilla striata polysaccharide solution.

[0101] 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL DMSO and added to the activated oxidized Bletilla striata polysaccharide solution. After stirring in the dark for 24 h, dialysis and freeze-drying were carried out to obtain methotrexate-Bletilla striata polysaccharide nanoparticles.

[0102] Example 4

[0103] 0.1 g methotrexate was dissolved in 5 mL DMSO to obtain a methotrexate solution.

[0104] 0.04 g 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.02 g N-hydroxysuccinimide were dissolved in 5 mL DMSO to obtain an activator solution.

[0105] The activated agent solution is added to the methotrexate solution, stirred at room temperature (25 °C) in the dark for 2 h, then 0.05 g of cystamine dihydrochloride is added, reacted at room temperature (25 °C) in the dark for 24 h, and the obtained reaction solution is slowly poured into a buffer solution with pH = 9.6 under stirring until yellow flocculent precipitate is generated; the solvent is removed by centrifugation for 3 times, and the buffer solution is cleaned by centrifugation with ultrapure water, and then freeze-dried to obtain a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark);

[0106] 1.0 g of bletilla striata polysaccharide and 25 mL of ultrapure water are stirred and mixed until completely dissolved, 0.4 g of sodium periodate is added, and oxidized at 25 °C in the dark for 8 h; 5 mL of ethylene glycol is added, and stirred (in the dark, at 25 °C) for 30 min, then the oxidation is terminated; the obtained reaction solution is loaded into a dialysis bag, dialyzed for 3 days, and then freeze-dried to obtain oxidized bletilla striata polysaccharide;

[0107] 0.02 g of the oxidized bletilla striata polysaccharide and 2 mL of 5% acetic acid solution are mixed and dissolved, then 0.02 g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 0.012 g of N-hydroxysuccinimide are added, stirred at room temperature in the dark for 3 h to obtain an activated oxidized bletilla striata polysaccharide solution;

[0108] 0.06 g of the methotrexate-cystamine dihydrochloride intermediate is dissolved in 6 mL of 5% acetic acid solution, then added to the activated oxidized bletilla striata polysaccharide solution, stirred in the dark for 36 h, and then dialyzed and freeze-dried to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0109] Example 5

[0110] 0.2 g of methotrexate, 0.08 g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and 0.04 g of N-hydroxysuccinimide are dissolved in 10 mL of DMSO, stirred at room temperature (25 °C) in the dark for 1 h under nitrogen protection, then 0.10 g of cystamine dihydrochloride is added, reacted at room temperature (25 °C) in the dark for 24 h, and the obtained reaction solution is slowly poured into a buffer solution with pH = 9.6 under stirring until yellow flocculent precipitate is generated; the solvent is removed by centrifugation for 3 times, and the buffer solution is cleaned by centrifugation with ultrapure water, and then freeze-dried to obtain a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark);

[0111] 1.0 g of bletilla striata polysaccharide and 25 mL of ultrapure water were stirred and mixed until completely dissolved, 0.4 g of sodium periodate was added, and oxidation was carried out at a temperature of 25 ℃ for 10 h in the dark, 5 mL of ethylene glycol was added to terminate the oxidation, and after stirring for 30 min (in the dark, at a temperature of 25 ℃), the obtained reaction solution was loaded into a dialysis bag, dialysis was carried out for 3 days, and after dialysis was completed, freeze-drying was carried out, to obtain oxidized bletilla striata polysaccharide;

[0112] After 0.01 g of the oxidized bletilla striata polysaccharide and 1 mL of DMSO were mixed and dissolved, 0.01 g of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and 0.006 g of N-hydroxysuccinimide were added, stirring was carried out at room temperature in the dark for 2 h, to obtain an activated oxidized bletilla striata polysaccharide solution;

[0113] After 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO, it was added to the activated oxidized bletilla striata polysaccharide solution, stirring was carried out in the dark for 20 h, and then dialysis and freeze-drying were carried out, to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0114] Test example

[0115] Fourier infrared spectrum detection: the bletilla striata polysaccharide, the oxidized bletilla striata polysaccharide, the methotrexate, the methotrexate-cystamine dihydrochloride intermediate, and the methotrexate-bletilla striata polysaccharide nanoparticles described in Example 1 were respectively mixed with a small amount of KBr, ground uniformly, and pressed into thin sheets. Scanning was carried out on the thin sheets by Fourier infrared spectrum in the wavelength range of 500-4000 cm -1 ;

[0116] Figure 1 The infrared spectrum of the bletilla striata polysaccharide (BSP), the oxidized bletilla striata polysaccharide (OBSP), the methotrexate (MTX), the methotrexate-cystamine dihydrochloride intermediate (MTX-ss-NH2), and the methotrexate-bletilla striata polysaccharide nanoparticles (MTX-ss-OBSP NPs) described in Example 1 was obtained by Figure 1 It can be known that the absorption peak of the stretching vibration of -OH in the BSP infrared spectrum is at 3410 cm -1 , the absorption peak of the stretching vibration of C-H in the sugar -CH2 or -CH3 is at 2912 cm -1 , the absorption peak of the bending vibration of O-H is at 1637 cm -1 , and the characteristic absorption peak of pyranose is at 1030 cm -1 ; compared with the BSP infrared spectrum, the OBSP infrared spectrum has an absorption peak of the stretching vibration of the C=O bond of the aldehyde group at 1700 cm -1 , indicating that the OBSP preparation is successful; the absorption peak of the stretching vibration of -NH2 in the MTX infrared spectrum is at 1644 cm -1 , and the absorption peak of the stretching vibration of -NH2 is at 1603 cm -1stretching vibration absorption peak of -COOH; the two peaks are characteristic absorption peaks of MTX. Compared with the infrared spectrum of MTX, the infrared spectrum of MTX-ss-NH2 has a weak amide I band (NC=O bond) stretching vibration absorption peak at 1733 cm -1 a bending vibration absorption peak of amide II band (CN-H bond) at 1553 cm -1 , which indicates that the carboxyl group of MTX forms an amide bond with the amino group of Cys, and MTX-ss-NH2 is successfully prepared; compared with the infrared spectrum of OBSP, the infrared spectrum of MTX-ss-OBSP has an absorption peak of carbonyl (C=O) at 1700 cm -1 disappears, and a stretching vibration absorption peak of imine bond (C=N) appears at 1636 cm -1 , which indicates that the aldehyde group of OBSP forms a Schiff base structure with the amino group; and a new absorption peak appears at 1450 cm -1 , which is due to the introduction of the benzene ring structure of MTX in the polymer; this indicates that MTX-ss-OBSP is successfully prepared.

[0117] Particle size, PDI, and Zeta potential detection: from Figure 2 , A (A is the transmission electron micrograph of methotrexate-baiji polysaccharide nanoparticles described in Example 1) can be known that the methotrexate-baiji polysaccharide nanoparticles described in Example 1 are observed by transmission electron microscopy to be uniform spherical shape and have good dispersity; the particle size, PDI, and Zeta potential of the methotrexate-baiji polysaccharide nanoparticles described in Example 1 are detected by using a Malvern particle size analyzer, from Figure 2 , B (B is the particle size distribution graph of the methotrexate-baiji polysaccharide nanoparticles described in Example 1) can be known that the average particle size of the methotrexate-baiji polysaccharide nanoparticles is 211 ± 4.12 nm, the PDI is 0.20 ± 0.00, and the Zeta potential is -42.57 ± 2.15.

[0118] ROS responsiveness (change in particle size and PDI) of nanoparticles: the particle size distribution and PDI of the methotrexate-baiji polysaccharide nanoparticles described in Example 1 in a phosphate buffer solution at pH = 7.4 and a phosphate buffer solution at pH = 7.4 + 1 mM H2O2 at specific time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d) are detected by using a Malvern particle size analyzer. Due to the presence of disulfide bonds, the nanoparticles theoretically have responsive cleavage behavior under H2O2 conditions, which is proved by changing the dispersion medium of the nanoparticles. From Figure 3 , A (A is the particle size distribution graph of the nanoparticles in different solutions) and Figure 3B (PDI change of nanoparticles in different solutions) in Figure 5 shows that when H2O2 is added to the PBS solution with pH = 7.4, the particle size of the nanoparticles decreases and the uniformity of the particle size distribution increases, indicating that the nanoparticles have ROS response capability.

[0119] In vitro release of methotrexate: The cumulative release rate of methotrexate in the methotrexate-baijiapu polysaccharide nanoparticles described in Example 1 was detected by the dialysis bag method. After the nanoparticles were fully dispersed in the release medium, they were loaded into the dialysis bag and suspended in 40 mL of PBS with pH = 7.4 and 40 mL of PBS with pH = 7.4 and 1 mM H2O2, respectively, and placed in a 37°C and 100 rpm / min shaker. At specific time points (1, 1.5 h, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h), 2 mL of supernatant was taken, and an equal amount of preheated release medium at 37°C was added. The released methotrexate in the supernatant was quantitatively analyzed by high performance liquid chromatography. Each experiment was repeated three times, and the results were expressed as mean ± standard deviation. The R 2 =0.9993 (Abs (302 nm) = 0.051 x [MTX] (μg· mL -1 ) + 0.001) calibration curve was established to determine the release amount of MTX in the sample. The in vitro release results of methotrexate-baijiapu polysaccharide nanoparticles are shown in Figure 4 . The release rate of methotrexate was faster within 24 h. The cumulative release rate of methotrexate-baijiapu polysaccharide nanoparticles in PBS with pH = 7.4 was 76.31 ± 3.00% at 108 h, and the cumulative release rate was 86.79 ± 1.09% under the action of PBS with pH = 7.4 and 1 mM H2O2 at 108 h, which proved that methotrexate-baijiapu polysaccharide nanoparticles had good release effect under the condition of H2O2.

[0120] CCK8 cell activity test: FLS cells were seeded in a 96-well plate at a density of 1.0 x 10 4 cells / well and incubated at 37°C, 5% CO2 for 24 h; FLS cells were treated with different concentrations (0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 9.0 μg / mL, 10.0 μg / mL, 12.0 μg / mL) of methotrexate, oxidized baijiapu polysaccharide, and methotrexate-baijiapu polysaccharide nanoparticles for 24 h, then 10 μL of CCK-8 reagent was added, and the reagent should be added slowly to avoid air bubbles, then incubated at 37°C for 30 min, and then the absorbance (A = 450 nm) value was detected at 450 nm wavelength by an enzyme-labeled instrument; Figure 5Figure of the effect of methotrexate, oxidized radix bistorta polysaccharide and methotrexate-radix bistorta polysaccharide nanoparticles on the viability of FLS cells described in Example 1, from Figure 5 It can be seen that, based on CCK8 analysis, methotrexate-radix bistorta polysaccharide nanoparticles at 10 μg / mL can inhibit the proliferation of FLS cells, and the inhibitory effect is weaker than that of free drug methotrexate at the same concentration, which is due to the negligible cytotoxicity of oxidized radix bistorta polysaccharide, which improves the biocompatibility of methotrexate-radix bistorta polysaccharide nanoparticles.

[0121] Live-dead cell staining experiment: using calcein (AM) / propidium iodide (PI) to evaluate the ability of methotrexate-radix bistorta polysaccharide nanoparticles described in Example 1 to kill FLS cells in vitro; FLS cells were cultured in 24-well plates for 24 h (6×10 4 cells / well), then incubated with complete culture medium, methotrexate described in Example 1, oxidized radix bistorta polysaccharide described in Example 1, methotrexate-radix bistorta polysaccharide nanoparticles described in Example 1 (concentration of methotrexate was 10 μg / mL) for 24 h. AM / PI staining was performed, and observed under a fluorescence microscope; Figure 6 Figure of the effect of methotrexate, oxidized radix bistorta polysaccharide and methotrexate-radix bistorta polysaccharide nanoparticles on the viability of FLS cells described in Example 1, from Figure 6 It can be seen that the methotrexate-radix bistorta polysaccharide nanoparticles killed FLS cells, and the degree of killing was stronger than that of free drug OBSP and weaker than that of MTX, which was consistent with the cytotoxicity results.

[0122] Scratch experiment: FLS cells were seeded in 24-well plates at a density of 2×10 5 cells / well, and placed in a 37°C incubator with 5% CO2 and complete culture medium for continuous culture. When the cell density reached more than 90%, a straight line was drawn in the center of the well with a 200 μL sterile gun head, and then washed twice with phosphate buffered saline (PBS) to smooth the edges of the scratch and remove floating cells. The image of the scratch site was obtained under an inverted microscope (×40) and recorded as 0 h. Drug solutions were prepared with serum-free culture medium, and the groups were as follows: control group, methotrexate group (methotrexate described in Example 1), oxidized radix bistorta polysaccharide group (oxidized radix bistorta polysaccharide described in Example 1), methotrexate-oxidized radix bistorta polysaccharide nanoparticle group (concentration of methotrexate was 10 μg / mL, methotrexate-oxidized radix bistorta polysaccharide nanoparticles described in Example 1). After 12 h and 24 h of intervention, the images of the scratch sites were obtained under an inverted microscope (×40) at the same site, and recorded as 12 h and 24 h, respectively. Among them, Figure 7The results of the effects of methotrexate, oxidized radix bistortae polysaccharide and methotrexate-radix bistortae polysaccharide nanoparticles on FLS cell migration are shown in the graphs (A is the detection of the effects of methotrexate, oxidized radix bistortae polysaccharide and methotrexate-radix bistortae polysaccharide nanoparticles on FLS cell migration, and B is the quantitative analysis graph of the effects of methotrexate, oxidized radix bistortae polysaccharide and methotrexate-radix bistortae polysaccharide nanoparticles on FLS cell migration), which are shown in Figure 7 It can be seen that the methotrexate group and the methotrexate-radix bistortae polysaccharide nanoparticle group have the most obvious inhibitory effect on the migration ability of FLS cells, followed by the oxidized radix bistortae polysaccharide group.

[0123] ROS scavenging experiment: an inverted fluorescence microscope was used to observe the effect of different drug treatments on the intracellular ROS level of FLS cells, and the specific operation was as follows: FLS cells were seeded in a 24-well plate at a density of 6x10 4 cells / well and cultured overnight. The original culture medium was removed, and complete culture medium was added to the blank group. The experimental and control groups were stimulated with the same amount of Rosup (final concentration 50 μg / mL) and incubated for 4 h. The whole culture was aspirated, and the cells were gently rinsed twice with PBS. The experimental group was added with 0.5 mL of complete culture medium containing methotrexate (methotrexate described in Example 1), oxidized radix bistortae polysaccharide (oxidized radix bistortae polysaccharide described in Example 1), and methotrexate-oxidized radix bistortae polysaccharide nanoparticles (concentration of methotrexate 10 μg / mL, methotrexate-oxidized radix bistortae polysaccharide nanoparticles described in Example 1), and incubated for 12 h. The old culture medium was removed and rinsed with PBS. Serum-free culture medium containing DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) was added, and the cells were incubated in a 37°C cell incubator for 30 min in the dark. After rinsing with PBS, an appropriate amount of PBS was added to each well, and the effect of different drug treatments on the intracellular ROS level of FLS cells was observed under an inverted fluorescence microscope. The fluorescence intensity was quantified using ImageJ software. The results are shown in Figure 8 A (A is the effect of methotrexate, oxidized radix bistortae polysaccharide and methotrexate-oxidized radix bistortae polysaccharide nanoparticles on intracellular ROS under an inverted fluorescence microscope) show that, compared with the control group, the green fluorescence signal in the methotrexate-oxidized radix bistortae polysaccharide nanoparticle treatment group at the same concentration decreased significantly. This indicates that methotrexate-oxidized radix bistortae polysaccharide nanoparticles reduce intracellular ROS, demonstrating significant antioxidant performance. The intracellular ROS level of the oxidized radix bistortae polysaccharide group also decreased. The fluorescence intensity of methotrexate was stronger than that of the control group, which may be because methotrexate is a folate antagonist that can affect cell metabolism through multiple pathways, produce oxidative stress, and increase ROS production. For Figure 8 B (B is the quantitative analysis graph) of the fluorescence quantitative statistical results also verifies this phenomenon.

[0124] To further study the ROS scavenging effect in cells, a flow cytometer was used for further determination. FLS cells were seeded in a 6-well plate at a density of 1x106 The cells were resuspended in serum-free medium after centrifugation, and then transferred to a flow tube. The intracellular DCF fluorescence signal was detected by selecting the FITC channel of the flow cytometer. Figure 9 As shown in A of FIG. 11 (A is a graph showing the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-oxidized Bletilla striata polysaccharide nanoparticles on intracellular ROS detected by flow cytometry), compared with the control group, methotrexate-Bletilla striata polysaccharide nanoparticles had the strongest ROS scavenging ability, followed by oxidized Bletilla striata polysaccharide. Figure 9 As shown in B of FIG. 11 (B is a quantitative analysis graph), methotrexate-Bletilla striata polysaccharide nanoparticles could scavenge the abnormally increased ROS in FLS cells. This result was consistent with the ROS results detected by an inverted fluorescence microscope.

[0125] Effect of methotrexate-Bletilla striata polysaccharide nanoparticles on macrophage polarization: mouse monocyte macrophage Raw264.7 cells were seeded in 6-well plates at a density of 1×10 6 After 24 h of adhesion culture, 10 μg / mL of lipopolysaccharide was added for 12 h, the culture medium was discarded, and the cells were washed with PBS. Then, the cells were treated with complete DMEM medium (control group), DMEM complete medium containing 10 μg / mL of methotrexate (methotrexate group), DMEM complete medium containing 10 μg / mL of oxidized Bletilla striata polysaccharide (oxidized Bletilla striata polysaccharide group), and DMEM complete medium containing 10 μg / mL of methotrexate-Bletilla striata polysaccharide nanoparticles (methotrexate-Bletilla striata polysaccharide nanoparticle group, the concentration of methotrexate was 10 μg / mL) for 24 h. The cells were washed with PBS, centrifuged (1000 rpm×5 min) to discard the supernatant, and then 100 μL of CD86 antibody (M1 marker) was added for 4°C incubation in the dark for 30 min. The mixture was mixed by blowing and centrifuged (1000 rpm×5 min) to discard the supernatant. The cells were resuspended with PBS, and then 4°C pre-cooled fixing solution was added for 1 h of incubation at room temperature in the dark. The cells were washed with PBS buffer, 500 μL of membrane-breaking solution was added, and then 100 μL of CD206 antibody (M2 marker) was added for 30 min of incubation at room temperature in the dark. The cells were centrifuged (1000 rpm×5 min) to discard the supernatant, washed with PBS buffer, resuspended with 300 μL of PBS, and then detected by flow cytometry. Figure 10 A and B of FIG. 12 (A is a graph showing the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles on the polarization of M1 macrophages to M2 macrophages, and B is a quantitative analysis graph), compared with the control group, methotrexate-Bletilla striata polysaccharide nanoparticles had the strongest ability to promote the polarization of M1 macrophages to M2 macrophages, followed by oxidized Bletilla striata polysaccharide. Figure 10It can be known that compared with the control group, the proportion of CD86+ macrophages in the methotrexate-baiji polysaccharide nanoparticle group and the oxidized baiji polysaccharide group is significantly reduced, and the proportion of CD206+ cells is increased. According to statistical analysis, the ratio of M2 / M1 cells in the methotrexate-baiji polysaccharide nanoparticle group and the oxidized baiji polysaccharide group is higher than that in the control group. The results show that the methotrexate-baiji polysaccharide nanoparticles can inhibit the M1 polarization of macrophages and promote the M2 polarization of macrophages.

[0126] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. It should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A type of methotrexate-Bletilla striata polysaccharide nanoparticle, characterized in that, It has the structure shown in Equation 1: Formula 1.

2. The method for preparing methotrexate-Bletilla striata polysaccharide nanoparticles according to claim 1, characterized in that, Includes the following steps: Methotrexate, a first activator, cystamine dihydrochloride and a first solvent are mixed and subjected to a light-protected reaction to obtain a methotrexate-cystamine dihydrochloride intermediate; Bletilla striata polysaccharide, sodium periodate and water were mixed and oxidized in the dark. Ethylene glycol was added to terminate the oxidation to obtain oxidized Bletilla striata polysaccharide. The oxidized Bletilla striata polysaccharide, the second activator, and the second solvent are mixed and activated to obtain an activated oxidized Bletilla striata polysaccharide solution. The methotrexate-cystamine dihydrochloride intermediate and the third solvent were mixed and then mixed with the activated oxidized Bletilla striata polysaccharide solution to carry out a Schiff base reaction, thereby obtaining the methotrexate-Bletilla striata polysaccharide nanoparticles.

3. The preparation method according to claim 2, characterized in that, Both the first activator and the second activator are 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide in the first activator is (0.04~0.08):(0.02~0.05). The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide in the second activator is (0.01~0.02):(0.006~0.012).

4. The preparation method according to claim 3, characterized in that, The mass ratio of methotrexate to 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the first activator is (0.1~0.2):(0.04~0.08). The mass ratio of methotrexate to cystamine dihydrochloride is (0.1~0.2):(0.05~0.1).

5. The preparation method according to claim 2, characterized in that, The mass ratio of Bletilla striata polysaccharide to sodium periodate is (1~2):(0.2~0.6); The ratio of Bletilla striata polysaccharide to ethylene glycol is (1.0~2.0) g : (5~10) mL.

6. The preparation method according to claim 2 or 5, characterized in that, The oxidation process in the dark is carried out at a temperature of 20℃~25℃ for 2~10 hours.

7. The preparation method according to claim 3, characterized in that, The mass ratio of the oxidized Bletilla striata polysaccharide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the second activator, and N-hydroxysuccinimide in the second activator is (0.01~0.02):(0.01~0.02):(0.006~0.012). The activation is carried out under light-protected conditions, at a temperature of 20-25°C, for a time of 1-3 hours.

8. The preparation method according to claim 2, characterized in that, The mass ratio of the methotrexate-cystamine dihydrochloride intermediate to the oxidized Bletilla striata polysaccharide is (0.03~0.06):(0.01~0.02).

9. The preparation method according to claim 2 or 8, characterized in that, The Schiff base reaction is carried out under light-protected conditions, at a temperature of 20-25°C, for a time of 18-36 hours.

10. The use of the methotrexate-Bletilla striata polysaccharide nanoparticles according to claim 1 or the methotrexate-Bletilla striata polysaccharide nanoparticles prepared by the preparation method according to any one of claims 2 to 9 in the preparation of a medicament for treating rheumatoid arthritis.

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