Targeted therapeutic drug for rheumatoid arthritis and preparation method thereof

Abstract

The invention discloses a rheumatoid arthritis targeted therapeutic drug and a preparation method thereof, and relates to the field of medicines. The invention relates to an acid response nano-particle CEC NPs, which is specifically composed of the following components: a carrier, which is an M2 type macrophage derived exosome; and loading the medicine. According to the acid response nano-particles based on the CXB and the CC-90011 loaded on the exosome derived from the M2 type macrophage, the M2Exo can specifically target an inflammatory environment, and the specific anti-inflammatory biological function of the M2Exo can be exerted; under the acidic condition, CEC NPs responds to release CE NPs and CC-90011, the CE NPs can repolarize M1 type macrophages and inhibit FLS activation to effectively relieve arthritis inflammatory response and synovial hyperplasia, and the CC-90011 can inhibit LSD1 to interfere the energy metabolism pathway of osteoclasts and inhibit osteoclast formation, so that the pathological bone resorption process is blocked, and bone erosion is relieved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a rheumatoid arthritis targeted therapeutic drug and a preparation method thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, the main symptoms of which are inflammatory cell infiltration, synovial inflammation, and bone erosion. According to statistics, approximately 0.5%-1.0% of the world's population suffers from rheumatoid arthritis and is troubled by it. If not treated in time, most patients will develop joint deformities and disability within 3 years. Commonly used therapeutic drugs in clinical practice include non-steroidal anti-inflammatory drugs, glucocorticoids, conventional disease-modifying anti-rheumatic drugs, and biological agents. However, these drugs have corresponding defects such as poor targeting and low bioavailability. In addition, although these drugs can relieve joint inflammation to a certain extent, they cannot effectively reduce damage to articular cartilage and improve joint structure.

[0003] Therefore, there is an urgent need to develop drugs that can treat RA inflammation while inhibiting bone erosion.

[0004] To this end, the present invention proposes a rheumatoid arthritis targeted therapeutic drug and a preparation method thereof Summary of the Invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a rheumatoid arthritis targeted therapeutic drug and a preparation method thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A targeted therapeutic drug for rheumatoid arthritis, which is acid-responsive nanoparticles CEC NPs, specifically composed of the following components:

[0008] The vector is an M2 macrophage-derived exosome;

[0009] The loaded drug comprises:

[0010] The cyclooxygenase inhibitor celecoxib CXB was encapsulated in M2 Exo to form CXB@Exo nanospheres CE NPs;

[0011] The osteoclast inhibitor CC-90011 is a novel oligomer that binds to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-hydrazone-polyethylene glycol 2000-N-hydroxysuccinimide (DSPE-Hyd-PEG 2000 -NHS) modified to form CC-linker (DSPE-Hyd-PEG2000 -CC-90011) and covalently linked to CE NPs.

[0012] Preferably: in the CC-linker, DSPE-Hyd-PEG 2000 The molar ratio of -NHS to CC-90011 is 1:2.

[0013] Preferably, the connection ratio of CE NPs to CC-linker is 10-100 μM CC-linker per 50 μM CE NPs, wherein the modification rate of 50 μM CC-linker reaches 53.1% (verified by flow cytometry).

[0014] Preferably, the acid-responsive nanoparticles CEC NPs have the following physicochemical properties:

[0015] Particle size: 100-200 nm (verified by transmission electron microscopy);

[0016] Zeta potential: -15 to -25 mV (detected by nanoparticle size analyzer);

[0017] Acidic response release: The release rate of CC-90011 at pH 6.5 was higher than that at pH 7.4 (verified by UV-visible spectroscopy).

[0018] A method for preparing a targeted therapeutic drug for rheumatoid arthritis, comprising the following steps:

[0019] S1: induce M2 macrophages;

[0020] S2: Extract exosomes from M2 macrophages;

[0021] S3: Synthesis of CE NPs (CXB@Exo);

[0022] S4: Preparation of CC-linker (DSPE-Hyd-PEG2000-CC-90011);

[0023] S5: Preparation of CEC NPs (CXB@Exo / CC-90011);

[0024] S6: In vivo validation of CEC NPs in mice.

[0025] Preferably, the step S1 includes the following steps:

[0026] S11: Mouse mononuclear macrophage cell line (RAW264.7) was cultured at 1×10 6 / well inoculated, cultured overnight in a medium containing 10% serum and 1% penicillin-streptomycin double antibody;

[0027] S12: Replace the serum-free medium containing 20 ng / mL interleukin-4 (IL-4) and induce for 24 hours.

[0028] Preferably, the step S2, which extracts the cell supernatant by differential centrifugation, specifically comprises the following steps:

[0029] S21: centrifugation at 300 g for 10 min to remove dead cells;

[0030] S22: centrifugation at 2000 g for 10 min to remove cell debris;

[0031] S23: centrifugation at 10,000 g for 30 min to remove impurities;

[0032] S24: Ultracentrifuge at 120,000 g for 2 h, add phosphate buffered saline (PBS) for resuspending, and store at -80°C.

[0033] Preferably, the step S3 includes the following steps:

[0034] S31: Cyclooxygenase inhibitor celecoxib CXB was mixed with M2 macrophage exosomes and repeatedly extruded through 400 nm and 200 nm polycarbonate membrane liposome extruders;

[0035] S32: Use a 50 kDa ultrafiltration centrifuge tube at 14,000 g for 30 min to remove free CXB and obtain purified CE NPs.

[0036] Preferably, the step S4 comprises the following steps:

[0037] S41: Dissolve 100 μM CC-90011 in 1 mL of pH 8.5 sodium bicarbonate solution and mix thoroughly;

[0038] S42: 50 μM DSPE-Hyd-PEG 2000 -NHS was added dropwise to the above solution;

[0039] S43: react at room temperature with magnetic stirring at 300 r / min for 24 h;

[0040] S44: Dialyze the solution in deionized water using a 500-1000 Da dialysis bag for 24 h, changing the water three times, and then freeze-dry to obtain a solid CC-linker.

[0041] Preferably, the step S5 comprises the following steps:

[0042] S51: CE NPs and CC-linker were mixed in 500 μL PBS and reacted at 40 °C for 2 h;

[0043] S52: The product CEC NPs were purified by 50 kDa ultrafiltration tube, resuspended in PBS and stored at -80 °C;

[0044] The step S6 comprises the following steps:

[0045] S61: First, the mice were immunized subcutaneously at the base of their tails with an emulsified mixture of bovine type II collagen solution and complete Freund's adjuvant.

[0046] S62: On day 21, mice were boosted with an emulsion of bovine type II collagen solution and incomplete Freund's adjuvant;

[0047] S63: Subsequently, CXB, CC-90011, CE, and CEC were administered via the tail vein, and a PBS positive control group and a healthy mouse negative control group were set up.

[0048] S64: Ankle joint samples were collected and fixed in 4% paraformaldehyde solution. The fixed samples were three-dimensionally reconstructed using Micro-CT scanning to generate a 3D rendering image of the ankle joint.

[0049] The beneficial effects of the present invention are:

[0050] The present invention is based on acid-responsive nanoparticles loaded with CXB and CC-90011 derived from M2 macrophage exosomes, where M2 Exo can specifically target the inflammatory environment and exert its own unique anti-inflammatory biological function; under acidic conditions, CEC NPs respond to release CE NPs and CC-90011, and at the same time, CE NPs can effectively reduce arthritis inflammation and synovial hyperplasia by repolarizing M1 macrophages and inhibiting the activation of FLS. CC-90011 interferes with the energy metabolism pathway of osteoclasts by inhibiting LSD1, inhibiting the formation of osteoclasts, thereby blocking the pathological bone resorption process and reducing bone erosion. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 middle, Figure 1 AC is the linker in DMSO-d6, CC-90011 and CC-linker 1 H-NMR spectrum.

[0052] Figure 2 middle, Figure 2 A is a schematic diagram of the synthesis of CE NPs. Figure 2 B is the UV-visible absorption spectra of CXB, M2Exo and CE NPs, Figure 2 CE was used to measure the absorbance of CXB, CE NPs, and free CXB in CE NPs in methanol solution at a wavelength of 254 nm using HPLC-UV.

[0053] Figure 3 middle, Figure 3 A is a schematic diagram of the preparation process of CEC NPs. Figure 3 B Flow cytometry analysis showed that after different concentrations of CC-linker-FITC (0, 10, 50, 100 μM) were co-incubated with CE NPs, the proportion of CC-linker-FITC modified CE NPs was shown;

[0054] Figure 4 middle, Figure 4 A is a schematic diagram of TEM images of M2 Exo, CE NPs and CEC NPs. Figure 4 B is a schematic diagram of Western blot analysis of exosome markers (TSG101, CD81) and macrophage markers (CD206, CD86, Arg-1) in different material groups (M2 Exo, CE NPs and CEC NPs) using GAPDH as an internal reference. Figure 4 C is the Zeta potential diagram of CC-linker, CE NPs and CEC NPs. Figure 4 D is a schematic diagram of the UV-visible absorption spectra of CC-linker, CE NPs, and CEC NPs;

[0055] Figure 5 Schematic diagram of the UV-visible absorption spectra of CC-90011 released by CEC NPs in the present invention in buffer solutions with different pH values ​​(6.5, 7.4);

[0056] Figure 6 Schematic diagram of the evaluation of bone erosion and osteophyte formation in the ankle joint after Micro-CT processing in the present invention. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0058] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," and "disposed" should be understood in a broad sense. For example, they may refer to fixed connection or disposition, detachable connection or disposition, or integral connection or disposition. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0059] Example

[0060] Induction of M1 and M2 macrophages

[0061] (1) RAW 264.7 cells were cultured at a volume of 1×10 6The cells were seeded into six-well plates and cultured in RAW264.7 culture medium containing 10% serum and 1% penicillin-streptomycin double antibody, and placed in a cell incubator overnight to adhere.

[0062] (2) The original culture medium was aspirated and the cells were washed twice with PBS to remove the residual culture medium. 1 μg / mL LPS and 20 ng / mL IL-4 were added and induced for 24 h to obtain M1 and M2 macrophages, respectively.

[0063] Extraction of exosomes derived from M2 macrophages

[0064] M2 macrophage culture supernatant was collected and exosomes (M2 Exo) were extracted using differential centrifugation. Dead cells were removed by centrifugation at 300g for 10 minutes, followed by 10 minutes at 2000g to remove cellular debris. The supernatant was carefully collected and centrifuged at 10,000g for 30 minutes to remove larger contaminants. Finally, the supernatant was centrifuged at 120,000g for 2 hours in an ultracentrifuge. The supernatant was discarded, and the M2 Exosomes were resuspended in PBS and stored at -80°C until further use. Dynamic light scattering was used to measure particle size and potential.

[0065] Synthesis of CXB@Exo nanospheres

[0066] Compound CXB was uniformly mixed with an excess of M2 Exo. The mixture was then repeatedly extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm using a liposome extruder to form uniformly sized CXB@Exo nanospheres (CE NPs). Following preparation, free CXB was removed by ultrafiltration at 14,000 g for 30 minutes using a 50 kDa molecular weight cutoff ultrafiltration centrifuge tube. Finally, the CXB encapsulation efficiency of the CE NPs was determined by high-performance liquid chromatography.

[0067] DSPE-Hyd-PEG 2000 -NHS modified drug CC-90011

[0068] Dissolve CC-90011 in DMSO to prepare a stock solution. Take 1 mL of sodium bicarbonate buffer at pH 8.5, add 100 μM CC-90011 stock solution, and mix thoroughly. Then, add DSPE-Hyd-PEG 2000-NHS is dissolved in DMSO to prepare a stock solution, and 50 μM of the stock solution is added dropwise to the above-mentioned mixed solution. Gentle stirring is maintained during the dropwise addition to ensure that the reaction system is evenly mixed. Subsequently, magnetic beads are added to the above-mentioned mixed solution, placed at room temperature, and stirred for 24 hours at a constant stirring speed (300 r / min) to ensure that the components in the solution are fully reacted. After 24 hours, the resulting mixed solution is transferred to a dialysis bag with a molecular weight cutoff of 500 to 1000 Da, placed in a 2L graduated cylinder containing 800 mL of deionized water, and a magnetic bead is added thereto at the same time. The graduated cylinder is placed on a stirrer and dialyzed at a speed of 300 r / min for 24 hours to remove free small molecule drugs in the solution. During this period, deionized water is replaced at 4 hours, 8 hours, and 12 hours to maintain dialysis efficiency and ensure the effective removal of small molecules. After dialysis, the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, marked accordingly, and the centrifuge tube was placed in a freeze dryer for freeze drying to obtain solid DSPE-Hyd-PEG. 2000 -CC-90011 (CC-linker) was stored at -80°C until use. Raman spectroscopy and H-NMR spectroscopy were used to verify the successful synthesis of the CC-linker.

[0069] Preparation of CXB@Exo / CC-90011 nanospheres

[0070] The synthesized CE NPs were mixed with CC-linker in 500 μL PBS and reacted at 40 °C for 2 h. The excess free small molecules were removed by 50 kDa ultrafiltration tube to obtain purified CXB@Exo / CC-90011 (CEC NPs) and resuspended in PBS and stored at -80 °C for use.

[0071] Characterization and functional verification

[0072] Laser confocal microscopy confirmed that CEC NPs were successfully loaded with drug CC-90011

[0073] First, DSPE-Hyd-PEG containing fluorescein isothiocyanate (FITC) was 2000-NHS reacts with CC-90011 to produce CC-linker-FITC. CE NPs are then synthesized and incubated with 1,1'-dioctyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DIL) dye for 30 minutes at room temperature in the dark. Excess DIL is removed by three ultrafiltration cycles. CE@DIL is then incubated with CC-linker-FITC for 2 hours to form fluorescent CECNPs, which are then ultrafiltered three times to remove free CC-linker-FITC. Finally, 10 μL of the product is plated on a glass slide, and the experimental results are observed using laser confocal microscopy.

[0074] Flow cytometry detection of the percentage of CE NPs modified with different concentrations of CC-linker-FITC

[0075] CE NPs were co-incubated with CC-linker-FITC at different concentrations (0, 10, 50, 100 μM) to synthesize CEC NPs. After purification, the percentage of CE NPs modified with CC-linker-FITC at different concentrations was detected by flow cytometry.

[0076] HPLC analysis of nanoparticles and their encapsulation efficiency

[0077] Take appropriate amount of CXB, CE NPs, CC-90011, DSPE-Hyd-PEG 2000 The drug loading percentage was calculated using the supernatant samples from ultrafiltration of -NHS, CC-linker, and CE NPs. A mobile phase of methanol and water (75:25, v / v) was used, and the column effluent was analyzed at a flow rate of 1 mL / min at 30 ± 0.5°C. UV detectors were set at 254 nm and 219 nm, respectively, with an injection volume of 20 μL. The encapsulation efficiency was calculated as follows: the drug concentration loaded on the nanoparticles was divided by the initial drug concentration, and the result was multiplied by 100%, i.e., encapsulation efficiency = (loaded and drug concentration on the nanoparticles / initial drug concentration) × 100%.

[0078] Transmission electron microscopy characterization of nanoparticles

[0079] An appropriate amount of M2 Exo, CE NPs, and CEC NPs suspension was carefully dropped onto a copper grid, followed by the addition of uranyl acetate for negative staining. After the sample was fully dried, the morphological changes of the exosomes before and after drug loading were observed using a transmission electron microscope and the particle size was recorded.

[0080] Particle size and Zeta potential

[0081] The M2 Exo, linker, CXB, CC-90011, CE NPs and CEC NPs solutions were diluted into 2 mL of ultrapure water, and their particle size and Zeta potential in water were detected using a nanoparticle size analyzer.

[0082] Raman spectroscopy

[0083] First, appropriate amounts of liquid samples (linker, CC-90011, and CC-linker) were dripped onto the surface of a gold-coated silicon plate. The plate was then placed in a fume hood to air-dry. The plate was then placed on the sample stage of a Raman spectrometer. The sample was scanned using a 638nm laser, located in the appropriate field of view. After the measurement, the data was exported and analyzed using Origin for plotting.

[0084] H NMR

[0085] 5 mg of CC-90011, linker, and CC-linker samples were weighed and dissolved in deuterated dimethyl sulfoxide. These dissolved samples were detected using a nuclear magnetic resonance spectrometer. After the detection, the data were exported and processed and analyzed using Origin software.

[0086] UV-visible spectroscopy detection of each nanoparticle and its acid-responsive release

[0087] Appropriate amounts of M2 Exo, CXB, CC-90011, linker, CC-linker, CE NPs, and CEC NPs were dispersed in deionized water, and the UV absorption spectra of each material were scanned using UV-Vis spectrophotometry. After measuring the characteristic peaks of each material, equal amounts of CEC NPs were dissolved in solutions at pH 6.5 and pH 7.4, respectively. The solutions were dialyzed using a dialysis bag with a molecular weight cutoff of 3000 Da to release CC-90011. The absorbance changes of CC-90011 at different time points were then measured using UV-Vis at a wavelength of 206 nm.

[0088] Exosome protein extraction

[0089] Take an exosome sample and add exosome lysis buffer at a 1:1 volume ratio. Mix thoroughly and place on ice for 10 minutes. Centrifuge at 12,000g for 15 minutes at 4°C. Collect the supernatant and analyze the exosome concentration using the BCA assay. Finally, add protein loading buffer at a 1:4 ratio. Mix thoroughly and boil at 95°C for 10 minutes. Store at -20°C until ready to use.

[0090] Cell protein extraction

[0091] Prepare cell lysis buffer at a 100:1 ratio of lysis buffer to protease inhibitor, add 150 μL to each well, and lyse on ice for 30 minutes. After centrifugation, collect the supernatant. After protein concentration is determined using a BCA kit, add protein loading buffer to the supernatant at a ratio of 1:4 to sample volume. Mix thoroughly, boil at 95°C for 10 minutes, and store at -20°C until ready to use.

[0092] Determine sample protein concentration using BCA

[0093] (1) Prepare a 0.5 mg / mL protein standard solution according to the instructions of the BCA kit.

[0094] (2) Take a 96-well plate and add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard solution to the plate in sequence, setting up three replicate wells for each concentration. After the sample is added, use diluent to make up the liquid volume of each well to 20 μL;

[0095] (3) Add the protein sample to be tested into the corresponding wells of a 96-well plate at a volume of 20 μL per well;

[0096] (4) Prepare BCA working solution accurately according to the instructions of the BCA kit. Then add 200 μL to each well and incubate at 37°C for 30 minutes.

[0097] (5) After the incubation, the OD value of each well was immediately measured using a microplate reader at a wavelength of 562 nm. A standard curve was drawn based on the results of the standard solution, and the concentration of each histone sample was finally calculated based on the standard curve.

[0098] Westernblot

[0099] (1) Gel preparation: Select a 10% PAGE gel according to the desired protein molecular weight;

[0100] (2) Protein electrophoresis: Adjust the protein loading amount according to the BCA results, 20 μg protein per well. Add electrophoresis buffer and set the voltage to 50 V. After the protein leaves the stacking gel, adjust the voltage to 110 V until the protein moves to the bottom edge of the gel.

[0101] (3) Transfer: Remove the gel and transfer it to the electrotransfer solution for soaking. Cut a PVDF membrane of the corresponding size and activate it in methanol solution for 1 minute. Then, place the transfer cassette with the black side facing down. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the order from bottom to top, and use a roller to roll away excess bubbles. Place the transfer cassette in the transfer tank, fill it with electrotransfer solution, and apply a constant current of 250mA. The transfer time is calculated based on 1 kDa corresponding to 1 minute.

[0102] (4) Blocking: Soak the PVDF membrane in 5% skim milk, shake slowly, and incubate at room temperature for 2 h;

[0103] (5) Primary antibody incubation: After blocking, wash the PVDF membrane three times with TBST buffer (7 min / time). Prepare the primary antibody in TBST solution at a ratio of 1:1000. After washing, incubate the membrane in the primary antibody solution at 4°C overnight. Recover the primary antibody and wash with TBST three times (7 min / time).

[0104] (6) Secondary antibody incubation: Place the washed PVDF membrane in a secondary antibody dilution buffer prepared with TBST (dilution ratio 1:2000) and incubate on a shaker at room temperature for 2 h. After incubation, recover the secondary antibody and wash with TBST three times, 7 min each time.

[0105] (7) Chemiluminescence: Liquid A and liquid B were prepared in a 1:1 ratio to prepare a luminescent solution. After washing the membrane, the luminescent solution was evenly added to the PVDF membrane and exposed using a chemiluminescence imager. The grayscale value was collected and analyzed using Image J.

[0106] Flow cytometry confirmed the successful induction of M1 and M2 macrophages

[0107] (1) Cell collection: Use a 1.5 mL EP tube to collect the induced cells, centrifuge at 300 g for 5 min, and then wash once with PBS buffer;

[0108] (2) Blocking: Add diluted Fc antibody and resuspend for blocking, centrifuge at 300g for 5 minutes, and remove the supernatant;

[0109] (3) CD86 staining: Dissolve 0.5 g of bovine serum albumin in 10 mL of PBS to prepare antibody diluent. Add 100 μL of CD86 (1:100) fluorescently labeled antibody to each tube and incubate at 4°C in the dark for 30 min.

[0110] (4) Fixation: After staining, centrifuge at 300 g for 5 min to remove the supernatant, wash twice with 500 μL PBS, add 100 μL 4% paraformaldehyde to each tube to resuspend the cells, and fix at room temperature for 30 min;

[0111] (5) Membrane disruption: by adding 30 μL Triton TM Prepare cell permeabilization buffer by adding X-100 to 10 mL of antibody diluent. After fixation, wash once with PBS, remove the PBS by centrifugation, and add 200 μL of permeabilization reagent for 10 minutes at room temperature.

[0112] (6) CD206 staining: Add CD206 (1:100) antibody to the permeabilization reagent to prepare a staining solution. After permeabilization, centrifuge and remove the supernatant. Add 100 μL of CD206 staining solution to each tube and incubate at room temperature in the dark for 60 min.

[0113] (7) On-line testing: Centrifuge to remove CD206 staining solution, wash twice with PBS, add 500 μL PBS to each tube, resuspend the cells, and then test on the instrument.

[0114] Biocompatibility verification of CEC NPs

[0115] Cytotoxicity assay

[0116] The CCK-8 kit was used to detect the cytotoxicity of CXB, M2 Exo, and CE NPs on macrophages and FLS cells. The following are the detailed steps for using CCK-8 to detect drug cytotoxicity:

[0117] (1) M1 and RA-FLS cells were cultured at 1×10 4 The cells were seeded into 96-well plates at a density of 100 μg / mL and allowed to adhere overnight.

[0118] (2) PBS, CXB, M2 Exo, and CE NPs were added to the cells and incubated for 24 h;

[0119] (3) After discarding the old culture medium, prepare CCK-8 working solution at a volume ratio of 10:1 between culture medium and CCK-8 reagent. After oscillation and mixing, add 100 μL of working solution to each well and return the cells to the cell culture incubator for an additional 30 min-1 h. Observe the color change regularly during the incubation period. Terminate the reaction when the solution changes from pink to orange-yellow.

[0120] (4) Use an enzyme-labeled instrument to measure the absorbance of each well at a wavelength of 450 nm.

[0121] Cellular uptake experiments

[0122] (1) M2 Exo and CE NPs were labeled with the fluorescent dye DIL, and the excess fluorescent dye was removed by ultrafiltration;

[0123] (2) RAW 264.7 and FLS cells were plated in 24-well plates and stimulated with LPS (1 μg / mL) for 24 h and 3 h to induce them into M1 and RA-FLS cells, respectively;

[0124] (3) After induction, the old culture medium was discarded, and the modified DIL-M2 Exo and DIL-CE NPs were added to the well plate and incubated with the cells for 1-4 h;

[0125] (4) The culture medium was aspirated at 1 h and 4 h of co-incubation, and the cells were washed three times with PBS to completely remove the exosomes and nanoparticles that were not taken up by the cells;

[0126] (5) Confocal microscopy: DAPI-containing mounting medium was used to stain the cell nuclei, and after air drying, CLSM was used to image and analyze the distribution of exosomes and nanoparticles in cells;

[0127] (6) Flow cytometric analysis: The cells were digested with trypsin to prepare a single-cell suspension, and then the fluorescence signal intensity in the cells was detected by flow cytometry to analyze the changes in fluorescence intensity at different time points.

[0128] Micro-CT experiment

[0129] (1) When establishing the collagen-induced arthritis (CIA) model in DBA mice, the primary immunization was performed by subcutaneous injection of an emulsified mixture of bovine type II collagen solution and complete Freund's adjuvant;

[0130] (2) On day 21, mice were boosted with an emulsion of bovine type II collagen solution and incomplete Freund's adjuvant;

[0131] (3) Seven days later, CXB, CC-90011, CE, and CEC were administered via the tail vein. A PBS positive control group and a healthy mouse negative control group were also established. At the end of the experiment, all mice were sacrificed, and ankle joint samples were collected and fixed in 4% paraformaldehyde solution.

[0132] (4) The fixed specimens were three-dimensionally reconstructed using Micro-CT scanning to generate 3D rendering images of the ankle joint to systematically evaluate the therapeutic effects of each treatment group.

[0133] result

[0134] To load CC-90011 onto CE NPs, CC-90011 was reacted with a linker via an amidation reaction between the amino group and the N-hydroxysuccinimide ester group (NHS group) to obtain a purified CC-linker. 1 H-NMR analysis was used to determine the chemical structures of CC-90011, linker and CC-linker. Figure 1 A) and CC-90011( Figure 1 B) compared to CC-linker( Figure 1 The amide hydrogen peak (δ 8.33) appearing in C) indicates the formation of an amide bond, which confirms the successful synthesis of the CC-linker.

[0135] After we successfully extracted M2 Exo, we loaded CXB into M2 Exo with the help of liposome extruder to form relatively uniform CE NPs ( Figure 2 A). UV-visible absorption spectrum analysis revealed that compared with CXB, CE NPs showed a characteristic UV absorption peak of CXB at a wavelength of 254 nm ( Figure 2 B), which indicates that CE NPs have been successfully synthesized. Further, the UV absorption peak of drug CXB at 254nm was used to analyze the encapsulation efficiency of CE NPs by HPLC. The results showed that the encapsulation efficiency of CXB in CE NPs reached 43.8% ( Figure 2 The above results show that we have successfully synthesized nanoparticles CENPs, which provides a basis for our subsequent experiments.

[0136] CC-90011 was linked to CE NPs by co-incubation using DSPE groups, and a CEC NPs complex was successfully constructed ( Figure 3 A). To optimize the synthesis conditions of CEC NPs and determine the optimal ratio of CE NPs to CC-linker, the CC-linker was labeled with a FITC fluorescent group, and then different concentrations of CC-linker-FITC were co-incubated with a fixed concentration of CE NPs. Flow cytometry results showed that after incubation at 37°C for 2 hours, the CC-linker-FITC labeling rate of CE NPs increased in a concentration-dependent manner. Among them, the fluorescence intensity of the 50μM and 100μM CC-linker-FITC treatment groups was the most significant, with positive labeling rates reaching 53.1% and 54.3%, respectively ( Figure 3 B).

[0137] Subsequently, the synthesis effect of CEC NPs was systematically verified and characterized. During the synthesis of CEC NPs, its morphological characteristics ( Figure 4 A) Expression of exosome-specific proteins ( Figure 4 B) and surface zeta potential ( Figure 4 C) remained stable without significant changes, which fully demonstrated that the NPs maintained the basic characteristics of exosomes and had good stability during the synthesis process. Then, the UV-Vis spectra of CEC NPs and the intermediates CE NPs and CC-linker required in the synthesis process were analyzed ( Figure 4D) Analysis found that the absorption peaks of CE NPs and CC-linker appeared in the UV-Vis spectrum of CEC NPs. These data fully demonstrate that the synthesis process of CEC NPs not only achieved effective loading, but also completely maintained the physical and chemical properties and biological characteristics of the nanoparticles. CEC NPs were dialyzed through a dialysis bag and the CC-90011 concentration in the dialysate was detected by UV-Vis to verify the responsiveness of CEC NPs to acidic environment. The results showed that compared with pH 7.4, the release rate of CC-90011 at pH 6.5 was significantly higher than that at pH 7.4 ( Figure 5 ).

[0138] After successfully confirming the significant anti-inflammatory and bone erosion inhibitory effects of CEC NPs in vitro, we further investigated their therapeutic efficacy in a collagen-induced arthritis (CIA) mouse model to evaluate their potential in vivo application. To evaluate the therapeutic efficacy, CIA mice received tail vein injections of different formulations (CXB, CC-90011, CE NPs, or CEC NPs) every three days starting on day 7 after booster immunization. PBS-treated CIA mice and healthy mice served as model and control groups, respectively. Micro-CT analysis on day 28 demonstrated that treatment with CXB, CC-90011, CE NPs, and CEC NPs reduced osteophytes and bone erosion in the model mice, but with varying efficacy. CC-90011 alone significantly inhibited bone erosion but had limited effect on osteophytes. In contrast, CE NPs not only enhanced the anti-inflammatory effects of the clinical drug CXB but also demonstrated a partial bone protective effect. When CC-90011 and CE NPs were co-loaded to form CEC NPs, the therapeutic effect was significantly improved, the degree of osteophytes and bone erosion was significantly reduced compared with the single-drug group, and the joint structure was restored to the level of the healthy group. This shows that CEC NPs achieved the optimal joint protection effect through synergistic targeted delivery and multiple mechanisms of action ( Figure 6 The results suggest that CC-90011 significantly improves bone erosion compared to CXB, and has greater potential and more significant clinical application advantages for the treatment of advanced RA.

[0139] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A targeted therapeutic drug for rheumatoid arthritis, which is acid-responsive nanoparticles CEC NPs, characterized in that: Specifically composed of the following components: The vector is exosomes derived from M2 macrophages; The loaded drug comprises: The cyclooxygenase inhibitor celecoxib CXB was encapsulated in M2 Exo to form CXB@Exo nanospheres CE NPs; The osteoclast inhibitor CC-90011 was modified with a linker molecule 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-hydrazone bond-polyethylene glycol 2000-N-hydroxysuccinimide to form a CC-linker and covalently linked to CE NPs.

2. The targeted therapeutic drug for rheumatoid arthritis according to claim 1, characterized in that: In the CC-linker, DSPE-Hyd-PEG 2000 The molar ratio of -NHS to CC-90011 is 1:

2.

3. The targeted therapeutic drug for rheumatoid arthritis according to claim 1, characterized in that: In the CC-linker, the connection ratio of CE NPs to CC-linker is: 10-100 μM CC-linker is connected to every 50 μM CE NPs.

4. The targeted therapeutic drug for rheumatoid arthritis according to claim 1, characterized in that: The acid-responsive nanoparticles CEC NPs have the following physicochemical properties: Particle size: 100-200 nm (verified by transmission electron microscopy); Zeta potential: -15 to -25 mV (detected by nanoparticle size analyzer); Acidic response release: The release rate of CC-90011 at pH 6.5 was higher than that at pH 7.

4.

5. A method for preparing a targeted therapeutic drug for rheumatoid arthritis, which is used to prepare a targeted therapeutic drug for rheumatoid arthritis according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: induce M2 macrophages; S2: Extract exosomes from M2 macrophages; S3: Synthesis of CE NPs; S4: Preparation of CC-linker; S5: Preparation of CEC NPs; S6: In vivo validation of CEC NPs in mice.

6. The method for preparing a targeted therapeutic drug for rheumatoid arthritis according to claim 5, characterized in that: The S1 step comprises the following steps: S11: Mouse mononuclear macrophage cells were cultured at 1×10 6 / well inoculated, cultured overnight in a medium containing 10% serum and 1% penicillin-streptomycin double antibody; S12: Replace the serum-free medium with 20 ng / mL interleukin-4 and induce for 24 h.

7. The method for preparing a targeted therapeutic drug for rheumatoid arthritis according to claim 5, characterized in that: The step S2, which extracts the cell supernatant by differential centrifugation, specifically comprises the following steps: S21: centrifugation at 300 g for 10 min to remove dead cells; S22: centrifugation at 2000 g for 10 min to remove cell debris; S23: centrifugation at 10,000 g for 30 min to remove impurities; S24: Ultracentrifuge at 120,000 g for 2 h, resuspend in phosphate buffer and store at -80°C.

8. The method for preparing a targeted therapeutic drug for rheumatoid arthritis according to claim 5, characterized in that: The S3 step comprises the following steps: S31: Cyclooxygenase inhibitor celecoxib CXB was mixed with M2 macrophage exosomes and then repeatedly extruded through 400 nm and 200 nm polycarbonate membrane liposome extruders; S32: Use 50 kDa ultrafiltration tubes to centrifuge at 14000g for 30 min to remove free CXB and obtain purified CE NPs.

9. The method for preparing a targeted therapeutic drug for rheumatoid arthritis according to claim 5, characterized in that: The S4 step comprises the following steps: S41: Dissolve 100 μM CC-90011 in 1 mL of pH 8.5 sodium bicarbonate solution and mix thoroughly; S42: 50 μM DSPE-Hyd-PEG 2000 -NHS was added dropwise to the above solution; S43: react at room temperature with magnetic stirring at 300 r / min for 24 h; S44: Dialyze the solution in deionized water using a 500-1000 Da dialysis bag for 24 h, changing the water three times, and then freeze-dry to obtain a solid CC-linker.

10. The method for preparing a targeted therapeutic drug for rheumatoid arthritis according to claim 5, characterized in that: The step S5 comprises the following steps: S51: CE NPs and CC-linker were mixed in 500 μL PBS and reacted at 40 °C for 2 h; S52: The product CEC NPs were purified by 50 kDa ultrafiltration tube, resuspended in PBS and stored at -80 °C; The step S6 comprises the following steps: S61: First, the mice were immunized subcutaneously at the base of their tails with an emulsified mixture of bovine type II collagen solution and complete Freund's adjuvant. S62: On day 21, mice were boosted with an emulsion of bovine type II collagen solution and incomplete Freund's adjuvant; S63: 7 days later, CXB, CC-90011, CE, and CEC were administered via the tail vein, and a PBS positive control group and a healthy mouse negative control group were set up. S64: Ankle joint samples were collected and fixed in 4% paraformaldehyde solution. The fixed samples were three-dimensionally reconstructed using Micro-CT scanning to generate a 3D rendering image of the ankle joint.

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