Macrophage membrane coated ROS responsive bionic nano drug delivery system, preparation and application

Abstract

The invention relates to the field of traditional Chinese medicine effective component new dosage form pharmaceutical preparations, and discloses an M2 type macrophage membrane coated ROS responsive bionic nano drug delivery system, preparation and application. Mesoporous silica is used as a core to load a traditional Chinese medicine effective component, namely tripterine, and lipid components, namely PEGylated distearoyl phosphatidyl ethanolamine (DSPE-PEG), dioleoyl lecithin (DOPC), cholesterol and the like, are used as raw materials to prepare a ROS-responsive lipid coating, so that ROS-responsive tripterine mesoporous silica is formed. And then realizing bionic fusion with the M2 type macrophage membrane on the outer layer through an ultrasonic coating technology to prepare the M2 type macrophage membrane coated ROS responsive bionic mesoporous silica nano drug delivery system. The invention can realize targeted therapy of rheumatoid arthritis, and has the advantages of biological safety and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to an M2 macrophage membrane-coated ROS-responsive bionic nano drug delivery system targeting rheumatoid arthritis, and its preparation and application. Background Art

[0002] Rheumatoid arthritis is a complex autoimmune chronic inflammatory disease with persistent synovial inflammation, progressive cartilage erosion, and even complications in other organs. The continued progression of rheumatoid arthritis eventually leads to irreversible joint deformity and disability, causing huge medical and economic burdens. Rheumatoid arthritis affects about 1% of adults worldwide, seriously affecting the quality of life. Studies have found that rheumatoid arthritis may be related to autoimmunity, genetics, infection, smoking and other factors. Rheumatoid arthritis is characterized by immune cell infiltration and synovial hyperplasia, leading to pannus formation, articular cartilage destruction and bone erosion. Infiltrating immune cells produce proinflammatory cytokines and stimulate fibroblast-like synoviocytes (FLS) to exert proinflammatory and tissue-destructive effects through aggressive migration and invasion and excessive production of pathogenic mediators (including chemokines, reactive oxygen species (ROS), receptor activators of NF-κB ligands, and metalloproteinases). At present, clinical treatments for rheumatoid arthritis are often accompanied by drug resistance and side effects, and the treatment effect is not ideal. Recent studies have shown that targeting inflammatory fibroblast-like synoviocytes is considered an effective method to restore synovial homeostasis to reverse bone and cartilage destruction, and triptolide has the potential to inhibit the abnormal function of inflammatory fibroblast-like synoviocytes.

[0003] Celastrol is a natural active product extracted from traditional Chinese medicine plants such as Tripterygium wilfordii and Celastrus orbiculatus, belonging to the Celastraceae family. It has a wide range of biological activities such as anti-inflammatory and antioxidant effects, and has shown good therapeutic effects in a variety of inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus. Celastrol is one of the most promising drug candidates in the clinical management of rheumatoid arthritis. It targets multiple signaling pathways involving NF-κB, ROS, DNA damage, cell cycle arrest, and apoptosis to inhibit inflammation and protect cartilage and bone from destruction. However, the clinical application of Celastrol is hampered by low solubility and systemic cytotoxicity, emphasizing the urgent need to develop innovative drug delivery vehicles.

[0004] Mesoporous silica nanoparticles have controllable particle morphology, orderly and regular pores, and adjustable pore size. They can encapsulate a large number of hydrophilic and hydrophobic drugs, and the nano surface is easily modified with a variety of functional groups and biomolecules, thus giving mesoporous silica nanoparticles imaging, targeting, long circulation, stimulus responsiveness and other characteristics, further broadening their applications. At the same time, the modified groups can be used as gating molecules to prevent drug leakage, thereby greatly reducing the toxic side effects of chemical drugs. Since tripterygium wilfordii is highly soluble in organic solvents, mesoporous silica nanoparticles can be loaded with a large amount of tripterygium wilfordii by the impregnation method. Unmodified mesoporous silica nanoparticles only have passive delivery capabilities, and their ability to deliver drugs to targeted arthritis sites is limited. The targeting is insufficient, and the exposed mesoporous silica nanoparticles are easily phagocytosed by the endothelial system after entering the body. The release of inflammatory factors in the lesions of rheumatoid arthritis attracts neutrophils, macrophages, etc. to rapidly aggregate, and through the interaction of adhesion molecules on the cell membrane surface, they are recruited to the inflamed joints. The delivery of tripterygium wilfordii using ROS-responsive mesoporous silica nanoparticles coated with M2 macrophage membranes can not only reduce the recognition and clearance of mesoporous silica nanoparticles by the endothelial system, but also actively target the synovial membrane of arthritis hyperplasia through adhesion molecules and chemokine receptors on the surface of M2 macrophage membranes, exert the pharmacological efficacy of tripterygium wilfordii, inhibit the migration and invasion of synovial fibroblasts, exert anti-inflammatory effects, and reverse the ratio of M1 macrophages to M2 macrophages in the lesion site.

[0005] The ROS-responsive lipid membrane coated on the M2 macrophage membrane acts as a gate switch. After entering the arthritis site, the high level of ROS in the activated synovial fibroblasts cleaves the lipid membrane, exposing the drug-loaded mesoporous silica pores, accelerating the release of celastrol, enhancing its efficacy in treating rheumatoid arthritis, and further reducing the migration and invasion of synovial fibroblasts and bone destruction in rheumatoid arthritis. At the same time, the inflammatory factor receptors and chemokine receptors on the surface of the M2 macrophage membrane, such as TNF-αR, IL-1R, CCR2, can neutralize the inflammatory factors TNF-α, IL-1β and chemokine CCL2 caused by rheumatoid arthritis, and reduce the level of inflammation. Therefore, the mesoporous silica nano-drug delivery system with ROS-responsive phospholipids coated on the M2 macrophage membrane as a gate switch has a wide range of clinical application value. Summary of the invention

[0006] In view of this, it is necessary to provide a ROS-responsive bionic nano drug delivery system coated with M2 macrophage membrane targeting rheumatoid arthritis, preparation and application. The purpose of the present invention is to construct a ROS-responsive bionic nano drug delivery system coated with M2 macrophage membrane targeting rheumatoid arthritis and a preparation method thereof, increase the targeting of traditional Chinese medicine preparations in rheumatoid arthritis lesions, and enhance their therapeutic effects.

[0007] A macrophage membrane-coated ROS-responsive bionic nano drug delivery system comprises encapsulated tripterygium wilfordii, a ROS-responsive phospholipid membrane, a mesoporous silica nanocarrier and an M2 macrophage membrane, wherein the mesoporous silica nanoparticles are prepared by a template method, the ROS-responsive phospholipid membrane is ultrasonically loaded, the tripterygium wilfordii is loaded by an immersion method, and the ROS-responsive phospholipid mesoporous silica nanoparticles and the M2 macrophage membrane are coated with an outer membrane by ultrasound.

[0008] Preferably, the M2 macrophage membrane is obtained by inducing differentiation of the RAW264.7 cell line through IL-4 and IL-13 stimulation, and expresses relevant adhesion molecules, inflammatory factor receptors and chemokine receptors; the M2 macrophage membrane is obtained by mechanical grinding and differential centrifugation extraction in sequence, and cell membrane vesicles are obtained by ultrasonic treatment; the relevant adhesion molecules, inflammatory factor receptors and chemokine receptors include LFA-1, TNF-αR, IL-1R and CCR2.

[0009] Preferably, the encapsulated tripterygium wilfordii is tripterygium wilfordii, an effective ingredient of the traditional Chinese medicine Tripterygium wilfordii, or its salt, and the components of the mesoporous silica nanocarrier are phospholipids, cholesterol and silica.

[0010] Preferably, the mesoporous silica silicon source is TEOS and MSN is prepared by a template method, hexadecyltrimethylammonium bromide (CTAB) is used as a template, tetraethoxysilane (TEOS) is added in an alkaline environment, the template is removed by centrifugal washing and high-temperature calcination to prepare MSN, and celastrol is encapsulated by an impregnation method using dichloromethane as a solvent.

[0011] Preferably, the phospholipid is one or a mixture of soybean lecithin, hydrogenated soybean lecithin, dioleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and their derivatives; the organic solvent is one or a mixture of ethanol and chloroform.

[0012] Preferably, the structure of mesoporous silica nanoparticles is that the mesoporous channels are arranged in an orderly hexagonal pattern and have a large specific surface area, and can load tripterygium wilfordii, with a corresponding encapsulation rate between 28% and 50%, and a drug loading between 5% and 19%.

[0013] Preferably, the method of coating the ROS-responsive phospholipid is ultrasound, with an ultrasound intensity of 100W to 500W and an ultrasound time of 5 to 30 minutes. The immersion method for encapsulating Celastrol was initially 12 to 48 hours and later extended to 24 hours.

[0014] Preferably, the encapsulated contents include ROS-responsive phospholipid DOPC, which has the ability to respond to ROS in inflammatory tissue cells.

[0015] Preferably, the mesoporous silica nanoparticles and the M2 macrophage membrane are fused with each other by ultrasound at 100W to 500W for 2 minutes.

[0016] Preferably, the entrapped ROS-responsive phospholipid DOPC lipid membrane ruptures upon encountering a high level of ROS, thereby accelerating the release of the entrapped drug under the condition of a high level of ROS.

[0017] Preferably, the average particle size of the mesoporous silica nanoparticles is 120 to 200 nm, and the particle size of the M2 macrophage membrane-coated ROS-responsive biomimetic nano-drug delivery system is 160 to 240 nm.

[0018] A method for preparing a macrophage membrane-coated ROS-responsive bionic nano drug delivery system comprises the following steps:

[0019] Step 1: extract M2 macrophage membranes, stimulate RAW264.7 cells with IL-4 and IL-13 to induce differentiation, obtain the expression of LFA-1, TNF-αR, IL-1R, and CCR2, and mechanically grind and extract the cell membranes by differential centrifugation and disperse them in double distilled water by ultrasonic dispersion;

[0020] Step 2, preparing mesoporous silica nanoparticles, preparing MSN by template method, using hexadecyltrimethylammonium bromide (CTAB) as template, adding tetraethoxysilane (TEOS) in alkaline environment, centrifugation washing and high-temperature calcination to remove the template to prepare MSN, and using dichloromethane as solvent to encapsulate tripterygium wilfordii;

[0021] Step 3, using phospholipids such as dioleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and their derivatives as raw materials, coating mesoporous silica by thin film hydration method and ultrasonic treatment to prepare ROS-responsive lipid membrane-coated mesoporous silica nanoparticles loaded with celastrol;

[0022] Step 4: The pre-prepared lipid membrane-coated mesoporous silica nanoparticles loaded with tripterygium wilfordii are co-incubated with M2 macrophage membranes and subjected to ultrasound to obtain a ROS-responsive bionic nano-drug delivery system.

[0023] Preferably, in step 1, RAW264.7 cells are passaged and expanded in a cell culture incubator with DMEM medium + 10% FBS + 1% PS, IL-4 and IL-13 are added to the cell culture medium to induce and stimulate the cells, and the cells are washed after centrifugation to remove the supernatant; the cells are resuspended with membrane extraction buffer, mechanically homogenized on ice, mixed with sucrose, centrifuged, and structures such as cell nuclei and mitochondria are removed, the supernatant is centrifuged, and the cell membrane is resuspended with distilled water after centrifugation, and freeze-dried and stored for later use.

[0024] Preferably, the DMEM medium + 8-15% FBS + 1% PS is passaged and expanded in a cell culture incubator at 37°C and a CO2 concentration of 5%, 10-50 ng / mL IL-4 and 10-50 ng / mL IL-13 are added to the cell culture fluid to induce the cells to stimulate the cells for 12-48 hours, the cells are centrifuged at 800-1500 rpm / min for 3-8 minutes to remove the supernatant, and then washed 2-6 times with PBS buffer, the temperature of the PBS buffer is 4°C, the speed of the centrifugal washing is 800-1500 rpm / min, and the washing time is 3-8 minutes;

[0025] In step 2, when tetraethoxysilane is added in an alkaline environment, it is added dropwise, and it takes 2 to 6 minutes for each 1 mL; celastrol is dissolved in dichloromethane, and mesoporous silica nanoparticles are added. After 12 to 48 hours, centrifugation is performed at 10,000 to 14,000 rpm for 3 to 10 minutes to obtain mesoporous silica nanoparticles loaded with celastrol.

[0026] Preferably, the membrane extraction buffer is prepared from 10 mM Tris, 1 mM MgCl2, 1 M sucrose and 1% protease inhibitor. After centrifugation, the cell membrane is resuspended in distilled water, and then freeze-dried and stored at -80°C for later use.

[0027] Preferably, in step 2, hexadecyltrimethylammonium bromide (CTAB) is weighed and dissolved in water containing sodium hydroxide solution, heated and stirred, anhydrous ethanol is added, tetraethyl silicate is added, stirred, cooled, centrifuged, washed, dried, and calcined to obtain mesoporous silica nanoparticles, and celastrol is dissolved in dichloromethane, mesoporous silica is added, stirred, centrifuged, the precipitate is collected, and dried to obtain mesoporous silica nanoparticles loaded with celastrol.

[0028] Preferably, in step three, dioleoyl phosphatidylcholine, polyethylene glycol-distearoyl phosphatidylethanolamine, and cholesterol are accurately weighed in a molar ratio of 58:5:38 and dissolved in chloroform to prepare blank liposomes by a thin film hydration method.

[0029] Preferably, in step three, the blank liposome membrane and the mesoporous silica nanoparticles are ultrasonicated at 100W to 500W for 5 to 30 minutes to obtain mesoporous silica nanoparticles with ROS-responsive lipid-gated switches.

[0030] The macrophage membrane-coated ROS-responsive bionic nano drug delivery system of the present invention is used for preparing drugs for targeted treatment of rheumatoid arthritis.

[0031] The invention uses tetraethoxysilicon source as raw material, hexadecyltrimethylammonium bromide as template agent and other raw materials to prepare mesoporous silica nanoparticles as nano cores to encapsulate tripterygium wilfordii, and lipid components such as polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG), dioleoylphosphatidylcholine (DOPC) and cholesterol are used as raw materials to prepare lipid membranes as ROS-responsive gating switches to form ROS-responsive mesoporous silica nanoparticles loaded with tripterygium wilfordii, and then bionic fusion is achieved with the outer M2 macrophage membrane through probe ultrasound technology, RAW264.7 cells are induced to differentiate with IL-4 and IL-13 to obtain M2 macrophages, and the cell membrane-coated ROS-responsive mesoporous silica nanoparticles loaded with tripterygium wilfordii (Cel@M2-L-MSN) are extracted to construct an M2 macrophage membrane-coated ROS-responsive bionic nano drug delivery system (Cel@M2-L-MSN). The bionic nano-drug delivery system can not only utilize the surface molecules of the M2 macrophage membrane to increase the biocompatibility of the system and reduce the phagocytosis of the endothelial system, but also enable the M2 macrophages to actively tend to inflammatory arthritis, target the synovium of rheumatoid arthritis, and deliver the effective ingredients of anti-inflammatory Chinese medicine to the affected joint lesions. It can also play the role of nanoparticles in slowly releasing drugs in the blood circulation, prolonging the drug action time, and accelerating the release of drugs in cells with high levels of ROS in inflammatory lesions.

[0032] In the present invention, the drug used is tripterygium wilfordii, an effective component of the Chinese herbal medicine tripterygium wilfordii. Studies have shown that tripterygium wilfordii has anti-inflammatory and synovial migration and invasion inhibitory effects. Tripterygium wilfordii is loaded into ROS-responsive mesoporous silica nanoparticles loaded with tripterygium wilfordii and coated with M2 macrophage membranes to increase drug aggregation in rheumatoid arthritis lesions and accelerate drug release, thereby increasing the efficacy of treating rheumatoid arthritis.

[0033] The present invention uses human synovial fibroblasts (MH7A) to simulate the synovial state of rheumatoid arthritis after being treated with lipopolysaccharide (LPS).

[0034] The present invention constructs a rat rheumatoid arthritis model (AIA) by subcutaneously injecting inactivated tuberculosis bacteria dispersed in mineral oil at the base of the tail, and evaluates the in vivo efficacy of the M2 macrophage membrane-coated ROS-responsive bionic nano-drug delivery system, which is common and recognized in the art.

[0035] The administration mode of the M2 macrophage membrane-coated ROS-responsive bionic nano drug delivery system prepared by the present invention is intravenous injection.

[0036] The advantages of the present invention are:

[0037] The prepared M2 macrophage membrane-coated ROS-responsive biomimetic nano-drug delivery system is different from other targeted nano-carriers modified with a single molecule or multiple molecules. The ROS-responsive biomimetic nano-drug delivery system has unique advantages in the treatment of rheumatoid arthritis:

[0038] 1. Using cytokines to stimulate the transformation of M0 macrophages into M2 macrophages, the protein molecules expressed on the cell surface can actively target the synovial tissue of rheumatoid arthritis;

[0039] 2. The ROS-responsive biomimetic mesoporous silica nanoparticles can effectively encapsulate tripterygium wilfordii by the impregnation method, and have a sustained release effect in normal blood circulation, prolonging the drug action time, and accelerate drug release in synovial cells of rheumatoid arthritis with high ROS levels;

[0040] 3. The ROS-responsive mesoporous silica nanoparticles loaded with tripterygium wilfordii coated with the M2 macrophage membrane have an inflammation neutralizing effect and enhance the therapeutic effect on rheumatoid arthritis;

[0041] 4. The ROS-responsive mesoporous silica nanoparticles loaded with tripterygium wilfordii are coated by the M2 macrophage membrane, which increases the biocompatibility of the system, reduces the phagocytosis of the endothelial system, and has high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The morphology, particle size and potential characterization of the ROS-responsive bionic nano-drug delivery system of mesoporous silica nanoparticles and M2 macrophage membranes in the present invention. In the figure, A is a transmission electron microscope image, and B and C are statistical graphs of the average particle size and Zeta potential, respectively.

[0043] Figure 2 A and B are drug loading time and Figure 2 C and D show the effect of dosing ratio on the drug loading capacity and encapsulation efficiency of tripterygium wilfordii; E shows the drug loading capacity and encapsulation efficiency of tripterygium wilfordii by the ROS-responsive bionic nanodrug delivery system coated with M2 macrophage membrane when the drug loading time is 24 h and the dosing ratio is 1:2 in the present invention.

[0044] Figure 3These are the qualitative detection results of surface protein expression of the nano-drug delivery system in the present invention. In the figure, A is the protein expression spectrum of the ROS-responsive bionic nano-drug delivery system of M2 macrophage cell membrane and M2 macrophage membrane-coated after induction of 10 ng / mL IL-4 and 10 ng / mL IL-13, B is the verification of its characteristic protein expression, C is the verification of the characteristic protein expression of the ROS-responsive bionic nano-drug delivery system of M2 macrophage cell membrane and M2 macrophage membrane-coated after induction of 25 ng / mL IL-4 and 25 ng / mL IL-13, and D is the verification of the characteristic protein expression of the ROS-responsive bionic nano-drug delivery system of M2 macrophage cell membrane and M2 macrophage membrane-coated after induction of 50 ng / mL IL-4 and 50 ng / mL IL-13.

[0045] Figure 4 The ROS responsiveness of the M2 macrophage membrane-coated ROS-responsive bionic nano drug delivery system of the present invention was verified. Simvastatin was used as a model drug, and the mesoporous silica nanoparticles loaded with simvastatin after modifying the lipid membrane released the drug in the presence or absence of hydrogen peroxide.

[0046] Figure 5 A is a confocal image of the targeting ability of the biomimetic mesoporous silica nanoparticles of the present invention to synovial fibroblasts, and B is the quantitative analysis of its mean fluorescence intensity.

[0047] Figure 6 In the present invention, A is the cytotoxicity of free tripterygium wilfordii at different concentrations and the ROS-responsive bionic nanodrug delivery system coated with M2 macrophage membrane on synovial fibroblasts; B is the cytotoxicity of free tripterygium wilfordii at different concentrations and the ROS-responsive bionic nanodrug delivery system coated with M2 macrophage membrane on synovial fibroblasts.

[0048] Figure 7 The effects of ROS-responsive mesoporous silica nanoparticles loaded with celastrol on the migration and invasion of synovial fibroblasts coated with M2 macrophages in the present invention, in which A and B are migration results in the cell scratch assay and invasion results in the transwell assay, respectively, and C and D are statistical results of the migration area in the migration assay and the number of cells in the invasion assay, respectively.

[0049] Figure 8 The inflammatory neutralization ability of the ROS-responsive bionic nano-drug delivery system coated with M2 macrophage membranes at different concentrations in the present invention on inflammatory factors (A) TNF-α and (B) IL-1β.

[0050] Fig. 9Figure A is an in vitro imaging picture of the main organs and hind limbs of the rheumatoid arthritis rat model 24 hours after the ROS-responsive bionic nanodrug delivery system with or without M2 macrophage membrane coating in the present invention, and B and C are quantitative fluorescence intensity graphs; Figure D is an in vivo imaging picture of the hind limbs of the rheumatoid arthritis rat model at different time points of the ROS-responsive bionic nanodrug delivery system with or without M2 macrophage membrane coating in the present invention, and E is a quantitative fluorescence intensity graph.

[0051] Fig.10 This is the therapeutic effect of the M2 macrophage membrane-coated ROS-responsive bionic nanodrug delivery system on rats with rheumatoid arthritis. In the figure, A is a gross picture of the ankle joint of the rat after treatment, B is the change in foot volume of the arthritis rat during the treatment, C is the arthritis severity score during the treatment, and D is the change in body weight of the arthritis rat during the treatment.

[0052] Fig.11 This is an imaging evaluation of the therapeutic effect of the M2 macrophage membrane-coated ROS-responsive bionic nanodrug delivery system on rats with rheumatoid arthritis. Figure A is the CT imaging result of the ankle joint of the rat after treatment, and Figure B is the ankle bone density (BMD) result of the rat after treatment.

[0053] Fig.12 This is a diagram showing the inflammation-alleviating effect of the M2 macrophage membrane-coated ROS-responsive bionic nano-drug delivery system of the present invention on rats with rheumatoid arthritis, wherein A, B, and C in the figure respectively represent the serum TNF-α, IL-1β, and IL-10 levels of the rats after treatment. DETAILED DESCRIPTION

[0054] In order to make the technical solution of the present invention easier to understand, the technical solution of the present invention is now clearly and completely described in the form of specific embodiments in conjunction with the accompanying drawings.

[0055] Example 1

[0056] Extraction and preparation of M2 macrophage membranes:

[0057] DMEM medium + 8-15% FBS + 1% PS was passaged and expanded in a cell culture incubator at 37°C and a CO2 concentration of 5%, and 10-50 ng / mL IL-4 and 10-50 ng / mL IL-13 were added to the cell culture medium to induce the cells to stimulate the cells for 12-48 hours, and the supernatant was removed by centrifugation at 800-1500 rpm / min for 3-8 minutes, and then washed 2-6 times with PBS buffer. The temperature of the PBS buffer was 4°C, the speed of the centrifugal washing was 800-1500 rpm / min, and the washing time was 3-8 minutes. In this Example 1, RAW264.7 cells were passaged and expanded in a cell culture incubator at 37°C and a CO2 concentration of 5%, and 10 ng / mL IL-4 and 10 ng / mL IL-13 were given in the cell culture medium. IL-13 stimulated cells for 24h, centrifuged at 1200rpm / min for 5min to remove the supernatant, and washed 3 times with PBS, the temperature was set to 4℃, the washing speed was set to 1200rpm / min, and the time was set to 5min. The prepared membrane extraction buffer was mixed with 10mM Tris, 1mM MgCl2 and 1% protease inhibitor, the cells were resuspended, mechanically homogenized on ice, centrifuged, the temperature was set to 4℃, the weight was set to 2000g, the time was set to 15min, the cell nucleus and other structures were removed, the supernatant was centrifuged again, the temperature was 4℃, the weight was set to 3000g, the time was set to 30min, the precipitate was taken and the cell membrane was resuspended with distilled water, and the cell membrane was freeze-dried and stored at -80℃ for later use.

[0058] Preparation of Mesoporous Silica Nanoparticles:

[0059] MSNs were prepared by a template method, with hexadecyltrimethylammonium bromide (CTAB) as a template, tetraethoxysilane (TEOS) added in an alkaline environment, centrifuged and washed, and calcined at high temperature to remove the template to prepare MSNs, and celastrol was encapsulated in dichloromethane as a solvent by an impregnation method. When tetraethoxysilane was added in an alkaline environment, it was added dropwise, and it took 2 to 6 minutes for every 1 mL; celastrol was dissolved in dichloromethane, and mesoporous silica nanoparticles were added. After 12 to 48 hours, it was centrifuged at 10000 to 14000 rpm for 3 to 10 minutes to obtain mesoporous silica nanoparticles loaded with celastrol. In this embodiment 1, MSNs were prepared according to the template method: hexadecyltrimethylammonium bromide (CTAB) was dissolved in ultrapure water, sodium hydroxide was added, the mixture was heated to 80°C in a water bath, anhydrous ethanol was added for 5 minutes, tetraethoxysilane (TEOS) was added, the reaction lasted for 2 hours, centrifuged, washed 3 times, dried, and calcined. The average particle size of the liposomes was determined by a particle size analyzer to be approximately 141 nm, and the zeta potential result was approximately -25 mV. Figure 1 .

[0060] Preparation of biomimetic mesoporous silica nanoparticles with ROS-responsive lipids as gate switches:

[0061] The lipid membrane was prepared by the thin film hydration method. Dioleoyl phosphatidylcholine, polyethylene glycol-distearoyl phosphatidylethanolamine, and cholesterol were accurately weighed in a molar ratio of 58:5:38 and dissolved in chloroform. The mixture was placed in a rotary evaporator at 50°C for 30 min to form a thin membrane. MSN was added, hydrated, ultrasonicated for 5-30 min, centrifuged at 12000 rpm for 10 min, washed, and dried to obtain ROS-responsive lipid-coated mesoporous silica nanoparticles. Dissolve tripterygium wilfordii in dichloromethane, add MSN, stir for 24h, centrifuge, take the precipitate, dry, and obtain mesoporous silica nanoparticles loaded with tripterygium wilfordii (Cel@MSN). Prepare ROS-responsive lipid-coated mesoporous silica nanoparticles loaded with tripterygium wilfordii (Cel@L-MSN) according to the above-mentioned thin film hydration method and fuse with the M2 macrophage membrane through probe ultrasound (100W, 2min), centrifuge and purify to obtain biomimetic mesoporous silica nanoparticles (Cel@M2-L-MSN) with ROS-responsive lipids as gated switches. Collect and quantify the free tripterygium wilfordii after centrifugation, and determine the free tripterygium wilfordii by ultraviolet spectrophotometer. According to the formula:

[0062] Encapsulation efficiency = (total tripterygium wilfordii - free tripterygium wilfordii) / total tripterygium wilfordii × 100%

[0063] Drug loading rate = (total tripterygium wilfordii - free tripterygium wilfordii) / ((total tripterygium wilfordii - free tripterygium wilfordii) + carrier mass) × 100%

[0064] like Figure 1 After coating, the pores of silica nanoparticles disappeared and the particle size increased. The particle size increased to about 180-220nm after the M2 macrophage membrane was coated with lipid mesoporous silica by a particle size analyzer. The original potential of the lipid mesoporous silica was about -11mV, but after coating with the negatively charged M2 macrophage membrane, the potential of Cel@M2-L-MSN was about -23mV. Figure 2, the formulation optimization of tripterygium wilfordii loaded: (Figure A, B) the dosage ratio of tripterygium wilfordii and mesoporous silica (1:4, 1:3, 1:2) and (Figure C, D) the drug loading time (12h, 24h, 48h) were used to optimize the formulation. The drug loading was 5%, 11%, 19%, and the encapsulation efficiency was 28%, 39%, and 50%, respectively. Figure E shows that when the dosage ratio of mesoporous silica was optimized to 1:2 and the drug loading time was 24h, the encapsulation efficiency of tripterygium wilfordii in Cel@MSN and Cel@M2-L-MSN reached 50%, and the drug loading rate was about 9%. Blank mesoporous silica, M2 macrophage membrane and biomimetic mesoporous silica were subjected to gel electrophoresis and Coomassie brilliant blue staining and Western blot. Figure 3 It can be observed that the biomimetic nano drug delivery system has related proteins consistent with the M2 macrophage membrane, and the biomimetic mesoporous silica retains the key proteins of the M2 macrophage membrane, such as LFA-1, TNF-αR, IL-1R and CCR2. At the same time, the expression of M2 macrophage membrane characteristic proteins in samples treated with IL-4 (25ng / mL) and IL-13 (25ng / mL) for 48h, centrifuged at 800rpm / min for 3min to remove the supernatant, and washed twice with PBS buffer was investigated; and the expression of M2 macrophage membrane characteristic proteins in samples treated with IL-4 (50ng / mL) and IL-13 (50ng / mL) for 12h, centrifuged at 1500rpm / min for 8min to remove the supernatant, and washed 6 times with PBS buffer were obtained, and similar results were obtained.

[0065] Example 2

[0066] Verification of ROS responsiveness of ROS-responsive nano-drug delivery system:

[0067] Dioleoylphosphatidylcholine, polyethylene glycol-distearoylphosphatidylethanolamine, and cholesterol were accurately weighed in a molar ratio of 58:5:38 and dissolved in chloroform to prepare a ROS-responsive nano drug delivery system (Cel@L-MSN) according to the above method.

[0068] The in vitro release behavior of simvastatin (SIM) in 5 μM H2O2 at 37 °C PBS was investigated by detecting the in vitro release of the drug. ROS-responsive mesoporous silica loaded with simvastatin, such as Figure 4 The ROS-responsive mesoporous silica loaded with simvastatin released nearly 70% of its drug content within 3 hours, while the ROS-responsive mesoporous silica nanodrug delivery system loaded with simvastatin in a hydrogen peroxide-free environment continued to release drug content until 12 hours, and the release curve became flat. This shows that the bionic nanodrug delivery system has a sustained release effect and can achieve the purpose of continuous drug delivery.

[0069] Example 3

[0070] Targeting ability of M2 macrophage membrane-coated biomimetic mesoporous silica to synovial fibroblasts:

[0071] The biomimetic mesoporous silica (Cel@M2-L-MSN) coated with M2 macrophage membrane was prepared according to the above method. MH7A cells were inoculated on the slides of 24-well plates and cultured overnight with culture medium containing 100 ng / mL LPS. Then, MSN-FITC, L-MSN-FITC, M2-MSN-FITC, and M2-L-MSN-FITC were co-incubated with MH7A cells for 4 hours. The concentration of nanoparticles in each group was kept at 30 μg / mL. After DAPI staining, the photos were taken under a confocal microscope. Figure 5 The increase in the nanofluorescence signal targeting MH7A is related to whether the nanoparticles are coated with the M2 macrophage membrane. This is due to the enhanced interaction between LFA-1 expressed by M2 macrophages and ICAM-1 on MH7A cells. Moreover, comparing the M2-MSN-FITC and M2-L-MSN-FITC groups, the modified lipid membrane does not affect the nanofluorescence signal targeting MH7A. The above results indicate that the surface proteins of the M2 macrophage membrane play a key role in the MH7A cell targeting process.

[0072] Example 4

[0073] Safety evaluation of ROS-responsive biomimetic nano-drug delivery system coated with M2 macrophage membrane:

[0074] The cell culture density of 96-well plates was 1 × 10 4 MH7A cells and RAW 264.7 cells were incubated with different concentrations (50, 100, 200, 400 and 800 ng / mL) of Cel@M2-L-MSN and Free Cel for 24 h, then gently rinsed with PBS, added with 10% Alamar Blue, and incubated at 37°C for 4 h. The absorbance was measured at 570 nm and 600 nm using a microplate reader, and the cell viability was calculated to evaluate the cytotoxicity of Cel@M2-L-MSN and Free Cel. Figure 6 As shown, the cell viability of both cells was over 90% at different concentrations, confirming that Cel@M2-L-MSN had no obvious toxicity to MH7A cells and RAW 264.7 cells at the predetermined dose.

[0075] Example 5

[0076] Evaluation of the inhibitory effect of ROS-responsive biomimetic nano-drug delivery system coated with M2 macrophage membrane on the migration and invasion of synovial fibroblasts:

[0077] For the migration experiment, MH7A cells were seeded into 6-well plates. When the cells reached 80-90% confluence, 5 of the wells were activated with LPS (1 μg / mL) for 12 hours. Next, a straight line was drawn in the 6-well plate with a pipette tip, rinsed with PBS, and cultured in serum-free medium. Free Cel, Cel@MSN, Cel@L-MSN, and Cel@M2-L-MSN (equivalent to 25 ng / mL Cel) were then added and incubated for 24 hours. The scratched area was observed and photographed using a microscope (CKX53, Olympus, Japan). The invasion experiment was similar to the migration experiment, and 1×10 4 MH7A cells were seeded in a 24-well plate and inoculated in a serum-free medium without matrix gel in the Transwell chamber. Complete medium containing 10% FBS was added to the lower chamber. Free Cel, Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN (equivalent to 25ng / mL Cel) were then added and incubated for 24h. The remaining cells in the chamber were gently wiped with a cotton swab, and the cells that migrated to the back through the membrane were fixed with 4% paraformaldehyde for 30min and stained with 0.1% crystal violet for 15min. The number of migrated cells was counted in three random fields under an optical microscope. Figure 7 , LPS stimulation significantly enhanced the migration and invasion ability of MH7A cells. Celastrol treatment significantly reduced the migration and invasion of cells. Compared with the Model group, free Cel, Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN all reduced the number of migrating and invasive cells. Cel@M2-L-MSN had a better inhibitory effect, which was due to the targeting of M2 and the ROS-responsive drug release.

[0078] Example 6

[0079] In vitro inflammation neutralization effect of biomimetic mesoporous silica coated with M2 macrophage membranes:

[0080] Cel@M2-L-MSN was diluted to a final concentration of 0, 32, 64, 128, 256, 512, 1024, and 2048 μg / mL and mixed with TNF-α and IL-1β, respectively. After incubation at 37°C for 1 h, the nanoparticles were removed by centrifugation at 12,000 rpm for 30 min at 4°C. The remaining TNF-α and IL-1β contents in the supernatant were detected by ELISA, and the binding ability of Cel@M2-L-MSN with TNF-α and IL-1β was determined to evaluate its inflammatory neutralization ability. Figure 8As shown, Cel@M2-L-MSN exhibited concentration-dependent binding ability to TNF-α and IL-1β. The half-maximal inhibitory concentration of Cel@M2-L-MSN for TNF-α was as low as 968.1μg / mL, and for IL-1β binding was as low as 376.4μg / mL, indicating that the nanosystem has a strong ability to neutralize inflammation.

[0081] Example 7

[0082] Targeted study of M2 macrophage membrane-coated biomimetic nano-drug delivery system on AIA model:

[0083] SD rats weighing about 180 g were subcutaneously injected at the base of their tails with 0.2 mg of heat-killed Mycobacterium tuberculosis (ground in an ice bath) dispersed in 100 μL of mineral oil. About 10-14 days later, the ankle joints of the rats became red and swollen, and the foot volume increased, which was considered a successful model construction and was used for subsequent experiments.

[0084] AIA model rats were randomly divided into 4 groups, 3 rats in each group, and Cy5, MSN-Cy5, L-MSN-Cy5, and M2-L-MSN-Cy5 were injected into the tail vein respectively. After 24 hours, the hind limbs and major organs (heart, liver, spleen, lung, and kidney) of the rats were taken for in vitro imaging using an in vivo imaging system (IVIS, USA), and their fluorescence intensity was calculated. Fig. 9 After in vitro treatment, obvious fluorescence signals were observed in the inflamed joints of AIA rats. The fluorescence intensity of M2-L-MSN-Cy5 in the inflamed joints at 6 h was 2.53 and 2.48 times that of the L-MSN-Cy5 group and MSN-Cy5, respectively.

[0085] Example 8

[0086] The therapeutic effect of M2 macrophage membrane-coated ROS-responsive biomimetic nanodrug delivery system on AIA model:

[0087] AIA rat modeling was performed as described in Example 7, and AIA model rats were randomly divided into 5 groups, 6 in each group. Starting from the 14th day of modeling, PBS, free Cel, Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN were injected into the tail vein at a dose of 1 mg / kg / 2 days of tripterygium wilfordii, and the drugs were administered once every two days for 7 times. Among them, the PBS group was used as the model group Model, and 6 healthy rats without modeling were selected as the control Control, and the remaining groups were named free Cel, Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN. During the treatment, arthritis scores were performed every 3 days, and the changes in rat foot volume were measured with a foot volume meter, and the changes in rat weight were measured. After the administration, all rats were anesthetized, and the inflammatory factors TNF-α, IL-1β and IL-10 were determined using an ELISA kit after blood was taken from the abdominal aorta; the corresponding hind feet were taken out and observed and bone density analyzed by Micro-CT imaging system.

[0088] The scoring rules for arthritis of the limbs and tail are as follows (the total score of the limbs and tail is the final score):

[0089] 0 points: no erythema or swelling;

[0090] 1 point: slight erythema and swelling;

[0091] 2 points: diffuse erythema and slight swelling;

[0092] 3 points: moderate erythema and swelling;

[0093] 4 points: severe erythema and swelling, and joint deformity.

[0094] like Fig.10 As shown in A, free Cel, Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN can significantly reduce the redness and swelling of arthritic feet. The arthritis score was used to comprehensively evaluate the disease progression of different treatments in each group. Fig.10 B, The change trend of the foot volume increase in each group was consistent with the change trend of the arthritis score. The results showed that compared with the model group, the swelling of the RA foot in the Cel treatment group was significantly reduced. Compared with the Cel group, the treatment effects of Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN were significantly improved. Cel@M2-L-MSN showed better treatment effect. Fig.10C, AIA rats in the model group showed natural disease progression, with a final score of 12.1 points, showing the most severe arthritis manifestations. The arthritis scores of AIA rats in the Cel@M2-L-MSN treatment group showed a slight upward trend, and the final average score was only 2.7 points, indicating the best treatment effect. Although not as good as the Cel@M2-L-MSN group, the treatment effects of the Cel@L-MSN and Cel@MSN groups were still relatively good, with average arthritis scores of 7.2 and 8 points, respectively. The progression of rheumatoid arthritis affects the body weight of rats. Fig.10 As shown in D, the weight gain of AIA rats was significantly slower than that of the normal control group. As the treatment time prolonged, the weight of mice in the Cel@M2-L-MSN group gradually became similar to that of the control group, and the weight gain trend was consistent with the treatment effect.

[0095] Bone erosion is a key pathological feature of rheumatoid arthritis and is often used as an indicator to assess the severity of the disease. Micro-CT was used to detect changes in bone tissue in each group of rats. Fig.11 As shown in the figure, compared with the control group, the inflamed ankle bone surface of the model group was rough and severely eroded, and the bone density (BMD) was significantly reduced. Rats in the model group and Free Cel group showed obvious severe bone destruction, rough bone surface, and severe bone erosion. In contrast, only relatively mild bone erosion was observed in the ankle joints of the Cel@MSN, Cel@L-MSN, and Cel@M2-L-MSN groups, and the degree of bone destruction decreased in turn, indicating that Cel@M2-L-MSN significantly reduced the degree of bone erosion in rheumatoid arthritis.

[0096] The levels of TNF-α, IL-1β and IL-10 in rat serum were detected by ELISA. Fig.12 As shown in the figure, the levels of serum TNF-α and IL-1β in AIA rats were significantly higher than those in normal rats. Compared with the model group, the levels of cytokines in the free Cel, Cel@MSN, Cel@L-MSN and Cel@M2-L-MSN groups were all reduced, while the change trend of the anti-inflammatory cytokine IL-10 in serum was the opposite. Among them, the Cel@M2-L-MSN group showed the most obvious decreases in TNF-α and IL-1β, the highest level of IL-10, and the most obvious anti-inflammatory effect.

[0097] It should be noted that the embodiments described herein are only partial embodiments of the present invention, rather than all implementations of the present invention, and the embodiments are only exemplary, and their role is only to provide a more intuitive and clear way to understand the content of the present invention, rather than to limit the technical solutions described in the present invention. Without departing from the concept of the present invention, all other implementation methods that can be thought of by ordinary technicians in this field without creative work, and other simple replacements and various changes to the technical solutions of the present invention, all belong to the protection scope of the present invention.

Claims

1. A macrophage membrane-coated ROS-responsive biomimetic nano-drug delivery system. Features: The macrophage membrane-coated ROS-responsive bionic nanodrug delivery system includes encapsulated tripterygium wilfordii, ROS-responsive phospholipid membrane, mesoporous silica nanocarrier and M2 macrophage membrane. The mesoporous silica nanoparticles are prepared by a template method, the ROS-responsive phospholipid membrane is loaded by ultrasound, the tripterygium wilfordii is loaded by an immersion method, and the ROS-responsive phospholipid mesoporous silica nanoparticles and M2 macrophage membrane are coated with an outer membrane by ultrasound.

2. The macrophage membrane-coated ROS-responsive biomimetic nano-drug delivery system according to claim 1, Features: The M2 macrophage membrane is obtained by inducing differentiation of the RAW264.7 cell line through IL-4 and IL-13, and after modification, the cell membrane surface overexpresses relevant adhesion molecules, inflammatory factor receptors and chemokine receptors; the M2 macrophage membrane is obtained by mechanical grinding and differential centrifugation extraction in sequence, and cell membrane vesicles are obtained by ultrasonic treatment; the relevant adhesion molecules, inflammatory factor receptors and chemokine receptors include LFA-1, TNF-αR, IL-1R and CCR2.

3. The macrophage membrane-coated ROS-responsive biomimetic nano drug delivery system according to claim 2, Features: The encapsulated drug is tripterygium wilfordii, an effective ingredient of the traditional Chinese medicine Tripterygium wilfordii. The lipid membrane components are phospholipids and cholesterol. The MSN nanocarrier component is silicon dioxide. The phospholipid is one or a mixture of soybean lecithin, hydrogenated soybean lecithin, dioleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and their derivatives; the organic solvent is one or a mixture of ethanol and chloroform.

4. The macrophage membrane-coated ROS-responsive biomimetic nano-drug delivery system according to claim 1, Features: The structure of mesoporous silica nanoparticles is that the mesopores are arranged in hexagonal order and have a large specific surface area, and can load tripterygium wilfordii, with a corresponding encapsulation rate between 28% and 50% and a drug loading between 5% and 19%.

5. The macrophage membrane-coated ROS-responsive biomimetic nano drug delivery system according to claim 1, Features: The lipid film coating method is a thin film hydration method and an ultrasonic method, the ultrasonic intensity is 100W to 500W, and the ultrasonic time is 5 to 30 minutes; The lipid-film-coated MSNs and macrophage membranes were ultrasonicated for 2-5 min to successfully coat the lipid-film-coated MSNs on the cell membranes.

6. The macrophage membrane-coated ROS-responsive biomimetic nano drug delivery system according to claim 1, Features: The average particle size of the mesoporous silica nanoparticles is 120-200 nm, the average particle size of the ROS-responsive mesoporous silica nanoparticles is 140-220 nm, and the particle size of the macrophage membrane-coated ROS-responsive bionic nano drug delivery system is 160-240 nm.

7. A method for preparing the macrophage membrane-coated ROS-responsive bionic nano drug delivery system as claimed in claim 1, Features: The preparation method of the macrophage membrane-coated ROS-responsive biomimetic nano drug delivery system comprises the following steps: Step 1: extracting M2 macrophage cell membranes, inducing differentiation of RAW264.7 cells with IL-4 and IL-13 to obtain M2 macrophages, mechanically grinding and extracting cell membranes by differential centrifugation, and dispersing them in double distilled water by ultrasonic dispersion; Step 2: MSNs were prepared by a template method, using hexadecyltrimethylammonium bromide (CTAB) as a template, adding tetraethoxysilane (TEOS) in an alkaline environment, centrifugation and high-temperature calcination to remove the template to prepare MSNs, and using dichloromethane as a solvent to encapsulate tripterygium wilfordii; Step 3, using phospholipids such as dioleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, cholesterol and their derivatives as raw materials, coating mesoporous silica by thin film hydration method and ultrasonic treatment to prepare ROS-responsive lipid membrane-coated mesoporous silica nanoparticles loaded with celastrol; Step 4: The pre-prepared lipid membrane-coated mesoporous silica nanoparticles loaded with tripterygium wilfordii are co-incubated with macrophage membranes and subjected to ultrasound to obtain a ROS-responsive bionic nano-drug delivery system.

8. The method for preparing the macrophage membrane-coated ROS-responsive bionic nano drug delivery system according to claim 7, Features: In step 1, RAW264.7 cells are passaged and expanded in a cell culture incubator with DMEM medium + 10% FBS + 1% PS, IL-4 and IL-13 are added to the cell culture medium to induce and stimulate the cells, and the cells are washed after centrifugation to remove the supernatant; the cells are resuspended with membrane extraction buffer, mechanically homogenized on ice, mixed with sucrose, centrifuged, and structures such as cell nuclei and mitochondria are removed. The supernatant is centrifuged, and the cell membrane is resuspended with distilled water after centrifugation, and freeze-dried and stored for later use.

9. The method for preparing the macrophage membrane-coated ROS-responsive bionic nano drug delivery system according to claim 8, Features: The DMEM medium + 8-15% FBS + 1% PS was incubated at 37°C, CO 2 Passaging and amplification in a cell culture incubator with a concentration of 5%, adding 10-50 ng / mL IL-4 and 10-50 ng / mL IL-13 to the cell culture medium to induce the cells to stimulate the cells for 12-48 hours, centrifuging at 800-1500 rpm / min for 3-8 minutes to remove the supernatant, and washing 2-6 times with PBS buffer, the temperature of the PBS buffer is 4°C, the speed of the centrifugal washing is 800-1500 rpm / min, and the washing time is 3-8 minutes; In step 2, when tetraethoxysilane is added in an alkaline environment, it is added dropwise, and it takes 2 to 6 minutes for each 1 mL; celastrol is dissolved in dichloromethane, and mesoporous silica nanoparticles are added. After 12 to 48 hours, centrifugation is performed at 10,000 to 14,000 rpm for 3 to 10 minutes to obtain mesoporous silica nanoparticles loaded with celastrol.

10. Use of the macrophage membrane-coated ROS-responsive bionic nano drug delivery system as claimed in claim 1, Features: Macrophage membrane-coated ROS-responsive biomimetic nanodrug delivery system is used to prepare drugs for the targeted treatment of rheumatoid arthritis.

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