Preparation and application of bionic cerium-based polyphenol nano-enzyme for treating rheumatoid arthritis

Abstract

The invention discloses preparation and application of bionic cerium-based polyphenol nano-enzyme for treating rheumatoid arthritis, and belongs to the field of medical preparations. The method comprises the following steps: respectively complexing cerium ions and tannic acid or epicatechin gallate to prepare cerium-based polyphenol nano-enzyme, and then preparing the cerium-based polyphenol nano-enzyme wrapped by a macrophage membrane. And loading the cerium-based polyphenol nano-enzyme wrapped by the macrophage membrane into the hydrogel to prepare the hyaluronic acid-based hydrogel. The cerium-based polyphenol nano-enzyme and the hyaluronic acid-based hydrogel are characterized, it is verified that the cerium-based polyphenol nano-enzyme and the hyaluronic acid-based hydrogel are successfully synthesized, the particle size of CeTA is about 22 nm, the particle size of M-CeTA is increased to 273 nm, the particle size of CeECG is about 95 nm, and the particle size of M-CeECG is increased to 292 nm. In-vitro experiments prove that the M-CeTA and the M-CeECG do not show obvious cytotoxicity when being less than 80 mu g / mL, and have an obvious inhibition effect on expression of cell inflammatory factors on inflammatory cells.

Description

Technical Field

[0001] The present invention belongs to the field of medical preparations, relates to the field of medical nanomaterials technology, and particularly relates to a preparation method and application of a biomimetic cerium-based polyphenol nanoenzyme for the treatment of rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease, which usually manifests as joint pain, swelling and stiffness. As of 2019, there were approximately 18 million RA patients globally, 70% of whom were women, and only 13 million RA patients could improve their severity through rehabilitation. RA can lead to a decline in physical function and skeletal muscle symptoms, resulting in a loss of labor force, bringing a great burden to families and society.

[0003] In the inflammatory tissues of rheumatoid arthritis, macrophages are activated into M1 macrophages, producing a large amount of inflammatory cytokines such as TNF-α and IL-6, and the ROS level is too high, resulting in oxidative stress. Therefore, converting M1 macrophages into M2 macrophages, clearing inflammatory cytokines at the inflammatory site, and clearing ROS are urgent problems to be solved in the treatment of rheumatoid arthritis. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation and application of a biomimetic cerium-based polyphenol nanoenzyme for the treatment of rheumatoid arthritis. First, endow the cerium-based polyphenol nanoenzyme with immune escape function and inflammatory targeting, and use membrane surface proteins to mediate active targeting to solve the problems of low clearance and low accumulation efficiency of nano-drugs in vivo; Second, construct a hyaluronic acid-based hydrogel, and use the ROS sensitivity of phenylborate ester bonds to achieve triggered release in the lesion microenvironment, prolong the retention time of drugs in the joint cavity, and reduce the risk of systemic toxicity; Third, based on the synergistic coordination of cerium ions with tannic acid or epigallocatechin gallate, endow CeTA and CeECG with superoxide dismutase and catalase dual activities, synchronously scavenge ROS and inhibit the AKT / NF-κB inflammatory pathway, and break the vicious cycle of oxidative stress-inflammation.

[0005] The technical solution of the present invention is as follows: A preparation method of a biomimetic cerium-based polyphenol nanoenzyme for the treatment of rheumatoid arthritis, comprising the following preparation steps: Step 1: Complex trivalent cerium ions with TA or ECG respectively to prepare CeTA or CeECG; Step 2: Wrap CeTA or CeECG with macrophage membranes to prepare M-CeTA or M-CeECG.

[0006] Furthermore, the preparation method further comprises the following preparation steps: Step 3: Load M-CeTA or M-CeECG into HA hydrogel respectively to prepare HA@M-CeTA hydrogel or HA@M-CeECG hydrogel.

[0007] Preferably, the steps for preparing M-CeTA are as follows: (I) Prepare CeTA: Add TA solution to cerium nitrate solution, adjust the pH of the mixed solution to 5 - 6, and stir well at 50 - 55 °C; Centrifuge to collect the precipitate and obtain CeTA through dialysis; The dosage ratio of TA in the TA solution to cerium nitrate in the cerium nitrate solution is 1:6 - 1:5; (II) Prepare M-CeTA: Mix macrophage cell membrane and CeTA at a mass ratio of 3:1 - 4:1, extrude using a liposome extruder, and freeze-dry to obtain M-CeTA.

[0008] Preferably, the steps for preparing CeECG are as follows: (I) Prepare CeECG: Add ECG solution to cerium nitrate solution, adjust the pH of the mixed solution to 5 - 6, and stir well at 50 - 55 °C; Centrifuge to collect the precipitate and obtain CeTA through dialysis; The dosage ratio of ECG in the ECG solution to cerium nitrate in the cerium nitrate solution is 1.5:1 - 1:1; (II) Prepare M-CeECG: Mix macrophage cell membrane and CeTA at a mass ratio of 3:1 - 4:1, extrude using a liposome extruder, and freeze-dry to obtain M-CeECG.

[0009] Preferably, the steps for preparing HA@M-CeTA hydrogel or HA@M-CeECG are as follows: (I) Prepare HA-TA: Dissolve HA in water, add NHS and EDC, adjust the pH value of the solution to 5 - 6, and add TA with stirring; Dialyze in water to obtain HA-TA; And prepare HA-PBA: Dissolve EDC and HOBt in DMSO, add to the aqueous solution of HA and 4-PBA, and adjust the pH value of the solution to 5 - 6; Stir, dialyze, and freeze-dry to obtain HA-PBA; (II) Prepare HA@M-CeTA hydrogel or HA@M-CeECG hydrogel: Dissolve M-CeTA or M-CeECG in water, and mix with the prepared HA-TA solution and HA-PBA solution to obtain HA@M-CeTA hydrogel or HA@M-CeECG hydrogel.

[0010] Preferably, in the steps for preparing HA-TA, the dosage ratio of HA, NHS, EDC, and TA is 4:4:1:1 - 4:4:1:2.

[0011] Preferably, in the step of preparing HA-PBA, the dosage ratio of HA, 4-PBA, EDC and HOBt is 1:1:1:1.

[0012] Preferably, in the step of preparing the HA@M-CeTA hydrogel or the HA@M-CeECG hydrogel, the dosage ratio of M-CeTA, HA-TA and HA-PBA is 1:1:1 to 1:1.5:1.5; the dosage ratio of M-CeECG, HA-TA and HA-PBA is 1:1:1 to 1:1.5:1.5.

[0013] Preferably, the preparation method of the macrophage cell membrane is as follows: RAW264.7 cells are collected with PBS, sonicated with a cell disruptor, sucrose is added, and the cell nuclei and unbroken cell precipitates are removed by centrifugation; the supernatant is centrifuged to obtain a precipitate containing macrophage cell membranes and residual proteins; the precipitate is washed and centrifuged to remove contaminating proteins to obtain macrophage cell membranes.

[0014] The biomimetic cerium-based polyphenol nanozyme prepared by the preparation method of the biomimetic cerium-based polyphenol nanozyme for the treatment of rheumatoid arthritis is used for the treatment of rheumatoid arthritis.

[0015] The mechanism of the present invention is as follows: A stable network structure is prepared through the coordination interaction between polyphenols and metal ions. Among them, polyphenol molecules have obvious anti-inflammatory activities. Cerium ions have multiple enzyme activities due to the characteristics of the mutual transformation and coexistence of Ce 3+ and Ce 4+ and can scavenge a variety of free radicals, playing antioxidant and anti-inflammatory roles. However, after metal polyphenol nanoparticles enter the human body, they will be rapidly cleared by the immunogenicity in the body, resulting in low drug efficiency. In the present invention, the macrophage cell membrane is used as the material of the biomimetic nano-drug delivery system, which can target the drug to the inflammatory site and avoid the recognition of the immune system. The biomimetic nano-drug delivery system of the present invention solves the problem that the cerium-based polyphenol nanozyme is rapidly cleared in the body. The HA hydrogel enables the drug to stay in the joint cavity for a long time and maintain a high drug concentration in the joint cavity. HA-TA and HA-PBA are crosslinked to prepare a hydrogel with phenylborate ester bonds, which responds to ROS release in the joint cavity. Doping the drug in the hydrogel can not only maintain the local drug concentration, reduce drug toxicity, but also lubricate the articular cartilage. After injecting the hyaluronic acid-based hydrogel into the ankle joint cavity of adjuvant-induced arthritis (AIA) rats, the cerium-based polyphenol nanozyme encapsulated by the macrophage cell membrane responds to ROS release. The macrophage cell membrane assists the cerium-based polyphenol nanozyme to escape immunity, actively accumulates at the inflammatory site, is absorbed by inflammatory cells, improves the inflammatory microenvironment, inhibits the occurrence of inflammation, and effectively treats RA.

[0016] Compared with the prior art, the present invention also has the following advantages: First, in the present invention, through macrophage cell membrane wrapping, the clearance rate of the nanozyme by the immune system is significantly reduced. Experiments show that the cell membrane-wrapped cerium-based polyphenol nanozyme has a higher enrichment efficiency at the inflammation site than the non-wrapped cerium-based polyphenol nanozyme.

[0017] Second, the phenylborate bond-crosslinked hyaluronic acid hydrogels (HA@M-CeTA, HA@M-CeECG) have ROS responsiveness and specifically release M-CeTA and HA@M-CeECG in the high-ROS microenvironment of the RA joint cavity. The reticular porous structure of the hydrogel can adsorb and protect the nanozyme to avoid the burst release effect.

[0018] Third, the CeTA and CeECG nanozymes have both superoxide dismutase (SOD) and catalase (CAT) activities, and the ROS scavenging efficiency is significantly better than that of single enzyme preparations.

[0019] Fourth, compared with Ce commonly used in the prior art 4+ , in the present invention, Ce 3+ is complexed with polyphenol molecules. Ce 3+ has strong reducing ability, can scavenge ROS, reduce oxidative stress damage, and Ce 3+ is relatively stable in an acidic environment and can continuously exert its antioxidant ability. In addition, Ce 3+ has low toxicity and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the particle size distribution and morphology diagram of the preparation prepared in the embodiment of the present invention. In the figure, A is used to show the particle size distribution of CeTA, B is used to show the particle size distribution of M-CeTA, C is used to show the particle size distribution of CeECG, D is used to show the particle size distribution of M-CeECG, E is the transmission electron microscopy image of CeTA (scale bar: 100 nm), and F is the transmission electron microscopy image of M-CeTA (scale bar: 500 nm).

[0021] Figure 2 In , A is the scanning electron microscopy image of the HA@M-CeTA hydrogel (scale bar: 10 μm), B is the scanning electron microscopy image of M-CeTA in HA@M-CeTA after magnification (scale bar: 1 μm), C is the scanning electron microscopy image of the HA@M-CeECG hydrogel (scale bar: 100 μm), and D is the scanning electron microscopy image of M-CeECG in HA@M-CeECG after magnification (scale bar: 1 μm).

[0022] Figure 3It is the structural characterization diagram of HA@M-CeTA prepared in the embodiment of the present invention. In the figure, A is the 1H NMR spectrum of HA, B is the 1H NMR spectrum of HA-TA, and C is the 1H NMR spectrum of HA-PBA.

[0023] Figure 4 It is the comparison diagram of the cytotoxicity of different preparations of TA. ([[]]END]] p <0.01, p <0.0001, n = 3).

[0024] Figure 5 It is the comparison diagram of the cytotoxicity of different preparations of ECG. ([[]]END]] p <0.0001, n = 3).

[0025] Figure 6 It is the comparison diagram of the influence of different preparations of TA on the cell supernatant level. ([[]]END]] p <0.01, p <0.0001, n = 3), A is the influence diagram of different preparations on the TNF-α level in the cell supernatant, and B is the influence diagram of different preparations on the IL-6 level in the cell supernatant.

[0026] Figure 7 It is the comparison diagram of the influence of different preparations of ECG on the cell supernatant level. ([[]]END]] p <0.05, p <0.001, p <0.0001, n = 3), A is the influence diagram of different preparations on the TNF-α level in the cell supernatant, and B is the influence diagram of different preparations on the IL-6 level in the cell supernatant.

[0027] Figure 8 It is the distribution of free DIR and HA@DIR in the rat body after ankle joint cavity injection. Specific implementation mode

[0028] The present invention will be further described below in conjunction with embodiments and experimental results.

[0029] Term Explanation: In the present invention, TA represents tannic acid. ECG represents epicatechin gallate.

[0030] PBS represents phosphate buffered saline. RAW264.7 cells refer to mouse monocyte macrophage leukemia cells. CeTA represents cerium-tannic acid nanozyme. CeECG represents cerium-epicatechin gallate nanozyme. M-CeTA represents macrophage membrane-coated cerium-tannic acid nanozyme. M-CeECG represents macrophage membrane-coated epicatechin gallate nanozyme. HA@M-CeTA represents hyaluronic acid-based hydrogel of M-CeTA. HA@M-CeECG represents hyaluronic acid-based hydrogel of M-CeECG. NHS represents N-hydroxysuccinimide. EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. 4-PBA represents 4-aminobenzeneboronic acid. HOBt represents 1-hydroxybenzotriazole. DMSO represents dimethyl sulfoxide. HA-TA represents tannic acid-modified hyaluronic acid. HA-PBA represents benzeneboronic acid-modified hyaluronic acid. BeNano 90 Zeta Nano Particle Size and Zeta Potential Analyzer is an optical detection system integrating BeNano 90 and BeNano Zeta. mM is a concentration unit, representing millimoles per liter, i.e., mmol / L.

[0031] Example 1. Preparation example of M-CeTA.

[0032] I. Preparation of CeTA.

[0033] Prepare a cerium nitrate solution (1.5 mM, solution A) and a TA solution (0.25 mM, solution B), and slowly and dropwise add an equal volume of solution B to solution A. Adjust the pH of the mixed solution to 5 with NaOH and stir at 50 °C for 3 hours. Centrifuge to collect the precipitate, disperse the precipitate in water and then centrifuge again to precipitate, repeating 3 times. After dialysis and freeze-drying, CeTA with the following structural formula is obtained.

[0034] .

[0035] II. Preparation of macrophage membrane.

[0036] RAW264.7 cells were collected with PBS, sonicated for 5 minutes using a cell disruptor, sucrose was added to a final concentration of 0.25 M, and centrifuged at 700×g for 10 minutes at 4 °C to remove nuclei and unbroken cell pellets. The supernatant was centrifuged at 14000×g for 30 minutes at 4 °C to obtain a pellet containing macrophage cell membranes and residual proteins. The pellet was washed 3 times with pre-cooled PBS and centrifuged to remove contaminating proteins, yielding macrophage cell membranes for wrapping onto CeTA. The protein concentration in the obtained cell membranes was determined using a BCA protein concentration assay kit.

[0037] III. Preparation of M-CeTA.

[0038] To wrap the isolated macrophage cell membranes onto CeTA, the macrophage cell membranes and CeTA were mixed at a mass ratio of 4:1, and extruded 50 times through 1 μm, 0.8 μm, and 0.4 μm polycarbonate membranes in sequence using a liposome extruder, followed by lyophilization to obtain M-CeTA.

[0039] Example 2. Preparation example of M-CeECG.

[0040] I. Preparation of CeECG.

[0041] A cerium nitrate solution (1.5 mM, solution A) and an ECG solution (0.25 mM, solution B) were prepared. Solution B was slowly added dropwise to solution A, with a volume ratio of solution A to solution B of 1:5. The pH of the mixed solution was adjusted to 5 with NaOH, and stirred at 50 °C for 3 hours. The precipitate was collected by centrifugation, redispersed in water and centrifuged again to precipitate, repeating 3 times. After dialysis and lyophilization, CeECG with the following structural formula was prepared.

[0042] 。

[0043] II. Preparation of macrophage cell membranes.

[0044] The method was the same as step II of Example 1.

[0045] III. Preparation of M-CeECG.

[0046] To wrap the isolated macrophage cell membranes onto CeECG, the macrophage cell membranes and CeECG were mixed at a mass ratio of 4:1, and extruded 50 times through 1 μm, 0.8 μm, and 0.4 μm polycarbonate membranes in sequence using a liposome extruder, followed by lyophilization to obtain M-CeECG.

[0047] Example 3. Preparation example of HA@M-CeTA and HA@M-CeECG hydrogels.

[0048] I. Preparation of HA-TA.

[0049] Dissolve 1000 mg of hyaluronic acid (HA) in 10 mL of deionized water, add 232 mg of NHS and 96 mg of EDC, adjust the pH value of the solution to 5.5, stir for 4 hours, then add 340 mg of TA, and continue stirring for 24 hours. Transfer the solution to a dialysis bag (MWCO: 14 kDa), dialyze in deionized water for 3 days, change the water every 12 hours, and then lyophilize to obtain HA-TA with the following structural formula.

[0050] 。

[0051] II. Preparation of HA-PBA.

[0052] Dissolve 1000 mg of HA in 10 mL of deionized water, and add 430 mg of 4-PBA to obtain a solution under continuous stirring. Dissolve 480 mg of EDC and 340 mg of HOBt in 2 mL of DMSO and add it to the above solution, adjust the pH value of the solution to 5, and stir at room temperature for 48 hours. Then dialyze and lyophilize to obtain hyaluronic acid modified with phenylboronic acid (HA-PBA).

[0053] 。

[0054] III. Preparation of HA@M-CeTA hydrogel.

[0055] Dissolve M-CeTA prepared in Example 1 in water (18 mg / mL), and prepare a 5% (w / w) HA-TA solution and a 5% (w / w) HA-PBA solution. Mix the above three solutions in equal volumes to prepare the HA@M-CeTA hydrogel.

[0056] IV. Preparation of HA@M-CeECG hydrogel.

[0057] Dissolve M-CeECG prepared in Example 2 in water (18 mg / mL), and at the same time prepare a 5% (w / w) HA-TA solution and a 5% (w / w) HA-PBA solution. Mix the above three solutions in equal volumes to prepare the HA@M-CeECG hydrogel.

[0058] Comparative Example 1. Preparation of HA hydrogel.

[0059] Prepare a 5% (w / w) HA-TA solution and a 5% (w / w) HA-PBA solution, and mix the above two solutions with deionized water in equal volumes to prepare the HA hydrogel.

[0060] The following are the detection, observation processes and results of CeTA, CeECG, M-CeTA, M-CeECG, HA@M-CeTA and HA@M-CeECG hydrogels.

[0061] 1. Take the prepared CeTA, CeECG, M-CeTA, and M-CeECG and dilute them with deionized water respectively. Use a BeNano 90 Zeta nanoparticle size and Zeta potential analyzer to measure the particle size and Zeta potential of the preparations. Among them, the Polydispersity Index (PDI) represents the aggregation degree of nanoparticles.

[0062] As Figure 1 shown in A of [reference], the hydrated particle size of CeTA is about 22 nm. As Figure 1 shown in B of [reference], the hydrated particle size of M-CeTA increases to 273 nm. As Figure 1 shown in C of [reference], the hydrated particle size of CeECG is about 95 nm. As Figure 1 shown in D of [reference], the particle size of M-CeECG increases to 292 nm.

[0063] 2. Dissolve CeTA and M-CeTA in deionized water respectively to obtain CeTA solution and M-CeTA solution. Use a syringe to drop the two solutions onto a copper mesh respectively, and dry the solvent with an infrared baking lamp. Place the fully dried electron microscope grid in a Transmission Electron Microscope (TEM) to observe and take scanning electron microscope pictures of CeTA and M-CeTA.

[0064] As Figure 1 shown in E and F of [reference], the particle size distributions of CeTA and M-CeTA are uniform, and the macrophage cell membrane is successfully wrapped onto CeTA.

[0065] 3. Cut the freeze-dried HA@M-CeTA and HA@M-CeECG hydrogels into thin slices respectively, fix them on a sample stage with conductive glue, spray gold, and use a Scanning Electron Microscope (SEM) to observe and take SEM pictures of HA@M-CeTA and HA@M-CeECG.

[0066] As Figure 2 shown in A of [reference], HA@M-CeTA has a uniform porous structure. As Figure 1 shown in B of [reference], the nanoparticles M-CeTA adsorbed onto the HA@M-CeTA hydrogel can be seen after magnification.

[0067] 4. Cut the freeze-dried HA@M-CeECG hydrogel into thin slices, fix it on a sample stage with conductive glue, spray gold, and use a scanning electron microscope to observe and take SEM pictures of HA@M-CeECG.

[0068] As Figure 2In C, HA@M-CeECG also has a uniform porous structure. As Figure 1 In D, after magnification, the nanoparticles M-CeECG adsorbed on the HA@M-CeECG hydrogel can be seen.

[0069] The following is the structural analysis process and results of HA@M-CeTA.

[0070] HA, HA-TA, and HA-PBA were respectively dissolved in heavy water (D 2 2O) and added to a nuclear magnetic tube, and scanned with a nuclear magnetic resonance spectrometer.

[0071] The nuclear magnetic resonance hydrogen spectra of HA, HA-TA, and HA-PBA are as Figure 3 shown. By comparing the nuclear magnetic resonance hydrogen spectra of HA-TA, HA-PBA, and HA, it was found that the peaks at 6.6 - 7.3 ppm in the nuclear magnetic resonance hydrogen spectrum of HA-TA are the aromatic rings or conjugated double bonds on TA, proving that TA was successfully connected to HA. The peaks at 6.7 - 7.2 ppm in the nuclear magnetic resonance hydrogen spectrum of HA-PBA are the hydrogens on the benzene ring of PBA, proving that PBA was successfully connected to HA.

[0072] The following is the cytotoxicity test process and results.

[0073] The cytotoxicity of CeTA and M-CeTA against RAW264.7 cells was investigated by the MTT method. 5000 RAW264.7 cells per well were seeded in a 96-well plate, and a blank group was set up without seeding cells. After the cells grew and adhered for 24 hours, the original medium was discarded, and media containing different concentrations (10, 40, 80 μg / mL) of CeTA and the corresponding concentrations of TA, Ce(NO 3 ) 3 ·6H 2 2O and M-CeTA were added as the dosing groups. At the same time, the Control group was added with the basal medium as the control group. After 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well in the dark, and after incubation for 4 hours, the original medium was discarded. 150 μL of DMSO was added to each well, and shaken at a constant temperature for 10 minutes to dissolve the formazan at the bottom. The absorbance of each well was measured at 490 nm with an enzyme-linked immunosorbent assay reader.

[0074] The results are as Figure 4 shown. After 24-hour cell culture with different preparations, the cell survival rate of each preparation showed a decreasing trend with the increase of the dosing concentration. Compared with TA alone and Ce(NO 3 ) 3 ·6H 2 2O, CeTA and M-CeTA had lower cytotoxicity. At 40 μg / mL, the cell survival rate of TA was 67.63%, Ce(NO3 ) 3 ·6H 2 The cell viability of TA was 54.08%, that of CeTA was 88.18%, and that of M-CeTA was 87.44%. When the concentration increased to 80 μg / mL, the cell viability of TA decreased to 56.48%, Ce(NO 3 ) 3 ·6H 2 O decreased to 29.73%, the cell viability of CeTA decreased to 77.26%, and the cell viability of M-CeTA decreased to 76.19%. At 80 μg / mL, the cell viabilities of the CeTA and M-CeTA groups also reached over 70%. Therefore, CeTA and M-CeTA have low toxicity and excellent biocompatibility to RAW264.7 cells.

[0075] The cytotoxicity of CeECG and M-CeECG to RAW264.7 cells was investigated by the same method. The results are as Figure 5 shown. After 24 h of cell culture with different preparations, the cell viabilities of each preparation decreased with the increase of the administration concentration. Compared with the individual ECG and Ce(NO 3 ) 3 ·6H 2 O, CeECG and M-CeECG had lower cytotoxicity. At 40 μg / mL, the cell viability of ECG was 63.22%, that of Ce(NO 3 ) 3 ·6H 2 O was 54.08%, that of CeECG was 88.16%, and that of M-CeECG was 85.86%. When the concentration increased to 80 μg / mL, the cell viability of ECG decreased to 53.97%, Ce(NO 3 ) 3 ·6H 2 O decreased to 29.73%, the cell viability of CeECG decreased to 75.93%, and the cell viability of M-CeECG decreased to 82.58%. At 80 μg / mL, the cell viabilities of the CeECG and M-CeECG groups also reached over 70%. Therefore, CeECG and M-CeECG have low toxicity and excellent biocompatibility to RAW264.7 cells.

[0076] The following is the experiment and results of the effect of M-CeTA on the levels of inflammatory factors in cell supernatant.

[0077] Inoculate 2×10 5RAW264.7 cells per well. After the cells adhered for 24 hours, the original medium was discarded, and the RAW264.7 cells were stimulated with 1 μg / mL LPS for 24 hours to induce differentiation. After discarding the original medium, 40 μg / mL free TA, CeTA, and M-CeTA with the same cerium concentration were added and incubated for 24 hours. Normal macrophages and an untreated LPS-stimulated model group were used as controls. The cell culture supernatant was collected, centrifuged at 1000 × g for 20 minutes at 4°C to remove impurities and cell debris, and the supernatant was retained. Subsequently, the concentrations of TNF-α and IL-6 in the supernatant of each group were detected according to the instructions of the TNF-α and IL-6 ELISA kits.

[0078] The results are as Figure 6 shown. Compared with the Control group, the contents of NO, inflammatory cytokines TNF-α and IL-6 produced by cells in the LPS group were significantly increased. The formulation group showed an inhibitory effect on the inflammatory cytokines TNF-α and IL-6. At the same concentration, M-CeTA exhibited the best property of inhibiting the production of TNF-α and IL-6 by cells ( p <0.0001).

[0079] The following are the experiments and results of the effects of M-CeECG on the levels of inflammatory factors in cell supernatants.

[0080] The effects of ECG, CeECG, and M-CeECG on the concentrations of TNF-α and IL-6 in cell supernatants were detected by the same method. The results are as Figure 7 shown. Compared with the Control group, the contents of NO, inflammatory cytokines TNF-α and IL-6 produced by cells in the LPS group were significantly increased. The formulation group showed an inhibitory effect on the inflammatory cytokines TNF-α and IL-6. At the same concentration, M-CeECG exhibited the best property of inhibiting the production of TNF-α and IL-6 by cells ( p <0.0001).

[0081] The following are the experiments and results of the effects of HA hydrogel on the in vivo distribution of drugs.

[0082] Dissolve 20 mg of poloxamer 188 in 7.5 mL of deionized water and preheat to 60°C. While stirring, add 1 mg of DIR dissolved in 0.5 mL of absolute ethanol dropwise to obtain DIR nanoparticles. Prepare HA@DIR hydrogel with the DIR solution. Inject free DIR and HA@DIR hydrogel into the joint cavities of rats respectively, and perform small animal in vivo imaging with a small animal imager at 0, 4, 8, 11, and 15 days respectively to observe and record the fluorescence distribution in vivo.

[0083] The results are as Figure 8As shown, the drug was distributed throughout the body on the 4th day, while the hydrogel-loaded drug was only distributed in the injected joint part, and there was no drug distribution in the internal organs. Compared with the free drug, the hydrogel significantly prolonged the residence time of the drug in the joint cavity, which could extend the action time of the drug and reduce the toxic and side effects of the drug.

Claims

1. A method for preparing a bionic cerium-based polyphenol nanozyme for the treatment of rheumatoid arthritis, characterized in that The method comprises the following preparation steps: Step 1, complexing trivalent cerium ions with TA or ECG to prepare CeTA or CeECG; Step 2: CeTA or CeECG is wrapped with macrophage membrane to prepare M-CeTA or M-CeECG.

2. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 1, characterized in that The following preparation steps are also included: Step 3: Load M-CeTA or M-CeECG into HA hydrogel to prepare HA@M-CeTA hydrogel or HA@M-CeECG hydrogel, respectively.

3. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 1 or 2, characterized in that The steps for preparing M-CeTA are as follows: (i) Preparation of CeTA: Add TA solution to cerium nitrate solution, adjust the pH of the mixed solution to 5-6, and stir thoroughly at 50-55°C; collect the precipitate by centrifugation, and obtain CeTA by dialysis; The ratio of TA in the TA solution to cerium nitrate in the cerium nitrate solution is 1:6~1:5; (ii) Preparation of M-CeTA: macrophage membrane and CeTA were mixed at a mass ratio of 3:1-4:1, extruded using a liposome extruder, and freeze-dried to obtain M-CeTA.

4. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 1 or 2, characterized in that The steps for preparing CeECG are as follows: (i) Preparation of CeECG: adding ECG solution to cerium nitrate solution, adjusting the pH of the mixed solution to 5-6, and stirring at 50-55 °C; collecting the precipitate by centrifugation, and obtaining CeTA by dialysis; The ratio of ECG in the ECG solution to cerium nitrate in the cerium nitrate solution is 1.5:1~1:1; (ii) Preparation of M-CeECG: Macrophage membrane and CeTA were mixed at a mass ratio of 3:1-4:1, extruded using a liposome extruder, and freeze-dried to obtain M-CeECG.

5. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 2, characterized in that The steps for preparing HA@M-CeTA hydrogel or HA@M-CeECG are as follows: (i) Preparation of HA-TA: Dissolve HA in water, add NHS and EDC, adjust the pH value of the solution to 5-6, add TA while stirring; dialyze in water to obtain HA-TA; and preparing HA-PBA: dissolving EDC and HOBt in DMSO, adding them to an aqueous solution of HA and 4-PBA, adjusting the pH value of the solution to 5-6; stirring, dialyzing, and freeze-drying to obtain HA-PBA; (ii) Preparation of HA@M-CeTA hydrogel or HA@M-CeECG hydrogel: dissolving M-CeTA or M-CeECG in water, and mixing the solution with the prepared HA-TA solution and HA-PBA solution to obtain HA@M-CeTA hydrogel or HA@M-CeECG hydrogel.

6. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 5, characterized in that: In the step of preparing HA-TA, the usage ratio of HA, NHS, EDC and TA is 4:4:1:1~4:4:1:

2.

7. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 5, characterized in that: In the step of preparing HA-PBA, the usage ratio of HA, 4-PBA, EDC and HOBt is 1:1:1:

1.

8. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 5, characterized in that: In the step of preparing HA@M-CeTA hydrogel or HA@M-CeECG hydrogel, the usage ratio of M-CeTA, HA-TA and HA-PBA is 1:1:1~1:1.5:1.5; the usage ratio of M-CeECG, HA-TA and HA-PBA is 1:1:1~1:1.5:1.

5.

9. The method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to claim 1, characterized in that The preparation method of the macrophage membrane is as follows: RAW264.7 cells are collected with PBS, sucrose is added after ultrasonication with a cell disruptor, and the cell nucleus and unbroken cell precipitates are removed by centrifugation; the supernatant is centrifuged to obtain a precipitate containing the macrophage membrane and residual protein; the precipitate is washed and centrifuged to remove contaminating proteins to obtain the macrophage membrane.

10. The bionic cerium-based polyphenol nanozyme prepared by the method for preparing the bionic cerium-based polyphenol nanozyme for treating rheumatoid arthritis according to any one of claims 1 to 9 is used for treating rheumatoid arthritis.

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