Cell membrane nanoparticle, preparation method and use thereof

Abstract

The present invention discloses a cell membrane nanoparticle and its preparation method and use. + T cells, cell membrane nanoparticles TaCD4M1-NP with ROS response. This nanoparticle can carry CD4 + T cells infiltrate the inflammatory site and rely on the membrane proteins of the cell membrane to achieve enrichment and retention in the inflammatory site, increase the drug concentration in the target site, and reduce toxic and side effects. By selecting a combination of anti-inflammatory drugs, methotrexate and dasatinib were selected for combined administration. The results showed that the combination had synergistic efficacy in various inflammatory models. Cell membrane nanoparticles loaded with MTX and DAS can effectively carry CD4 + T cells, which infiltrate inflammatory sites such as the colon in ulcerative colitis (UC) and the joints in rheumatoid arthritis (RA), significantly enhance the efficacy of drugs against UC and RA, providing a new approach for the treatment of inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to cell membrane nanoparticles and a preparation method and application thereof. Background Art

[0002] Cell membrane-encapsulated nanoparticles, while retaining the properties of the original core nanoparticles, can enhance the physiological functions of the original cells, such as enhancing infiltration of lesions or adsorption of cytokines by relying on proteins on the cell membrane surface. Furthermore, cell membrane nanoparticles can prolong their circulation in the body by utilizing surface proteins such as CD47 and CD24, preventing endocytosis.

[0003] Introducing additional proteins into the cell membrane through physical binding or chemical modification can enhance the functionality of cell membrane nanoparticles. Modifying additional proteins can chemically attach the target protein to the cell membrane surface using a linker, but this approach may affect the function of the original surface protein. Alternatively, the target protein can be linked to a lipophilic carbon chain, such as DSPE, leveraging the hydrophobicity of the lipophilic carbon chain to insert into the phospholipid bilayer of the cell membrane. Genetic engineering techniques, which modify target cells to express specific proteins, have been applied to the development of cell membrane nanoparticles. This approach not only preserves the original cell membrane function but also allows for stable expression of the target protein on the cell membrane surface. For example, engineered cells expressing the α4 protein, which is then combined with the original β1 protein on the cell membrane surface to form the inflammatory receptor very late-appearing antigen-4 (VLA-4), are extracted from the cell membrane and prepared into nanoparticles targeting vascular cell adhesion molecule-1 (VCAM-1) at sites of inflammation for the treatment of pneumonia. There are also studies that use genetic engineering to make T cells overexpress programmed cell death protein 1 (PD-1), and after preparing them into cell membrane nanoparticles, they exert anti-tumor effects by blocking the programmed cell death 1 ligand 1 (PD-L1) of tumor cells.

[0004] Immune cells have natural inflammation targeting capabilities, such as neutrophils, macrophages, T cells, etc. In early inflammation, CD4 + T helper 1 (Th1) cells and neutrophils infiltrate the site of injury and release pro-inflammatory cytokines. In the late stage of inflammation, regulatory T (Treg) cells and Th2 cells proliferate and inhibit the inflammatory process by producing anti-inflammatory cytokines, such as transforming growth factor-β (TGF-β) and IL-10. In arthritis and colitis, a large number of CD4 +T cells infiltrate inflamed tissues. However, if inflammation remains uncontrolled, overactivated immune cells, such as macrophages, secrete proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6. These further recruit immune cells from the peripheral blood to the site of inflammation, exacerbating the severity of inflammation and creating a microenvironment high in reactive oxygen species (ROS). Therefore, leveraging the deep tissue penetration ability of immune cells at the site of inflammation can enhance the targeting and penetration of nanocarriers into inflamed tissues. This is the origin of the present invention. Summary of the Invention

[0005] In order to solve at least one of the technical problems in the background technology of the present invention, the present invention provides cell membrane nanoparticles and a preparation method and use thereof.

[0006] The technical solution of the present invention is: One object of the present invention is to provide a method for preparing cell membrane nanoparticles, comprising the following steps:

[0007] Drug-loaded nanoparticle preparation: Methotrexate (MTX) and dasatinib (DAS) were linked to hyaluronic acid (HA) using a reactive oxygen species-sensitive copper thiol acetal crosslinker (TK) to synthesize HA-TK-MTX or HA-TK-DAS polymers, which self-assembled into drug-loaded nanoparticles in saline. Cell membrane preparation: Using genetic engineering techniques, a macrophage cell line stably expressing aCD4 single-chain antibody scFv on its cell surface was constructed. The macrophages were polarized to the M1 phenotype through infection, and the cell membranes were subsequently extracted to obtain CD4-targeted cell membranes. Cell membrane nanoparticle preparation: The cell membranes and drug-loaded nanoparticles were co-extruded to produce cell membrane nanoparticles. It should be noted that the drug-loaded nanoparticles used in this step are HA-TK-MTX and HA-TK-DAS.

[0008] Another object of the present invention is to provide a method for preparing different cell membrane nanoparticles, comprising the following steps:

[0009] Drug-loaded nanoparticle preparation: MTX and DAS were linked to hyaluronic acid using a reactive oxygen species-sensitive copper thiol crosslinker to synthesize HA-TK-MTX or HA-TK-DAS polymers, which self-assembled into drug-loaded nanoparticles in saline. Cell membrane preparation: Macrophages were polarized to the M1 phenotype, and the cell membranes were extracted. NHS-functionalized DSPE-PEG was then reacted with a CD4 monoclonal antibody to produce DSPE-PEG-aCD4, which was then inserted into the M1 cell membrane to create CD4-targeted cell membranes. Cell membrane nanoparticle preparation: Cell membranes and drug-loaded nanoparticles were co-extruded to produce cell membrane nanoparticles. It should be noted that the drug-loaded nanoparticles used in this step are HA-TK-MTX and HA-TK-DAS.

[0010] Another object of the present invention is to provide cell membrane nanoparticles TaCD4M1-NP prepared according to the above-mentioned preparation method.

[0011] Another object of the present invention is to provide a use of the cell membrane nanoparticles described above in preparing a drug for treating inflammatory diseases.

[0012] The present invention screens out a new drug combination with synergistic anti-inflammatory effect, and uses dasatinib, which is used to treat leukemia, to treat inflammatory diseases, and innovatively uses methotrexate and dasatinib in combination to treat inflammatory diseases. It also innovatively designs a drug that can carry endogenous CD4 + T cell drug delivery system using CD4 + The in vivo distribution characteristics of T cells and their chemotactic properties towards inflammatory sites enable targeted drug delivery and efficient treatment of inflammatory diseases such as rheumatoid arthritis and ulcerative colitis, providing new methods and new ideas for the treatment of inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the synthesis process and mechanism of action of cell membrane nanoparticles according to an embodiment of the present invention ( Figure 1 A is the synthetic route; Figure 1 B is a diagram of the mechanism of action); Figure 2 The data are as follows: In vitro inhibition data of MTX and DAS on immune cells (data are expressed as mean ± standard deviation (n = 3)); Figure 3 Schematic diagram of the anti-inflammatory effect of combined administration of MTX and DAS in mice with collagen-induced arthritis (CIA) ( Figure 3 A is the experimental flow chart; Figure 3 B is a picture of paw swelling; Figure 3 C is the arthritis index data; Figure 3 D is the weight curve of mice; Figure 3 E is the serum IL-6 concentration; Figure 3 F is the serum IL-1β concentration; Figure 3 G is the serum TNF-α concentration; data are mean ± SD (n = 4-5), ns means no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 4 The anti-inflammatory effect of combined administration of MTX and DAS in the mouse model of acute inflammation induced by lipopolysaccharide ( Figure 4 A is the experimental flow chart; Figure 4 B is the bioluminescence image of mice and inflammation sites; Figure 4C is the fluorescence intensity of the inflammatory site; data are expressed as mean ± standard deviation (n = 8) and analyzed by two-tailed unpaired Student's t-test. ns means no significant difference, *P < 0.05, **P < 0.01); Figure 5 The middle figure shows the anti-inflammatory effect of combined administration of MTX and DAS in Zymosan A induced arthritis (ZIA) mice ( Figure 5 A is the experimental flow chart; Figure 5 B is the relative circumference diagram of the joint; Figure 5 C is a weight curve graph; Figure 5 D is the synovial inflammatory factor; data are expressed as mean ± standard deviation (n = 5), ns means no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 6 Schematic diagram of the anti-inflammatory effect of combined administration of MTX and DAS in ulcerative colitis (UC) mice ( Figure 6 A is the experimental flow chart; Figure 6 B is the colon length diagram; Figure 6 C is the colon length graph; Figure 6 D is the body weight curve; data are expressed as mean ± standard deviation (n = 5), ns means no significant difference, *P < 0.05, **P < 0.01, ****P < 0.0001); Figure 7 The polymer synthesis route of the present invention is ( Figure 7 A is the synthesis of HA-TK; Figure 7 B is the synthesis of HA-TK-MTX; Figure 7 C is the synthesis of carboxylated DAS; Figure 7 D is the synthesis of HA-TK-DAS); Figure 8 The NMR characterization data diagram ( Figure 8 A is the characterization of HA-TK; Figure 8 B is the characterization of HA-TK-MTX; Figure 8 C is the characterization of carboxylated DAS; Figure 8 D is the characterization of HA-TK-DAS); Figure 9 Figure 2 shows the polarization of macrophages and the targeting of CD4+ T cells and inflammation by aCD4M1-NP. Figure 9 A is a flow chart of macrophage polarization; Figure 9 B is the expression level of CD86; Figure 9 C is the expression level of CD206; Figure 9 D is the synthetic route map; Figure 9 E is polarized macrophages in vitro with CD4 + Schematic diagram of T cell binding; Figure 9 F is the flow chart of animal experiments; Figure 9 G is the fluorescence image of the inflammation site; Figure 9 H is the fluorescence intensity graph of the inflammatory site; data are expressed as mean ± standard deviation (n = 2-5), ns means no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 10 For the construction of engineered cells ( Figure 10 A is a schematic diagram of lentiviral infection; Figure 10 B is the infection efficiency; Figure 10 C is the expression of CD86; Figure 10 D is the expression of PSGL-1; Figure 10 E is the expression of LFA-1; data are expressed as mean ± SD (n = 3), ns means no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 11 For the characterization of TaCD4M1-NP ( Figure 11 A is the TEM image of nanoparticles, a is NP, b is TaCD4M1-NP, the scale bar is 100 nm; Figure 11 B is the particle size and potential data; Figure 11 C is the PAGE characterization of membrane proteins; Figure 11 D is a release curve diagram); Figure 12 CD4 of TaCD4M1-NP + T cell targeting ( Figure 12 A is the confocal detection data; Figure 12 B is with CD4 + T cell binding rate; Figure 12 C is the experimental flow chart; Figure 12 D is a flow scatter plot; Figure 12 E is CD4 + T cells and CD8 + The binding ratio of T cells; data are expressed as mean ± SD (n = 3), ****P < 0.0001); Figure 13 The inflammatory targeting of TaCD4M1-NP ( Figure 13 A is the flow chart of animal experiments; Figure 13 B is a fluorescent image of the colon; Figure 13 C is the fluorescence intensity of the colon; Figure 13 D is the flow chart of animal experiments; Figure 13 E is the fluorescence image of the inflammation site; Figure 13 F is the fluorescence intensity of the inflammatory site; data are expressed as mean ± standard deviation (n = 5), ns means no significant difference, *P < 0.05, **P < 0.01, ****P < 0.0001); Figure 14 The efficacy of the cell membrane nanoparticles of the present invention in the collagen-induced arthritis (CIA) mouse model ( Figure 14 A is the experimental flow chart; Figure 14 B is the arthritis score; Figure 14 C is the change in body weight; Figure 14 D is the paw thickness; Figure 14 E is a picture of a rotating cage; Figure 14 F is the maximum speed; Figure 14 G is the running time; Figure 14 H is the concentration of IL-6, IL-1β, and TNF-α in the synovium; Figure 14 I represents the concentration of IL-6, IL-1β, and TNF-α in serum; data are expressed as mean ± SD (n = 6); ns indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 15 Pathological sections of ankle joint and Micro-CT images of paw ( Figure 15 A: H&E staining; Figure 15 B is Safranin O-fast green staining; Figure 15 C is Masson staining; Figure 15 D is Micro-CT image of the hind paw; scale bar is 100 μm); Figure 16 The efficacy of the cell membrane nanoparticles of the present invention in the ulcerative colitis (UC) mouse model ( Figure 16 A is the flow chart of animal experiments; Figure 16 B is weight change; Figure 16 C is the disease activity index; Figure 16 D is fecal occult blood test; Figure 16 E is the intestinal permeability test; Figure 16 F is a picture of the colon; Figure 16 G is colon length; data are expressed as mean ± SD (n = 6), ns means no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 17 The efficacy of the cell membrane nanoparticles of the present invention in the Zymosan A-induced arthritis (ZIA) mouse model ( Figure 17 A is the flow chart of animal experiments; Figure 17 B is the relative circumference of the joint; Figure 17 C is the weight curve; Figure 17 D is the concentration of IL-6, IL-1β, and TNF-α in the synovium; Figure 17E is the concentration of IL-6, IL-1β, and TNF-α in serum; data are expressed as mean ± SD (n = 6), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); Figure 18 Safety evaluation of the cell membrane nanoparticles of the present invention ( Figure 18 A is a hemolysis test; Figure 18 B is the flow chart of animal experiments; Figure 18 C is the activity of ALT and AST in the liver; Figure 18 D is routine blood analysis; Figure 18 E is an organ section, the scale bar is 100 μm; data are expressed as mean ± standard deviation (n = 3 or 6), ns means no significant difference). DETAILED DESCRIPTION

[0014] The above solution will be further described below with reference to the accompanying drawings and specific embodiments.

[0015] The preparation method of the cell membrane nanoparticles of the present invention is as follows ( Figure 1 A): ① Using a reactive oxygen species (ROS)-sensitive thioketal (TK) crosslinker, MTX and DAS were linked to biosafe, biodegradable hyaluronic acid (HA) to synthesize HA-TK-MTX and HA-TK-DAS polymers, respectively. These polymers self-assemble in saline or water to form ROS-responsive nanoparticles. ② Cell membranes expressing CD4-targeting proteins were prepared using two different methods. DSPE-PEG-aCD4 was prepared and aCD4 was linked to the cell membrane surface via DSPE intercalation. The first method involved genetic engineering to construct a macrophage cell line that stably expressed the aCD4 single-chain antibody fragment (scFv) on its cell surface. Following construction, the macrophages were polarized to the M1 phenotype, and the cell membranes were extracted. The second method involved modifying the CD4 monoclonal antibody (aCD4) with distearoylphosphatidylethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide (DSPE-PEG2000-NHS, DP-NHS). Third, the aCD4-expressing macrophage membrane was coated on the surface of HA-TK-MTX / DAS nanoparticles using coextrusion using a liposome extruder.

[0016] The working principle of the cell membrane nanoparticles of the present invention is as follows ( Figure 1 B): ① After intravenous administration, the nanoparticles rely on anti-CD4 to target and carry CD4 + T cells, which rely on their inflammatory chemotaxis to carry nanoparticles to penetrate into the inflammatory site. ② Without CD4+ The free nanoparticles of T cells can rely on the P-selectin glycoprotein ligand-1 (PSGL-1) and lymphocyte function associated antigen-1 (LFA-1) on the cell membrane surface to bind to the P-selectin (P-selectin) and intercellular cell adhesion molecule-1 (ICAM-1) ligands of diseased blood vessels respectively, and are enriched in the inflammatory site. ③ The nanoparticles in the inflammatory site respond to the high ROS in the environment and release two immunosuppressive drugs to synergistically inhibit immune cells. The cell membrane nanoparticles carry CD4 + The T cell drug delivery ladder system can be carried out by CD4 + T cells achieve targeted drug delivery to the site of inflammation, inhibit the activity of various immune cells in the site of inflammation, eliminate inflammatory factors and reactive oxygen species in the lesion site, and significantly inhibit the inflammatory response.

[0017] Experimental methods

[0018] 1.1 Reagents: Fetal bovine serum, RPMI 1640, and Dulbecco's Methionine Medium (DMEM) were purchased from Gibco (USA). Penicillin and streptomycin were purchased from Wuhan Punosai Life Science Co., Ltd. Sodium pyruvate and non-essential amino acids were purchased from Beijing Solaibao Technology Co., Ltd. Hyaluronic acid (HA, 10 kDa) was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. Luminol, lucigenin, succinic anhydride, methotrexate (MTX), DMAP, NHS, and EDC were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. ACK red blood cell lysis buffer, fecal occult blood test kit, and dasatinib (DAS) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Alamar Blue was purchased from Shanghai Yisheng Biotechnology Co., Ltd. Ficoll-paqueplus monocyte separation medium was purchased from Cytiva (USA). Mouse CD4, CD3, and CD28 monoclonal antibodies were purchased from Bio X Cell (USA). DSPE-PEG2000-NHS was purchased from Shanghai Pengshuo Biotechnology Co., Ltd. RIPA lysis buffer, DiO dye, Hoechst 33342, BCA kit, and lipopolysaccharide (LPS) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. IFN-γ was purchased from Peprotech, USA. CD4 +T cell negative selection kit was purchased from STEMCELL, Canada. Mouse IL-6, IL-1β, and TNF-α ELISA kits and Lipo3000 were purchased from Thermo Fisher Scientific, USA. Phosphotungstic acid negative staining solution was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. H₂O₂ was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Dextran sulfate sodium (DSS, 40 kDa) was purchased from Shanghai Xibao Biotechnology Co., Ltd. Zymosan A was purchased from Sigma, Germany. TK-NH₂ and FITC-dextran (4 kDa) were purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. ALT and AST detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Bovine type II collagen, complete Freund's adjuvant (CFA), and incomplete Freund's adjuvant (IFA) were purchased from Chondrex, USA.

[0019] 1.2 Experimental Animals: CD4 + T cells were isolated from the spleens of C57BL / 6 mice and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids. Raw264.7, J774A.1, and HEK-293T cells were purchased from Zhejiang Zhongchu Biotechnology Co., Ltd. and cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin.

[0020] New Zealand rabbits were purchased from Pizhou Xiaohe Technology Development Co., Ltd. Six- to eight-week-old male C57BL / 6, male DBA / 1, and female BALB / c mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (Laboratory Animal Production License: SCXK (Su) 2021-0013) and housed in the SPF animal facility of Soochow University (Laboratory Animal Use License: SYXK (Su) 2016-0050) under conditions of 20 ± 2°C humidity, 50 ± 5%, and 12 h of light per day. Mice were acclimated for 1–3 days before the start of the formal experiments. All experiments were conducted under the approval and supervision of the Soochow University Animal Ethics Committee (Approval No.: SUDA20200622A03).

[0021] 1.3 Inhibitory drug screening in Raw264.7 macrophages

[0022] Raw264.7 cells were seeded in black-rimmed, clear-bottomed 96-well plates. When the cell density reached 40–50%, various concentrations of methotrexate (MTX) or dasatinib (DAS) were added to each well and cultured in a cell culture incubator. Each concentration was replicated in quadruplicate. Positive wells contained cells but no drug, negative wells contained pure culture medium but no cells, and experimental wells contained cells and drug. When the cell density in positive wells reached 80–90%, 10 μL of Alamar Blue was added to each well. The cells were incubated in a dark incubator for 2 h. Fluorescence intensity (Ex = 545 nm, Em = 590 nm) was measured using a microplate reader (M1000 Pro, Tecan, Switzerland). Cell viability was calculated according to Equation 1.

[0023] Formula 1

[0024] 1.4 CD4 + T cell inhibitory drug screening

[0025] Primary CD4 + T cells were extracted using a negative selection kit. Male C57BL / 6 mice were euthanized with CO2, and the spleen was dissected and placed on a cell strainer for grinding. After lysing red blood cells, the target cells were isolated using a kit. + T cells were resuspended in RPMI 1640 medium supplemented with murine IL-2 (10 ng / mL) and IL-7 (1 ng / mL) and transferred to 6-well plates coated with anti-CD3 (1 μg / mL) and anti-CD28 (5 μg / mL). After 3 days of culture, cells were purified by Ficoll-Paque Plus gradient centrifugation for subsequent experiments.

[0026] CD4 + T cells were seeded in a 96-well plate with a black edge and a clear bottom, with 1×10 cells per well. 6 Then, different concentrations of MTX or DAS were added and incubated in a cell culture incubator, with four replicates for each concentration. After 12 hours, 10 μL of Alamar Blue was added to each well. Incubate in the dark for 2 hours in a cell culture incubator. Fluorescence intensity was measured with a microplate reader, and cell viability was calculated according to Equation 1.

[0027] 1.5 Anti-inflammatory effect of combined administration of MTX and DAS in mice with collagen-induced arthritis

[0028] Male DBA / 1 mice were immunized twice intraperitoneally with bovine type II collagen to induce arthritis (CIA) at the base of the tail. On day 0, bovine type II collagen and CFA were mixed at a 1:1 ratio by volume. A milky, creamy, oil-in-water emulsion was prepared using a high-speed homogenizer (Vs-35S, Wuxi Woxin Instrument Manufacturing Co., Ltd.) on ice. 100 μL of this emulsion was injected intradermally at the base of the tail of each mouse. On day 21, a second oil-in-water emulsion was prepared using the same method (CFA was replaced with IFA). On day 26, mice developed mild redness and swelling in the paws and were divided into four groups: ① CIA, ② MTX, ③ DAS, and ④ MTX + DAS, with 5 mice in each group. Drugs were administered intravenously via the tail vein on days 26, 29, 32, 35, and 38. The MTX group received 50 μg of MTX, the DAS group received 50 μg of DAS, and the MTX+DAS group received 50 μg of MTX and 50 μg of DAS. During treatment, the survival of the mice was observed, their body weights were recorded, and their paw swelling was scored and photographed. On day 42, the mice were anesthetized with isoflurane gas, and blood was collected from the retroorbital venous plexus. Serum levels of IL-6, IL-1β, and TNF-α were measured according to the ELISA kit instructions.

[0029] 1.6 Anti-inflammatory effect of combined administration of MTX and DAS in mice with LPS-induced inflammation

[0030] On day 0, an LPS-induced inflammation model was established in the right hind paw of female BALB / c mice. LPS (50 μg) was dissolved in sterile PBS and injected subcutaneously into each mouse's paw. On day 1, redness and swelling appeared in the paw of the mice, and the mice were divided into four groups: ① LPS model group, ② MTX group, ③ DAS group, and ④ MTX + DAS group, with 8 mice in each group. On days 2 and 4, LPS was administered via the tail vein. The MTX group received 50 μg MTX, the DAS group received 50 μg DAS, and the MTX + DAS group received 50 μg MTX and 50 μg DAS. On day 5, each mouse was intraperitoneally injected with a mixture of 200 μg Luminol and 50 μg Lucigenin. Bioluminescence was measured 5 minutes later using an IVIS imager (IVISLumina III, Caliper Life Sciences, USA).

[0031] 1.7 Anti-inflammatory effect of combined administration of MTX and DAS in mice with zymosan A-induced arthritis

[0032] On day 0, Zymosan A was dissolved in sterile PBS. Male C57BL / 6 mice were anesthetized with isoflurane gas and 20 μL (30 μg) of Zymosan A solution was injected into the knee joint cavity through the patellar ligament to induce arthritis (ZIA). On day 1, the knee joints of mice were red and swollen. The mice were divided into four groups: ① ZIA group, ② MTX group, ③ DAS group, and ④ MTX + DAS group, with 5 mice in each group. Drugs were administered via the tail vein on days 1, 3, and 5. The MTX group received 50 μg MTX, the DAS group received 50 μg DAS, and the MTX + DAS group received 25 μg MTX and 25 μg DAS. The mice were observed daily for survival and body weight. The length and width of the knee joints were measured with a vernier caliper, and the circumference was calculated.

[0033] On day 6, mice were euthanized with CO2, and synovial tissue from the ankle joints was dissected and homogenized in 1 mL of RIPA strong lysis buffer in an ice bath. The mixture was lysed at 4°C for 1 hour and centrifuged at 12,000g at 4°C for 20 minutes. The supernatant was then collected. The protein concentration of the supernatant was determined using a BCA kit, and the concentrations of IL-6, IL-1β, and TNF-α were determined using ELISA kits.

[0034] 1.8 Anti-inflammatory effect of combined administration of MTX and DAS in mice with dextran sulfate sodium-induced colitis

[0035] Male C57BL / 6 mice were given free access to water containing 2.5% dextran sodium sulfate (40 kDa) to induce colitis, a model of UC. On day 0, mice were divided into four groups: ① UC, ② MTX, ③ DAS, and ④ MTX+DAS, with 5 mice in each group. From days 0 to 5, mice had free access to water containing 2.5% dextran sodium sulfate. Drugs were administered intravenously through the tail vein on days 1, 3, and 5. The MTX group received 60 μg of MTX, the DAS group received 60 μg of DAS, and the MTX+DAS group received 30 μg of MTX and 30 μg of DAS. The survival of the mice was observed daily, and body weights were recorded. On day 6, mice were euthanized by CO2, and the colon was dissected and photographed to record length. Approximately 1 cm of colon was cut, homogenized in RIPA strong lysis buffer, and centrifuged to obtain the supernatant. Protein concentration was determined using a BCA kit, and interleukin-6 concentration in the supernatant was determined using an ELISA kit.

[0036] 1.9 Synthesis and Characterization of HA-TK-MTX / DAS

[0037] HA (0.2 mmol) was dissolved in deionized water by sonication, and EDC (0.4 mmol) and NHS (0.4 mmol) were added. The reaction was stirred at room temperature for 1 h to activate the carboxyl groups. TK-NH2 (0.2-1 mmol) was then added. The reaction was stirred at room temperature for 24 h in the dark and under nitrogen. After the reaction, the product was dialyzed against deionized water and freeze-dried to obtain HA-TK. MTX (0.2 mmol), EDC (0.4 mmol), and NHS (0.4 mmol) were dissolved in DMSO and stirred at room temperature for 1 h to activate the carboxyl groups. HA-TK (0.2-0.4 mmol) was dissolved in formamide, and the carboxyl-activated MTX was added. The reaction was stirred at room temperature for 24 h in the dark and under nitrogen. After the reaction, the product was dialyzed against water, 50% ethanol, and DMSO, and freeze-dried to obtain HA-TK-MTX. Unmodified DAS (0.3 mmol), succinic anhydride (1.2 mmol), and DMAP (0.3 mmol) were dissolved in DMF (N,N-dimethylformamide) and stirred at room temperature for 24 h in the dark and under nitrogen. After the reaction, the reaction solution was pumped dry, re-dissolved in acetonitrile, and separated using a semi-HPLC (P0050, Beijing Huideyi Technology Co., Ltd.) (mobile phase: 35% acetonitrile and 65% water, detection wavelength: 320 nm) to obtain carboxylated DAS. HA-TK-DAS was synthesized using the same method as HA-TK-MTX. The product was dissolved in deuterated DMSO and characterized by 1H NMR (Unity Inova 400M, Varian, USA).

[0038] 1.10 Synthesis of aCD4M1-NPs

[0039] Anti-CD4 and DSPE-PEG2000-NHS were added to a low-protein adsorption tube at molar ratios of 1:0, 1:5, 1:10, and 1:25. The protein concentration was adjusted to 1 mg / mL with sterile PBS. The pH was adjusted to 8.5 by adding 10 μL of 1 mol / L NaHCO₃ per 100 μL of solution. The reaction was carried out on a four-dimensional rotator (BE-1100, Kylin-Bell, China) at room temperature for 12 hours. The product, DSPE-PEG2000-anti-CD4, was obtained after ultrafiltration.

[0040] J774A.1 macrophages were cultured with 10 ng / mL LPS and 10 ng / mL IFN-γ for 24 hours to polarize them to the M1 phenotype. Cell membranes were extracted using a cell membrane extraction kit, and protein concentration was determined using a BCA assay. 50 μg of DSPE-PEG2000-aCD4 was added to each 1 mg of membrane protein. The cells were incubated at 37°C for 30 minutes on a thermostatic shaker (THZ-103B, Shanghai Yiheng Scientific Instrument Co., Ltd.) at 300 rpm. Free DSPE-PEG2000-aCD4 was removed by centrifugation at 16,000 g for 10 minutes at 4°C. HA-TK-MTX / Cy5 was added at a 1:1 ratio of cell membrane protein to nanoparticle mass. Nanoparticles (M1-NPs) and aCD4M1-NPs with a particle size of approximately 200 nm were prepared using a liposome extruder (LF-1, Avestin, Canada).

[0041] 1.11 aCD4M1-NP CD4 + T cell and inflammation targeting

[0042] CD4 was isolated from mouse spleen using a negative selection kit + T cells were then added with the prepared M1-NPs and aCD4M1-NPs and incubated at 37°C for 15 minutes. Free nanoparticles were then removed by centrifugation and analyzed by flow cytometry (FACSCalibur Aria III, BD Biosciences, USA).

[0043] An inflammatory model was established by subcutaneously injecting 50 μg of LPS into the paw of female BALB / c mice. The animals were divided into five groups: ①des-NP group, ②M0-NP group, ③M1-NP group, ④aCD4M1-NP group, and ⑤aCD4 pre-depletion group, with 4-5 mice in each group. On the first day, each mouse in the aCD4 pre-depletion group was injected with 20 μg of anti-CD4 antibody into the tail vein to eliminate CD4 in the body. + T cells. On day 2, mice were administered intravenously through the tail vein. 12 hours later, fluorescence intensity at the modeling site was measured using an IVIS imager (IVIS Lumina III, Caliper Life Sciences, USA) (Ex = 745 nm, Em = 800 nm). des represents the cell membrane protein disruption group; cell membranes were prepared by sonication at 600 W for 30 minutes. M0 represents normal J774A.1 cells.

[0044] 1.12 Construction of genetically engineered cells expressing anti-CD4 protein

[0045] Insert the target gene sequence into the pFucci plasmid vector. The pFucci-CMV-anti-CD4-puro plasmid is constructed by tandemly linking the mCD8α leader, anti-mCD4 scFv fragment (clone 2C11), mCD28 hinge + mCD28TM, P2A, and Thy1.1 into the pFucci-CMV-puro vector to obtain the Anti-CD4 scFv-m CD28-P2A-Thy1.1 gene sequence as shown in SEQ ID NO: 1:

[0046]

[0047] 1.13 Characterization of TaCD4M1-NPs

[0048] Membranes of J774A.1 cells genetically engineered to express anti-CD4 protein were extracted and prepared using a liposome extruder at a 1:1 ratio of cell membrane protein mass to nanoparticle mass. TaCD4M1-NPs were measured using a Malvern particle size analyzer (ZS90, Malvern Instruments Ltd., UK) for particle size and potential. Nanoparticle morphology was observed using a transmission electron microscope (HT7700, Hitachi, Japan) after negative staining with phosphotungstic acid. Cell membranes were extracted from the prepared TaCD4M1-NPs using a cell membrane extraction protocol and compared with freshly extracted cell membranes for characterization using polyacrylamide gel electrophoresis (PAGE). TaCD4M1-NPs were placed in a 3500 kDa dialysis bag, sealed, and placed in a 50 mL centrifuge tube. H2O2 was added to final concentrations of 500 μM and 1 mM, and the tubes were shaken at 37°C on a constant temperature shaker at 300 rpm. 100 μL of the solution was taken out at 0, 0.5, 1, 2, 3, 4, 6, 12, 24, 48, 72, 96, and 120 h, and the contents of MTX and DAS in the solution were detected by ultraviolet spectrophotometer (UV2600i, Shimadzu Instrument Co., Ltd., Japan).

[0049] 1.14 TaCD4M1-NP on CD4 + Targeting of T cells and sites of inflammation

[0050] M0-NP, M1-NP and TaCD4M1-NP were added to CD4 + T cells were incubated in the dark for 15 min. Hoechst 33342 and DiO were used to stain the cell nucleus and cell membrane, and the cells were detected by confocal microscopy (AIR HD25, ABI, USA). + T cells were reacted at 37°C for 15 min, centrifuged after the reaction, and detected by flow cytometry.

[0051] Male C57BL / 6 mice were allowed to freely drink water containing 2.5% dextran sulfate to induce colitis, a model of UC. On day 6, the mice showed significant weight loss and hematochezia, and were divided into 6 groups: ①PBS group, ②free drug group, ③M0-NP group, ④M1-NP group, ⑤TaCD4M1-NP group, and ⑥aCD4 pre-depletion group, with 5 mice in each group. In the aCD4 pre-depletion group, each mouse was injected with 20 μg of mouse-derived anti-CD4 antibody into the tail vein to eliminate CD4 in the body. +T cells were administered via tail vein on day 7. Six hours later, mice were euthanized with CO2, and the colons were dissected, weighed, and imaged using IVIS (Ex = 620 nm, Em = 670 nm).

[0052] Male C57BL / 6 mice were injected intra-articularly with zymosan A to establish an acute arthritis model (ZIA). On day 1, the knee joints of the mice were swollen and red. The mice were then divided into five groups: ① PBS group, ② drug-free group, ③ M1-NP group, ④ TaCD4 M1-NP group, and ⑤ aCD4 pre-depletion group, with 5 mice in each group. On day 1, each mouse in the aCD4 pre-depletion group received a tail vein injection of 20 μg of anti-CD4 antibody. On day 2, drug administration was repeated intravenously. IVIS imaging (Ex = 620 nm, Em = 670 nm) was performed 6 hours later.

[0053] 1.15 Evaluation of the therapeutic effect of TaCD4M1-NP on CIA mice

[0054] Male DBA / 1 mice were immunized twice intraperitoneally with bovine type II collagen to induce arthritis (CIA) at the base of the tail. On day 0, bovine type II collagen and CFA were mixed at a 1:1 ratio by volume. A milky, creamy, oil-in-water emulsion was prepared using a high-speed homogenizer (Vs-35S, Wuxi Woxin Instrument Manufacturing Co., Ltd.) on ice. 100 μL of this emulsion was injected intradermally at the base of the tail of each mouse. On day 21, a second oil-in-water emulsion was prepared using the same method (CFA was replaced with IFA). On day 26, mice developed mild redness and swelling in the paws and were divided into six groups: ①CIA group, ②Healthy group, ③Free drug group, ④M0-NP group, ⑤M1-NP group, and ⑥TaCD4M-NP group, with 6 mice in each group. On days 26, 29, 32, 35, and 38, different cell membrane nanoparticles were prepared and administered intravenously to each mouse at a dose of 30 μg MTX + 30 μg DAS. During treatment, the mice were observed for survival and their body weights were recorded. On day 39, the mice were tested for locomotion and paw grip. Before formal testing, they underwent adaptive training. After the adaptive training, the mice were placed in a custom rotating cage and the rotation speed was gradually increased to 70 rpm. The rotation speed at which the mouse fell was recorded. The rotation speed was then maintained at 25 rpm, and the time the mouse remained in the cage was recorded.

[0055] On day 40, paw thickness was measured in each group, and blood was collected to determine serum levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α. Synovial tissue was dissected from the modeling site, homogenized, and lysed with RIPA buffer. Synovial concentrations of the proinflammatory cytokines IL-6, IL-1β, and TNF-α were determined. Mice were euthanized, and hind paws were dissected and imaged and analyzed using Micro-CT (SkyScan 1176, Bruker, Germany). Paws were fixed with paraformaldehyde, embedded in paraffin, and sectioned. Tissue sections were stained with hematoxylin and eosin (H&E), safranin O-fast green (SF), and Masson's trichrome.

[0056] 1.16 Evaluation of the therapeutic effect of TaCD4M1-NP in UC mice

[0057] Colitis was induced in male C57BL / 6 mice by freely drinking water containing 2.5% dextran sulfate. On day 0, the mice were divided into six groups: ①UC group, ②Healthy group, ③Drug-free group, ④M0-NP group, ⑤M1-NP group, and ⑥TaCD4M1-NP group, with six mice in each group. On days 1, 3, and 5, different cell membrane nanoparticles were prepared and administered intravenously via the tail vein at a dose of 30 μg MTX + 30 μg DAS per mouse. The survival of the mice was observed daily, and their body weight was recorded. The disease activity index (DAI) was also calculated based on the clinical symptoms of colitis. DIA consists of three components: weight loss (0, <1%; 1, 1-5%; 2, 5-10%; 3, 10-15%; 4, >15%), stool looseness (0, normal stool; 2, loose stool; 4, watery diarrhea), and hematochezia (0, normal; 2, mild hematochezia; 4, severe hematochezia). Each component was scored independently, and the total score was calculated. On day 5, mice were gavaged with FITC-dextran (4 kDa) at 0.6 mg / g body weight to assess intestinal integrity. Four hours after gavage, mice were anesthetized with isoflurane, and blood was collected from the retroorbital venous plexus. Fluorescence intensity (Ex = 480 nm, Em = 530 nm) was measured using a microplate reader (M1000 Pro, Tecan, Switzerland). On day 6, fresh feces were collected and hematochezia was assessed using a fecal occult blood test kit. Mice were euthanized by CO2, and the colon was dissected and photographed.

[0058] 1.17 Evaluation of therapeutic efficacy in ZIA mice

[0059] Zymosan A was injected intra-articularly into the knee joints of male C57BL / 6 mice to establish a ZIA model. On day 1, the mice developed redness and swelling in the knee joints and were divided into six groups: ① ZIA group, ② Healthy group, ③ Free drug group, ④ M0-NP group, ⑤ M1-NP group, and ⑥ TaCD4M1-NP group, with six mice in each group. On days 1, 3, and 5, different cell membrane nanoparticles were prepared and administered intravenously via the tail vein at a dose of 37.5 μg MTX + 12.5 μg DAS per mouse. The mice were observed daily for survival and body weight. The length and width of the knee joints were measured with a vernier caliper, and the circumference was calculated. On day 6, blood was collected from the mice under isoflurane anesthesia, and serum levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α were measured. Mice were then euthanized with CO2, and synovial tissue was dissected from the modeling site to measure the concentrations of the proinflammatory cytokines IL-6, IL-1β, and TNF-α.

[0060] 1.18 Safety Evaluation of TaCD4M1-NP

[0061] 4 mL of rabbit blood was collected and added to twice the volume of PBS. The blood was centrifuged at 10,000 g and 4°C for 5 minutes. The supernatant was discarded and the sample was resuspended in PBS. Washing was repeated five times, and the final dilution was made to 40 mL with PBS. For every 200 μL of red blood cell solution, 800 μL of nanoparticle solution of varying concentrations was added. The sample with PBS was used as the negative control group, and the sample with water was used as the positive control group. Each sample was repeated three times. After the sample was allowed to stand at room temperature for 4 hours, it was centrifuged at 10,000 g and 4°C for 3 minutes. The degree of hemolysis was observed by photographing. The supernatant was collected and the absorbance at 577 nm was measured, with 655 nm as the reference wavelength. The hemolysis rate was calculated according to Equation 2.

[0062] Formula 2

[0063] On day 0, DBA / 1 male mice were divided into two groups: ① Healthy group and ② TaCD4M1-NP group, with 6 mice in each group. On days 0, 2, and 4, 30 μg of MTX + 30 μg of DAS were administered intravenously via the tail vein. The mice were observed daily for survival, and their body weights were recorded. On day 5, blood was collected for routine blood analysis. Mice were euthanized, and the hearts, livers, spleens, lungs, and kidneys were dissected and analyzed by H&E staining. The livers were ground and assayed for ALT and AST activity using a kit.

[0064] 2 Experimental results

[0065] 2.1 Inhibitory effects of MTX and DAS on macrophages or T cells in vitro

[0066] The effects of MTX and DAS on macrophages and CD4 + The inhibitory effect of MTX on T cells. MTX is a folic acid analog that affects the synthesis of tetrahydrofolate by inhibiting the activity of dihydrofolate reductase, thereby interfering with the biosynthesis of DNA. It is clinically used to treat rheumatoid arthritis and cancer. DAS is a tyrosine kinase inhibitor that is clinically used to treat leukemia and cytokine storm caused by CAR-T therapy. The results showed that MTX can significantly inhibit the activity of macrophage Raw264.7, IC 50 was 1.453 μM, and DAS had poor inhibitory activity against Raw264.7 ( Figure 2 ); DAS can significantly inhibit CD4 + T cell activity, IC 50 is 0.5708μM, while MTX has an inhibitory effect on CD4 + IC of T cells 50 11.283 mM ( Figure 2 ).

[0067] 2.2 Therapeutic effects of combined administration of MTX and DAS on different mouse inflammatory models

[0068] Collagen-induced chronic arthritis model (CIA, a commonly used rheumatoid arthritis model): The experimental process of combined drug treatment of CIA mice is as follows: Figure 3 As shown in A. After five treatments, compared with the CIA group, the three treatment groups could reduce the swelling of the mouse paws to varying degrees ( Figure 3 B). On day 42, compared with the RA group, the combined drug group could significantly reduce the RA index from 13.6 points in the CIA group to 8.3 points; although the RA index of the MTX group and the DAS group was also reduced compared with the CIA group, there was no statistical difference ( Figure 3 C). The body weight curve showed a statistical difference similar to that of the RA index. The body weight of the combined drug group was significantly increased compared with the CIA group, but there was no statistical difference between the MTX group and the DAS group and the CIA group ( Figure 3 C). The results of serum inflammatory factor concentrations showed that the combined drug group could significantly reduce the concentrations of IL-6, IL-1β, and TNF-α compared with the CIA group. Although the MTX group and DAS group could also inhibit the concentrations of pro-inflammatory factors to a certain extent compared with the CIA group, the effect was lower than that of the combined drug group ( Figure 3 E to Figure 3 G). Among them, the efficacy of the combined drug group in reducing serum IL-6 was 1.9 times that of the MTX group and 3.2 times that of the DAS group ( Figure 3 E); The efficacy of reducing serum IL-1β was 1.4 times that of the MTX group and 8.6 times that of the DAS group ( Figure 3 F); The efficacy of reducing serum TNF-α was 2.2 times that of the MTX group and 3 times that of the DAS group ( Figure 3 G). In summary, MTX and DAS showed a certain degree of synergistic effect in the treatment of CIA mouse model.

[0069] Lipopolysaccharide (LPS)-induced acute inflammation model: The experimental process of combined drug treatment of LPS mice is as follows: Figure 4 As shown in A. Luminol and Lucigenin can be used to indicate the degree of inflammation, and the intensity of inflammation is proportional to the fluorescence intensity. After two injections, the degree of inflammation in each treatment group was reduced to varying degrees. As can be seen from the paw fluorescence images, the LPS model group had the strongest paw inflammation, while the combined treatment group significantly reduced paw inflammation ( Figure 4 B). The efficacy of the combined drug group in reducing inflammation was 3.5 times that of the MTX group and 1.9 times that of the DAS group ( Figure 4 C). In summary, MTX and DAS showed a certain degree of synergistic effect in the treatment of LPS mouse model.

[0070] Zymosan A induced acute arthritis model (ZIA): The experimental process of combined drug treatment of ZIA mice is as follows Figure 5 As shown in A. After three treatments, all five treatment groups improved the symptoms of the mice to varying degrees, relieving knee joint swelling and reducing serum inflammatory factor levels, but none of them improved the mice's body weight. The combined MTX+DAS (40μg+20μg) treatment effect was stronger than the other combined treatment groups and the single-drug group, significantly reducing knee joint swelling and the levels of pro-inflammatory cytokines in the synovium compared to the ZIA group ( Figure 5 B and Figure 5 D). Among them, the effect of the MTX+DAS (40μg+20μg) group in inhibiting synovial IL-6 was 1.5 times that of the MTX group and 2.7 times that of the DAS group; the effect of inhibiting synovial IL-1β concentration was 1.7 times that of the MTX group and 4.28 times that of the DAS group; the effect of inhibiting synovial TNF-α concentration was 1.8 times that of the MTX group and 1.7 times that of the DAS group ( Figure 5 D). In summary, MTX and DAS showed a certain degree of synergistic effect in the treatment of ZIA mouse model.

[0071] Dextran sodium sulfate-induced ulcerative colitis model (UC): The experimental process of combined drug administration to treat UC mice is as follows: Figure 6As shown in A. After 3 injections, each drug group could significantly increase the length of the mice's colon, among which the combined drug group had the strongest effect, extending the colon length from 4.2cm in the UC group to 5.6cm ( Figure 6 B and Figure 6 C). Each drug group can reduce the level of inflammatory factors in the colon. The combined drug group's efficacy in reducing IL-6 in the colon is 2.4 times that of the MTX group and 1.9 times that of the DAS group, which is significantly better than the single drug group ( Figure 6 D). The combined drug group can also significantly improve the weight loss of mice ( Figure 6 E). In summary, MTX and DAS showed good synergistic effects in the treatment of UC mouse model.

[0072] 2.3 Synthesis and characterization of HA-TK-MTX / DAS

[0073] Each repeating disaccharide unit of HA contains a free carboxyl group, and the ROS-sensitive TK linker has amino groups at both ends. Therefore, EDC and NHS can be used as catalysts to react the terminal amino groups of HA and TK linker through esterification reaction ( Figure 7 A). MTX contains free carboxyl groups, so it can react with HA-TK-NH2 to connect MTX to the polymer carrier to prepare a ROS-responsive drug ( Figure 7 B). Since HA is highly hydrophilic and MTX is highly lipophilic, self-assembled nanoparticles with MTX inside and HA outside will be formed in water. The same method can be used to prepare nanoparticles loaded with DAS. However, since the original drug DAS (unmodified DAS) has no free carboxyl group and only one hydroxyl group, the hydroxyl group can be modified to a carboxyl group. The specific method is to use DMAP and TEA as catalysts to react with succinic anhydride to introduce the carboxyl group ( Figure 7 C), and then prepare HA-TK-DAS ( Figure 7 D).

[0074] The results of the nuclear magnetic resonance spectrum showed that 1.3ppm was the methyl proton of the N-acetyl group on the HA skeleton, 1.5~2.5ppm was the characteristic proton peak of the methylene group in the HA skeleton; 1.6ppm was the methyl proton peak of TK-NH2; after HA was connected to TK, the nuclear magnetic resonance spectrum showed the characteristic peak of the methyl proton of TK, proving that TK was successfully connected to the HA skeleton ( Figure 8 A). 7.0ppm and 8.0ppm are the proton peaks on the benzene ring of MTX, and 8.5ppm is the proton peak of the amino groups on the side of the two carboxyl groups; after the reaction of MTX and HA-TK, the product showed the characteristic proton peak of MTX, proving that the connection was successful ( Figure 8B). The methylene proton peak of the unmodified Dasa is 4.0 ppm. After modification with succinic anhydride, the proton peak shifts downfield to 4.4 ppm due to the electron-withdrawing effect of the carboxyl group, proving that succinic anhydride is successfully attached to the hydroxyl group. Figure 8 C). 7.25ppm is the characteristic proton peak of DAS benzene ring, and 8.5ppm is the characteristic proton peak of primary ammonia hydrogen linked to the benzene ring; after DAS reacts with HA-TK, the product shows the characteristic proton peak of DAS, proving that the connection is successful ( Figure 8 D).

[0075] 2.4 Preparation and Inflammation Targeting of aCD4M1-NPs

[0076] LPS and IFN-γ were used to induce polarization of M0 macrophages to M1, and IL-4 and IL-13 were used to induce polarization of macrophages to M2a. Figure 9 A). After J774A.1 cells were induced with LPS and IFN-γ for 24 h or 48 h, the expression of CD86, a characteristic protein of M1 macrophages, was increased ( Figure 9 B). After J774A.1 cells were induced with IL-4 and IL-13 for 24 or 48 hours, the expression of CD206, a characteristic protein of M2a macrophages, was significantly increased ( Figure 9 C).

[0077] Can carry peripheral blood CD4 + Nanoparticle design for T cells Figure 9 D. DSPE-PEG2000-NHS reacts with anti-CD4 (aCD4) protein to synthesize DSPE-PEG-aCD4. After DSPE-PEG-aCD4 is co-incubated with the cell membrane, the aCD4 protein is anchored to the phospholipid bilayer of the cell membrane through DSPE. The prepared nanoparticles are then co-extruded with the cell membrane to allow the cell membrane to encapsulate the nanoparticles, thus preparing a CD4-carrying nanoparticle. + T cell membrane nanoparticles aCD4M1-NP. Flow cytometry results showed that aCD4M1-NP can target CD4 through surface aCD4 + T cells, 96.3% of T cells can be targeted and bound by cell membrane nanoparticles, while M1-NP without aCD4 target protein has a significantly weaker binding to T cells ( Figure 9 E). Inflammation targeting experiments of aCD4M1-NP were conducted in an LPS-induced acute inflammation mouse model ( Figure 9F). The results showed that all cell membrane nanoparticles could be targeted to the inflammatory site. Among them, M1-NP had a stronger targeting effect than des-NP (cell membrane protein inactivated group) and M0-NP. However, aCD4M1-NP had the strongest effect, with a targeting efficiency 1.6 times that of M1-NP, 2.2 times that of M0-NP, and 2.9 times that of des-NP. + After T cell clearance, the enrichment of aCD4M1-NP in the inflammatory site was significantly reduced, which was comparable to that of the M1-NP group, proving that the efficient inflammatory targeting of aCD4M1-NP is indeed due to the CD4 + T cells ( Figure 9 G and Figure 9 H).

[0078] 2.5 Synthesis and characterization of TaCD4M1-NPs

[0079] The expression of aCD4 on the cell membrane surface by DSPE-PEG-aCD4 insertion requires repeated preparation, has low insertion efficiency, and low aCD4 expression. More importantly, DSPE-PEG-NHS modification requires reaction with the active amino group of aCD4, which may affect the activity of aCD4. Therefore, genetic engineering can be used to make J774A.1 cells stably express aCD4 protein. First, the target gene is designed, which is composed of mCD8α leader, anti-mCD4 scFv fragment (clone 2C11), mCD28 hinge, mCD28TM, P2A and tag protein Thy1.1 in series, and inserted into the pFucci-CMV-puro vector. A three-plasmid system is used to construct a lentivirus ( Figure 10 A). Then, J774A.1 cells were infected with lentivirus to construct engineered cells that stably expressed aCD4 protein. Flow cytometry results showed that the cell positive rate reached 68% ( Figure 10 B). Interestingly, after viral infection, macrophages polarize to the M1 phenotype and highly express the inflammatory adhesion molecules PSGL-1 and LFA-1 ( Figure 10 CE).

[0080] The cell membrane of the cells was extracted and co-extruded with drug-loaded nanoparticles to prepare cell membrane-encapsulated nanoparticles TaCD4M1-NP. TEM (scanning electron microscopy) results showed that HA nanoparticles (NPs) were spherical with a particle size of approximately 140 nm; TaCD4M1-NPs showed a spherical core-shell structure with a particle size of approximately 150 nm. Figure 11A). The hydrated particle size of cell membrane vesicles (TaCD4M1) is approximately 158 nm, with a zeta potential of -22 mV; the hydrated particle size of HA nanoparticles (NP) is approximately 165 nm, with a zeta potential of -17 mV; the hydrated particle size of coated nanoparticles (TaCD4M1-NP) increases slightly to 178 nm, with a zeta potential of -22 mV, which is lower than that of NP and consistent with that of cell membrane vesicles ( Figure 11 B). PAGE results showed that the membrane proteins of TaCD4M1-NP were consistent with those of TaCD4M1 vesicles, proving that the cell membrane was successfully coated with nanoparticles ( Figure 11 C). TaCD4M1-NP has a good ROS response. Under the condition of 500μM H2O2, 46.7% of MTX and 34.6% of DAS were released in 24 hours, but only 7.9% was released in PBS. ( Figure 11 D).

[0081] 2.6 CD4 of TaCD4M1-NP + T cell and inflammation targeting

[0082] Extraction of CD4 from mouse spleen + T cells were co-incubated with M0-NP, M1-NP and TaCD4M1-NP. Confocal microscopy results showed that M0-NP and M1-NP had a small amount of binding to the cell membrane surface, while TaCD4M1-NP nanoparticles (red) could obviously bind to T cells and co-localize with the cell membrane (green), proving that TaCD4M1-NP nanoparticles can rely on the anti-CD4 protein on the membrane surface to bind to CD4 + T cell binding ( Figure 12 A). Flow cytometry results showed a similar trend, TaCD4M1-NP could bind to CD4 + T cells significantly bound to the cells, while M1-NP only bound to a small amount of cells ( Figure 12 B). CD4 + T cells and CD8 + After the T cells were mixed in a 1:1 ratio, the two nanoparticles were added in sequence to explore their targeting ( Figure 12 C). Flow cytometry results showed that M1-NP would bind to CD8 + T and CD4 + T cells bind nonspecifically, while TaCD4M1-NP specifically targets CD4 + T cells, the binding rate is 99.9% ( Figure 12 D). Flow cytometry histogram results showed that TaCD4M1-NP can significantly target CD4 compared with M1-NP. + T cells, while CD8 + T cell targeting was poor, with no difference between the two nanoparticles ( Figure 12 E). Therefore, even in the presence of CD8 + In the presence of T cells, TaCD4M1-NP can also precisely target CD4 through the anti-CD4 protein on the cell membrane. + T cells.

[0083] The experimental process of targeting the inflamed colon of UC model mice is as follows: Figure 13 As shown in A. The results showed that the free drug group had little accumulation in the colon, while the M0-NP, M1-NP, and aCD4 pre-depletion groups all had a certain degree of targeting in the colon; among them, the TaCD4M1-NP group had the strongest colon targeting, which was 2.4 and 1.8 times that of the M1-NP group and the aCD4 pre-depletion group ( Figure 13 B and Figure 13 C). The fluorescence intensity of the target site of the M1-NP group and the aCD4 pre-depletion group is similar, indicating that after the T cells in the body are eliminated, TaCD4M1-NP cannot carry CD4 + T cells, and its targeting effect was similar to that of the M1-NP group without anti-CD4 protein ( Figure 13 C). Experimental procedures for targeting inflamed joints in ZIA mice. Figure 13 The results showed that the TaCD4M1-NP group had the strongest targeting, while the M1-NP group and the aCD4 pre-depletion group had similar targeting. The targeting of the TaCD4M1-NP group was 1.8 and 1.6 times that of the M1-NP group and the aCD4 pre-depletion group ( Figure 13 E and Figure 13 F)

[0084] 2.7 Evaluation of the therapeutic effect of TaCD4M1-NP on CIA mice

[0085] The experimental process of TaCD4M1-NP treatment of CIA mice is as follows Figure 14 As shown in A. The RA index results showed that the three cell membrane nanoparticle groups (M0-NP, M1-NP and TaCD4M1-NP) could significantly reduce the RA index compared with the free drug group. Among them, the TaCD4M1-NP group had the best efficacy, with the RA index below 5 points ( Figure 14B). Body weight results showed that the body weight of each drug-treated group increased to a certain extent compared with the CIA group, indicating that all groups had a certain therapeutic effect. On day 40, compared with the CIA group, the body weight of the free drug group increased to a certain extent, but there was no statistical difference. The body weight of the M0-NP, M1-NP, and TaCD4M1-NP groups all increased significantly, among which the TaCD4M1-NP group had the least weight loss ( Figure 14 C). The results of mouse paw thickness showed that compared with the CIA group, the free drug group inhibited paw thickening by 22.5%, the M0-NP group inhibited it by 56.3%, the M1-NP group inhibited it by 73.8%, and the TaCD4M1-NP group inhibited it by 90.0% ( Figure 14 D).

[0086] When the paws of mice swell, their motor and grasping abilities will decrease. In order to better evaluate the therapeutic effects of each group of drugs, the behavioral abilities of mice were measured, namely the rotating cage test ( Figure 14 E). When the speed was gradually increased to 70 rpm, compared with the CIA group, the motor ability (paw grasping ability) of the free drug group only recovered 11.9%, the M0-NP group recovered 34.3%, the M1-NP group recovered 55.7%, and the TaCD4M1-NP group recovered 72.8%. The effect of the TaCD4M1-NP group in restoring the motor ability of mice was 6.1, 2.1, and 1.3 times that of the free drug group, M0-NP group, and M1NP group, respectively ( Figure 14 F). When the fixed speed was 25 rpm, the effect of TaCD4M1-NP group in restoring the exercise capacity of mice was 3.8, 1.5 and 1.4 times that of free drug group, M0-NP group and M1-NP group respectively ( Figure 14 G).

[0087] ELISA was used to measure the concentrations of inflammatory cytokines in the ankle synovium and serum of CIA mouse models. In the synovium, compared with the CIA group, all treatment groups reduced the concentrations of proinflammatory cytokines IL-6, IL-1β, and TNF-α to varying degrees, with the TaCD4M1-NP group showing the strongest effect ( Figure 14 H). Similarly, in the serum, the anti-inflammatory trend of each treatment group was similar to that in the synovium, and the concentrations of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were reduced to varying degrees, with the TaCD4M1-NP group having the strongest effect ( Figure 14 I). The efficacy of the TaCD4M1-NP group in inhibiting inflammatory factors was 11.4 times that of the free drug group, 2.1 times that of the M0-NP group, and 1.4 times that of the M1-NP group.

[0088] H&E section staining showed that the synovium in the CIA group showed malignant proliferation, with lining cells disappearing due to severe inflammation, and a large number of immune cells infiltrating into the stromal cells. The pannus formed invaded the joint cavity, causing necrosis and erosion of the cartilage surface. The synovium in the Healthy group showed normal morphology, with 2 to 3 layers of lining cells, no thickening, no proliferation of synovial stromal cells, and no inflammatory cell infiltration. The synovium in the Free Drug group showed massive proliferation of synovial stromal cells and massive infiltration of immune cells, indicating inflammation. The synovium in the M0-NP group showed thickening of synovial lining cells, and inflammatory cells infiltrated the entire synovium. The synovium in the M1-NP group showed 3 to 4 layers of synovial lining cells, with partial proliferation of stromal cells. The TaCD4M1-NP group showed 2 to 3 layers of lining cells, no proliferation of stromal cells, and no inflammatory cell infiltration in the synovium. Figure 15 A). SF slices show uneven coloration of the entire ankle cartilage in the CIA group, with severe surface cartilage degeneration. Cartilage morphology and thickness were normal in the Healthy group, appearing red. Cartilage in the free drug group and the M0-NP group showed significant degeneration and decreased thickness. Cartilage structure was normal in the M1-NP group, but overall thickness was decreased. Cartilage structure and thickness were normal in the TaCD4M1-NP group ( Figure 15 B). As can be seen from the Masson slices, the synovial fibrosis of mice in the CIA group was severe and blue; the synovial structure of the Healthy group was normal with no fibrosis; the synovial fibrosis of the free drug group and the M0-NP group was severe; the synovial fibrosis of the M1-NP group and the TaCD4M1-NP group was mild ( Figure 15 C). Therefore, the TaCD4M1-NP group can significantly improve the pathological structure of the synovium, reduce the infiltration of inflammatory cells and the proliferation of synovial stromal cells, and prevent synovial fibrosis and chondrocyte degeneration.

[0089] Micro-CT images show that all joints of mice in the CIA group showed bone erosion, especially in the ankle and finger joints, with rough bone surfaces and partial bone loss. The paw bones in the Healthy group were normal in morphology, with smooth and intact surfaces. The finger and ankle joints in the free drug and M0-NP groups showed severe bone erosion, with partial bone loss. The finger and ankle joints in the M1-NP group showed rough surfaces, but no bone loss, and bone erosion was improved. The finger and ankle joints in the TaCD4M1-NP group had normal structures, smooth bone surfaces, and only some finger joints had rough surfaces ( Figure 15 D).

[0090] 2.8 Evaluation of the therapeutic effect of TaCD4M1-NP on UC mice

[0091] The experimental process of TaCD4M1-NP treatment of UC mice is as follows Figure 16As shown in A. The weight change curves showed that the three groups of coated nanoparticles (M0-NP, M1-NP and TaCD4M1-NP) all showed a certain ability to inhibit weight loss, among which the TaCD4M1-NP group had the strongest efficacy ( Figure 16 B). The mice in the model group began to have mild diarrhea and bloody stools on the second day, suffered severe weight loss on the fourth day, and severe diarrhea and bloody stools on the fifth day; the free drug group, M0-NP group, and M1-NP group all experienced varying degrees of moderate to severe diarrhea, bloody stools, and weight loss; the TaCD4M1-NP group only experienced mild bloody stools on the third day, and no severe loose stools, bloody stools, or weight loss occurred. The results of the disease activity index (DAI) showed that the DIA index of the mice in the UC group began to rise rapidly from the third day, reaching 11.3 on the sixth day; the free drug group showed a similar trend to that of the UC group; the M0-NP, M1-NP, and TaCD4M1-NP groups all showed a certain inhibitory effect, among which the TaCD4M1-NP group had the strongest effect, significantly inhibiting the DIA index compared with the free drug, M0-NP, and M1-NP groups ( Figure 16 C).

[0092] When colon epithelial cells are severely damaged, it will cause ulceration and bleeding, and symptoms such as bloody stools will appear. The severity of colitis can be determined by detecting the blood content in the feces. The results showed that the feces of the mice in the Healthy group had no color reaction, indicating that the mice's intestines were healthy and normal, and there was no bleeding; the feces of the mice in the UC group were dark brown, indicating that the mice had severe intestinal bleeding; the feces of the free drug group were darker blue-green, indicating that the mice had intestinal bleeding; the feces of the mice in the M0-NP group and the M1-NP group were blue-green, which was lighter in color than the free drug group, indicating that the degree of bleeding was alleviated, but the intestine still had ulcers; the feces of the TaCD4M1-NP group were lighter blue-green, indicating that the integrity of the intestine was restored to the greatest extent and the bleeding was the weakest ( Figure 16 D).

[0093] When the colon is damaged, intestinal integrity is destroyed and permeability is enhanced. Therefore, the degree of colon damage can be determined by gavage with FITC-Dextran and detecting the FITC-Dextran content in the blood. After mice were gavage with FITC-Dextran, the serum of the mice in the Healthy group showed almost no fluorescence; the serum fluorescence intensity of the UC group increased significantly, 5.1 times that of the Healthy group, indicating that the intestinal damage was severe and the integrity of the epithelial cells was destroyed; although the serum fluorescence intensity of the free drug group decreased to a certain extent compared with the UC group, there was no significant difference, indicating that the ability of the free drug to restore intestinal integrity was weak; the three groups of coated nanoparticles (M0-NP, M1-NP, TaCD4M1-NP) all showed a certain therapeutic effect, and the fluorescence intensity decreased significantly compared with the free drug group, indicating that the therapeutic effect was significantly improved after being prepared into nanoparticles; the TaCD4M1-NP group had the strongest therapeutic effect, and the serum fluorescence intensity was significantly reduced compared with the M0-NP and M1-NP groups ( Figure 16 E).

[0094] Shortened colon length is a prominent feature of the DSS-induced UC model. The colon length of the healthy group was 7.7 cm, while that of the UC group was significantly reduced to only 4.2 cm. After drug treatment, colon length recovered to a certain extent. The recovery capacity of colon length in the M0-NP, M1-NP, and TaCD4M1-NP groups was 2.9, 3.6, and 5.6 times that of the drug-free group, respectively. Figure 16 F and Figure 16 G).

[0095] 2.9 Evaluation of the therapeutic effect of TaCD4M1-NP on ZIA mice

[0096] The experimental process of TaCD4M1-NP treatment of ZIA mice is as follows Figure 17 As shown in A. The knee joint swelling curve of mice showed that compared with the ZIA group, the M0-NP, M1-NP and TaCD4M1-NP groups could significantly reduce the knee joint circumference, among which the aCD4M1-NP group had the lowest knee joint swelling ( Figure 17 B). The weight change curves showed that the weight of mice in both the M1-NP and TaCD4M1-NP groups was significantly restored, but the TaCD4M1-NP group had a stronger effect in restoring the weight of mice ( Figure 17 C). In the synovium, compared with the ZIA group, each treatment group could reduce the concentrations of inflammatory cytokines IL-6, IL-1β, and TNF-α to varying degrees, with the TaCD4M1-NP group having the strongest effect ( Figure 17D). Similar trends were shown in serum. Each treatment group could reduce the concentrations of inflammatory cytokines IL-6, IL-1β, and TNF-α to varying degrees, with the TaCD4M1-NP group showing the strongest effect ( Figure 17 E). The ability of the TaCD4M1-NP group to reduce proinflammatory cytokines was 3.6 times that of the free drug group, 1.6 times that of the M0-NP group, and 1.2 times that of the M1-NP group.

[0097] 2.10 Safety Evaluation of TaCD4M1-NP

[0098] The safety of TaCD4M1-NP was investigated by hemolysis experiments. No hemolysis was observed in the PBS group, while red blood cells in the H2O group were hemolyzed, with the solution turning from clear to red. No significant hemolysis was observed at different concentrations of TaCD4M1-NP, with the hemolysis rate at 1000 μg / mL being only 2.4% ( Figure 18 A).

[0099] To further investigate the in vivo safety of TaCD4M1-NP nanoparticles, the experimental procedure is as follows Figure 18 As shown in B. After three injections of TaCD4M1-NP nanoparticles in DBA / 1 mice, the ALT and AST activities in the liver were not significantly different from those in the PBS group, demonstrating that TaCD4M1-NP nanoparticles have no liver toxicity ( Figure 18 C). Blood biochemical analysis results showed that after injection of TaCD4M1-NP nanoparticles, the number of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) did not change significantly; the proportion of lymphocytes, monocytes, and neutrophils in the white blood cells did not change significantly; and the concentration of hemoglobin in the blood (HGB) and mean corpuscular hemoglobin (MCH) did not change significantly ( Figure 18 D). Blood biochemistry results showed that TaCD4M1-NP did not affect the composition of cells in the blood and the morphological function of red blood cells. Organ section results showed that in the heart sections of the TaCD4M1-NP group, the muscle fiber striations were clear, without degeneration or necrosis; in the liver sections, the liver lobules of each group were intact and normal, and the liver cells were arranged neatly, without deformation or sclerosis; in the spleen sections, the splenic bodies and periarterial lymphoid sheaths of each group were clear, without degeneration; in the lung sections, the alveoli of each group were loose and without lesions; in the kidney sections, the renal tubules of each group were clear, and the renal capsules were not hypertrophied ( Figure 18E). Organ sections demonstrate the favorable in vivo safety profile of TaCD4M1-NP. In summary, TaCD4M1-NP demonstrates a strong safety profile, with no significant toxicity observed after in vivo administration.

[0100] 3. Summary

[0101] The present invention synthesizes CD4 + T cells, cell membrane nanoparticles TaCD4M1-NP with ROS response. The nanoparticles can "hitch a ride" on CD4 + T cells, while relying on homologous targeting of the cell membrane, achieve drug enrichment at the site of inflammation, increase drug concentration at the target site, and reduce systemic toxic side effects. We also selected a combination of anti-inflammatory drugs, including methotrexate (MTX), a drug with strong inhibitory effects on macrophage activity, and dasatinib (DAS), originally used to treat leukemia and with strong inhibitory effects on T cell activity. Results showed that this combination had a synergistic therapeutic effect in models of zymosan A-induced arthritis (ZIA), collagen-induced arthritis (CIA), ulcerative colitis (UC), and LPS-induced acute inflammation, enhancing anti-inflammatory effects and providing new approaches and new ideas for the treatment of inflammatory diseases.

Claims

1. A method for preparing cell membrane nanoparticles, characterized in that: The following steps are involved: Preparation of drug-loaded nanoparticles: Methotrexate and dasatinib were linked to hyaluronic acid using a reactive oxygen species-sensitive copper thiol crosslinker to synthesize HA-TK-MTX or HA-TK-DAS polymers, which self-assembled into drug-loaded nanoparticles in saline. Cell membrane preparation: Using genetic engineering technology, a macrophage cell line that stably expresses aCD4 single-chain antibody scFv on the cell surface is constructed. The infection process polarizes the macrophages to the M1 type, and then the cell membrane is extracted to obtain the CD4-targeted cell membrane; Preparation of cell membrane nanoparticles: Cell membrane and drug-loaded nanoparticles are co-extruded to prepare cell membrane nanoparticles.

2. The preparation method according to claim 1, characterized in that The specific method for constructing engineered cells stably expressing aCD4 protein in the cell membrane preparation step is as follows: A target gene was designed, consisting of the mCD8α leader, anti-mCD4 scFv fragment clone 2C11, mCD28 hinge, mCD28TM, P2A, and tag protein Thy1.1, all concatenated. The gene sequence is shown in SEQ ID NO: 1 and inserted into a plasmid vector to construct a virus. The virus was then used to infect J774A.1 cells to create engineered cells that stably express the aCD4 protein.

3. A method for preparing cell membrane nanoparticles, characterized in that: The following steps are involved: Preparation of drug-loaded nanoparticles: Methotrexate and dasatinib were linked to hyaluronic acid using a reactive oxygen species-sensitive copper thiol crosslinker to synthesize HA-TK-MTX or HA-TK-DAS polymers, which self-assembled into drug-loaded nanoparticles in saline. Cell membrane preparation: Macrophages are polarized to the M1 cell type, and the cell membranes are extracted. DSPE-PEG functionalized with NHS is then reacted with a CD4 monoclonal antibody to produce DSPE-PEG-aCD4. DSPE-PEG-aCD4 is then inserted into the M1 cell membrane to obtain a CD4-targeted cell membrane. Preparation of cell membrane nanoparticles: Cell membrane and drug-loaded nanoparticles are co-extruded to prepare cell membrane nanoparticles.

4. The preparation method according to claim 1 or 3, characterized in that The synthesis steps of HA-TK-MTX are as follows: HA was dissolved in deionized water by ultrasonication, and EDC and NHS were added and stirred at room temperature for 1 h to activate the carboxyl groups; Add TK-NH2, protect from light, protect under nitrogen, and stir at room temperature for 24h; After the reaction, HA-TK was dialyzed against deionized water and freeze-dried to obtain HA-TK; MTX, EDC, and NHS were dissolved in DMSO and stirred at room temperature for 1 h to activate the carboxyl groups; HA-TK was dissolved in formamide, and then MTX with activated carboxyl groups was added. The mixture was stirred at room temperature for 24 h in the presence of nitrogen and protected from light. After the reaction, the solution was dialyzed with water, 50% ethanol and DMSO in sequence and freeze-dried to obtain HA-TK-MTX.

5. The preparation method according to claim 1 or 3, characterized in that The synthesis steps of HA-TK-DAS are as follows: Unmodified DAS, succinic anhydride, and DMAP were dissolved in DMF, protected from light and nitrogen, and stirred at room temperature for 24 h; After the reaction, the reaction solution was dried by oil pump, redissolved in acetonitrile, and separated by Semi-HPLC with a mobile phase of 35% acetonitrile and 65% water and a detection wavelength of 320 nm to obtain carboxylated DAS. HA was dissolved in deionized water by ultrasonication, and EDC and NHS were added and stirred at room temperature for 1 h to activate the carboxyl groups; Add TK-NH2, protect from light, protect under nitrogen, and stir at room temperature for 24h; After the reaction, HA-TK was dialyzed against deionized water and freeze-dried to obtain HA-TK; Carboxylated DAS, EDC, and NHS were dissolved in DMSO and stirred at room temperature for 1 h to activate the carboxyl groups, thereby obtaining carboxyl-activated DAS. HA-TK was dissolved in formamide, and then carboxyl-activated DAS was added. The mixture was protected from light and nitrogen, and stirred at room temperature for 24 h. After the reaction, the solution was dialyzed with water, 50% ethanol and DMSO in sequence and freeze-dried to obtain HA-TK-DAS.

6. Cell membrane nanoparticles TaCD4M1-NP prepared according to the preparation method of cell membrane nanoparticles according to any one of claims 1 to 5.

7. Use of the cell membrane nanoparticles according to claim 6 for preparing a drug for treating inflammatory diseases, wherein the inflammatory diseases include rheumatoid arthritis and ulcerative colitis.

8. The use according to claim 7, characterized in that The cell membrane nanoparticles can carry CD4 in vivo + T cells infiltrate into the inflammatory site and release drugs in response to ROS, significantly reducing the levels of pro-inflammatory cytokines in serum and inflammatory sites.

9. A pharmaceutical composition for treating inflammatory diseases, characterized in that: The pharmaceutical composition comprises cell membrane nanoparticles prepared by the preparation method according to any one of claims 1 to 5, and contains methotrexate and dasatinib, wherein the weight ratio of methotrexate to dasatinib is 1:(0.5~2).

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