CN116327700A - Methotrexate nano-drug-carrying system, preparation method thereof and application thereof in treating rheumatoid arthritis - Google Patents
Methotrexate nano-drug-carrying system, preparation method thereof and application thereof in treating rheumatoid arthritis Download PDFInfo
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Abstract
The invention discloses a methotrexate nano-drug-loading system, a preparation method thereof and application of the methotrexate nano-drug-loading system in treating rheumatoid arthritis, and particularly relates to a mannose-modified reduction-responsive vesicle-coated methotrexate for treating the rheumatoid arthritis. The methotrexate nano drug-loading system disclosed by the invention is delivered to macrophages, nano drugs enter the macrophages at the inflammation part to play a role of the drugs, and M1M is converted into M2M; in an RA mouse model, man-PMTX can reach and stay in an inflammatory joint in a targeting way, M1 pro-inflammatory macrophages in the body are converted into M2 anti-inflammatory macrophages, and the release of pro-inflammatory cytokines is reduced, meanwhile, the release of anti-inflammatory cytokines is promoted, the inflammatory microenvironment in the inflammatory joint is properly regulated, and the RA progress is quickly slowed down. The Man-PMTX therapeutic results show that it can accumulate effectively in RA joints, rapidly alleviate inflammation, with low side effects, indicating that it is a simple and effective treatment for RA.
Description
Technical Field
The invention relates to a nano-drug, in particular to a mannose-modified reduction-responsive vesicle-entrapped methotrexate for treating rheumatoid arthritis.
Background
Rheumatoid Arthritis (RA) is a systemic autoimmune disease, and some clinical antirheumatic arthritis drugs can control inflammation and pain caused by RA, but cannot effectively prevent the formation and development of bone destruction, which causes pain in joints, severe swelling in joint parts, joint deformity and even permanent irreversible injury in RA patients. Some RA drugs have been approved for clinical use, where Methotrexate (MTX) can achieve an overall anti-inflammatory effect on RA at low doses, and its specific therapeutic mechanism is not yet fully studied. MTX is a hydrophilic small molecule drug with some drug treatment drawbacks, such as: short circulation time, low bioavailability, slow onset of action, long-term sustained treatment, and easy occurrence of adverse reaction and bone erosion. In view of the therapeutic shortcomings of MTX, the prior art has presented a number of MTX entrapped nanomedicines that can be delivered to the affected site for antirheumatic treatment by active and passive targeting, such as a human serum albumin-based MTX loaded nanomedicine (mtx@hsa NMs) for SPARC targeted RA treatment. The preparation method of the MTX nano-drug disclosed in the prior art needs to be simplified, and the treatment effect needs to be improved.
Disclosure of Invention
The existing MTX-entrapped liposome, nano-particles and other medicaments enter the inflammation part in a passive transportation mode, but the treatment effect of the nano-systems needs to be improved. The invention discloses a new mannose-modified reduction-responsive vesicle-entrapped methotrexate, which has excellent effects of eliminating leg inflammation of RA mice, slowing down inflammatory infiltration of synovium at diseased joints, protecting cartilage and regulating and controlling immune microenvironment at joints.
The invention adopts the following technical scheme:
a methotrexate nanodrug delivery system comprising a polymer vesicle and methotrexate; the polymer comprises PEG-P (TMC-DTC) -Y, PEG-P (CL-DTC) -Y or PEG-P (LA-DTC) -Y, Y being a cationic fragment; further, the polymer also comprises X-PEG-P (TMC-DTC), X-PEG-P (CL-DTC) or X-PEG-P (LA-DTC), X is a modifying molecule; preferably, X is mannose.
The invention prepares a block polymer by copolymerizing a DTC monomer containing a disulfide five-membered ring, other monomers (TMC, CL, LA) and polyethylene glycol, combines a cationic segment or a modified molecule to obtain a polymer for embedding methotrexate, wherein the molecular weight of the polymer is 10-50 kg/mol, the molecular weight of a PEG segment in the polymer is 2-10 kg/mol, the molecular weight of the cationic segment is 0.1-2 kg/mol, and the molecular weight of the DTC segment is 1-5 kg/mol. Preferably, the molecular weight of the polymer is 20 to 35 kg/mol and the molecular weight of the DTC segment in the polymer is 1.5 to 4 kg/mol.
The invention discloses a preparation method of the methotrexate nano-drug-loading system, which takes polymer and methotrexate as raw materials to prepare the methotrexate nano-drug-loading system; wherein the polymer is a polymer containing a cation fragment and comprises PEG-P (TMC-DTC) -Y, PEG-P (CL-DTC) -Y or PEG-P (LA-DTC) -Y; or the polymer is a polymer containing a cationic fragment and a polymer containing a modified molecule (including X-PEG-P (TMC-DTC), X-PEG-P (CL-DTC) or X-PEG-P (LA-DTC)), wherein the molar ratio of the polymer containing the cationic fragment to the polymer containing the modified molecule is 1:0-0.5, and the polymer containing the modified molecule is not 0. Specifically, methotrexate is loaded into a polymer vesicle by utilizing electrostatic interaction, so as to obtain the methotrexate nano-drug delivery system.
The invention also discloses a methotrexate nano-drug-carrying system freeze-dried powder, which is obtained by mixing the methotrexate nano-drug-carrying system with a freeze-drying protective agent and then freeze-drying; the specific freeze-drying method is the prior art.
The invention discloses an application of the methotrexate nano-drug-carrying system or the methotrexate nano-drug-carrying system freeze-dried powder in preparing a medicine for treating rheumatoid arthritis. Furthermore, the invention also discloses application of the methotrexate nano-drug-loading system or the methotrexate nano-drug-loading system freeze-dried powder in preparing a drug with repolarized M1M macrophage as M2M macrophage; or in the preparation of a medicament for recovering joints and synovium and reducing synovial inflammatory cell infiltration; or in the preparation of a medicament for inhibiting Dendritic Cell (DC) activation; or in the preparation of a medicament for protecting articular cartilage and bone tissue; or in the preparation of anti-inflammatory drugs; or in the preparation of a medicament for promoting TGF-beta secretion; or in the preparation of anti-autoimmune disease drugs.
The prior art discloses MTX loaded nanomedicines (mtx@hsa NMs) based on serum albumin and targeting molecules such as folic acid and polypeptides for SPARC targeted RA treatment. Macrophages, T lymphocytes and synovial fibroblasts are all the inducers of RA inflammation. Macrophages, especially M1-type macrophages (M1M), play a major role in the course of RA and can secrete several pro-inflammatory cytokines such as tumor necrosis factor- α (TNF- α), interleukin-6 (IL-6) and interleukin-1β (IL-1β). M2-type macrophages (M2M) can secrete anti-inflammatory cytokines and can repair damaged tissue. The methotrexate nano drug-loading system disclosed by the invention is delivered to macrophages, nano drugs enter the macrophages at the inflammatory part to play a role of the drugs, M1M is converted into M2M, and the process effectively improves the curative effect of MTX on RA. Compared with the prior MTX nano-drug, the invention has the advantages of biodegradability, convenient preparation, strong reproducibility and small particle size. In an RA mouse model, man-PMTX can reach and stay in an inflammatory joint in a targeting way, M1 pro-inflammatory macrophages in the body are converted into M2 anti-inflammatory macrophages, and the release of pro-inflammatory cytokines is reduced, meanwhile, the release of anti-inflammatory cytokines is promoted, the inflammatory microenvironment in the inflammatory joint is properly regulated, and the RA progress is quickly slowed down. The Man-PMTX therapeutic results show that it can accumulate effectively in RA joints, rapidly alleviate inflammation, with low side effects, indicating that it is a simple and effective treatment for RA.
Drawings
FIG. 1 is a schematic diagram of Man-PMTX active targeting therapy RA. And after intravenous injection of Man-PMTX, actively targeting and accumulating to RA joints, remodelling RA immune microenvironment, converting M1M into M2M, inhibiting the secretion of proinflammatory cytokines, and relieving the progress of RA.
FIG. 2 is a hydrogen nuclear magnetic resonance chart (400 MHz, CDCl) of polymers (A) PEG-P (TMC-DTC) -NPC and (B) PEG-P (TMC-DTC) -sp 3 ）。
FIG. 3 is a graph of hydrogen nuclear magnetic resonance spectra (DMSO) of polymers (A) NHS-PEG-P (TMC-DTC) (400 MHz) and (B) Man-PEG-P (TMC-DTC) (600 MHz)d 6 ）。
Fig. 4 is a physicochemical characterization of PMTX and Man-PMTX. (A) Particle size distribution of PMTX and ManPMTX with 10%, 20%, 30% mannose. Particle size distribution of PMTX and Man-PMTX (B) at day 0 and day 30 after preparation and (C) particle size distribution in PB containing 10% FBS for 48 hours. (D) Release profile of Man-PMTX in PB with and without 10 mM GSH (n=3). (E) Hemolysis of mouse erythrocytes by PMTX, man-PMTX and free MTX, positive control is 1% triton, pure water, and negative control is normal saline.
FIG. 5 shows the uptake of Man-PMTX by LPS stimulated RAW 264.7 cells and its repolarization. (A) CLSM images after 4 hours incubation of RAW 264.7 cells with PMTX or Man-PMTX, with FITC-labeled MTX and Cy 5-labeled polymer vesicles (5 μg Cy5/mL and 14 μg FITC/mL), scale bar: 50. and [ mu ] m. (B) Flow cytometry detected the M2M content (cd206+, n=3) of RAW 264.7 cells after incubation with free MTX, PMTX or Man-PMTX (MTX: 20 μg/mL) for 24 h and semi-quantitative analysis thereof (C).
Fig. 6 shows activation and repolarization of LPS-stimulated BMDMs after incubation with PBS, PMTX or Man-PMTX (MTX: 20 μg/mL, n=4) for 24 h. (a) a typical flow cytometer fluorescence intensity profile; (B) The ratio of M1M to total cells and (C) the ratio of M2M/M1M.
FIG. 7 shows the concentration of cytokines (A) TNF- α, (B) IL-1β, (C) IL-10 in the medium after incubation of LPS-stimulated BMDMs with PBS, PMTX or ManPMTX (MTX: 20 μg/mL, n=4) for 24 h.
FIG. 8 is the establishment of a mouse model for RA (500. Mu.g zymosan/knee joint) and the targeting of PMTX and Man-PMTX in ZIA mice (Cy 5: 0.3. Mu.g/M.TX: 4 mg/kg, n=3). (a) apparent swelling of the ZIA mouse joints; zymosan injection for 12 hours before and after mice (B) knee joint diameter and (C) concentration of IL-6 and TNF- α in plasma (n=6). (D) In vivo fluorescence imaging and semi-quantitative analysis of RA mice after intravenous injection of Cy 5-labeled PMTX or Man-PMTX. Ex vivo fluorescence images of joints and major organs 24 hours after injection (E) and ex vivo imaging of hind legs and fluorescence semi-quantitative analysis (F).
Fig. 9 is the effect of Man-PMTX dose and number of doses on RA efficacy in anti-mice (first needle dose set to day 0, n=5). PBS and free MTX groups were used as controls. (A) Experimental arrangement. (B) inflammation and (C) changes in healthy lateral knee joint width. (D) Inflammation and (E) changes in knee circumference on healthy sides.
FIG. 10 is the effect of Man-PMTX dosing and number of doses on RA mouse cytokine secretion. The concentrations of TNF- α in plasma on day three (a), day six (B) and day nine (C) (n=5), and the concentrations of TNF- α and (E) IL-6 at joint sites (D) on day nine (n=3) were treated.
FIG. 11 is treatment of RA mice with Man-PMTX, PMTX or free MTX. (A) Experimental arrangement. Intravenous injection (MTX: 4 mg/kg) was performed twice on days 0 and 3, healthy mice and PBS were used as controls. (B) Changes in RA knee joint broadband and knee circumference (n=7) and (C) concentrations of IL-6 and TGF- β in day 7 plasma (n=7); the relative expression levels (E) of four pro-inflammatory cytokines (D) and their mRNA (n=3) in the RA joints of mice.
FIG. 12 is a histological analysis of H & E sections of the major organs of mice after day 7 of treatment as shown in FIG. 11A for Man-PMTX, PMTX and free MTX. Scale bar: 50. and [ mu ] m.
Fig. 13 is the activation and repolarization of RA joint macrophages in mice by Man-PMTX (n=3). (A) Immunofluorescent staining images and semi-quantitative analysis of M2M marker (cd206+) on RA joint sections of mice 7 days after treatment according to fig. 11A (white arrow points to synovium). Scale bar: 100. and [ mu ] m. (B) Flow cytometry examined the M2M (CD11b+F4/80+CD206+) content and the M2M/M1M ratio (4 mg MTX/kg) in all cells of the RA-joint after injection of one needle.
FIG. 14 is a histological morphological analysis of RA joints at day 7 after MTX, PMTX and Man-PMTX treatment as in FIG. 11A. (A) H&E. Histological images of safranin fast green and masson staining, the scale is 50 [ mu ] m; (B) Micro-CT images and structural parameters (Tb.Sp, tb.N and BV/TV); wherein white arrows represent bone erosion (n=3), xp<0.05, **p<0.01, ***p<0.001。
Description of the embodiments
The invention discloses a method for actively targeting MTX delivery to RA by using macrophage to target mannose-modified polymer nanovesicles (Man-PMTX), which can rapidly relieve inflammatory RA symptoms and reduce systemic toxicity. In forming such mannose-functionalized self-crosslinking polymers, man-PMTX achieves high and robust MTX loading and uniform size (50 nm). Man-PMTX activates anti-inflammatory M2 type macrophages, reducing secretion of TNF-alpha and IL-1β. Referring to fig. 1, in the RA mouse model, man-PMTX is able to target and stay in the inflammatory joint, convert M1 pro-inflammatory macrophages in vivo into M2 anti-inflammatory macrophages, and reduce pro-inflammatory cytokine release while promoting anti-inflammatory cytokine release, properly regulate the inflammatory microenvironment in the inflammatory joint, and rapidly slow RA progression. The Man-PMTX therapeutic results show that it can accumulate effectively in RA joints, rapidly alleviate inflammation, with low side effects, indicating that it is a simple and effective treatment for RA.
Methotrexate disodium (MTX.2Na, 99%, dalian Meen Biotechnology development Co., ltd.), water-soluble crystalline powder, molecular weight 498.4g/mol. Cy5-NHS (98%, lumiprobe Corp.), mannose hydrochloride (98%, J)&K) Glutathione (GSH, 99%,roche), zymosan a (Sigma) and lipopolysaccharide (LPS, sigma-aldrich) were used directly after purchase. PEG-P (TMC-DTC) -spimine @Mn=5.0- (16.2-2.0) -0.2 kg/mol) and Man-PEG-P (TMC-DTC)M n The block copolymer having a Man functionalization of approximately 100% was a prior art product, having a =0.2-7.5- (15.5-2.2) kg/mol, and the chemical structure characterization is shown in fig. 2 and 3. Recombinant proteins IFN-gamma and M-CSF were purchased from PeproTech. IL-6, TNF- α, IL-1β, TGF- β and IL-10 ELISA kits were purchased from Invitrogen corporation. Fluorescein-labeled antibodies, including CD80-APC, CD86-PE, CD11c-FITC, CD11b-FITC, CD206-APC, F4/80-PE, CD3-APC, CD4-PE, CD8-FITC, and PE-Cy5-MHC-II, were purchased from Biolegend for flow cytometry. PrimeScript RT kit and SYBR premix Ex Taq kit were purchased from Takara. TNF- α, IL-6, IL-1β and IL-10 primers were synthesized from the Ji Ma gene. All kits, antibodies and proteins were used in accordance with the instructions.
The molecular weight, distribution and surface potential of the polymer vesicles were determined using a Zetasizer-Nano-ZS (Malven Instruments, UK). The drug loading and encapsulation efficiency of Man-PMTX were determined by UV-Vis (HITACHI), which measures mainly the absorbance at 298 nm. Whereas drug release by Man-PMTX was tested by High Performance Liquid Chromatography (HPLC) on a reverse C18 column (4.6x150 mM, 5 μm) with mobile phase a acetonitrile, phase B aqueous phase (20 mM PB, pH: 7.2), mobile phase ratio 8:92 (v/v), flow rate 1 mL/min, MTX wavelength detected 302 nm. Multifunctional enzyme-labeled instrument (Varioskan LUX, thermo Scientific) was used for detection of cytokine concentration in enzyme-linked immunosorbent assay (ELISA) test and to determine absorbance of formazan formed at 570 nm by living cells and MTT reagent. The relevant analyses of the flow experiments were all determined using a BD FACSVerse flow cytometer (united states). Fluorescence images were tested using confocal laser scanning microscopy CLSM (lycra, germany). For the microcomputer tomography measurement, the structure of bone tissue was evaluated by a device (micro-CT, skyScan 1176, aartselaar, belgium) using the following settings: 65kv, 385 mA and 1 mm Al filter. Three-dimensional reconstruction (3D), bone trabecular region, bone volume fraction (BV/TV), bone trabecular spacing (tb.sp) less Liang Shuliang (tb.n) was performed using NRecon software. Fluorescence imaging in mice was determined using a near infrared fluorescence imaging system (IVIS, lumina II; caliper, mass., USA).
Mouse mononuclear macrophage RAW 264.7 was purchased from Shanghai department of science cell bank. Bone Marrow Derived Macrophages (BMDM) are extracted from mouse bone marrow. The specific operation steps are as follows: mice were sacrificed by cervical scission, sprayed with alcohol or soaked throughout the body, and transferred into an ultra clean bench. The limbs of the mice are taken out by clean forceps, muscles are removed, the mice are soaked in PBS, the mice are transferred into a biosafety cabinet in a cell house in a sealing way, and cells in the bone marrow are rewashed into a centrifuge tube by PBS by adopting a perfusion method. Subsequently, the extracted impurities were removed by using a nylon mesh, and the washed cells were centrifuged (1500 rpm,5 min) to remove the supernatant. 4 mL red blood cell lysate was added to the lower layer cells to lyse the red blood cells, and after 5 minutes, 8 mL PBS was added to terminate. Centrifuging, removing supernatant, adding 1640 medium containing 25 ng/mL M-CSF, and culturing at 3-4X10 6 The density of the wells/wells was spread in 6-well plates, half-changed after 3 days of culture, and full-changed after 6 days to obtain BMDM.
Mice from which BMDM was extracted and used in animal experiments were female C57BL/6 mice of 12 weeks of age, which were purchased from Kwangsi laboratory animals Co.
To establish a mouse model of rheumatoid arthritis, zymosan (Zymosan a) was resuspended in 3 mL sterile physiological saline, then boiled and homogeneously emulsified by sonication. The emulsified zymosan was then injected into the left knee joint of the mice, and 50 μl of sterile physiological saline was injected into the other knee joint as a control. Blood was taken 12 hours after modeling and the concentrations of TNF- α and IL-6 were determined using ELISA kits. The height (a, anterior-posterior diameter) and width (b, left-right diameter) of the knee joint of the mouse were measured with a vernier caliper at predetermined time points, and the knee joint circumference (L) was calculated according to the following formula:
All animal experimental procedures were approved by the university of su laboratory animal center and the committee for animal care and use of su university, and all animal research protocols were in compliance with the guidelines for laboratory animal care and use.
Statistical analysis of the data in the present invention employed mean ± Standard Deviation (SD). Statistical differences between groups were analyzed by One way ANOVA methodp<0.05 indicates significant differences, high significant differences are expressed asp<0.01 and%p<0.001.
Example one preparation and characterization of Polymer drug-loaded vesicles
The preparation and characterization of the polymer drug-loaded vesicles are similar to the information previously disclosed by the applicant, and the invention is briefly described below. PEG-P (TMC-DTC) -sp [ ], andMn=5.0- (16.2-2.0) -0.2 kg/mol) and Man-PEG-P (TMC-DTC)Mn=0.2 to 7.5- (15.5 to 2.2) kg/mol) were mixed in different molar ratios (9/1, 8/2 or 7/3) and Man-PMTX was prepared by self-assembly and disulfide cross-linking. The two polymers of Man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) -sp were dissolved in DMF respectively and then mixed in a molar ratio of 1:9 to give a polymer solution. To 900. Mu.L of HEPES buffer solution (5 mM, pH 6.6) containing 0.3 mg methotrexate sodium salt (MTX.2 Na) at room temperature under normal stirring, 100. Mu.L of polymer solution (10 mg/mL) was added, followed by shaking (200 rpm) 12 h in a shaking table at 37℃and then transferring to a dialysis bag (MWCO: 3500) for dialysis for 24 hours to remove the organic solvent and the free drug, and the dialysis medium was HEPES (5 mM, pH 6.6) and PB (10 mM, pH 7.4) in this order; man-PMTX was obtained.
According to the method, only PEG-P (TMC-DTC) -sp is used as a polymer, and Man-PEG-P (TMC-DTC) is not added to obtain the drug-carrying vesicle PMTX; according to the method, man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) -sp are mixed according to different molar ratios, so that the targeting drug-carrying vesicles with different targeting molecule contents are obtained. According to the above method, no MTX was added to obtain empty vesicles.
Man-PMTX the Drug Loading Efficiency (DLE) and Drug Loading Capacity (DLC) of MTX were determined by UV-visible spectrophotometry at 302 nm and were calculated as follows:
wherein PEM L ，PEM I And Ps represent the amount of drug entrapped, the amount of drug administered, and the amount of polymer, respectively.
In the study of in vitro drug release by Man-PMTX, the vesicle concentration was set at 500 ng/mL. It was added to a release bag (MWCO 12000), PBS at pH 7.4 of 15 mL and PBS containing 10 mM GSH (pH 7.4) were added separately outside the release bag, and used to simulate the in vivo cytoplasmic reducing environment, and the solution was placed in a shaking table (200 rpm) at 37 ℃. The 5 mL dialysate was withdrawn at 0.25, 0.5, 1, 2, 4, 6 hours, respectively, with the corresponding 5 mL dialysate being replenished after each withdrawal. The concentration in each removal medium was determined by HPLC and the cumulative drug release at the corresponding time point was calculated (n=3, mean ± standard deviation SD).
Biosafety of Man-PMTX. The biological safety of MTX and the nano-drug loaded with MTX in vivo circulation is verified by adopting a hemolytic experiment, groups with different gradient concentrations (6.25-800 mug/mL MTX) are designed, and a pure water group and a 1% Triton group are simultaneously set as positive controls. After taking out fresh blood of the mice, centrifuging and washing the mice for 3 to 5 times by using normal saline until no obvious red cell fragments are observed in the supernatant, taking out red cells at the lower layer, and diluting the red cell suspension into 2% by using PBS. 300 mu L of red blood cell suspension is taken and added into an EP tube, 300 mu L of MTX, PMTX and Man-PMTX are respectively added, after the same concentration gradient (6.25,12.5,25,50,100,200,400,800 mu g/mL) is set for each group of samples and uniformly mixed, positive control and negative control are set, PBS is added into the positive control, and 1% Triton (Triton X-100) is added into the negative control. The samples were then incubated in a shaker at 37℃for 3 hours. And obtaining a supernatant by centrifugation, measuring the absorbance of the supernatant at 398 and nm by using a multifunctional enzyme-labeled instrument, and respectively calculating the hemolysis rate. From the experimental results, the absorbance of the free MTX group or the nano-drug PMTX and Man-PMTX group is measured by a multifunctional enzyme-labeling instrument, and the hemolysis rate is less than 5% after calculation by a formula. Even if the drug concentration is as high as 800 mug/mL, the drug still has a low hemolysis rate. This result shows that the drug-loaded nano-vesicles have higher biological safety in blood.
Fig. 4 is a physicochemical characterization of PMTX and Man-PMTX. Wherein, (A) PMTX and ManPMTX containing 10%, 20% and 30% mannose have particle size distribution. Particle size distribution of PMTX and Man-PMTX (B) at day 0 and day 30 after preparation and (C) particle size distribution in PB containing 10% FBS for 48 hours. (D) Release profile of Man-PMTX in PB with and without 10 mM GSH (n=3). (E) Hemolysis of mouse erythrocytes by PMTX, man-PMTX and free MTX, positive control is 1% triton, pure water, and negative control is normal saline. The measurement of DLS shows that the sizes of Man-PMTX and PMTX are uniform, the particle size is 51+/-4 nm, the size distribution of nano vesicles is narrow (PDI 0.14), the particle size and the distribution are unchanged when the nano vesicles are stored in a refrigerator at 4 ℃ for 1 month, the original particle size and the original distribution can be kept when the nano vesicles are simulated in vivo environment with 10% FBS, and the results show that the nano drug loaded with MTX shows better stability. The drug loading of MTX in PMTX and Man-PMTX was measured by UV-vis spectrometer (302 nm) and was as high as 18.5 wt% (encapsulation efficiency 80.1%, table 1). Man-PMTX showed a reductive responsiveness to start releasing drug after addition of 10 mM GSH and release about 100% of MTX within 6 hours, in contrast to only 10% in a release solution that mimics the in vivo physiological environment without GSH addition, this reductive responsiveness also provides more potential for macrophage-targeted therapy. As a contrast, the release amount of the existing MTX-CPP33-RCCPs and MTX-Anis-RCCPs within 10 hours is less than 80%, and the requirements of rapidly relieving inflammatory RA symptoms and reducing systemic toxicity cannot be met.
a Measuring by an ultraviolet spectrophotometer; b DLS assay in PB c Zetasizer Nano-ZS assay.
Example two in vitro experiments
Endocytosis experiments of Man-PMTX vesicles. RAW 264.7 cells were seeded on glass coverslips in 24 well plates (1X 10) 5 cells/well). 24. After an hour, LPS (100 ng/mL) was added and after 4 hours Cy 5-labelling was addedFor 4 hours, 4% paraformaldehyde fixed for 15 minutes, DAPI stained for 3 minutes (three washes with PBS per step) and then observed using a Confocal Laser Scanning Microscope (CLSM). Uptake of Man-PMTX by RAW 264.7 cells was observed using CLSM technique with Cy 5-labeled polymer and FITC-labeled MTX.
Repolarization of macrophages by Man-PMTX vesicles. The macrophage cell line RAW 264.7 cells stimulated with LPS and primary macrophage BMDM mimic activated macrophages in the inflammatory microenvironment. RAW 264.7 cells were cultured at 5X 10 5 The density of individual/wells was evenly spread in a 12-well plate. After 24 hours, LPS (100 ng/mL) was added, while Free MTX, PMTX or Man-PMTX was added and incubated for 24 hours. After scraping with a cell scraper, the cells were dispersed in 100. Mu.L of PBS to give a single cell suspension. APC-anti-CD206 antibody was added and incubated at 4℃for 30 min. Cells were centrifuged (1000 rpm,3 min), the medium was removed, and the concentration of cytokines was measured using ELISA kit, respectively. Cells were washed three times with PBS and finally dispersed in 500 μl PBS, and then analyzed for proportion of M2 type macrophages using flow cytometry.
FIG. 5 shows the uptake of Man-PMTX by LPS stimulated RAW 264.7 cells and its repolarization. Wherein, (a) CLSM images after incubation of RAW 264.7 cells with PMTX or Man-PMTX for 4 hours, polymer vesicles labeled with FITC MTX and Cy5 (5 μg Cy5/mL and 14 μg FITC/mL), scale bar: 50. and [ mu ] m. (B) Flow cytometry detected the M2M content (cd206+, n=3) of RAW 264.7 cells after incubation with free MTX, PMTX or Man-PMTX (MTX: 20 μg/mL) for 24 h and semi-quantitative analysis thereof (C). From experimental results, the Cy5 and FITC fluorescence in the cytoplasm was more intense in the Man-PMTX treated RAW 264.7 cells compared to the PMTX group. After LPS-stimulated RAW 264.7 cells were treated with free MTX, PMTX or Man-PMTX, the proportion of M2 type macrophages (M2M) was significantly increased, and thus the proportion of M1 type macrophages (M1M) was decreased
BMDM cells were plated in 12-well plates (1X 10) 6 cells/well)) overnight. LPS (100 ng/mL) was added and IFN-. Gamma. (20 ng/mL) incubated with PBS, free MTX or MTX-loaded polymer vesicles (Free MTX, PMTX or Man-PMTX) for 24 hours, the otherOne group was not treated at all and incubated with PBS as a control. The culture media were collected and the concentration of cytokines was measured separately using ELISA kit. BMDM cells in the 12-well plate were scraped with a cell scraper and dispersed in 100. Mu.L of PBS. APC-anti-CD206, FITC-anti-CD11b and PE-anti-F4/80 antibody were added and incubated at 4℃for 30 min. Cells were centrifuged (1000 rpm/min,3 min), washed 3 times with PBS, dispersed in 500 μl PBS, and immediately analyzed by flow cytometry for the proportion of macrophages of type M1 and type M2. Fig. 6 shows activation and repolarization of LPS-stimulated BMDMs after incubation with PBS, PMTX or Man-PMTX (MTX: 20 μg/mL, n=4) for 24 h. Wherein, (a) a typical flow cytometer fluorescence intensity profile; (B) The ratio of M1M to total cells and (C) the ratio of M2M/M1M. BMDM was co-stimulated with LPS and IFN-gamma, and flow cytometry showed a significantly higher M2M (F4/80+CD206+) ratio for Man-PMTX treated BMDM than for PMTX and PBS groups. The increase in the proportion of M2M in Man-PMTX treated BMDM from 41.97% to 52.35% and the decrease in M1M from 58% to 50% compared to PBS group suggests a significant increase in anti-inflammatory M2M while the proportion of M1M decreases. It is particularly worth mentioning that the M2M/M1M ratio of the Man-PMTX treated group is significantly improved compared to the other groups, with an increase in M2M/M1M from 0.7 to 1.0, which indicates that in vitro experiments, pro-inflammatory M1M can be re-polarized to anti-inflammatory M2M under the effect of Man-PMTX. At the same time, the M2M of BMDM stimulated by Man-PMTX was also significantly improved (52.35% and 37.83%, respectively), M1M was reduced from 55% to 50%, and M2M/M1M was also significantly improved, compared to PMTX treatment groups, and the PMTX and Man-PMTX groups were 0.77 and 1.0, respectively, which indicated that Man-PMTX promoted more macrophage uptake and had better repolarization.
Man-PMTX anti-inflammatory effect on macrophages in vitro. Changes in cytokine concentrations in cell culture media after PMTX and Man-PMTX treatment of BMDM induced by LPS and IFN-gamma were investigated. From the cytokine experimental results of ELISA assays (FIG. 7), man-PMTX treated BMDM reduced TNF- α concentration from 110 pg/mL to 80 pg/mL, PMTX group to 95 pg/mL, IL-1β concentration from 1100 pg/mL to 420 pg/mL, and PMTX group to 633 pg/mL. The anti-inflammatory cytokine IL-10 was increased from 41 pg/mL to 57 pg/mL. This result shows that Man-PMTX has a significant anti-inflammatory effect relative to PBS control and PMTX groups, which can effectively reduce the concentration of pro-inflammatory cytokines while increasing the concentration of anti-inflammatory cytokines.
Example three animal experiments
Establishment of ZIA model and in vivo biological distribution of Man-PMTX. According to the conventional method, a zymosan-induced rheumatoid arthritis model (ZIA model, FIG. 8A) of mice was established, and it was observed that the knee joint appeared to be significantly red and swollen after 12 hours of injection of zymosan at the left knee joint of mice. Blood from ZIA model mice was tested by ELISA kit to determine the concentrations of pro-inflammatory cytokines (TNF-alpha and IL-6) in the blood and the concentrations of pro-inflammatory cytokines in the blood were found to increase substantially after modeling for 12 hours, with TNF-alpha and IL-6 concentrations increasing to 120 μg/mL and 175 μg/mL, respectively (FIG. 8C). At the same time the vernier caliper measures the swelling of its knee joint from 4.8 mm to 6 mm (fig. 8B). The disease state of the rheumatoid arthritis model is met, and thus the model animal is used for subsequent experiments.
In vivo targeting of Man-PMTX to ZIA mice was studied by near infrared fluorescence imaging techniques. PMTX and Man-PMTX are conventional methods by chemical coupling labeling Cy 5. After 24 h of ZIA mice model was established, 200. Mu.L (Cy 5: 3. Mu.g/min) of Cy5-PMTX and Cy5-Man-PMTX were injected into ZIA mice via the tail vein. After Cy 5-labeled NMs injections 2, 4, 6, 8, 12 h, cy5 fluorescence images were obtained by IVIS. 24 h, killing the mice to obtain main viscera, and taking out two hind legs for in vitro imaging. 24 hours after the ZIA mouse model was established, drug-loaded vesicles PMTX and Man-PMTX labeled with Cy5 were injected into the body of the ZIA mouse model (Cy 5:3 [ mu ] g/MTX: 4 mg/kg) by tail vein injection after the swelling of the legs of the mouse reached a maximum. The enrichment of fluorescence in mice was then observed by in vivo fluorescence imaging at each time point. From the results of the fluorescence imaging experiments (fig. 8D), cy5 fluorescence was already observed significantly at the left leg knee from 2 hours, whereas over time, the PMTX group significantly decreased in fluorescence until 12 hours, with no significant fluorescence observed at the knee site in the mice. The Man-PMTX group remained high in Cy5 enrichment at the knee site and did not fade until 10 hours, as did the fluorescence quantification results. At 24 hours, mice were sacrificed and their major viscera and two hind legs were removed for in vitro fluorescence imaging (fig. 8e & f). From the figure, the fluorescence intensity of Cy5 on the modeling side was higher in the mice of the group than on the healthy side, and the fluorescence enrichment on the modeling side was significantly higher in the mice of the Man-PMTX group than in the PMTX group.
From the above experimental results, man-PMTX can achieve a long-lasting enrichment of inflammatory sites in vivo. Meanwhile, the method can be used for repolarizing the M1M enriched in the RA into M2M, and can inhibit the release of pro-inflammatory cytokines and improve the concentration of the anti-inflammatory cytokines.
Dose of Man-PMTX for treatment of ZIA mice. RA mice were divided into five groups (n=3) and the left leg knee joints were induced to develop a pronounced red swelling with zymosan, respectively, and TNF- α concentrations were determined for each group 24 hours after induction. After 24 hours modeling of the ZIA mice, the mice were divided into 5 groups (n=5) and three times total were given by tail vein injection of PBS, free MTX (4 mg/kg) and Man-PMTX (doses of 2 mg/kg, 4 mg/kg, 8 mg/kg, respectively) every three days. The changes of the knee joint diameter and leg circumference before and after treatment are measured by a vernier caliper. Blood was taken at day 3 of dosing to determine the concentration of TNF- α cytokine in serum and at days 6 and 9 to determine the concentration of IL-6 cytokine in serum. After the end of the treatment, mice were sacrificed on day 9, their synovial cartilage was removed, immersed in 1 mL PBS, and ground with a homogenizer. The supernatant was removed after centrifugation by a centrifuge (3000 rpm), and the concentrations of pro-inflammatory cytokines (TNF-. Alpha.and IL-6) at the synovial cartilage were determined using ELISA kit. Fig. 9 is the effect of Man-PMTX dose and number of doses on RA efficacy in anti-mice (first needle dose set to day 0, n=5). PBS and free MTX groups were used as controls. (A) Experimental arrangement. (B) inflammation and (C) changes in healthy lateral knee joint width. (D) Inflammation and (E) changes in knee circumference on healthy sides. According to the leg circumference change, no obvious swelling occurs at the knee joint on the unmodeled side, the leg circumference and the width change are not obvious, the knee joint on the modeled side and the leg circumference are obviously swollen on the 0 th day, the PBS group still has obvious swelling after administration, the swelling of the administration group is slowed down, then the swelling of each group starts to drop, but the alleviation speed of the PBS group is obviously lower than that of the administration group. It can be seen that all groups had a significant effect on relief from swelling, with groups 4 mg/kg and 8 mg/kg being the most effective and there being no statistical difference between the two groups. IL-6 in the serum was examined with ELISA kit on day 3 (FIG. 10A) where IL-6 was not significantly changed, whereas on day six the dosed group had significantly decreased TNF- α relative to the serum of the PBS group (FIG. 10B), and the 4 mg/kg and 8 mg/kg decreases were significant and were not statistically different from the concentration of TNF- α in the serum of healthy mice. On day 9, TNF- α concentrations were significantly reduced in serum from all dosing groups, and PBS group was also significantly reduced (fig. 10C). This result indicated that ZIA model mice had a regression in inflammation at day 9, but relatively decreased TNF- α concentrations in the Man-PMTX-administered group, further indicating inhibition of the secretion of pro-inflammatory cytokines by ZIA mice by Man-PMTX. It also shows that after two administrations, the symptoms of RA mice can be significantly improved, and the concentration of the proinflammatory cytokines in serum can be reduced. Mice were sacrificed on day ten, cartilage and synovium at their leg knee joints were removed, soaked with 500 μl PBS, and the supernatants were centrifuged after grinding with a homogenizer to determine cytokines TNF- α and IL-6 in the joints (fig. 10d & e). From the TNF- α concentration, the TNF- α concentration at the joint decreased after administration, with a decrease of 8 mg/kg being most pronounced, to a concentration substantially consistent with that of the healthy group. The concentration of IL-6 was such that the drug concentration was 4 mg/kg, which was already the same as that of IL-6 in the healthy group. The invention selects 4 mg/kg as the proper concentration for administration, and carries out subsequent experiments.
Treatment study of ZIA mice by Man-PMTX. ZIA mice were divided into four groups (PBS, free MTX, PMTX, man-PMTX, dose: 4 mg/kg) and given every third day by tail vein injection, two total needles, and the mice were sacrificed on day 7 (FIG. 11A). Healthy and PBS groups served as negative and positive controls, respectively. Mouse knee joint diameter, circumferenceThe length (fig. 11B) and cytokine concentration (fig. 11C) were substantially consistent with the results of the above experiments, with the knee joint on the modeled side of the mice beginning to be significantly red and swollen after 12 hours of zymosan induction, followed by the start of dosing. The results showed that the leg swelling of the PMTX and Man-PMTX treated RA mice was significantly reduced, the knee joint diameter and circumference were significantly reduced on day 6, to a level similar to healthy mice. The concentration of the cytokines in serum will generally be one of the key indicators of therapeutic efficacy, and the concentrations of the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine TGF-beta in mouse serum will be determined at day 6. The IL-6 concentration was significantly reduced in the PMTX and Man-PMTX groups compared to the PBS group, and the TGF-beta concentration was increased, with Man-PMTX results similar to healthy mice with no statistical difference. Researchers have found that TGF- β can stimulate mesenchymal stem cells to differentiate into osteoblasts and cartilage, contributing to the ability of tissue repair. Although currently clinical treatment of RA mainly uses MTX (5-25 mg/week, oral), intravenous injection of free MTX has little effect on cytokine concentration from the results of the treatment experiments. RA joint synovium and cartilage were collected at day 7, homogenized with physiological saline, and the concentrations of the typical M1M markers TNF- α, IL-1β, IFN- γ and IL-6 were detected with ELISA kit, while quantified with BCA quantification of protein concentration (fig. 11D). The results showed that all of these M1M markers were significantly reduced (/ v) after tail vein injection of Man-PMTX compared to PBS group p) Is also well below the non-targeted PMTX group and is comparable to healthy mouse levels. The results are consistent with cytokine results in plasma. In addition, RT-PCR detection of the relative mRNA expression levels of TNF-. Alpha., IL-6 and IL-1. Beta. Genes in the inflammatory joints of the Man-PMTX group and PMTX group showed the same trend as cytokines (FIG. 11E). Whereas the expression of the M2M marker IL-10 gene was significantly higher than in the PBS group. The result shows that the Man-PMTX has a regulating effect on the inflammatory related cytokines, can effectively reduce the proinflammatory cytokines and related gene levels and simultaneously improve the anti-inflammatory cytokines and related gene levels. These results confirm the effectiveness of Man-PMTX for the treatment of RA mice inflammation. FIG. 12 is H of the major organs of the mice after day 7 of treatment as shown in FIG. 11A for Man-PMTX, PMTX and free MTX&Histological score of E sectionsAnd (5) separating. Scale bar: 50. and [ mu ] m.
Man-PMTX modulation of immune microenvironment. After 24 hours modeling of the ZIA mice, the mice were divided into 3 groups (n=3), and PBS, free MTX, and Man-PMTX (MTX: 4 mg/kg) were injected through tail veins, respectively, while healthy mice were set as control groups. After 24 hours of injection, mice were sacrificed, the diseased site synovial cartilage was removed, immersed in 1 mL PBS, homogenized, centrifuged, and the supernatant was discarded and ACK-split red was used for 5 min. Then, the cells were washed twice with PBS, and after counting by a cell counting plate, APC-anti-CD206, FITC-anti-CD11b and PE-anti-F4/80 antibody were added and incubated at 4℃for 30 min. Cells were centrifuged (1000 rpm, 3 min), washed 3 times with PBS, dispersed in 500 μl PBS, and immediately analyzed for the proportion of M1-type and M2-type macrophages by flow cytometry. Immunofluorescence analysis was further performed on the treated bone joint sections of mice for the RA joint M2M marker (CD206+), and the fluorescence intensity of the Man-PMTX and PMTX groups CD206 was enhanced compared with that of the PBS group (FIG. 13A), especially the Man-PMTX synovial site M2M was more, showing that M2M was more abundant and well positioned in the RA joint. These results demonstrate that Man-PMTX injections can selectively accumulate in RA joints, significantly target activated macrophages, and further down-regulate M1M and pro-inflammatory cytokines, repolarizing M1M to M2M, secreting more anti-inflammatory cytokines, thereby eliminating inflammation. The large amount of M2M accumulates in the synovium relative to the Man-PMTX group, which, although currently used clinically for RA treatment (5-25 mg/week, oral administration), has a much lower efficacy in reducing inflammation, producing anti-inflammatory macrophages and cytokines, etc., resulting in the inability to protect the synovium and bone with its treatment. To further explore the anti-inflammatory mechanism of Man-PMTX in vivo, RA mice were studied for activation and polarization of macrophages in inflammatory joints after receiving single dose Man-PMTX treatment. The results showed that intravenous injection of Man-PMTX resulted in a dramatic decrease in total macrophage numbers in RA joints (from about 18% to about 6.9%) compared to PBS group (fig. 13B). Notably, the M2M ratio at RA joints in Man-PMTX treated group increased from 13.9% to 53.7% in PBS group. M2M has been reported to secrete anti-inflammatory cytokines, chemokines, protecting tissues from bone damage.
Effect of Man-PMTX on RA mouse joint synovium. Since RA is commonly damaged by bone, it is desirable to explore the inflammatory conditions of synovial cartilage at joint sites in RA mice after treatment and repair of the injury. The RA joint sites of MTX formulation treated mice were stained for H & E, safranin fast green or maroon to analyze tissue injury, immune cell infiltration, vascular growth, bone injury or collagen fiber distribution. After 24 hours of ZIA mice modeling, they were divided into 4 groups of at least 10 mice each. PBS, MTX, PMTX, man-PMTX was injected by tail vein every 3 days. Levels of pro-inflammatory cytokines (TNF- α, IL-6, IL-1β) in serum before and after treatment were detected by ELISA kit (n=3). Proinflammatory cytokine and protein levels in knee arthritic tissue extracts were detected using ELISA and BCA methods (n=3). The daily changes in knee diameter and leg circumference before and after treatment were measured using vernier calipers (n=7). Body weight was measured every 2 days. After the end of the treatment, the mice were sacrificed on day 7 and the major viscera and the diseased hind limbs of the mice were removed. The synovial cartilage of the affected part of the mice was removed, three of which were immersed in PBS, and the protein content thereof was measured using micro BCA, while the cytokine contents (TNF-. Alpha., IL-6, IL-1. Beta. And IFN-. Gamma.) were measured using ELISA kit. And 3, extracting total RNA at the synovial cartilage by using TRIzol extract, purifying and drying. cDNA was synthesized from total RNA using a high-capacity cDNA reverse transcription kit according to the instructions. The synthesized cDNA, primers and SYBR Premix Ex Taq II (Takara, japan) were mixed and run on a real-time PCR system (Bio-Rad CFX connect). 3 affected leg bones of each group of mice were taken out, fixed with paraformaldehyde and sectioned for CD206 immunofluorescence staining, H & E, safranine fast-green and masson staining, and used for evaluation of immune microenvironment, synovial cartilage inflammatory infiltration and damage to synovial cartilage. Where PBS, man-PMTX treatment and healthy groups remained to continue to be observed and sacrificed after the third week, RA affected hind limbs were removed, 4% paraformaldehyde fixed for one week, and tibia and femur scanning imaging was performed on an animal computed tomography scanner. Fig. 14A shows that PBS group has severe synovial cavity destruction and severe immune cell infiltration, while Man-PMTX treatment group is significantly improved, indicating that it can effectively restore joints and synovium, reducing synovial inflammatory cell infiltration. Safranin fast green staining images showed significant glycosaminoglycan loss in RA mice cartilage, whereas this severe articular cartilage degeneration and damage was not found in Man-PMTX treated joints, which had smooth and intact cartilage, substantially consistent with joints of healthy mice. However, free MTX and PMTX did not alleviate bone damage from H & E and safranin fast green staining analysis. Masson staining showed that Man-PMTX was effective in inhibiting inflammatory attack and reducing fibrosis. The primer sequences used are shown in Table 2.
Furthermore, micro-CT evaluation was performed on knee joints by quantitative histomorphometric imaging methods, and the effect of Man-PMTX injection on joint site bone erosion and bone loss was analyzed (fig. 14B). The constructed CT image shows that the RA joint bone erosion of the PBS mice is serious, and the Man-PMTX injection group can obviously relieve the bone injury and the Tb is obviously reduced. Sp increases, BV/TV and Tb increases. N (×p), the series of data is all close to the data of the tibia of healthy mice. Man-PMTX can effectively prevent RA-related bone injury and cartilage injury, can obviously reduce M1M and pro-inflammatory cytokines, promotes M1M repolarization to M2M and simultaneously promotes TGF-beta secretion. M2M and TGF- β have been reported to protect tissue from damage, stimulating differentiation of mesenchymal stem cells into osteoblasts and cartilage.
In clinic, RA treatment (5-25 mg/week, oral) is much less effective in reducing inflammation, producing anti-inflammatory macrophages and cytokines, etc., resulting in the inability to protect synovial membranes and bone with the treatment. In order to better treat the rheumatoid arthritis, the invention prepares the ManPMTX nano-drug of mannose-modified macrophages, and realizes stable entrapment of MTX. In vivo fluorescence imaging demonstrated that mannose-mediated Man-PMTX nanomaterials could be delivered to arthritic joints and achieve a long-lasting enrichment following systemic administration in ZIA mice. RA treatment experiments show that the Man-PMTX (4 mg/kg) significantly improves the therapeutic effect of MTX compared with free MTX and PMTX, but only causes slight systemic side effects, and the Man-PMTX nano-drug can realize longer retention and enrichment in arthritic joints. In addition, immunofluorescence of joint sections suggests that Man-PMTX has the ability to repolarize M1M to M2M, causing macrophages to reduce the secretion of pro-inflammatory cytokines while increasing the secretion of anti-inflammatory cytokines at RA. The treated bone slices and Micro-CT show that the Man-PMTX protects cartilage and reduces infiltration of inflammation in joints, and bone loss is reduced. Thus, the present study provides a rational and innovative approach to RA treatment based on Man-PMTX NMs with attractive clinical therapeutic potential.
Claims (10)
1. A methotrexate nanodrug delivery system, which is characterized by comprising a polymer vesicle and methotrexate; the polymer comprises PEG-P (TMC-DTC) -Y, PEG-P (CL-DTC) -Y or PEG-P (LA-DTC) -Y, Y being a cationic fragment.
2. The methotrexate nanodrug delivery system according to claim 1, wherein the polymer further comprises X-PEG-P (TMC-DTC), X-PEG-P (CL-DTC) or X-PEG-P (LA-DTC), X being a modifying molecule.
3. The methotrexate nanodrug delivery system according to claim 2, wherein X is mannose.
4. The methotrexate nanodrug delivery system according to claim 1, wherein the molecular weight of the polymer is 10-50 kg/mol, the molecular weight of the PEG segment in the polymer is 2-10 kg/mol, the molecular weight of the cationic segment is 0.1-2 kg/mol, and the molecular weight of the DTC segment is 1-5 kg/mol.
5. The method for preparing the methotrexate nano-drug delivery system according to claim 1, wherein the polymer and the methotrexate are used as raw materials to prepare the methotrexate nano-drug delivery system.
6. The method for preparing the methotrexate nanodrug delivery system according to claim 5, wherein the polymer is a polymer containing a cationic fragment; or the polymer is a polymer containing cation fragments and a polymer containing modified molecules, and the molar ratio of the polymer containing cation fragments to the polymer containing modified molecules is 1:0-0.5, excluding 0.
7. The method for preparing the methotrexate nanodrug delivery system according to claim 5, wherein the methotrexate is loaded into the polymer vesicles by electrostatic interaction to obtain the methotrexate nanodrug delivery system.
8. The methotrexate nano-drug-carrying system freeze-dried powder is prepared by mixing the methotrexate nano-drug-carrying system according to claim 1 with a freeze-drying protective agent and then freeze-drying the mixture.
9. The methotrexate nano-drug delivery system or the methotrexate nano-drug delivery system freeze-dried powder of claim 1 is applied to the preparation of the medicine for treating rheumatoid arthritis.
10. The methotrexate nanodrug delivery system or the methotrexate nanodrug delivery system freeze-dried powder of claim 1 for preparing a medicament with repolarized M1M macrophages as M2M macrophages; or in the preparation of a medicament for recovering joints and synovium and reducing synovial inflammatory cell infiltration; or in the preparation of a medicament for inhibiting dendritic cell activation; or in the preparation of a medicament for protecting articular cartilage and bone tissue; or in the preparation of anti-inflammatory drugs; or in the preparation of a medicament for promoting TGF-beta secretion; or in the preparation of anti-autoimmune disease drugs.
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