Preparation method and application of bionic nanoparticles for treating rheumatoid arthritis

Abstract

The invention aims to provide a preparation method and application of bionic nanoparticles for treating rheumatoid arthritis, and belongs to the technical field of preparation of nano biomedical materials.According to the nanoparticles, hollow carbon spheres serve as carriers, platinum particles are loaded through an impregnation method, and through a bionic modification strategy of a macrophage membrane, the bionic nanoparticles are prepared. The invention constructs a bionic nano-particle for treating rheumatoid arthritis. The nanoparticles can generate singlet oxygen under the condition of no light stimulation, realize cascade generation of singlet oxygen by virtue of catalase-like activity and photo-thermal performance of the nanoparticles, and efficiently kill abnormally proliferated synovial fibroblasts at the rheumatoid arthritis part so as to treat rheumatoid arthritis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobiomedical material preparation, and particularly relates to a preparation method of a biomimetic nanoparticle for rheumatoid arthritis treatment and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease, and its main features are chronic synovitis and vascular pannus formation, which further leads to the progressive destruction of joint cartilage and bone structure, and eventually causes joint deformity and loss of function. The disease seriously affects the quality of life of patients and brings heavy economic burden to society. At present, the clinical treatment of RA mainly relies on non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs such as methotrexate), glucocorticoids and biological agents (such as anti-TNF-α antibodies). These drugs can control the disease to some extent, but still have many limitations. Because the traditional drug delivery method is systemic administration, only a small amount of drug can be enriched in the inflammatory joint, and most of the drugs are distributed in non-target tissues and organs, which limits the therapeutic effect. The non-targeted drugs are easy to cause damage to healthy tissues, for example, methotrexate can cause bone marrow suppression and liver toxicity, long-term use of glucocorticoids can cause osteoporosis, metabolic disorders, etc., and biological agents significantly increase the risk of opportunistic infections. In addition, some drugs have poor water solubility or poor stability, and are easily removed or degraded in the body circulation, making it difficult to maintain an effective therapeutic concentration at the lesion site. After long-term drug use, some patients may experience a decrease or loss of drug response. In order to overcome the above challenges, a nanodrug delivery system has emerged. By loading drugs into nanoparticles (such as liposomes, polymer nanoparticles PLGA, mesoporous silica, etc.), the drugs can be passively targeted to the inflammatory joint tissue with increased vascular permeability to some extent through the enhanced permeability and retention (EPR) effect, providing a new idea for improving drug efficacy and reducing side effects.

[0003] However, traditional nanoparticles still face major obstacles when used in vivo. Because the surface of the nanoparticles is easily recognized and quickly removed by the body's immune system (especially the mononuclear phagocyte system MPS), the blood circulation time is short, which limits the opportunity of the nanoparticles to reach the target. The EPR effect alone has limited targeting efficiency, and the complex microenvironment (such as high expression of various chemokines and adhesion molecules) in the RA lesion area requires the delivery system to have stronger active recognition and binding ability.

[0004] In recent years, biomimetic nanotechnology has brought revolutionary breakthroughs to solve the above problems. This technology modifies the surface of synthetic nanoparticles by mimicking the structure and function of natural biological components (such as cells and proteins), giving them excellent biocompatibility and intelligent targeting ability. Among them, cell membrane biomimetic technology is considered as a promising strategy. For example, by wrapping the cell membrane derived from red blood cells, macrophages, platelets and other cells on the surface of the nanoparticle core, it can inherit the multiple biological functions of the source cells, effectively avoiding phagocytosis by macrophages, and greatly extending its in vivo circulation half-life. The receptors expressed on the membrane surface can make the nanoparticles actively sense and chemotaxis to the tissue site, achieving active targeting. Although biomimetic nanoparticles show great potential, their application in RA treatment is still in the early stages. How to efficiently and stably prepare biomimetic nanoparticles with uniform size, complete membrane wrapping and high drug loading, and verify their high efficiency in targeting, significant therapeutic effect and excellent safety in complex RA pathological environment, are key technical problems that need to be solved by those skilled in the art. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of biomimetic nanoparticles for rheumatoid arthritis treatment and its application. The biomimetic nanoparticles prepared by the method can generate singlet oxygen without external light stimulation, and utilize its catalase-like activity (CAT) and photothermal properties to achieve enhanced release of singlet oxygen. After being coated with macrophage membranes, the targeting ability and biological safety of the nanoparticles are improved, providing a new strategy for the clinical treatment of rheumatoid arthritis with nanomaterials.

[0006] The present application adopts the following technical solutions: A preparation method of biomimetic nanoparticles for rheumatoid arthritis treatment, comprising the following steps: S1, anhydrous ethanol, ammonia solution and deionized water are added to a glass container in proportion, and the solution is fully mixed at room temperature by rapid stirring to obtain a mixed solution one; S2, tetraethyl orthosilicate is slowly dropped into the mixed solution one of step S1, and the solution is continuously stirred rapidly, and the solution gradually changes from transparent to milky white, to obtain a mixed solution two; S3, resorcinol solid is added to the mixed solution two of step S2, and is ultrasonically dispersed to fully dissolve the solid, and is continuously stirred rapidly to obtain a mixed solution three; S4, formaldehyde solution is slowly and uniformly dropped into the mixed solution three of step S3, and after 10 minutes of continuous rapid stirring, the solution gradually changes to light brown, and after 24 hours of medium speed stirring at room temperature, the solution gradually changes from light brown to yellow brown, to obtain a mixed solution four; S5, the mixed solution of S4 is centrifuged at high speed, the supernatant is discarded, and the precipitate is washed with anhydrous ethanol / deionized water at a ratio, centrifuged at high speed for 5 times, and separated into solid and liquid to obtain a yellow-brown precipitate; S6, the yellow-brown precipitate obtained in S5 is dried at 70 DEG C for 12-18h to obtain a yellow-brown solid, which is put into a jade mortar and ground to obtain a yellow-brown powder; S7, the yellow-brown powder in S6 is added into a crucible boat, calcined at 600 DEG C for 30min and at 900 DEG C for 3h in a nitrogen atmosphere to obtain a black powder, which is C nanoparticles; S8, the C nanoparticles in S7 are added into a hydrofluoric acid solution, stirred rapidly, washed with deionized water by centrifugation for 5 times, and separated into solid and liquid to obtain a black precipitate, which is a hollow C nanoparticle; S9, chloroplatinic acid is dissolved in deionized water, ultrasonically dispersed to fully dissolve, then slowly added into the black precipitate solution obtained in S8, and stirred rapidly at constant temperature until the solution is completely evaporated to dryness, and the obtained black powder is gradiently dried at constant temperature to obtain a black powder; S10, the black powder obtained in S9 is resuspended in deionized water, ultrasonically dissolved, and reduced by rapid stirring in a hydrogen stream to obtain CP nanoparticles; S11, the solid CP nanoparticles are dissolved in a PBS buffer, ultrasonically dispersed, mixed with macrophage membrane vesicles at a ratio, and repeatedly extruded through poly carbonate membranes with different pore sizes to obtain the cascade self-enhanced non-light singlet oxygen generating biomimetic nanoparticles CP@mem.

[0007] Further, the volume ratio of the anhydrous ethanol, the ammonia solution and the deionized water in S1 is 60-70mL: 10-20mL: 1.5-3mL; wherein the mass concentration of the ammonia solution is 25%; the stirring speed is 880r / min, and the stirring time is 15min.

[0008] Further, the volume of the tetraethyl orthosilicate in S2 is 1-1.73mL, and the stirring speed is 880r / min; the stirring time is 15min.

[0009] Further, the mass of the resorcinol in S3 is 0.4-0.6g, the ultrasonic dispersion instrument is used for dissolving and dispersing, the frequency is 80kHz, the time is 10min; the stirring speed is 880r / min, and the stirring time is 15min.

[0010] Further, the volume of the formaldehyde solution in S4 is 0.56-0.84mL, the mass concentration is 37%, the stirring time is 10min; the stirring speed is 500r / min, and the stirring time is 24h.

[0011] Further, the speed of the high-speed centrifugation in S5 is 8000 r / min, and the time is 8 min; the volume ratio of the anhydrous ethanol to the deionized water is 1:3.

[0012] Further, the mass concentration of the hydrofluoric acid solution in S8 is 10%, the stirring speed is 880 r / min, and the time is 6-12 h; the speed of the high-speed centrifugation is 12000 r / min, and the time is 120 min.

[0013] Further, the concentration of the chloroplatinic acid solution in S9 is 0.01 M, the dosage is 6.76-8.12 muL, the ultrasonic dispersion frequency is 80 kHz, and the time is 10 min; the stirring speed is 880 r / min, the stirring temperature is 50 DEG C, and the stirring time is 12 h; the gradient drying is carried out in a constant-temperature blast drying oven, and the drying is carried out at 60 DEG C for 30 min and at 100 DEG C for 6 h.

[0014] Further, the ultrasonic dispersion frequency in S10 is 80 kHz, and the time is 10 min; the hydrogen flow is provided by a hydrogen generator, the stirring speed is 500 r / min, and the time is 30-60 min.

[0015] Further, the ultrasonic dispersion frequency in S11 is 80 kHz, and the time is 15 min; the mass ratio of the CP nanoparticles to the macrophage membrane vesicles is 1:5, the poly carbonate membrane pore size is 400 mu m and 200 mu m respectively, and the extrusion is carried out 20 times respectively. The nanoparticles are hollow spherical structures, are uniformly distributed, and have a hydration particle size of 150-200 nm.

[0016] The application of the bionic nanoparticles in the preparation of a kit for treating rheumatoid arthritis.

[0017] The beneficial effects of the application are as follows: The application provides a kit for treating rheumatoid arthritis, which comprises nanoparticles, the nanoparticles are hollow carbon spheres as carriers, platinum particles are loaded on the surfaces of the nanoparticles by an immersion method, and the nanoparticles are uniformly dispersed, have uniform particle sizes, and can generate singlet oxygen without external light stimulation by coating macrophage membranes. The nanoparticles are gathered to a lesion site by using the chemotaxis and homologous targeting performance of the macrophage membranes, first react with oxygen in the lesion site to generate singlet oxygen to achieve a killing effect.

[0018] Further, the catalytic decomposition of hydrogen peroxide produced by the catalase-like activity of the nanoparticles at the lesion site generates oxygen, which in turn provides sufficient raw material for the generation of singlet oxygen, thereby promoting the generation of singlet oxygen. In addition, the thermal effect of the nanoparticles generated under laser irradiation further promotes the generation of singlet oxygen, ultimately achieving a cascade self-enhanced light-free generation of singlet oxygen to treat rheumatoid arthritis by killing abnormally proliferated synovioblasts. The nanoparticles can achieve efficient and powerful killing effect of abnormally proliferated synovioblasts by using a simple and easy-to-operate preparation method. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Transmission electron microscopy images of the nanoparticles synthesized in Example 1 of the present application before and after modification by macrophage membrane vesicles (the large image is before modification, and the small image is after modification).

[0020] Figure 2 Hydrated particle size and potential analysis of the nanoparticles synthesized in Example 1 of the present application before and after modification by macrophage membrane vesicles.

[0021] Figure 3 Gel electrophoresis-Comassie brilliant blue staining map of the nanoparticles synthesized in Example 1 of the present application.

[0022] Figure 4 Photothermal performance of the nanoparticles synthesized in Example 1 of the present application.

[0023] Figure 5 Performance of the nanoparticles synthesized in Example 1 of the present application in catalyzing the decomposition of hydrogen peroxide (H2O2).

[0024] Figure 6 Singlet oxygen generation performance of the nanoparticles synthesized in Example 1 of the present application.

[0025] Figure 7 Cascade-enhanced singlet oxygen generation performance of the nanoparticles synthesized in Example 1 of the present application.

[0026] Figure 8 In vivo photoacoustic imaging image of the nanoparticles synthesized in Example 1 of the present application after modification by macrophage membrane vesicles (mem) and polyvinylpyrrolidone (PVP).

[0027] Figure 9 In vivo anti-rheumatoid arthritis treatment effect image of the nanoparticles synthesized in Example 1 of the present application. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0029] Embodiment 1 A preparation method of a biomimetic nanoparticle for rheumatoid arthritis treatment, comprising the following steps: S1, 70 mL of anhydrous ethanol (EtOH), 10 mL of deionized water (ddH2O) and 3 mL of 25% ammonia solution (NH3·H2O) were added into a 200 mL glass container, respectively, and stirred at a speed of 880 r / min for 15 min at room temperature to fully mix the solutions; S2, 1.73 mL of tetraethyl orthosilicate (TEOS) was slowly added into the mixed solution of step S1, and the solution was continuously stirred at a speed of 880 r / min for 15 min, so that the solution gradually changed from transparent to milky white; S3, 0.4 g of resorcinol was added into the mixed solution of step S2, and the mixed solution was placed in an ultrasonic disperser to fully dissolve the solid, with an ultrasonic frequency of 80 kHz and a time of 10 min. Then the mixed solution was continuously stirred at a speed of 880 r / min for 15 min; S4, 0.56 mL of formaldehyde solution with a mass concentration of 37% was added into the mixed solution of step S3 at a speed of 3 s / drop, and the solution gradually changed to light brown after being continuously stirred at a speed of 880 r / min for 10 min. The solution was stirred at a speed of 500 r / min for 24 h at room temperature to obtain a yellow-brown liquid; S5, the mixed solution obtained in S4 was centrifuged at a high speed of 8000 r / min, and after the supernatant was discarded, the precipitate was washed with anhydrous ethanol: deionized water = 10 mL: 30 mL for 5 times to obtain a yellow-brown precipitate; S6, the yellow-brown precipitate obtained in S5 was placed in a constant-temperature air drying oven, and dried at 70℃ for 12 h to obtain a yellow-brown solid. The yellow-brown solid was fully ground in an agate mortar to obtain a yellow-brown powder; S7, the yellow-brown powder in S6 was added into a crucible boat, and calcined at 600℃ for 30 min and at 900℃ for 3 h under a nitrogen atmosphere in a tube furnace to obtain a black powder, which was C nanoparticles; S8, the black powder in S7 was added into a 10% hydrofluoric acid solution with a concentration of 1 mg / mL, and stirred at a speed of 880 r / min for 6 h. Deionized water was added and centrifuged at a speed of 12000 r / min for 20 min. The washing was repeated for 5 times, and the solid-liquid separation was performed to obtain a black precipitate, which was a hollow C nanoparticle. S9, dissolve chloroplatinic acid in deionized water to make its concentration 0.01M, 80kHz ultrasonic dispersion for 10min to make it fully dissolved, take 6.76μL to slowly add to the black precipitate aqueous solution obtained in S8 at a speed of 5s / drop, stir in a 50℃ constant temperature water bath at a speed of 880r / min until the liquid is completely evaporated, the black powder obtained is dried in a constant temperature air drying oven at 60℃ for 30min, 100℃ for 6h; S10, resuspend the black powder obtained in S9 in deionized water, 80kHz ultrasonic dissolution for 10min, stirring in the hydrogen stream of hydrogen generator at 500r / min for 30min, to obtain CP nanoparticles; S11, dissolve the solid CP nanoparticles in PBS, 80kHz ultrasonic dispersion for 15min, mix with macrophage membrane vesicles according to the ratio of 1mg: 5mg, then repeatedly extrude in a cell extruder 400μm polycarbonate membrane for 20 times, then repeatedly extrude in a 200μm polycarbonate membrane for 20 times, to obtain the cascade self-enhanced non-light singlet oxygen generation biomimetic nanoparticles CP@mem.

[0030] Example 2 A preparation method of a biomimetic nanoparticle for rheumatoid arthritis treatment, comprising the following steps: S1, take anhydrous ethanol (EtOH) 65mL, deionized water (ddH2O) 15mL, 25% ammonia solution (NH3·H2O) 2.75mL into a 200mL glass container, stir at a speed of 880r / min for 15min at room temperature to make the solution fully mixed; S2, take tetraethyl orthosilicate (TEOS) 1.15mL and slowly drop into the mixed solution of step S1, continue to stir at a speed of 880r / min for 15min, the solution gradually changes from transparent to milky white; S3, weigh 0.45g of resorcinol and add it to the mixed solution of step S2, put the mixed solution into an ultrasonic disperser to make the solid fully dissolved, the ultrasonic frequency is 80kHz, the time is 10min. Then continue to stir the mixed solution at a speed of 880r / min for 15min; S4, add 0.6mL of formaldehyde solution with a mass concentration of 37% to the mixed solution of step S3 at a speed of 3s / drop, after 880r / min continuous stirring for 10min, the solution gradually changes to light brown, stir at a speed of 500r / min for 24h at room temperature, to obtain a yellow-brown liquid; S5, centrifuge the mixed solution obtained in S4 at a high speed of 8000r / min, discard the supernatant, and wash the precipitate with anhydrous ethanol: deionized water=15mL: 45mL for 5 times to obtain a yellow-brown precipitate; S6, the yellow brown precipitate obtained in S5 was placed in a constant temperature air drying oven, and dried at 70℃ for 12h to obtain a yellow brown solid, which was ground in a marver mortar to obtain a yellow brown powder; S7, the yellow brown powder in S6 was placed in a crucible boat, and calcined at 600℃ for 30min and at 900℃ for 3h in a tube furnace under nitrogen atmosphere to obtain a black powder, which was C nanoparticles; S8, the black powder in S7 was added to a 10% hydrofluoric acid solution according to a concentration of 1mg / mL, stirred at a speed of 880r / min for 8h, centrifuged at a speed of 12000r / min for 20min, and washed repeatedly for 5 times to obtain a black precipitate, which was a hollow C nanoparticle; S9, chloroplatinic acid was dissolved in deionized water to obtain a concentration of 0.01M, and ultrasonically dispersed at a frequency of 80kHz for 10min to fully dissolve, 7μL of which was slowly added to the black precipitate solution obtained in S8 at a speed of 5s / drop, and stirred in a constant temperature water bath at 50℃ at a speed of 880r / min until the liquid was completely evaporated, and the obtained black powder was dried at 60℃ for 30min and at 100℃ for 6h in a constant temperature air drying oven; S10, the black powder obtained in S9 was resuspended in deionized water, ultrasonically dissolved at a frequency of 80kHz for 10min, and stirred at a speed of 500r / min in a hydrogen stream generated by a hydrogen generator for 40min to obtain CP nanoparticles; S11, the solid CP nanoparticles were dissolved in PBS, ultrasonically dispersed at a frequency of 80kHz for 15min, mixed with macrophage membrane vesicles at a ratio of 10mg: 50mg, and repeatedly extruded through a 400μm polycarbonate membrane in a cell extruder for 20 times, and then repeatedly extruded through a 200μm polycarbonate membrane for 20 times to obtain the cascade self-enhanced non-light singlet oxygen generating biomimetic nanoparticles CP@mem.

[0031] Example 3 A preparation method of a biomimetic nanoparticle for rheumatoid arthritis treatment, comprising the following steps: S1, 60mL of anhydrous ethanol (EtOH), 20mL of deionized water (ddH2O), and 2.5mL of 25% ammonia solution (NH3·H2O) were added to 200mL glass containers respectively, and stirred at a speed of 880r / min for 15min to fully mix the solutions; S2, 1.25mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise into the mixed solution of step S1, and the solution was continuously stirred at a speed of 880r / min for 15min until it changed from transparent to milky white; S3, 0.5 g of resorcinol was weighed and added to the mixed solution of step S2, and the mixed solution was placed in an ultrasonic disperser to fully dissolve the solid. The ultrasonic frequency was 80 kHz, and the time was 10 min. Then the mixed solution was continuously stirred at a speed of 880 r / min for 15 min; S4, 0.65 mL of 37% formaldehyde solution was added to the mixed solution of step S3 at a speed of 3 s / drop, and after stirring at 880 r / min for 10 min, the solution gradually turned light brown. Then it was stirred at a speed of 500 r / min for 24 h at room temperature to obtain a yellow-brown liquid; S5, the mixed solution obtained in S4 was centrifuged at a high speed of 8000 r / min. After discarding the supernatant, the precipitate was washed with a mixture of anhydrous ethanol and deionized water (20 mL:60 mL) for 5 times to obtain a yellow-brown precipitate; S6, the yellow-brown precipitate obtained in S5 was placed in a constant-temperature air drying oven and dried at 70°C for 12 h to obtain a yellow-brown solid. The solid was ground in a jade mortar to obtain a yellow-brown powder; S7, the yellow-brown powder in S6 was added to a crucible boat and calcined in a tube furnace under a nitrogen atmosphere at 600°C for 30 min and at 900°C for 3 h to obtain a black powder, which was C nanoparticles; S8, the black powder in S7 was added to a 10% hydrofluoric acid solution at a concentration of 1 mg / mL and stirred at a speed of 880 r / min for 9 h. Deionized water was added and centrifuged at 12000 r / min for 20 min. The washing was repeated for 5 times, and the solid-liquid separation was performed to obtain a black precipitate, which was a hollow C nanoparticle; S9, chloroplatinic acid was dissolved in deionized water to a concentration of 0.01 M. It was fully dissolved by ultrasonic dispersion at 80 kHz for 10 min. 7.5 μL was slowly added to the black precipitate aqueous solution obtained in S8 at a speed of 5 s / drop. It was stirred in a constant-temperature water bath at 50°C at a speed of 880 r / min until the liquid was completely evaporated. The obtained black powder was dried in a constant-temperature air drying oven at 60°C for 30 min and at 100°C for 6 h; S10, the black powder obtained in S9 was resuspended in deionized water and dissolved by ultrasonic dispersion at 80 kHz for 10 min. It was stirred in a hydrogen stream generated by a hydrogen generator at a speed of 500 r / min for 45 min to obtain CP nanoparticles; S11, the solid CP nanoparticles were dissolved in PBS and dispersed by ultrasonic dispersion at 80 kHz for 15 min. After mixing with macrophage membrane vesicles at a ratio of 4 mg:20 mg, the mixture was repeatedly extruded through a 400 μm polycarbonate membrane in a cell extruder for 20 times and then repeatedly extruded through a 200 μm polycarbonate membrane for 20 times to obtain the cascade self-enhanced non-light singlet oxygen generation biomimetic nanoparticles CP@mem.

[0032] Example 4 A method for preparing a biomimetic nanoparticle for rheumatoid arthritis treatment, comprising the following steps: S1, take 63 mL of anhydrous ethanol (EtOH), 17 mL of deionized water (ddH2O), 2.25 mL of 25% ammonia solution (NH3·H2O) into a 200 mL glass container, stir at room temperature at 880 r / min for 15 min, and mix the solution thoroughly; S2, take 1.35 mL of tetraethyl orthosilicate (TEOS) and slowly drop it into the mixed solution of step S1, continue to stir at 880 r / min for 15 min, and the solution gradually changes from transparent to milky white; S3, weigh 0.53 g of resorcinol and add it to the mixed solution of step S2, put the mixed solution into an ultrasonic disperser to fully dissolve the solid, the ultrasonic frequency is 80 kHz, and the time is 10 min. Then continue to stir the mixed solution at 880 r / min for 15 min; S4, add 0.7 mL of 37% formaldehyde solution to the mixed solution of step S3 at a speed of 3 s / drop, and after 880 r / min continuous stirring for 10 min, the solution gradually changes to light brown, and stir at 500 r / min for 24 h at room temperature, to obtain a yellow-brown liquid; S5, centrifuge the mixed solution obtained in S4 at a high speed of 8000 r / min, discard the supernatant, and then wash the precipitate with anhydrous ethanol: deionized water = 12 mL: 36 mL for 5 times to obtain a yellow-brown precipitate; S6, put the yellow-brown precipitate obtained in S5 into a constant temperature air drying oven, and dry at 70°C for 12 h to obtain a yellow-brown solid, then put it into an agate mortar and grind it thoroughly to obtain a yellow-brown powder; S7, put the yellow-brown powder in S6 into a crucible boat, and calcine it in a tube furnace under nitrogen atmosphere at 600°C for 30 min and at 900°C for 3 h to obtain a black powder, which is C nanoparticles; S8, add the black powder in S7 to a 10% hydrofluoric acid solution at a concentration of 1 mg / mL, stir at 880 r / min for 10 h, centrifuge at 12000 r / min for 20 min after adding deionized water, repeat the washing for 5 times, and then separate the solid and liquid to obtain a black precipitate, which is a hollow C nanoparticle; S9, dissolve chloroplatinic acid in deionized water to make its concentration 0.01M, 80kHz ultrasonic dispersion for 10min to make it fully dissolved, take 7.75μL to slowly add to the black precipitate aqueous solution obtained in S8 at a speed of 5s / drop, in a 50℃ constant temperature water bath with a stirring speed of 880r / min until the liquid is completely evaporated, the black powder obtained is dried in a constant temperature air drying oven at 60℃ for 30min, 100℃ for 6h; S10, resuspend the black powder obtained in S9 in deionized water, 80kHz ultrasonic dissolution for 10min, stirring in the hydrogen stream of hydrogen generator at 500r / min for 50min to obtain CP nanoparticles; S11, dissolve the solid CP nanoparticles in PBS, 80kHz ultrasonic dispersion for 15min, mix with macrophage membrane vesicles according to the ratio of 20mg: 100mg, then repeatedly extrude in a cell extruder 400μm polycarbonate membrane for 20 times, then repeatedly extrude in a 200μm polycarbonate membrane for 20 times to obtain the cascade self-enhanced non-light singlet oxygen generation biomimetic nanoparticles CP@mem.

[0033] Example 5 Preparation of a biomimetic nanoparticle for rheumatoid arthritis treatment: S1, take anhydrous ethanol (EtOH) 67mL, deionized water (ddH2O) 13mL, 25% ammonia solution (NH3·H2O) 2mL into a 200mL glass container, stir at a speed of 880r / min for 15min at room temperature to make the solution fully mixed; S2, take tetraethyl orthosilicate (TEOS) 1.55mL and slowly drop into the mixed solution of step S1, continue to stir at a speed of 880r / min for 15min, the solution gradually changes from transparent to milky white; S3, weigh 0.55g of resorcinol and add it to the mixed solution of step S2, put the mixed solution into an ultrasonic disperser to make the solid fully dissolved, the ultrasonic frequency is 80kHz, the time is 10min. Then continue to stir the mixed solution at a speed of 880r / min for 15min; S4, add 0.7mL of 37% formaldehyde solution to the mixed solution of step S3 at a speed of 3s / drop, after 880r / min continuous stirring for 10min, the solution gradually changes to light brown, stir at a speed of 500r / min for 24h at room temperature to obtain a yellow-brown liquid; S5, centrifuge the mixed solution obtained in S4 at a high speed of 8000r / min, discard the supernatant, and wash the precipitate with anhydrous ethanol: deionized water=25mL: 75mL for 5 times to obtain a yellow-brown precipitate; S6, the yellow brown precipitate obtained in S5 was placed in a constant temperature air drying oven, and dried at 70°C for 12h to obtain a yellow brown solid, which was ground in a marver mortar to obtain a yellow brown powder; S7, the yellow brown powder in S6 was added to a crucible boat, and calcined at 600°C for 30min and at 900°C for 3h in a tube furnace under nitrogen atmosphere to obtain a black powder, which was C nanoparticles; S8, the black powder in S7 was added to a 10% hydrofluoric acid solution at a concentration of 1mg / mL, stirred at 880r / min for 11h, and centrifuged at 12000r / min for 20min with the addition of deionized water, and the washing was repeated for 5 times, and the solid-liquid separation was performed to obtain a black precipitate, which was hollow C nanoparticles; S9, chloroplatinic acid was dissolved in deionized water to obtain a concentration of 0.01M, and ultrasonic dispersion was performed at 80kHz for 10min to fully dissolve the solution, 8μL of which was slowly added to the black precipitate aqueous solution obtained in S8 at a speed of 5s / drop, and stirred in a constant temperature water bath at 50°C at a speed of 880r / min until the liquid was completely evaporated, and the obtained black powder was dried in a constant temperature air drying oven at 60°C for 30min and at 100°C for 6h; S10, the black powder obtained in S9 was resuspended in deionized water, and ultrasonic dissolution was performed at 80kHz for 10min, and stirring was performed at 500r / min in the hydrogen stream of a hydrogen generator for 40min to obtain CP nanoparticles; S11, the solid CP nanoparticles were dissolved in PBS, and ultrasonic dispersion was performed at 80kHz for 15min, and mixed with macrophage membrane vesicles at a ratio of 3mg: 15mg, and then repeatedly extruded through a cell extruder 400μm polycarbonate membrane for 20 times, and then repeatedly extruded through a 200μm polycarbonate membrane for 20 times to obtain the cascade self-enhanced non-light singlet oxygen generation biomimetic nanoparticles CP@mem.

[0034] Example 6 A preparation method of a biomimetic nanoparticle for rheumatoid arthritis treatment, comprising the following steps: S1, 68mL of anhydrous ethanol (EtOH), 12mL of deionized water (ddH2O) and 1.5mL of 25% ammonia solution (NH3·H2O) were added to 200mL glass containers respectively, and stirred at a speed of 880r / min for 15min to fully mix the solutions; S2, 1.65mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise into the mixed solution of step S1, and the stirring was continued at a speed of 880r / min for 15min, and the solution gradually changed from transparent to milky white; S3, 0.6 g of resorcinol was weighed and added to the mixed solution of step S2, and the mixed solution was placed in an ultrasonic disperser to fully dissolve the solid. The ultrasonic frequency was 80 kHz, and the time was 10 min. Then the mixed solution was continuously stirred at a speed of 880 r / min for 15 min; S4, 0.84 mL of 37% formaldehyde solution was added to the mixed solution of step S3 at a speed of 3 s / drop, and after stirring at 880 r / min for 10 min, the solution gradually turned light brown. Then it was stirred at a speed of 500 r / min for 24 h at room temperature to obtain a yellow-brown liquid; S5, the mixed solution obtained in S4 was centrifuged at a high speed of 8000 r / min. After discarding the supernatant, the precipitate was washed with anhydrous ethanol: deionized water = 30 mL: 90 mL for 5 times to obtain a yellow-brown precipitate; S6, the yellow-brown precipitate obtained in S5 was placed in a constant temperature air drying oven and dried at 70°C for 12 h to obtain a yellow-brown solid. The solid was ground in a jade mortar to obtain a yellow-brown powder; S7, the yellow-brown powder in S6 was added to a crucible boat and calcined at 600°C for 30 min and at 900°C for 3 h under a nitrogen atmosphere in a tube furnace to obtain a black powder, which was C nanoparticles; S8, the black powder in S7 was added to a 10% hydrofluoric acid solution at a concentration of 1 mg / mL and stirred at a speed of 880 r / min for 12 h. Deionized water was added and centrifuged at 12000 r / min for 20 min. The washing was repeated for 5 times, and the solid-liquid separation was performed to obtain a black precipitate, which was a hollow C nanoparticle; S9, chloroplatinic acid was dissolved in deionized water to a concentration of 0.01 M. It was fully dissolved by ultrasonic dispersion at 80 kHz for 10 min. 8.12 μL was slowly added to the black precipitate aqueous solution obtained in S8 at a speed of 5 s / drop. It was stirred in a constant temperature water bath at 50°C at a speed of 880 r / min until the liquid was completely evaporated. The obtained black powder was dried in a constant temperature air drying oven at 60°C for 30 min and at 100°C for 6 h; S10, the black powder obtained in S9 was resuspended in deionized water and dissolved by ultrasonic dispersion at 80 kHz for 10 min. It was stirred in a hydrogen stream generated by a hydrogen generator at 500 r / min for 60 min to obtain CP nanoparticles; S11, the solid CP nanoparticles were dissolved in PBS and dispersed by ultrasonic dispersion at 80 kHz for 15 min. After mixing with macrophage membrane vesicles at a ratio of 5 mg: 25 mg, the mixture was repeatedly extruded through a 400 μm polycarbonate membrane in a cell extruder for 20 times, and then repeatedly extruded through a 200 μm polycarbonate membrane for 20 times to obtain the cascade self-enhanced non-light singlet oxygen generation biomimetic nanoparticles CP@mem.

[0035] The nanoparticles prepared in Example 1 were dissolved in PBS and administered by intravenous injection through the mouse tail to detect the imaging performance and therapeutic effect of the nanoparticles.

[0036] Figure 1 The transmission electron microscopy images of the nanoparticles synthesized in Example 1 before and after modification by macrophage membrane vesicles. As can be seen from the images, the synthesized nanoparticles are hollow spherical structures, and the coating layer can be clearly observed after coating by macrophage membrane vesicles (inserted small image).

[0037] Figure 2 The hydrated particle size and Zeta potential images of the nanoparticles synthesized in Example 1. As can be seen from the images, the hydrated particle size of the synthesized CP nanoparticles is ~211 nm, and the Zeta potential is -15 mV. The hydrated particle size of the CP@mem nanoparticles obtained after modification by macrophage membrane vesicles is ~247 nm, and the Zeta potential is -22 mV.

[0038] Figure 3 The gel electrophoresis-Comassie brilliant blue staining images of the nanoparticles synthesized in Example 1 after coating by macrophage membrane vesicles. As can be seen from the images, the expression of cell membrane proteins can be clearly observed in the nanoparticles modified by cell membranes.

[0039] Figure 4 The photothermal performance of the nanoparticles synthesized in Example 1. As can be seen from the images, the nanoparticles have a concentration-dependent thermal performance under 1064 nm laser irradiation.

[0040] Figure 5 The performance of the nanoparticles synthesized in Example 1 in catalyzing the decomposition of hydrogen peroxide. As can be seen from the images, the nanoparticles have the performance of catalyzing the decomposition of hydrogen peroxide (H2O2) to generate oxygen.

[0041] Figure 6 The singlet oxygen generation performance of the nanoparticles synthesized in Example 1. As can be seen from the images, the intensity of the ultraviolet-visible light characteristic peak (410 nm) of DPBF rapidly decreases with the action time of CP@mem, indicating that DPBF is irreversibly oxidized by the singlet oxygen generated by CP@mem in the dark.

[0042] Figure 7 The effect image of the cascade-enhanced singlet oxygen generation performance of the nanoparticles synthesized in Example 1. As shown in the figure, under the combined action of H2O2 and H2O2+1064 nm laser (Laser), the oxidation effect of CP@mem and DPBF after five minutes of action is obviously enhanced, indicating the promoting effect of H2O2 and 1064 nm laser on the generation of singlet oxygen.

[0043] Figure 8In vivo photoacoustic imaging images of the nanoparticles synthesized in Example 1 of the present application after modification by macrophage membrane vesicles (mem) and polyvinylpyrrolidone (PVP). As shown in the figure, after administration into the tail vein of the rheumatoid arthritis model mice, the photoacoustic signal of the arthritis site gradually decreased after the signal was enhanced with the extension of time, and the nanoparticles modified by macrophage membrane vesicles had an earlier enrichment time and stronger photoacoustic signal intensity than the PVP-modified nanoparticles.

[0044] Figure 9 In vivo anti-rheumatoid arthritis treatment effect images of the nanoprobe synthesized in Example 1 of the present application. As shown in the figure, the rheumatoid arthritis model mice were treated in different ways, and compared with the H2O2 alone group, the effect of the combination therapy group (H2O2+ Laser) was obviously better than that of the single treatment group.

[0045] Based on the above results, it can be concluded that the present application adopts a cell membrane biomimetic strategy to construct a new type of nanoparticle, which can actively target and enrich in the rheumatoid arthritis (RA) lesion area. The nanoparticle can continuously generate singlet oxygen without external stimulation, effectively killing the abnormally proliferating synovial fibroblasts in the RA site, thereby achieving precise treatment of the disease. In addition, the particle has a catalase-like activity, which can catalyze the decomposition of the highly expressed hydrogen peroxide (H2O2) in the inflammatory microenvironment to produce oxygen to further promote the generation of singlet oxygen. Combined with its excellent photothermal performance, the nanoparticle can form a thermal effect locally, synergistically enhancing the yield of singlet oxygen, and ultimately realizing a cascade self-enhanced non-light singlet oxygen generation system. This system effectively removes the abnormally proliferating synovial fibroblasts, achieving effective intervention of rheumatoid arthritis. The entire preparation process is simple and reliable, and has good clinical application prospects.

[0046] The above only details the preferred embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application, and all such changes shall be included in the protection scope of the present application.

Claims

1. A method for the preparation of biomimetic nanoparticles for the treatment of rheumatoid arthritis, characterized by: Comprise the following steps: S1, anhydrous ethanol, ammonia solution and deionized water are added into a glass container in proportion, stirred rapidly at room temperature, and the solution is fully mixed to obtain a mixed solution one; S2, slowly drop the tetraethyl orthosilicate into the mixed solution one of step S1, continue to stir rapidly, the solution gradually changes from transparent to milky white, to obtain a mixed solution two; S3, add the solid resorcinol to the mixed solution two of step S2, ultrasonic dispersion, make the solid fully dissolved, continue to stir rapidly, to obtain a mixed solution three; S4, slowly and uniformly drop the formaldehyde solution into the mixed solution three of step S3, continue to stir rapidly for 10 min, then the solution gradually changes to light brown, medium speed stirring at room temperature for 24 h, then the solution gradually changes from light brown to yellow brown, to obtain a mixed solution four; S5, high speed centrifugation of the mixed solution four of S4, after discarding the supernatant, the precipitate is washed with anhydrous ethanol / deionized water in proportion, high speed centrifugation for 5 times, solid-liquid separation, to obtain yellow brown precipitate; S6, the yellow brown precipitate obtained in S5 is dried at 70 DEG C for 12-18 h, to obtain yellow brown solid, put into a marvelet mortar and grind thoroughly, to obtain yellow brown powder; S7, the yellow brown powder in S6 is added into a crucible boat, calcined at 600 DEG C for 30 min and at 900 DEG C for 3 h in nitrogen atmosphere, to obtain black powder, namely C nanoparticles; S8, the C nanoparticles in S7 are added into a hydrofluoric acid solution, stirred rapidly, washed with deionized water by centrifugation for 5 times, solid-liquid separation, to obtain black precipitate, namely hollow C nanoparticles; S9, chloroplatinic acid is dissolved in deionized water, ultrasonic dispersion to make it fully dissolved, then slowly drop into the black precipitate aqueous solution obtained in S8, constant temperature rapid stirring until the solution is completely evaporated, the obtained black powder is gradient constant temperature dried, to obtain black powder; S10, the black powder obtained in S9 is resuspended in deionized water, ultrasonic dissolution, reduced in hydrogen stream with rapid stirring, to obtain CP nanoparticles; the ultrasonic dispersion frequency in S10 is 80 kHz, time 10 min; the hydrogen stream is provided by a hydrogen generator, stirring speed is 500 r / min, time 30-60 min; S11, the solid CP nanoparticles are dissolved in PBS buffer, ultrasonic dispersion, mixed with macrophage membrane vesicles in proportion, repeatedly extruded through polycarbonate membrane with different pore size gradient for several times, to obtain the cascade self-enhanced non-light singlet oxygen generation biomimetic nanoparticles CP@mem.

2. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The volume ratio of the anhydrous ethanol, ammonia solution and deionized water in S1 is 60-70 mL: 10-20 mL: 1.5-3 mL; wherein, the mass concentration of the ammonia solution is 25%; the stirring speed is 880 r / min, and the stirring time is 15 min.

3. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The volume of the tetraethyl orthosilicate in S2 is 1-1.73 mL, and the stirring speed is 880 r / min; the stirring time is 15 min.

4. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The mass of the resorcinol in S3 is 0.4-0.6 g, the ultrasonic dispersion instrument is used for dissolution and dispersion, the frequency is 80 kHz, the time is 10 min; the stirring speed is 880 r / min, and the stirring time is 15 min.

5. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The volume of the formaldehyde solution in S4 is 0.56-0.84 mL, the mass concentration is 37%, the stirring time is 10 min, the stirring speed is 500 r / min, and the stirring time is 24 h.

6. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The speed of high-speed centrifugation in S5 is 8000 r / min, and the time is 8 min. The volume ratio of anhydrous ethanol to deionized water is 1:

3.

7. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The mass concentration of the hydrofluoric acid solution in S8 is 10%, the stirring speed is 880 r / min, the time is 6-12 h, the speed of high-speed centrifugation is 12000 r / min, and the time is 120 min.

8. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The concentration of the chloroplatinic acid solution in S9 is 0.01 M, the amount used is 6.76-8.12 μL, the ultrasonic dispersion frequency is 80 kHz, the time is 10 min, the stirring speed is 880 r / min, the stirring temperature is 50 °C, the stirring time is 12 h, the gradient drying is carried out in a constant-temperature blast drying oven, the drying at 60 °C is carried out for 30 min, and the drying at 100 °C is carried out for 6 h.

9. A method of preparing a biomimetic nanoparticle for the treatment of rheumatoid arthritis as claimed in claim 1, wherein: The ultrasonic dispersion frequency in S11 is 80 kHz, the time is 15 min, the mass ratio of CP nanoparticles to macrophage membrane vesicles is 1:5, the pore size of the polycarbonate membrane is 400 μm and 200 μm respectively, and the extrusion is carried out 20 times respectively.

10. The use of the biomimetic nanoparticles prepared by the preparation method in claim 1 in the preparation of a kit for treating rheumatoid arthritis.

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