Methotrexate-bletilla striata polysaccharide nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of rheumatoid arthritis medicines, in particular to methotrexate-bletilla striata polysaccharide nanoparticles as well as a preparation method and application thereof. The invention provides methotrexate-bletilla striata polysaccharide nanoparticles. The methotrexate-bletilla striata polysaccharide nanoparticles have a structure as shown in a formula 1. The nanoparticle is formed by connecting methotrexate and bletilla striata polysaccharide through a disulfide bond, and theoretically, the nanoparticle can respond to high active oxygen stimulation in a rheumatoid arthritis microenvironment to realize drug release. In addition, the research proves that the nanoparticles can effectively inhibit proliferation and migration of fibroblast-like synovial cells, adjust polarization of M1 type macrophages to M2 type macrophages and relieve synovial inflammation. The methotrexate-bletilla striata polysaccharide nanoparticles are simple in preparation process operation and convenient to use, and have good development and utilization prospects. Formula 1

Description

Technical Field

[0001] The present invention relates to the technical field of rheumatoid arthritis drugs, and in particular to methotrexate-bletilla striata polysaccharide nanoparticles and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting approximately 0.5% to 1.0% of the global population. Women, in particular, are at two to three times the risk of developing the disease compared to men. RA is characterized by severe inflammation at the synovial joints, leading to unbearable joint pain, swelling, and stiffness, and can even lead to disability and premature death. Methotrexate (MTX) is the preferred antirheumatic drug for the treatment of RA, primarily administered orally or by injection. These two routes of administration present numerous challenges. First, oral MTX bioavailability varies among individuals, and absorption is easily affected by food and the gastrointestinal environment. It can also cause systemic adverse reactions such as gastrointestinal reactions and liver and kidney dysfunction. Second, intravenous MTX administration can pose systemic toxic risks, including mucositis (such as oral ulcers) and hepatotoxicity and renal toxicity. Third, oral or intravenous administration lacks targeted targeting, resulting in insufficient drug accumulation in affected joints, necessitating higher doses to achieve efficacy, further exacerbating side effects. Fourth, traditional delivery methods cannot adjust the release rate according to the severity of joint inflammation. Studies have found that the onset of RA inflammation is closely associated with M1 / M2 macrophage imbalance, abnormal proliferation of fibroblast-like synoviocytes, and high levels of reactive oxygen species (ROS). Therefore, based on the specific characteristics of the RA microenvironment, it is reasonable to develop a multifunctional nanoparticle for intra-articular injection that maintains the M1 / M2 macrophage balance, inhibits abnormal proliferation of fibroblast-like synoviocytes in RA patients, and responds to ROS stimulation to achieve on-demand drug release to effectively treat rheumatoid arthritis. This nanoparticle can also be used for local drug delivery in diseases with a high reactive oxygen species pathological microenvironment. Summary of the Invention

[0003] In view of this, the present invention aims to provide methotrexate-Bletilla striata polysaccharide nanoparticles and their preparation method and application. The methotrexate-Bletilla striata polysaccharide nanoparticles are responsive to microenvironmental ROS.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a methotrexate-Bletilla striata polysaccharide nanoparticle having a structure shown in Formula 1: Formula 1.

[0005] The present invention also provides a method for preparing the methotrexate-Bletilla striata polysaccharide nanoparticles described in the above technical solution, comprising the following steps: Mixing methotrexate, a first activator, cystamine dihydrochloride and a first solvent, and performing a reaction in the dark to obtain a methotrexate-cystamine dihydrochloride intermediate; Mixing bletilla striata polysaccharide, sodium periodate and water, performing light-protected oxidation, and then adding ethylene glycol to terminate the oxidation to obtain oxidized bletilla striata polysaccharide; mixing the oxidized bletilla striata polysaccharide, a second activator, and a second solvent to activate the mixture, thereby obtaining an activated oxidized bletilla striata polysaccharide solution; The methotrexate-cystamine dihydrochloride intermediate and the third solvent are mixed, and then mixed with the activated oxidized bletilla striata polysaccharide solution, and subjected to a Schiff base reaction to obtain the methotrexate-bletilla striata polysaccharide nanoparticles.

[0006] Preferably, the first activator and the second activator are both 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).

[0007] Preferably, 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).

[0008] Preferably, the mass ratio of the bletilla striata polysaccharide to sodium periodate is (1-2): (0.2-0.6); The dosage ratio of the bletilla striata polysaccharide and ethylene glycol is (1.0-2.0) g: (5-10) mL.

[0009] Preferably, the temperature of the light-avoiding oxidation is 20° C. to 25° C., and the time is 2 to 10 hours.

[0010] Preferably, 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 in a dark environment at a temperature of 20-25° C. for 1-3 hours.

[0011] Preferably, the mass ratio of the methotrexate-cystamine dihydrochloride intermediate to the oxidized bletilla striata polysaccharide is (0.03-0.06): (0.01-0.02).

[0012] Preferably, the Schiff base reaction is carried out in the dark, at a temperature of 20-25° C., and for 18-36 hours.

[0013] The present invention also provides the use of the methotrexate-Bletilla striata polysaccharide nanoparticles described in the above technical solution or the methotrexate-Bletilla striata polysaccharide nanoparticles prepared by the preparation method described in the above technical solution in the preparation of drugs for treating diseases with a pathological microenvironment with high reactive oxygen species.

[0014] The present invention provides a methotrexate-Bletilla striata polysaccharide nanoparticles (MTX-ss-OBSP NPs) having a structure shown in Formula 1: Formula 1.

[0015] The methotrexate-Bletilla striata polysaccharide nanoparticles described in this invention are responsive to microenvironmental ROS. Formed by disulfide bonds linking methotrexate and Bletilla striata polysaccharide, these nanoparticles can release the drug in response to the highly reactive oxygen species (ROS) present in the rheumatoid arthritis microenvironment. Furthermore, research has demonstrated that these nanoparticles can effectively inhibit the proliferation and migration of fibroblast-like synoviocytes, regulate the polarization of M1 macrophages to M2 macrophages, and alleviate synovial inflammation. The preparation process of these methotrexate-Bletilla striata polysaccharide nanoparticles is simple, easy to use, and has promising prospects for development and utilization.

[0016] Compared with methotrexate or Bletilla striata polysaccharide, preparing the two into methotrexate-Bletilla striata polysaccharide nanoparticles has the following advantages: on the one hand, the particle size of methotrexate-Bletilla striata polysaccharide nanoparticles is less than 300nm, 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 is a high molecular compound, and its solution is difficult to enter the cell to exert pharmacological effects. Preparing it into nanoparticles can not only more easily enter the cell to eliminate the high level of ROS in the cell to improve synovial inflammation, but also regulate M2 macrophage polarization, thereby slowing the progression of rheumatoid arthritis.

[0017] The present invention also provides a method for preparing methotrexate-Bletilla striata polysaccharide nanoparticles according to the above technical solution, comprising the following steps: mixing methotrexate, a first activator, cystamine dihydrochloride, and a first solvent, and performing a light-proof reaction to obtain a methotrexate-cystamine dihydrochloride intermediate; mixing Bletilla striata polysaccharide, sodium periodate, and water, performing a light-proof oxidation, and then adding ethylene glycol to terminate the oxidation to obtain oxidized Bletilla striata polysaccharide; mixing the oxidized Bletilla striata polysaccharide, a second activator, and a second solvent, and performing activation to obtain an activated oxidized Bletilla striata polysaccharide solution; mixing the methotrexate-cystamine dihydrochloride intermediate and a third solvent, and then mixing with the activated oxidized Bletilla striata polysaccharide solution, and performing a Schiff base reaction to obtain the methotrexate-Bletilla striata polysaccharide nanoparticles. The preparation method of the present invention has a simple preparation process and readily available raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The infrared spectra of Bletilla striata polysaccharide (BSP), oxidized Bletilla striata polysaccharide (OBSP), methotrexate (MTX), methotrexate-cystamine dihydrochloride intermediate (MTX-ss-NH2) and methotrexate-Bletilla striata polysaccharide nanoparticles (MTX-ss-OBSP NPs) described in Example 1 are shown; Figure 2 The transmission electron micrograph and particle size distribution diagram of the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1; Figure 3 This is the ROS responsiveness graph (particle size and PDI changes) of the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1; Figure 4 This is the in vitro release graph of methotrexate from the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1; Figure 5 This is a graph showing the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles on FLS cell viability as described in Example 1; Figure 6 This is a graph showing the live and dead cell staining results of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1; Figure 7 This is a graph showing the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles on FLS cell migration as described in Example 1; Figure 8 This is a graph showing the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles on ROS in FLS cells observed under an inverted fluorescence microscope as described in Example 1; Figure 9 This is a graph showing the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles on ROS in FLS cells as detected by flow cytometry as described in Example 1; Figure 10 This is a graph showing the results of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles promoting the polarization of M1 macrophages to M2 macrophages as described in Example 1. DETAILED DESCRIPTION

[0019] The present invention provides a methotrexate-Bletilla striata polysaccharide nanoparticle having a structure shown in Formula 1: Formula 1.

[0020] The present invention also provides a method for preparing the methotrexate-Bletilla striata polysaccharide nanoparticles described in the above technical solution, comprising the following steps: Mixing methotrexate, a first activator, cystamine dihydrochloride and a first solvent, and performing a reaction in the dark to obtain a methotrexate-cystamine dihydrochloride intermediate; Mixing bletilla striata polysaccharide, sodium periodate and water, performing light-protected oxidation, and then adding ethylene glycol to terminate the oxidation to obtain oxidized bletilla striata polysaccharide; mixing the oxidized bletilla striata polysaccharide, a second activator, and a second solvent to activate the mixture, thereby obtaining an activated oxidized bletilla striata polysaccharide solution; The methotrexate-cystamine dihydrochloride intermediate and the third solvent are mixed, and then mixed with the activated oxidized bletilla striata polysaccharide solution, and subjected to a Schiff base reaction to obtain the methotrexate-bletilla striata polysaccharide nanoparticles.

[0021] In the present invention, the preparation process of the methotrexate-Bletilla striata polysaccharide nanoparticles is shown in Formula 2: Formula 2.

[0022] In the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0023] The invention mixes methotrexate, a first activator, cystamine dihydrochloride and a first solvent, and performs a light-shielding reaction to obtain a methotrexate-cystamine dihydrochloride intermediate.

[0024] In the present invention, the first activator preferably includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is preferably (0.04-0.08):(0.02-0.05), more preferably (0.05-0.07):(0.03-0.04). In an embodiment of the present invention, the mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide can be 0.04:0.02. In the present invention, the function of the first activator is to activate the carboxyl group.

[0025] In the present invention, the first solvent preferably includes dimethyl sulfoxide (DMSO), an acetic acid solution with a mass concentration of 3% to 5%, or a sodium bicarbonate solution with a mass concentration of 6% to 8%. In an embodiment of the present invention, the first solvent may be DMSO.

[0026] In the present invention, the mixing preferably includes mixing methotrexate and part of the first solvent to obtain a methotrexate solution; mixing the first activator and the remaining first solvent to obtain a first activator solution; adding cystamine dihydrochloride after mixing the methotrexate solution and the first activator solution; or the mixing preferably includes mixing methotrexate, the first activator and the first solvent and then adding cystamine dihydrochloride.

[0027] When the mixing comprises mixing methotrexate and a portion of the first solvent to obtain a methotrexate solution; mixing the first activator and the remaining first solvent to obtain a first activator solution; and adding cystamine dihydrochloride after mixing the methotrexate solution and the first activator solution, the present invention does not impose any particular restrictions on the preparation of the methotrexate solution and the first activator solution, and processes well known to those skilled in the art can be employed. In the present invention, the mixing of the methotrexate solution and the first activator solution preferably involves adding the first activator solution to the methotrexate solution and then stirring in the dark. The stirring in the dark is preferably performed at room temperature (specifically, in the range of 20-25°C) and for 1-3 hours. In embodiments of the present invention, the stirring in the dark can be performed at 25°C and for 2 hours. The present invention does not impose any particular restrictions on the process for adding cystamine dihydrochloride, and processes well known to those skilled in the art can be employed. The present invention does not impose any particular restrictions on the ratio of the portion of the first solvent to the remaining first solvent, and mixing can be performed in any ratio.

[0028] When the mixing comprises mixing methotrexate, a first activator, and a first solvent, followed by the addition of cystamine dihydrochloride, the present invention does not impose any particular restrictions on the mixing process, and procedures well known to those skilled in the art may be employed. After the mixing is completed, the present invention further preferably includes stirring in the dark, preferably at room temperature (specifically, a temperature range of 20-25°C) and for 18-24 hours. In embodiments of the present invention, the stirring in the dark may be at 25°C and for 24 hours. The present invention does not impose any particular restrictions on the addition of cystamine dihydrochloride, and procedures well known to those skilled in the art may be employed.

[0029] In the present invention, the mass ratio of methotrexate to 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the first activator is preferably (0.1-0.2):(0.04-0.08), more preferably 0.1:0.04.

[0030] In the present invention, the mass ratio of methotrexate to cystine dihydrochloride is preferably (0.1-0.2):(0.05-0.1), more preferably (0.12-0.18):(0.06-0.09). In an embodiment of the present invention, the mass ratio of methotrexate to cystine dihydrochloride can be 0.1:0.05.

[0031] In the present invention, the usage ratio of methotrexate to the first solvent is preferably 0.1 g:(5 mL-10 mL), preferably 0.1 g:5 mL. In an embodiment of the present invention, the usage ratio of methotrexate to the first solvent can be 0.1 g:5 mL.

[0032] In the present invention, the temperature of the light-shielding reaction is preferably 20-25°C, more preferably 25°C; the time is preferably 18-24h, more preferably 24h. In an embodiment of the present invention, the temperature of the light-shielding reaction can be 25°C and the time can be 24h. In the present invention, the light-shielding reaction is preferably carried out in a protective atmosphere, and the protective atmosphere is preferably one of a nitrogen atmosphere, an argon atmosphere and a helium atmosphere, more preferably a nitrogen atmosphere. In an embodiment of the present invention, the protective atmosphere can be a nitrogen atmosphere.

[0033] In the present invention, the light-avoiding reaction is a reaction in which the carboxyl group in the methotrexate and the amino group in the cystamine dihydrochloride form an amide bond, thereby causing an amidation reaction.

[0034] After the light-proof reaction is completed, the present invention further preferably includes pouring the obtained reaction solution into a buffer solution, stirring until a yellow flocculent precipitate is produced, and then centrifuging, washing and freeze-drying in sequence. In the present invention, the pH value of the buffer solution is preferably 9.4 to 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 have any special restrictions on the stirring process, and a process well known to those skilled in the art can be used. The present invention does not have any special restrictions on the centrifugal process, and a 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, the centrifugation is repeated 3 times to remove the solvent. In the present invention, the detergent used for washing is preferably ultrapure water, and the washing method is preferably centrifugation; the drying is preferably freeze-drying, and the present invention does not have any special restrictions on the freeze-drying process, and a process well known to those skilled in the art can be used.

[0035] After obtaining the methotrexate-cystamine dihydrochloride intermediate, the present invention mixes bletilla striata polysaccharide, sodium periodate and water, performs light-proof oxidation, and then adds ethylene glycol to terminate the oxidation to obtain oxidized bletilla striata polysaccharide.

[0036] In the present invention, the water is preferably ultrapure water.

[0037] In the present invention, the mixing process is preferably to mix the bletilla striata polysaccharide and water and then add sodium periodate. In the present invention, the mixing of the bletilla striata polysaccharide and water is preferably carried out under stirring conditions. The present invention has no particular limitation on the stirring process and can be carried out using a process well known to those skilled in the art.

[0038] In the present invention, the mass ratio of the Bletilla striata polysaccharide to sodium periodate is preferably (1-2):(0.2-0.6), more preferably (1.0-1.5):(0.3-0.5). In an embodiment of the present invention, the mass ratio of the Bletilla striata polysaccharide to sodium periodate can be 1:0.4.

[0039] In the present invention, the mass ratio of the Bletilla striata polysaccharide to water is preferably (1-2) g: (20-60) mL, more preferably (1-1.5) g: (25-35) mL. In an embodiment of the present invention, the mass ratio of the Bletilla striata polysaccharide to water can be 1 g: 25 mL.

[0040] In the present invention, the temperature of the light-avoiding oxidation is preferably 20-25°C, more preferably 25°C; the time of the light-avoiding oxidation is preferably 2-10 hours, more preferably 6 hours. In an embodiment of the present invention, the temperature of the light-avoiding oxidation can be 25°C, and the time can be 6 hours.

[0041] In the present invention, the mass ratio of the Bletilla striata polysaccharide to the volume ratio of ethylene glycol is preferably 1.0-2.0 g:5-10 mL, more preferably 1.0-1.5 g:5-7.5 mL. In an embodiment of the present invention, the mass ratio of the Bletilla striata polysaccharide to the volume ratio of ethylene glycol can be 1 g:5 mL.

[0042] In the present invention, the condition for terminating the oxidation is preferably stirring for 0.5 to 1 hour.

[0043] In the present invention, after the termination oxidation is completed, the hydroxyl groups in the Bletilla striata polysaccharide are converted into aldehyde groups.

[0044] After the termination of the oxidation, the present invention also preferably includes dialysis and drying performed in sequence; the dialysis is preferably performed using a dialysis bag for 3 days; the drying is preferably freeze-drying; the present invention does not have any special limitations on the freeze-drying process, and the process well known to those skilled in the art can be used.

[0045] After obtaining the oxidized Bletilla striata polysaccharide, the present invention mixes the oxidized Bletilla striata polysaccharide, a second activator and a second solvent to perform activation, thereby obtaining an activated oxidized Bletilla striata polysaccharide solution.

[0046] In the present invention, the second activator is preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is preferably (0.01-0.02):(0.006-0.012). In an embodiment of the present invention, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide can be 0.01:0.006.

[0047] In the present invention, the second solvent preferably includes ultrapure water or DMSO. In an embodiment of the present invention, the second solvent may be ultrapure water.

[0048] In the present invention, the mixing preferably includes mixing the oxidized Bletilla striata polysaccharide and the second solvent and then adding the second activator. The present invention does not have any special restrictions on the mixing process of the oxidized Bletilla striata polysaccharide and the second solvent and the adding process of the second activator, and can be carried out using a process well known to those skilled in the art.

[0049] In the present invention, the activation is preferably carried out under conditions of light protection and stirring. The present invention does not have any particular limitations on the conditions of light protection and stirring, and conditions familiar to those skilled in the art can be used. In the present invention, the activation temperature is preferably 20°C to 25°C, more preferably 25°C; the activation time is preferably 1 hour to 3 hours, more preferably 2 hours. In an embodiment of the present invention, the activation temperature can be 25°C and the activation time can be 2 hours.

[0050] After obtaining the activated oxidized Bletilla striata polysaccharide solution, the present invention mixes the methotrexate-cystamine dihydrochloride intermediate and a third solvent, and then mixes the mixture with the activated oxidized Bletilla striata polysaccharide solution to perform a Schiff base reaction to obtain the methotrexate-Bletilla striata polysaccharide nanoparticles.

[0051] In the present invention, the third solvent preferably includes DMSO, an acetic acid solution with a mass concentration of 3% to 5%, or a sodium bicarbonate solution with a mass concentration of 6% to 8%. In an embodiment of the present invention, the third solvent may be DMSO.

[0052] In the present invention, 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, more preferably 0.03 g: 3 mL.

[0053] The present invention does not have any special limitation on the mixing process, and the mixing process may be carried out using a process well known to those skilled in the art.

[0054] In the present invention, the mass ratio of the methotrexate-cystamine dihydrochloride intermediate to oxidized Bletilla striata polysaccharide is preferably (0.03-0.06):(0.01-0.02), more preferably (0.04-0.05):(0.012-0.017). In an embodiment of the present invention, the mass ratio of the methotrexate-cystamine dihydrochloride intermediate to oxidized Bletilla striata polysaccharide can be 0.03:0.01.

[0055] In the present invention, the Schiff base reaction is preferably carried out under conditions of light protection and stirring. The present invention does not have any special restrictions on the conditions of light protection and stirring, and can be carried out using a process well known to those skilled in the art. In the present invention, the temperature of the Schiff base reaction is preferably 20-25°C, more preferably 25°C; the time of the Schiff base reaction is preferably 18-36 h, more preferably 20-30 h. In an embodiment of the present invention, the temperature of the Schiff base reaction can be 25°C and the time can be 24 h.

[0056] The present invention also provides the use of the methotrexate-Bletilla striata polysaccharide nanoparticles described in the above technical solution, or the methotrexate-Bletilla striata polysaccharide nanoparticles prepared by the preparation method described in the above technical solution, in the preparation of a medicament for treating diseases with a pathological microenvironment characterized by a high reactive oxygen species. In the present invention, the disease with a pathological microenvironment characterized by a high reactive oxygen species preferably includes rheumatoid arthritis. The present invention does not impose any particular limitations on the method of use, and methods well known to those skilled in the art may be used.

[0057] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0058] Example 1 Dissolve 0.1 g of methotrexate in 5 mL of DMSO to obtain a methotrexate solution; 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; The activator solution was added to the methotrexate solution, stirred in the dark at room temperature (25°C) for 2 h, and then 0.05 g of cystamine dihydrochloride was added. The mixture was reacted in the dark at room temperature (25°C) for 24 h. Under stirring, the obtained reaction solution was slowly poured into a pH=9.6 buffer solution until a yellow flocculent precipitate was produced. The solvent was removed by centrifugation three times, and the buffer solution was washed clean by centrifugation with ultrapure water, and freeze-dried to obtain a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark). 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 25°C in the dark for 6 h. 5 mL of ethylene glycol was added and stirring was continued (at 25°C in the dark) for 30 min before terminating the oxidation. The resulting reaction solution was placed in a dialysis bag and dialyzed for 3 days. After dialysis, the solution was freeze-dried to obtain oxidized Bletilla striata polysaccharide. After dissolving 0.01 g of the oxidized Bletilla striata polysaccharide and 1 mL of ultrapure water, 0.01 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.006 g of N-hydroxysuccinimide were added, and the mixture was stirred in the dark at room temperature for 2 h to obtain an activated oxidized Bletilla striata polysaccharide solution; 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO and added to the activated oxidized bletilla striata polysaccharide solution. After stirring for 24 h in the dark, the solution was dialyzed and freeze-dried to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0059] Example 2 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 and stirred in the dark at room temperature (25°C) for 1 h. 0.10 g of cystamine dihydrochloride was then added and the mixture was reacted in the dark at room temperature (25°C) for 24 h. The resulting reaction solution was slowly poured into a pH 9.6 buffer solution while stirring until a yellow flocculent precipitate was produced. The solvent was removed by centrifugation three times, and the buffer solution was washed cleanly with ultrapure water by centrifugation. The mixture was then freeze-dried to obtain a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark). 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 25°C in the dark for 2 h. 5 mL of ethylene glycol was added to terminate the oxidation, and the mixture was stirred for 30 min (in the dark, at 25°C). The resulting reaction solution was placed in a dialysis bag and dialyzed for 3 days. After dialysis, the mixture was freeze-dried to obtain oxidized Bletilla striata polysaccharide. After dissolving 0.01 g of the oxidized Bletilla striata polysaccharide and 1 mL of ultrapure water, 0.01 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.006 g of N-hydroxysuccinimide were added, and the mixture was stirred in the dark at room temperature for 2 h to obtain an activated oxidized Bletilla striata polysaccharide solution; 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO and added to the activated oxidized bletilla striata polysaccharide solution. After stirring for 18 h in the dark, the solution was dialyzed and freeze-dried to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0060] Example 3 0.1 g of methotrexate, 0.04 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 0.02 g of N-hydroxysuccinimide were dissolved in 10 mL of DMSO and stirred in the dark at room temperature (25°C) for 2 h. 0.05 g of cystamine dihydrochloride was then added and the mixture was reacted in the dark at room temperature (25°C) for 24 h. The resulting reaction solution was slowly poured into a pH 9.6 buffer solution while stirring until a yellow flocculent precipitate was produced. The solvent was removed by centrifugation three times, and the buffer solution was washed cleanly with ultrapure water by centrifugation. The mixture was then freeze-dried to obtain the methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark). 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 the mixture was oxidized at 25°C in the dark for 4 h. 5 mL of ethylene glycol was added and the mixture was stirred (at 25°C in the dark) for 30 min before terminating the oxidation. The resulting reaction solution was placed in a dialysis bag and dialyzed for 3 days. After the dialysis was completed, the mixture was freeze-dried to obtain oxidized Bletilla striata polysaccharide. After dissolving 0.01 g of the oxidized Bletilla striata polysaccharide and 1 mL of DMSO, 0.01 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.006 g of N-hydroxysuccinimide were added, and the mixture was stirred in the dark at room temperature for 3 h to obtain an activated oxidized Bletilla striata polysaccharide solution; 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO and added to the activated oxidized bletilla striata polysaccharide solution. After stirring for 24 h in the dark, the solution was dialyzed and freeze-dried to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0061] Example 4 Dissolve 0.1 g of methotrexate in 5 mL of DMSO to obtain methotrexate solution; Dissolve 0.04 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.02 g of N-hydroxysuccinimide in 5 mL of DMSO to obtain an activator solution; The activator solution was added to the methotrexate solution, stirred in the dark at room temperature (25°C) for 2 h, and then 0.05 g of cystamine dihydrochloride was added. The mixture was reacted in the dark at room temperature (25°C) for 24 h. Under stirring, the obtained reaction solution was slowly poured into a pH=9.6 buffer solution until a yellow flocculent precipitate was produced. The solvent was removed by centrifugation three times, and the buffer solution was washed clean by centrifugation with ultrapure water, and freeze-dried to obtain a methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark). 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 the mixture was oxidized at 25°C in the dark for 8 h. 5 mL of ethylene glycol was added and the mixture was stirred (at 25°C in the dark) for 30 min before terminating the oxidation. The resulting reaction solution was placed in a dialysis bag and dialyzed for 3 days. After the dialysis was completed, the mixture was freeze-dried to obtain oxidized Bletilla striata polysaccharide. After dissolving 0.02 g of the oxidized Bletilla striata polysaccharide and 2 mL of 5% acetic acid solution, 0.02 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.012 g of N-hydroxysuccinimide were added, and the mixture was stirred in the dark at room temperature for 3 h to obtain an activated oxidized Bletilla striata polysaccharide solution; 0.06 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 6 mL of 5% acetic acid solution and added to the activated oxidized bletilla striata polysaccharide solution. After stirring for 36 h in the dark, the solution was dialyzed and freeze-dried to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0062] Example 5 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. The mixture was stirred in the dark for 1 h at room temperature (25 °C) under nitrogen protection. Then, 0.10 g of cystamine dihydrochloride was added. The mixture was reacted in the dark for 24 h at room temperature (25 °C). The resulting reaction solution was slowly poured into a pH 9.6 buffer solution while stirring until a yellow flocculent precipitate was produced. The solvent was removed by centrifugation three times, and the buffer solution was washed cleanly with ultrapure water by centrifugation. The mixture was freeze-dried to obtain the methotrexate-cystamine dihydrochloride intermediate (light yellow powder, stored in the dark). 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 the mixture was oxidized at 25°C in the dark for 10 h. 5 mL of ethylene glycol was added to terminate the oxidation. After stirring for 30 min (in the dark, at 25°C), the resulting reaction solution was placed in a dialysis bag and dialyzed for 3 days. After dialysis, the mixture was freeze-dried to obtain oxidized Bletilla striata polysaccharide. After dissolving 0.01 g of the oxidized Bletilla striata polysaccharide and 1 mL of DMSO, 0.01 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.006 g of N-hydroxysuccinimide were added, and the mixture was stirred in the dark at room temperature for 2 h to obtain an activated oxidized Bletilla striata polysaccharide solution; 0.03 g of the methotrexate-cystamine dihydrochloride intermediate was dissolved in 3 mL of DMSO and added to the activated oxidized bletilla striata polysaccharide solution. After stirring for 20 h in the dark, the solution was dialyzed and freeze-dried to obtain methotrexate-bletilla striata polysaccharide nanoparticles.

[0063] Test Case Fourier transform infrared spectroscopy: The polysaccharide of Bletilla striata, oxidized polysaccharide of Bletilla striata, methotrexate, methotrexate-cystamine dihydrochloride intermediate, and methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1 were mixed and ground evenly with a small amount of KBr, and pressed into thin slices. The results were analyzed by Fourier transform infrared spectroscopy at a wavelength of 500-4000 cm -1 Scan the range; Figure 1 The infrared spectra of Bletilla striata polysaccharide (BSP), oxidized Bletilla striata polysaccharide (OBSP), methotrexate (MTX), methotrexate-cystamine dihydrochloride intermediate (MTX-ss-NH2) and methotrexate-Bletilla striata polysaccharide nanoparticles (MTX-ss-OBSP NPs) described in Example 1 are shown in FIG. Figure 1 It can be seen that in the BSP infrared spectrum, 3410 cm -1 The stretching vibration absorption peak of -OH is at 2912 cm -1 The stretching vibration peak of CH in sugars -CH2 or -CH3 is 1637 cm -1 The bending vibration absorption peak of OH is at 1030 cm -1 The OBSP infrared spectrum shows a characteristic absorption peak at 1700 cm-1, compared with the BSP infrared spectrum. -1 The stretching vibration absorption peak of the C=O bond of the aldehyde group appeared at 1644 cm -1 The stretching vibration absorption peak of -NH2 is 1603 cm -1 The stretching vibration absorption peak of -COOH is at 1733 cm; these two peaks are the characteristic absorption peaks of MTX. -1 A weak stretching vibration absorption peak of amide I band (NC=O bond) appeared at 1553 cm -1The bending vibration absorption peak of amide II band (CN-H bond) appeared at 1700 cm, indicating that the carboxyl group of MTX formed an amide bond with the amino group of Cys, and MTX-ss-NH2 was successfully prepared. The infrared spectrum of MTX-ss-OBSP was compared with that of OBSP. -1 The absorption peak of carbonyl (C=O) disappeared, and the absorption peak at 1636 cm -1 The stretching vibration absorption peak of the imine bond (C=N) appeared at 1450 cm -1 The emergence of a new absorption peak is due to the introduction of the benzene ring structure of MTX into the polymer, indicating that MTX-ss-OBSP was successfully prepared.

[0064] Particle size, PDI, Zeta potential detection: by Figure 2 As shown in A (A is the transmission electron microscopy image of the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1), the transmission electron microscopy observation showed that the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1 were uniformly spherical and well dispersed. The particle size, PDI, and Zeta potential of the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1 were measured using a Malvern particle size analyzer. Figure 2 As shown in Figure B (B is the particle size distribution diagram of the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1), the average particle size of the methotrexate-Bletilla striata polysaccharide nanoparticles is 211±4.12 nm, the PDI is 0.20±0.00, and the Zeta potential is -42.57±2.15.

[0065] ROS responsiveness of nanoparticles (particle size and PDI changes): A Malvern particle size analyzer was used to measure the particle size distribution and PDI of the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1 in pH = 7.4 phosphate buffer and pH = 7.4 phosphate buffer + 1mM H2O2 at specific time points (0d, 1d, 2d, 3d, 4d, 5d, 6d, 7d). Due to the presence of disulfide bonds, the nanoparticles theoretically exhibit responsive cleavage behavior under H2O2 conditions. This experiment demonstrated this by changing the dispersion medium of the nanoparticles. Figure 3 A in (A is the particle size distribution of nanoparticles in different solutions) and Figure 3 As shown in Figure B (PDI change diagram of nanoparticles in different solutions), when H2O2 was added to the PBS solution with pH = 7.4, the particle size of the nanoparticles decreased and the uniformity of the particle size distribution increased, indicating that the nanoparticles have ROS response ability.

[0066] In vitro release of methotrexate: The dialysis bag method was used to detect the cumulative release rate of methotrexate from the methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1. After the nanoparticles were fully dispersed in the release medium, they were placed in a dialysis bag and suspended in 40 mL of PBS with pH = 7.4 and PBS with pH = 7.4 + 1 mM H2O2, respectively, and placed on a shaker at 37°C and 100 rpm / min. At specific time points (1, 1.5h, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48h, 60h, 72h, 84h, 96h, 108h), 2 ml of the supernatant was taken and supplemented with an equal amount of release medium preheated to 37°C. The methotrexate released in the supernatant was quantitatively analyzed by high performance liquid chromatography. Each experiment was repeated 3 times, and the results were expressed as mean ± standard deviation. 2 =0.9993(Abs(302nm)=0.051×[MTX](μg·mL -1 The release amount of MTX in the sample was determined by using a calibration curve of 0.001 + 0.001. Figure 4 The release rate of methotrexate was rapid within 24 hours. The cumulative release rate of methotrexate-Bletilla striata polysaccharide nanoparticles in pH 7.4 phosphate buffer was 76.31±3.00% over 108 hours. In pH 7.4 phosphate buffer + 1 mM H2O2, the cumulative release rate was 86.79±1.09% over 108 hours, demonstrating that methotrexate-Bletilla striata polysaccharide nanoparticles have a good release effect under H2O2 conditions.

[0067] CCK8 cell activity test: FLS cells were cultured at 1.0×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and incubated at 37°C in 5% CO2 for 24 h. FLS cells were treated with methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-Bletilla striata polysaccharide nanoparticles at 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, and 12.0 μg / mL) for 24 h. Then, 10 μL of CCK-8 reagent was added. The reagent should be injected slowly to avoid bubbles. The cells were then incubated at a constant temperature of 37°C for 30 min. After the incubation period, the absorbance was measured at a wavelength of 450 nm (A = 450 nm) using a microplate reader. Figure 5 The graph shows the effects of methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles on FLS cell viability as described in Example 1. Figure 5It can be seen that based on CCK8 analysis, 10 μg / mL methotrexate-Bletilla striata polysaccharide nanoparticles can inhibit the proliferation of FLS cells, and the inhibitory effect is weaker than that of the free drug methotrexate at the same concentration. This is because the negligible cytotoxicity of oxidized Bletilla striata polysaccharide improves the biocompatibility of methotrexate-Bletilla striata polysaccharide nanoparticles.

[0068] Live-dead cell staining experiment: Calcein (AM) / propidium iodide (PI) was used to evaluate the ability of methotrexate-Bletilla striata 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 The cells were then incubated with complete culture medium, methotrexate described in Example 1, oxidized Bletilla striata polysaccharide described in Example 1, and methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1 (at a methotrexate concentration of 10 μg / mL). The cells were stained with AM / PI and observed under a fluorescence microscope. Figure 6 The live and dead cell staining results of methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles described in Example 1 (wherein, A is the AM / PI calcein staining of FLS by methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles, and B is the quantitative analysis of AM / PI calcein staining of FLS by methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles), Figure 6 It can be seen that the methotrexate-Bletilla striata polysaccharide nanoparticles killed FLS cells, and the killing degree was stronger than that of the free drug OBSP and weaker than that of MTX, which was consistent with the cytotoxicity results.

[0069] Scratch test: FLS cells were cultured at a rate of 2 × 10 5 Cells were seeded at a density of 10 cells / well in a 24-well plate and cultured in complete medium in a 5% CO2, 37°C incubator until the cells reached a density of over 90%. A straight line was drawn in the center of the well using a 200μL sterile pipette tip. The wells were washed twice with phosphate-buffered saline (PBS) to smooth the edges of the scratch and remove floating cells. The scratch site was then imaged under an inverted microscope (×40) and marked as 0h. The drug solution was prepared in serum-free medium and divided into the following groups: control group, methotrexate group (methotrexate as described in Example 1), oxidized Bletilla striata polysaccharide group (oxidized Bletilla striata polysaccharide as described in Example 1), and methotrexate-oxidized Bletilla striata polysaccharide nanoparticle group (methotrexate concentration of 10μg / mL, methotrexate-oxidized Bletilla striata polysaccharide nanoparticles as described in Example 1). After 12h and 24h of intervention, images of the scratch site were obtained at the same site under an inverted microscope (×40) and marked as 12h and 24h, respectively. Figure 7The results of the effects of methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles on FLS cell migration described in Example 1 are shown (A is the detection of methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles on FLS cell migration, and B is the quantitative analysis of methotrexate, oxidized Bletilla striata polysaccharide and methotrexate-Bletilla striata polysaccharide nanoparticles on FLS cell migration). Figure 7 It can be seen that the methotrexate group and the methotrexate-Bletilla striata polysaccharide nanoparticles group had the most obvious inhibitory effect on the migration ability of FLS cells, followed by oxidized Bletilla striata polysaccharide.

[0070] ROS scavenging experiment: An inverted fluorescence microscope was used to observe the effects of different drug treatments on the ROS levels in FLS cells. The specific operation was as follows: FLS cells were cultured at a rate of 6×10 4 Cells were seeded at a density of 100 cells / well in a 24-well plate 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 an equal volume of Rosup (final concentration of 50 μg / mL) and incubated for 4 hours. The entire culture medium was aspirated, and the cells were gently rinsed twice with PBS. The experimental group was treated with 0.5 mL of complete culture medium containing methotrexate (methotrexate described in Example 1), oxidized Bletilla striata polysaccharide (oxidized Bletilla striata polysaccharide described in Example 1), and methotrexate-oxidized Bletilla striata polysaccharide nanoparticles (10 μg / mL methotrexate concentration, described in Example 1) and cultured for a further 12 hours. The old culture medium was removed, and the cells were rinsed with PBS. Serum-free culture medium containing DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) was added, and the cells were incubated in a dark incubator at 37°C for 30 minutes. After rinsing with PBS, an appropriate amount of PBS was added to each well. The effects of different drug treatments on ROS levels in FLS cells were observed using an inverted fluorescence microscope. The fluorescence intensity was quantified using ImageJ software. Figure 8 As shown in Figure A (A is the result of the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-oxidized Bletilla striata polysaccharide nanoparticles on intracellular ROS under an inverted fluorescence microscope), compared with the control group, the green fluorescence signal in the group treated with the same concentration of methotrexate-oxidized Bletilla striata polysaccharide nanoparticles decreased significantly. This shows that methotrexate-oxidized Bletilla striata polysaccharide nanoparticles reduced intracellular ROS, demonstrating significant antioxidant properties. The intracellular ROS level in the oxidized Bletilla striata polysaccharide group was also reduced. The fluorescence intensity of methotrexate was stronger than that of the control group, probably because methotrexate is a folic acid antagonist that can affect cell metabolism through multiple pathways, produce oxidative stress, and lead to increased ROS generation. For Figure 8 The statistical results of fluorescence quantification in Figure B (B is the quantitative analysis diagram) also verified this phenomenon.

[0071] To further study the ROS scavenging effect in cells, flow cytometry was used for further determination. FLS cells were cultured at 1x106 Cells were seeded at a density of 100 cells / well in a 6-well plate. After the same treatment method as above, serum-free culture medium was added for washing and centrifugation. The cells were then resuspended in serum-free culture medium. Finally, the cell suspension was transferred to a flow cytometer and the FITC channel of the flow cytometer was selected to detect the intracellular DCF fluorescence signal. Figure 9 A in the figure (A is the flow cytometry test result of the effects of methotrexate, oxidized Bletilla striata polysaccharide, and methotrexate-oxidized Bletilla striata polysaccharide nanoparticles on intracellular ROS) shows that compared with the control group, methotrexate-Bletilla striata polysaccharide nanoparticles have the strongest ability to scavenge ROS, followed by oxidized Bletilla striata polysaccharide; Figure 9 As shown in Figure B (B is a quantitative analysis graph), methotrexate-Bletilla striata polysaccharide nanoparticles can eliminate the abnormally elevated ROS in FLS cells. This result is consistent with the ROS results detected by inverted fluorescence microscopy.

[0072] Effects of methotrexate-Bletilla striata polysaccharide nanoparticles on macrophage polarization: Mouse mononuclear macrophage Raw264.7 cells were cultured at a rate of 1×10 6 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured for 24 h. LPS was added for pretreatment for 12 h, the culture medium was discarded, and the cells were washed with PBS. Then, ordinary DMEM complete medium (control group), DMEM complete medium containing 10 μg / mL of methotrexate as described in Example 1 (methotrexate group), DMEM complete medium containing 10 μg / mL of oxidized Bletilla striata polysaccharide as described in Example 1 (oxidized Bletilla striata polysaccharide group), and DMEM complete medium containing 10 μg / mL of methotrexate-Bletilla striata polysaccharide nanoparticles as described in Example 1 (methotrexate-Bletilla striata polysaccharide nanoparticle group, the concentration of methotrexate was 10 μg / mL) were added for treatment for 24 h. Wash the cells with PBS, centrifuge (1000 rpm × 5 min), discard the supernatant, add 100 μL CD86 antibody (M1 marker), incubate at 4 ° C in the dark for 30 min, pipette to mix, centrifuge (1000 rpm × 5 min), discard the supernatant, resuspend the cells with PBS, add 4 ° C pre-cooled fixative, and incubate at room temperature in the dark for 1 hour. Add PBS buffer to wash, add 500 μL permeabilization solution, and then add 100 μL CD206 antibody (M2 marker), incubate at room temperature in the dark for 30 min, centrifuge (1000 rpm × 5 min), discard the supernatant. Wash with PBS buffer, add 300 μL PBS to resuspend the cells, and detect on a flow cytometer. Figure 10 The graphs show that methotrexate, oxidized bletilla striata polysaccharide and methotrexate-bletilla striata polysaccharide nanoparticles promote the polarization of M1 macrophages to M2 macrophages as described in Example 1 (A shows the graph of methotrexate, oxidized bletilla striata polysaccharide and methotrexate-bletilla striata polysaccharide nanoparticles promoting the polarization of M1 macrophages to M2 macrophages, and B shows the quantitative analysis graph). Figure 10Compared with the control group, the proportion of CD86+ macrophages in the methotrexate-Bletilla striata polysaccharide nanoparticle and oxidized Bletilla striata polysaccharide groups was significantly reduced, while the proportion of CD206+ cells was increased. Statistical analysis showed that the M2 / M1 cell ratio was higher in the methotrexate-Bletilla striata polysaccharide nanoparticle and oxidized Bletilla striata polysaccharide groups than in the control group. These results suggest that methotrexate-Bletilla striata polysaccharide nanoparticles can inhibit macrophage M1 polarization and promote macrophage M2 polarization.

[0073] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation thereto. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications shall also be considered within the scope of protection of the present invention.

Claims

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

2. The method for preparing methotrexate-Bletilla striata polysaccharide nanoparticles according to claim 1, characterized in that: The following steps are involved: Mixing methotrexate, a first activator, cystamine dihydrochloride and a first solvent, and performing a reaction in the dark to obtain a methotrexate-cystamine dihydrochloride intermediate; Mixing bletilla striata polysaccharide, sodium periodate and water, performing light-protected oxidation, and then adding ethylene glycol to terminate the oxidation to obtain oxidized bletilla striata polysaccharide; mixing the oxidized bletilla striata polysaccharide, a second activator, and a second solvent to activate the mixture, thereby obtaining an activated oxidized bletilla striata polysaccharide solution; The methotrexate-cystamine dihydrochloride intermediate and the third solvent are mixed, and then mixed with the activated oxidized bletilla striata polysaccharide solution, and subjected to a Schiff base reaction to obtain the methotrexate-bletilla striata polysaccharide nanoparticles.

3. The preparation method according to claim 2, wherein The first activator and the second activator are both 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, wherein 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, wherein The mass ratio of the bletilla striata polysaccharide to sodium periodate is (1-2): (0.2-0.6); The dosage ratio of the bletilla striata polysaccharide and 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 temperature of the light-proof oxidation is 20° C. to 25° C., and the time is 2 to 10 hours.

7. The preparation method according to claim 3, wherein 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 in a dark environment at a temperature of 20-25° C. for 1-3 hours.

8. The preparation method according to claim 2, wherein 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, wherein The Schiff base reaction is carried out in a dark environment at a temperature of 20-25° C. and a reaction time of 18-36 hours.

10. 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 drugs for treating diseases with a pathological microenvironment of high reactive oxygen species.

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