Injectable magneto-electric composite anti-inflammatory microspheres and preparation and application thereof

Abstract

The application discloses injectable magneto-electric composite anti-inflammatory microspheres, and a preparation and application thereof. The microspheres can be recognized by macrophages in a mode and phagocytosed into the inside of the macrophages. Meanwhile, the phagocytosis of the macrophages to the microspheres can be strengthened by an externally applied magnetic field, and the macrophages are promoted to change to an anti-inflammatory polarization direction. Therefore, the anti-inflammatory polarization effect of the macrophages can be regulated by regulating the strength of the external magnetic field, inflammation can be eliminated, and tissue repair can be promoted. The injection preparation prepared by dispersing the microspheres in physiological saline is injected into a mouse body, and experiments prove that the injection preparation can effectively relieve rheumatoid arthritis. Therefore, the injectable magneto-electric composite anti-inflammatory microspheres can be used for preparing a medicine for treating an inflammatory disease and preparing an anti-inflammatory medicine for wound repair. The preparation process of the application is simple and easy to realize, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the biomedical field, and in particular to a magnetoelectric composite anti-inflammatory microsphere. Background Art

[0002] Inflammation is a protective response to pathogens or endogenous factors. If inflammation remains uncontrolled or becomes chronic, it can often lead to disease and even induce systemic immune-inflammatory responses. For example, rheumatoid arthritis (RA) is one of the most common immune-mediated inflammatory diseases and is a persistent and difficult-to-treat condition. Without timely and effective treatment, chronic inflammation of the joint lining (synovium) can lead to severe joint damage, disability, and work impairment. A key characteristic of RA is its ability to affect multiple joints simultaneously, manifesting as symmetrical joints and aggressive arthritis of the limbs. Severe cases can even lead to irreversible damage and deformity of articular cartilage, compromising patients' daily quality of life. The pathogenesis of RA involves multiple cytokines and cells. Furthermore, during wound repair processes such as osseointegration and bone repair, or tissue regeneration, the presence of implant materials, acting as foreign bodies in the body, inevitably triggers a corresponding inflammatory response, impacting the relationship between the material and bone tissue. Therefore, reducing the release of inflammatory factors and limiting the inflammatory response are crucial in disease treatment and wound repair.

[0003] Currently, there are three main types of commonly used anti-inflammatory materials: 1. Materials with intrinsic anti-inflammatory properties, such as hyaluronic acid, chitosan, collagen, hydrogels, and metal nanoparticles; 2. Materials used for implant coatings, such as glycosaminoglycans, hyaluronic acid, keratin, and magnesium ion coatings; and 3. Materials used for anti-inflammatory drug delivery, such as hydrogel scaffolds, hydroxymethylcellulose films, and chitosan-encapsulated microparticles. The first two are purely anti-inflammatory materials and have a slow onset of action; the third delivers anti-inflammatory drugs with inaccurate release. Moreover, once these three materials enter the human body, the onset and effectiveness of their effects cannot be externally controlled, making them ineffective in achieving effective anti-inflammatory effects. Summary of the Invention

[0004] In light of this, the present invention aims to provide injectable magnetoelectric composite anti-inflammatory microspheres, their preparation, and use. These microspheres can be pattern-recognized by macrophages and engulfed within them. Simultaneously, an externally applied magnetic field can enhance macrophage phagocytosis of the microspheres and induce macrophages to shift toward anti-inflammatory polarization. Therefore, by externally controlling the magnetic field intensity, the anti-inflammatory polarization effect of macrophages can be modulated, eliminating inflammation and promoting tissue repair.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, the present invention provides an injectable magnetoelectric composite anti-inflammatory microsphere, which is formed by extracellular matrix microsheets (ECM) coated on the outer layer of polyvinylidene fluoride trifluoroethylene (PVTF) / cobalt ferrite (CFO) composite microspheres.

[0007] In some specific examples of the present invention, the size of the microchip is 1 to 20 μm.

[0008] In a second aspect of the present invention, the present invention provides a method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres, comprising the following steps:

[0009] (1) A polyvinylidene fluoride trifluoroethylene (PVTF) solution formed by polyvinylidene fluoride trifluoroethylene in N,N-dimethylformamide and a cobalt ferrite (CFO) dispersion formed by cobalt ferrite nanoparticles in ethanol are mixed and ultrasonically dispersed to obtain a PVTF / CFO mixed dispersion, wherein the mass ratio of polyvinylidene fluoride trifluoroethylene to cobalt ferrite is (20-100):1; deionized water with a volume ratio of (0.5-2):1 and the PVTF / CFO mixed dispersion are placed in the lower layer and the upper layer of the same container, respectively, and the phases are separated and allowed to stand to obtain a precipitate;

[0010] (2) dispersing the precipitate with ethanol and placing it in a hydrothermal reactor, followed by hydrothermal reaction at (110-140)°C for 0.5-2h, separating, and obtaining a solid phase as the hydrothermal product;

[0011] (3) placing the hydrothermal product obtained in step (2) in an ethanolamine aqueous solution, heating in a water bath at (70-90)° C. for 10-60 min, washing, and sterilizing to obtain a modified product;

[0012] (4) taking cells that have grown into complete sheets, preparing extracellular matrix sheets by repeated freezing and thawing, and breaking the complete sheets into micro-sheets by vibration during the thawing process to obtain extracellular matrix micro-sheets, wherein the size of the extracellular matrix micro-sheets is 1 to 20 μm;

[0013] (5) Mixing the modified product and the extracellular matrix microsheet in a mass ratio of (3-8):1 in ultrapure water, adjusting the pH to 6-8, adding genipin for cross-linking, heating in a water bath at (30-45)°C for 3-6 hours, washing, separating, and obtaining a precipitate, which is the target product;

[0014] The feed ratio of the sum of the mass of the modified product and the extracellular matrix microsheet to ultrapure water is (4-9) mg:10 mL, and the mass of genipin is 1-5% of the sum of the mass of the modified product and the extracellular matrix microsheet.

[0015] In some specific examples of the present application, in step (1), the ultrasonic dispersion time is 0.5-2 h.

[0016] In some specific examples of the present application, in step (1), the phase separation standing time is 1-3 h.

[0017] In some specific examples of the present application, in step (1), the PVDF- Trifluoroethylene solution is prepared by adding PVDF- Trifluoroethylene solution powder into N, N-dimethylformamide solvent at a ratio of (0.1-3) g: 25 mL, and stirring to obtain a uniform mixture; preferably, the stirring is magnetic stirring, and the stirring time is 2-6 h.

[0018] The CFO dispersion liquid is prepared by adding CFO nanoparticles into ethanol at a ratio of (2-60) mg: 15 mL, and ultrasonic dispersion to obtain a uniform mixture; preferably, the ultrasonic dispersion time is 0.5-1 h.

[0019] In some specific examples of the present application, in step (1), the volume ratio of the PVTF solution and the CFO dispersion liquid is (1-3): 1.

[0020] In some specific examples of the present application, in step (2), the ratio of the precipitate to ethanol is (10-500) mg: 20 mL.

[0021] In some specific examples of the present application, in step (3), the concentration of the ethanolamine aqueous solution is 14-16 mol / L, and the ratio of the hydrothermal product to the ethanolamine aqueous solution is (0.1-2) g: 50 mL.

[0022] In some specific examples of the present application, in step (3), the cleaning is deionized water cleaning, or cleaning with hydrogen peroxide solution followed by deionized water cleaning. Preferably, the mass concentration of the hydrogen peroxide solution is 3%.

[0023] In some specific examples of the present application, in step (3), the sterilization refers to dispersing the cleaned product in a 75% (volume concentration) ethanol solution, and soaking for 0.5-2 h.

[0024] In some specific examples of the present application, in step (5), the mass of genipin is 3% of the total mass of the modified product and the extracellular matrix microsheet.

[0025] In some specific examples of the present application, in step (5), the cleaning is centrifugal cleaning with ultrapure water for several times.

[0026] In some specific examples of the present invention, the separation is centrifugal separation, and the speed of centrifugal separation is 8000 rpm and the time is 5 minutes.

[0027] The injectable magnetoelectric composite anti-inflammatory microspheres prepared by the present invention can be pattern-recognized and phagocytosed by macrophages, and have a magnetoelectric coupling effect under a low-frequency alternating magnetic field of 50 Hz. The externally controlled magnetic field can enhance the phagocytosis of the microspheres by macrophages, and can also cause the microspheres to generate an electric potential response inside the macrophages, prompting the macrophages to shift towards anti-inflammatory polarization. Alternating magnetic fields of different intensities have different anti-inflammatory polarization effects on macrophages. Changes in the intensity of the externally controlled magnetic field can regulate the anti-inflammatory polarization effect of macrophages, thereby achieving a controllable anti-inflammatory effect, eliminating inflammation and promoting tissue repair. Experiments in which the injection preparation prepared by dispersing the microspheres in physiological saline and injecting them into mice have shown that it can effectively alleviate rheumatoid arthritis. Therefore, in the third aspect of the present invention, the present invention also provides the use of the injectable magnetoelectric composite anti-inflammatory microspheres in the preparation of drugs for treating immune-mediated inflammatory diseases, as well as the use in the preparation of anti-inflammatory drugs for wound repair.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The injectable magnetoelectric composite anti-inflammatory microspheres of the present invention have significantly improved biocompatibility, can be well dispersed in the aqueous system, and are more easily recognized and phagocytosed by macrophages and enter the interior of macrophages; under the application of an external magnetic field, the pattern recognition and phagocytosis of macrophages are further enhanced, and the surface of the microspheres generates an electric potential response, inducing the flow of calcium ions inside the macrophages, prompting them to polarize in the anti-inflammatory direction, eliminating inflammation and promoting tissue repair. Therefore, the anti-inflammatory polarization effect of macrophages can be regulated by externally regulating the magnetic field intensity, achieving controllable anti-inflammatory.

[0030] (2) A significant advantage of the present invention is the easily operable regulation mechanism of the external magnetic field, which enables the anti-inflammatory effect inside the cells to be precisely controlled by external means. Specifically, by selectively turning on the external magnetic field to stimulate the function of macrophages at specific time points, precise intervention in the inflammatory response is achieved. In addition, the present invention also allows for the flexible selection of the magnetic field intensity according to the desired biological effect to optimize the anti-inflammatory level of macrophages, thereby achieving the desired therapeutic effect.

[0031] (3) The preparation method of the injectable magnetoelectric composite anti-inflammatory microspheres of the present invention is simple and easy to implement, which is conducive to promotion and application.

[0032] (4) The anti-inflammatory effect of the injectable magnetoelectric composite anti-inflammatory microspheres of the present invention is achieved through the three steps of "ECM recognition", "cell phagocytosis" and "magnetoelectric response" in vitro, which sequentially regulate the phagocytosis of macrophages and cellular immune polarization under an alternating magnetic field. Moreover, since the microspheres have good dispersibility in the aqueous phase, the microspheres can be used as biomedical injectable microspheres for clinical injection targeting lesions. This type of magnetoelectrically coupled bioactive microspheres has brought about an innovative method for in vivo injection anti-inflammatory. By adjusting the application time and intensity of the external magnetic field, the microspheres can accurately and efficiently exert their anti-inflammatory effects inside the cells and regulate the cellular immune microenvironment. This undoubtedly provides an innovative non-invasive regulation scheme for cell therapy and broadens the application prospects of external magnetic field regulation of electroactive materials in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A It is the Fourier transform infrared spectrum of the product obtained by step (1) and the product obtained by step (2) in Example 1.

[0034] Figure 1B It is a differential thermal scanning analysis chart of the product obtained in step (1) and the product obtained in step (2) in Example 1.

[0035] Figure 2A and Figure 2B They are scanning electron microscope images of the product obtained in step (1) and the product obtained in step (2) in Example 1, respectively.

[0036] Figure 3 This is the X-ray diffraction analysis diagram of the microspheres obtained in step (2) of Example 1.

[0037] Figure 4A and Figure 4B The X-ray photoelectron spectroscopy analysis diagrams are respectively performed on the microspheres obtained in step (2) and the product obtained in step (3) in Example 1.

[0038] Figure 5 This is a scanning electron microscope image of the target product obtained in step (5) of Example 1.

[0039] Figure 6 This is the Fourier transform infrared spectrum of the target product obtained in step (5) of Example 1.

[0040] Figure 7 This is a result diagram of the surface potential of the target product obtained in Example 1 measured by an electrometer under the action of a magnetic field.

[0041] Figure 8 This is a laser confocal fluorescence microscope observation image of the target product obtained in Example 1 after being phagocytosed by macrophages.

[0042] Figure 9It is a flow cytometry analysis diagram of the target product obtained in Example 1 and the final products obtained in Comparative Examples 1 to 3 after being phagocytosed by macrophages under the action of a magnetic field.

[0043] Figure 10 This is a flow cytometry analysis diagram of the polarization state of macrophages detected after the target product obtained in Example 1 and the final product obtained in Comparative Example 3 were phagocytosed by macrophages under the action of a magnetic field.

[0044] Figure 11 This is a flow cytometry analysis diagram of the polarization state of macrophages detected after the target product obtained in Example 1 was phagocytosed by macrophages under the action of magnetic fields of different intensities.

[0045] Figure 12 It is a comparison of HE tissue staining with and without the action of a magnetic field after the target product obtained in Example 1 and the final product obtained in Comparative Example 2 were injected into the ankle joints of rheumatoid arthritis mice. DETAILED DESCRIPTION

[0046] In order to better illustrate the present invention and facilitate understanding of the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is subject to the claims.

[0047] In the following examples and comparative examples, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased on the market. For example, PVTF powder is produced by Piezotech, France. FC30, a vinylidene fluoride / trifluoroethylene copolymer, has a molar ratio of 7:3 between vinylidene fluoride and trifluoroethylene. CFO nanoparticles were purchased from Sigma. Extracellular matrix sheets were derived from NIH3T3 cells purchased from the Chinese Academy of Sciences Cell Bank. BMDM macrophages were purified from the bone marrow of SPF-grade C57BL / 6 mice purchased from the Zhejiang Provincial Laboratory Animal Center. BMDM macrophages can also be purchased commercially.

[0048] BMDM macrophage culture steps: Place mouse bone marrow cells in a centrifuge tube, add 3 mL of red blood cell lysis buffer, place in an ice-water mixing bath to lyse for 3 minutes to purify macrophage precursors, centrifuge at 1300 rpm for 3 minutes, take the bottom cells and add 4 mL of αMEM culture medium for culture, and place in a constant temperature and humidity incubator at a temperature of 37°C and a carbon dioxide concentration of 5%. M-CSF induction factor needs to be added to the culture medium to induce macrophage maturation, and the final concentration of the induction factor added is 30 ng / mL.

[0049] The steps of preparing the extracellular matrix sheet by repeated freeze-thaw method are as follows: 10 6 NIH3T3 cells are taken in a culture dish, 10 mL of DMEM high-sugar medium is added, and the culture dish is placed in a constant-temperature and constant-humidity incubator with a temperature of 37°C and a carbon dioxide concentration of 5%. The medium is changed every 2 days, and a cell sheet is obtained after 7 days of culture. After the medium is discarded, the cell sheet is washed twice with ultrapure water, each time with (3-5) mL. The cell sheet is frozen at -80°C for 1-5 h, then thawed at room temperature, and the liquid is discarded. New ultrapure water is added, and the volume is still (3-5) mL. The freeze-thaw process is repeated more than 3 times to remove the cells. Then, a water solution containing DNase and RNase (the concentration of each enzyme is 0.1 mg / mL) is added to remove the residual DNA and RNA on the cell sheet, and an extracellular matrix sheet is obtained.

[0050] Sample preparation and testing scheme of flow cytometry: during the maturation stage of macrophage culture, the target product microspheres are added to the culture medium for phagocytosis, and the addition amount is 1 mg / 10 6 cells. After a period of culture in the incubator under the experimental conditions, the cells are taken out, washed twice with PBS to remove unphagocytosed microspheres, each time for 3 min. The cells are incubated and digested with trypsin at 37°C twice, each time for 6 min. The cells are collected by blowing and centrifuged in a flow cytometry special round-bottom centrifuge tube at 1500 rpm for 5 min each time. After collection, the cells are sequentially dyed with dyes corresponding to the experimental conditions, each time for 30 min. After centrifugation, the cells are collected in a centrifuge tube, and 0.5 mL of PBS is added to each tube to suspend the cells uniformly, and then the cells are tested on the machine. Except for the dyeing agent required by the experimental conditions, the macrophages are specifically dyed with PE-F4 / 80. The target product microspheres have spontaneous fluorescence with a center wavelength of about 700 nm, and do not need additional dyeing. The flow cytometry detection parameter is the fluorescence intensity value of a single cell in each fluorescence channel. In this experiment, each group of samples is tested three times, and the effective cell amount for each test is 10 5 .

[0051] Example 1

[0052] (1) 1 g of PVTF powder is added to 25 mL of N,N-dimethylformamide (DMF), and magnetic stirring is performed for 3 h to obtain a PVTF solution. 20 mg of CFO nanoparticles is added to 15 mL of ethanol, and ultrasonic dispersion is performed for 1 h to obtain a CFO dispersion. The PVTF solution and the CFO dispersion are mixed to obtain 40 mL of a PVTF / CFO mixed dispersion, and ultrasonic dispersion is continued for 1 h. 10 mL of deionized water is placed in the lower layer of a test tube, and 10 mL of the PVTF / CFO mixed dispersion is placed in the upper layer of the test tube. After standing for phase separation for 2 h, centrifugation is performed at 8000 rpm for 5 min, and the precipitate is retained;

[0053] (2) The precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor, then heated in a muffle furnace at 120°C for 2 h and centrifuged at 8000 rpm for 5 min. The solid phase obtained by separation was the hydrothermal product.

[0054] (3) The hydrothermal product obtained in step (2) was placed in 50 mL of a 16 mol / L ethanolamine aqueous solution, heated in a 37° C. water bath for 30 min, then oxidized and cleaned with a 3% mass concentration hydrogen peroxide solution, then washed with deionized water, and separated by centrifugation at 8000 rpm for 5 min; the separated solid phase product was dispersed in a 75% volume concentration ethanol solution, soaked for 1 h for sterilization and disinfection, and the solid phase obtained by centrifugation was the modified product;

[0055] (4) Cells that have grown into complete sheets in a culture dish are taken and extracellular matrix sheets are prepared by repeated freeze-thaw at -80°C / 25°C. During the thawing process, the complete sheets are broken into micro-sheets by vibration. The size of the micro-sheets is 1 to 20 μm.

[0056] (5) 30 mg of the modified product obtained in step (3) and 5 mg of the extracellular matrix microsheets obtained in step (4) were mixed in an ultrapure aqueous solution, the pH was adjusted to 6-8, 1.05 mg of genipin was added for cross-linking, and the mixture was heated in a water bath at 37°C for 5 h. The mixture was then washed several times by centrifugation with ultrapure water, and the precipitate obtained by separation was the target product.

[0057] Characterization and identification:

[0058] The product obtained in step (1) and the product obtained in step (2) of this embodiment 1 were subjected to Fourier transform infrared spectroscopy comparison analysis, and the FTIR graphs thereof are as follows: Figure 1A As shown in the two spectrum curves, No treat corresponds to the product obtained in step (1), and Heated corresponds to the product obtained in the hydrothermal reaction in step (2). Figure 1A Medium, 885cm -1 and 1189cm -1 The absorption peak at 1403 cm corresponds to the mixed mode of CH2 rocking and CF2 asymmetric stretching vibration. -1 The absorption peaks at 766cm correspond to the stretching vibration of CF2 and the stretching vibration mode of CC. These characteristic peaks are all characteristic peaks of the infrared spectrum of PVTF; -1 The absorption peak at 475 cm is the characteristic peak of α phase. -1 and 1289cm -1 The absorption peak at 1430 cm is the characteristic peak of β phase. -1 The absorption peak at 509 cm is the characteristic peak of γ phase. -1 and 847cm -1The absorption peak is a mixed characteristic peak of the β phase and the γ phase, and the absorption peak of the crystalline phase in the Heated spectrum is higher. This shows that the main components of the product obtained in step (1) and the product obtained in step (2) are both PVTF, and the product obtained in step (2) has better crystallinity.

[0059] The product obtained in step (1) (No treatment) and the product obtained in step (2) (Heated) in Example 1 were subjected to DSC differential scanning calorimetry analysis. The DSC graphs are shown in FIG. Figure 1B As shown in the two spectrum curves. Figure 1B In the figure, the ferroelectric phase transition peak is around 100°C, and the crystalline phase melting peak is in the range of 140-160°C. It can be seen that the product obtained in step (1) does not have a clear ferroelectric phase, and the peak height and peak area of ​​the crystalline phase melting peak are both smaller than those of the product obtained in step (2). Therefore, the product obtained after the hydrothermal treatment in step (2) has higher ferroelectricity and crystallinity.

[0060] The surface morphologies of the product obtained in step (1) (No treatment) and the product obtained in step (2) (Heated) in Example 1 were tested using a scanning electron microscope. The SEM results were as follows: Figure 2A and Figure 2B shown. Figure 2A and Figure 2B It can be seen that the product obtained in step (1) and the product obtained in step (2) are both microspheres, the size of the former microspheres is (20-1000) nm, and the size of the latter microspheres is (400-600) nm; moreover, compared with the product obtained in step (1), the surface crystallization of the microspheres obtained in step (2) is obvious, and the particle size is more uniform, with an average particle size of about 500 nm.

[0061] The microspheres obtained in step (2) of Example 1 were subjected to X-ray diffraction analysis, and the XRD pattern thereof was as follows: Figure 3 The spectrum curve is shown in FIG. Among them, the peak at 19.1° corresponds to the (020) α-phase crystal plane in PVTF, the peak at 19.6° corresponds to the (200) β-phase crystal plane in PVTF, the peak at 35.0° corresponds to the strongest peak (311) crystal plane in CFO, and the peak at 40.5° corresponds to the (220) δ-phase crystal plane in PVTF. It can be seen that the product obtained in step (2) is a mixed crystalline microsphere of PVTF and CFO.

[0062] The microspheres obtained in step (2) and the product obtained in step (3) of this embodiment 1 were subjected to X-ray photoelectron spectroscopy analysis, and their XPS graphs were as follows: Figure 4A and Figure 4BAs shown. Among them, the microspheres obtained in step (2) are marked as "before modification" and the product obtained in step (3) is marked as "after modification". By comparison, it can be found that before and after the modification, the content of N element on the surface of the microspheres increased from 0.15% to 6.52%, the content of O element increased from 0.98% to 9.93%, and the content of F element decreased from 46.63% to 24.05%. It can be seen that the surface group modification effect of the product obtained in step (3) is obvious, the content of amino and hydroxyl groups increases, and the content of fluorine atoms decreases.

[0063] The product obtained in step (5) was analyzed for functional groups using Fourier transform infrared spectroscopy. The FTIR spectrum is shown in FIG. Figure 5 Among them, the peaks related to PVTF identification are marked in green, namely 1401cm -1 , 1176cm -1 , 1095cm -1 and 888cm -1 The peaks related to the PVTF ferroelectric phase are marked in blue and are 1261 cm -1 β phase, 842 cm -1 and 512cm -1 β and γ phases, 436 cm -1 The γ phase of the extracellular matrix is ​​marked with magenta, and the peaks related to proteins are 1638 cm -1 Amide I band, 1550 cm -1 Amide II band, 1461 cm -1 CN stretching vibration peak, 1261 cm -1 Amide III band, 1022 cm -1 CO stretching vibration peak, 802 cm -1 It can be seen that the target product obtained in step (5) not only maintains the ferroelectric phase of PVTF, but also keeps the protein conformation of the extracellular matrix intact.

[0064] The surface morphology of the target product obtained in step (5) was tested using a scanning electron microscope, and the SEM image thereof is as follows: Figure 5 As shown, in the target product, the extracellular matrix microsheets formed a good surface coating on the PVTF / CFO microspheres.

[0065] In summary, the target product is a PVTF / CFO composite extracellular matrix microsphere, which is formed by coating the outer layer of the PVTF / CFO composite microsphere with extracellular matrix microsheets. Among them, polyvinylidene fluoride trifluoroethylene (PVTF) is the main body, cobalt ferrite (CFO) nanoparticles are distributed in the PVTF main body, and the outermost layer is coated with extracellular matrix (ECM).

[0066] Performance testing:

[0067] In an alternating magnetic field with a frequency of 50 Hz, the magnetoelectric properties of the target product obtained in Example 1 were tested using an electrometer. The specific method is as follows: the target product obtained in Example 1 was cooled and condensed into a film with a thickness of approximately 130 nm during a hydrothermal process. Silver was plated on a 0.5 cm*0.5 cm area in the center of both sides of the film. Wires were connected to the positive and negative electrodes of the electrometer as the two poles of the voltage test, and the film was placed in a magnetic field for testing. The results are shown in FIG. Figure 7 As shown. Figure 7 Analysis shows that when no magnetic field is applied, the potential is almost 0; when the magnetic field strength is 47.1Gs, the potential is about 38mV (~38mV); when the magnetic field strength is 94.2Gs, the potential is about 71mV (~71mV); when the magnetic field strength is 188.3Gs, the potential is about 134mV (~134mV); when the magnetic field strength is 282.5Gs, the potential is about 247mV (~247mV). It can be seen that when the applied magnetic field strength continues to increase, the corresponding surface potential fluctuation also increases and the relationship is almost linear, indicating that the target product has a magnetoelectric coupling effect under the condition of a low-frequency AC magnetic field of 50Hz, and its magnetoelectric coupling coefficient is about 0.67V / (cm·Oe).

[0068] Laser confocal fluorescence microscopy was used to characterize the recognition and phagocytosis of the target product microspheres obtained in Example 1 by BMDM macrophages. The specific method is as follows: in the mature stage of macrophage culture, the target product microspheres were added to the culture medium for phagocytosis, and the addition amount was 1 mg / 10 6 The cells were cultured in the incubator for 6 hours and then taken out. They were washed twice with PBS to remove the unphagocytosed microspheres. The cytoskeleton was stained with FITC-phalloidin and the nucleus was stained with DAPI. The cells were observed under a microscope. The target product microspheres spontaneously fluoresced in red and no additional staining was required. Figure 8 As shown. Figure 8 In the image, the blue color represents the cell nucleus stained with DAPI, the green color represents the macrophage cytoskeleton stained with FITC, and the red color represents the target product microspheres. This shows that the target product microspheres have passed the surface recognition mechanism of the macrophage and have been successfully engulfed and taken up by the macrophage.

[0069] Comparative Example 1

[0070] (1) Add 1 g of PVTF powder to 25 mL of N,N-dimethylformamide (DMF) and stir magnetically for 3 h to obtain a PVTF solution; add 20 mg of CFO nanoparticles to 15 mL of ethanol and ultrasonically disperse for 1 h to obtain a CFO dispersion; mix the above PVTF solution and CFO dispersion to obtain 40 mL of PVTF / CFO mixed dispersion, and continue ultrasonically dispersing for 1 h; take 10 mL of deionized water and place it in the lower layer of the test tube, take 10 mL of the above PVTF / CFO mixed dispersion and place it in the upper layer of the test tube, let it stand for 2 h to separate the phases, centrifuge at 8000 rpm for 5 min, and retain the precipitate;

[0071] (2) The precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor, then heated in a muffle furnace at 120°C for 2 h and centrifuged at 8000 rpm for 5 min. The solid phase obtained by separation was the hydrothermal product.

[0072] (3) The hydrothermal product obtained in step (2) was placed in 50 mL of a 16 mol / L ethanolamine aqueous solution, heated in a 37° C. water bath for 30 min, then oxidized and cleaned with a 3% (mass concentration) hydrogen peroxide solution, washed with deionized water, and centrifuged at 8000 rpm for 5 min; the separated solid phase product was dispersed in a 75% (volume concentration) ethanol solution, soaked for 1 h for sterilization and disinfection, and the solid phase obtained by centrifugation was the modified product.

[0073] Comparative Example 2

[0074] (1) Add 1 g of PVTF powder to 25 mL of N,N-dimethylformamide (DMF) and stir magnetically for 3 h to obtain a PVTF solution; add 20 mg of CFO nanoparticles to 15 mL of ethanol and ultrasonically disperse for 1 h to obtain a CFO dispersion; mix the above PVTF solution and CFO dispersion to obtain 40 mL of PVTF / CFO mixed dispersion, and continue ultrasonically dispersing for 1 h; take 10 mL of deionized water and place it in the lower layer of the test tube, take 10 mL of the above PVTF / CFO mixed dispersion and place it in the upper layer of the test tube, let it stand for 2 h to separate the phases, centrifuge at 8000 rpm for 5 min, and retain the precipitate;

[0075] (2) The precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor, then heated in a muffle furnace at 120°C for 2 h and centrifuged at 8000 rpm for 5 min. The solid phase obtained by separation was the hydrothermal product.

[0076] (3) The hydrothermal product obtained in step (2) was placed in 50 mL of a 16 mol / L ethanolamine aqueous solution, heated in a 37°C water bath for 30 min, then washed with deionized water and centrifuged at 8000 rpm for 5 min; the separated solid phase product was dispersed in a 75% (volume concentration) ethanol solution, soaked for 1 h for sterilization and disinfection, and the solid phase obtained by centrifugation was the modified product.

[0077] Comparative Example 3

[0078] (1) Add 1 g of PVTF powder to 25 mL of N,N-dimethylformamide (DMF) and stir magnetically for 3 h to obtain a PVTF solution; add 25 mL of PVTF solution to 15 mL of ethanol to obtain 40 mL of PVTF dispersion, and mix ultrasonically for 1 h; take 10 mL of deionized water and place it in the lower layer of the test tube, take 10 mL of the above PVTF dispersion and place it in the upper layer of the test tube, let it stand for 2 h to separate the phases, centrifuge at 8000 rpm for 5 min, and retain the precipitate;

[0079] (2) The precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor, then heated in a muffle furnace at 120°C for 2 h, centrifuged at 8000 rpm for 5 min, and the solid phase was separated as the hydrothermal product;

[0080] (3) The hydrothermal product obtained in step (2) was placed in 50 mL of a 16 mol / L ethanolamine aqueous solution, heated in a 37°C water bath for 30 min, then washed with deionized water and centrifuged at 8000 rpm for 5 min; the separated solid phase product was dispersed in a 75% (volume concentration) ethanol solution, soaked for 1 h for sterilization, and the solid phase obtained by centrifugation was the modified product;

[0081] Example 2

[0082] (1) Add 0.1 g of PVTF powder to 25 mL of N,N-dimethylformamide (DMF) and stir magnetically for 3 h to obtain a PVTF solution; add 2 mg of CFO nanoparticles to 15 mL of ethanol and ultrasonically disperse for 0.5 h to obtain a CFO dispersion; mix the above PVTF solution and CFO dispersion to obtain 40 mL of PVTF / CFO mixed dispersion, and continue ultrasonically dispersing for 0.5 h; take 5 mL of deionized water and place it in the lower layer of the test tube, take 10 mL of the above PVTF / CFO mixed dispersion and place it in the upper layer of the test tube, let it stand for 1 h to separate the phases, centrifuge at 8000 rpm for 5 min, and retain the precipitate;

[0083] (2) The precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor. The mixture was then heated in a muffle furnace at 120°C for 0.5 h and centrifuged at 8000 rpm for 5 min. The solid phase obtained by separation was the hydrothermal product.

[0084] (3) The hydrothermal product obtained in step (2) was placed in 50 mL of a 14 mol / L ethanolamine aqueous solution, heated in a 37°C water bath for 10 min, then oxidized and cleaned with a 3% mass concentration hydrogen peroxide solution, washed with deionized water, and centrifuged at 8000 rpm for 5 min; the separated solid phase product was dispersed in a 75% volume concentration ethanol solution, soaked for 0.5 h for sterilization, and the solid phase obtained by centrifugation was the modified product;

[0085] (4) Cells grown into complete sheets in a culture dish were used to prepare extracellular matrix sheets by repeated freeze-thawing at -80°C to 25°C. During the thawing process, the complete sheets were broken into micro-sheets with a size of 1 to 20 μm by vibration.

[0086] (5) Take 15 mg of the modified product obtained in step (3) and 5 mg of the extracellular matrix microsheet obtained in step (4), mix them in an ultrapure water solution, adjust the pH to 6-8, add 0.6 mg of genipin for cross-linking, heat in a 37°C water bath for 3 h, then use ultrapure water centrifugation to wash several times, and finally separate the precipitate to obtain the target product.

[0087] The same characterization method as in Example 1 was adopted to identify the target product obtained in Example 2 as PVTF / CFO composite extracellular matrix microspheres.

[0088] Example 3

[0089] (1) 3.0 g of PVTF powder was added to 25 mL of N,N-dimethylformamide (DMF) and magnetically stirred for 3 h to obtain a PVTF solution; 60 mg of CFO nanoparticles were added to 15 mL of ethanol and ultrasonically dispersed for 2 h to obtain a CFO dispersion; the above PVTF solution and CFO dispersion were mixed to obtain 40 mL of PVTF / CFO mixed dispersion, and ultrasonic dispersion was continued for 1 h; 20 mL of deionized water was placed in the lower layer of the test tube, and 10 mL of the above PVTF / CFO mixed dispersion was placed in the upper layer of the test tube. After standing for 3 h to separate the phases, the mixture was centrifuged at 8000 rpm for 5 min to retain the precipitate;

[0090] (2) The precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor, then heated in a muffle furnace at 120°C for 2 h and centrifuged at 8000 rpm for 5 min. The solid phase obtained by separation was the hydrothermal product.

[0091] (3) The hydrothermal product obtained in step (2) was placed in 50 mL of a 16 mol / L ethanolamine aqueous solution, heated in a 37° C. water bath for 60 min, then oxidized and cleaned with a 3% mass concentration hydrogen peroxide solution, washed with deionized water, and centrifuged at 8000 rpm for 5 min; the separated solid phase product was dispersed in a 75% volume concentration ethanol solution, soaked for 2 h for sterilization, and the solid phase obtained by centrifugation was the modified product;

[0092] (4) Cells grown into complete sheets in a culture dish were used to prepare extracellular matrix sheets by repeated freeze-thawing at -80°C to 25°C. During the thawing process, the complete sheets were broken into micro-sheets with a size of 1 to 20 μm by vibration.

[0093] (5) 40 mg of the modified product obtained in step (3) and 5 mg of the extracellular matrix microsheet obtained in step (4) were mixed in an ultrapure aqueous solution, the pH was adjusted to 6-8, 1.35 mg of genipin was added for cross-linking, and the mixture was heated in a water bath at 37°C for 6 h. The mixture was then washed several times by centrifugation with ultrapure water, and the precipitate obtained by separation was the target product.

[0094] The same characterization method as in Example 1 was adopted to identify the target product obtained in Example 2 as PVTF / CFO composite extracellular matrix microspheres.

[0095] Cell experiments

[0096] Flow cytometry was used to characterize the phagocytosis of different groups of microspheres by BMDM macrophages under the influence of a magnetic field. Figure 9 As shown, the product obtained in Comparative Example 3 is recorded as P, the product obtained in Comparative Example 2 is recorded as PM, the product in Comparative Example 1 is recorded as PH, and the product in Example 1 is recorded as EPC. The applied magnetic field strength is 188.3Gs, the applied frequency is 50Hz, and the applied time is 10min. In each group of bar graphs, the light color on the left indicates no magnetic field is applied, and the dark color on the right indicates the magnetic field is applied. The statistical data is the percentage of cells with positive 700nm fluorescence labeling under the PE-F4 / 80 macrophage labeling positive condition, that is, the percentage of macrophages containing target product microspheres. From Figure 9 As can be seen from the comparison of each group: compared with the case without the magnetic field, the phagocytosis of each microsphere by macrophages was enhanced under the influence of the magnetic field, and the most significant improvement was seen in the EPC group with the surface modified with extracellular matrix. This indicates that the external controlled magnetic field can enhance the phagocytosis of microspheres by macrophages, and that the extracellular matrix can promote the pattern recognition and phagocytosis of microspheres by macrophages.

[0097] Flow cytometry was used to characterize the changes in macrophage polarization after BMDM macrophages phagocytosed different groups of microspheres under the influence of a magnetic field. Figure 10 The M2 / M1 ratio was calculated by dividing the average fluorescence intensity of the different channels. The ratio represented the degree of anti-inflammatory polarization of the macrophages. The macrophages without microspheres were set as the control group and marked as 0. The product of the comparative example 3 was set as P, and the product of the example 1 was set as EPC. The results are shown in Figure 10 It can be seen that the M2 / M1 value of the EPC group was significantly higher than that of the P group and the control group 0, regardless of whether a magnetic field was applied. In the P group and the control group 0, the M2 / M1 value did not change significantly before and after the magnetic field was applied. In the EPC group, the M2 / M1 value was significantly improved after the magnetic field was applied. This shows that for the EPC group, the surface modification of the extracellular matrix can promote the anti-inflammatory polarization of the macrophages, and the external magnetic field can act on the microspheres of the EPC group, further promoting the anti-inflammatory polarization of the macrophages.

[0098] The flow cytometry was used to characterize the changes in the polarization of the BMDM macrophages after phagocytosis of the microspheres under the influence of different intensity magnetic fields, as shown in Figure 11 The magnetic field intensity was 0 Gs, 47.1 Gs, 94.2 Gs and 188.3 Gs, the magnetic field frequency was 50 Hz, and the application time was 10 min. The results show that compared with the control group, after the macrophages phagocytose the target product microspheres of the example 1, the anti-inflammatory polarization of the macrophages is significantly improved under the influence of different intensity alternating magnetic fields. The magnetic field intensity of 94.2 Gs is the best magnetic field intensity for promoting the inflammatory effect.

[0099] It can be seen that when the magneto-electric composite anti-inflammatory microspheres of the application are recognized and phagocytosed by the macrophages into the cells, the microspheres will produce a magneto-electric coupling effect of magnetic signal-stress-strain-electric signal under the action of the external magnetic field, forming a microelectric field on the surface of the microspheres, thereby affecting the concentration of calcium ions in the macrophages and regulating the immune polarization effect. Therefore, by adjusting the signal intensity parameters of the external magnetic field, magneto-electric bioactive microspheres with different electrical properties can be obtained, thereby regulating the immune behavior of the macrophages in the cells.

[0100] In vivo experiment

[0101] The anti-inflammatory effect of the target product of the example 1 in the animal body was observed by performing HE tissue staining on the feet of the rheumatoid arthritis mice, as shown in Figure 12As shown. Among them, rheumatoid arthritis mice adopted CAIA induction mode, and LPS was injected into the abdominal cavity on the 4th day after the injection of collagen combined with antibodies into the tail vein. This time was recorded as the 0th day of the start of inflammation. Next, on the 2nd day, the ankle joint was injected with a microsphere preparation (the concentration of the preparation was 0.5 mg microspheres / 20uL normal saline, and the dose was 0.5 mg microspheres / 20g mouse body weight). Then, the magnetic field was applied three times at 2.5, 3, and 3.5 days respectively, and samples were taken for detection on the 7th day. Among them, the healthy mouse group was recorded as NoRA, the rheumatoid arthritis mouse group was recorded as RA, the mouse group injected with the microspheres obtained from comparative example 2 was recorded as PM, the mouse group injected with the microspheres obtained from example 1 was recorded as EPC, and the mouse group treated with a magnetic field on this basis was recorded as EPC+Mag. From Figure 12 It can be seen that starting from the RA group, the number of dark inflammatory cells in the bone marrow gradually decreased from left to right, and the number of EPC+Mag group was similar to that of the NoRA normal group, indicating that the target product obtained in the embodiment can effectively alleviate rheumatoid arthritis.

[0102] In summary, the injectable magnetoelectric composite anti-inflammatory microspheres prepared by the present invention can be pattern-recognized and phagocytosed by macrophages, and have a magnetoelectric coupling effect under the condition of a low-frequency alternating magnetic field of 50 Hz. The external controlled magnetic field can enhance the phagocytosis of the microspheres by macrophages, and can also cause the microspheres to generate an electric potential response inside the macrophages, prompting the macrophages to switch to an anti-inflammatory polarization direction. Alternating magnetic fields of different intensities have different anti-inflammatory polarization effects on macrophages. Changes in the intensity of the external controlled magnetic field can regulate the anti-inflammatory polarization effect of macrophages, thereby playing a controllable anti-inflammatory role, eliminating inflammation and promoting tissue repair. Experiments in which the injection preparation prepared by dispersing the microspheres in physiological saline and injecting them into mice have shown that it can effectively alleviate rheumatoid arthritis. Therefore, in the third aspect of the present invention, the present invention also provides the use of the injectable magnetoelectric composite anti-inflammatory microspheres in the preparation of drugs for treating immune-mediated inflammatory diseases, as well as the use in the preparation of anti-inflammatory drugs for wound repair.

[0103] It can be seen that the objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. The embodiments may be modified as desired without departing from the principles described. Therefore, the present invention includes all variations within the spirit and scope of the claims.

Claims

1. An injectable magnetoelectric composite anti-inflammatory microsphere, characterized in that: It is formed by extracellular matrix microsheets coated on the outer layer of polyvinylidene fluoride trifluoroethylene / cobalt ferrite composite microspheres.

2. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 1, comprising the following steps: (1) A polyvinylidene fluoride trifluoroethylene solution formed by polyvinylidene fluoride trifluoroethylene in N,N-dimethylformamide and a cobalt ferrite dispersion formed by cobalt ferrite nanoparticles in ethanol are mixed and ultrasonically dispersed to obtain a PVTF / CFO mixed dispersion, wherein the mass ratio of polyvinylidene fluoride trifluoroethylene to cobalt ferrite is (20-100):1; deionized water with a volume ratio of (0.5-2):1 and the PVTF / CFO mixed dispersion are placed in the lower layer and the upper layer of the same container respectively, and the phases are separated and allowed to stand to obtain a precipitate; (2) The precipitate is dispersed with ethanol and placed in a hydrothermal reactor, followed by a hydrothermal reaction at (110-140) °C for 0.5-2 h, followed by separation, and the resulting solid phase is the hydrothermal product; (3) placing the hydrothermal product obtained in step (2) in an ethanolamine aqueous solution, heating in a water bath at (70-90)°C for 10-60 min, washing, and sterilizing to obtain a modified product; (4) Taking cells that have grown into complete sheets, using a repeated freeze-thaw method to prepare extracellular matrix sheets, and using a vibration method during the thawing process to break the complete sheets into microsheets to obtain extracellular matrix microsheets, wherein the size of the extracellular matrix microsheets is 1 to 20 μm; (5) Mix the modified product and the extracellular matrix microsheet in a mass ratio of (3-8):1 in ultrapure water, adjust the pH to 6-8, add genipin for cross-linking, heat in a water bath at (30-45) °C for 3-6 h, wash and separate, and obtain a precipitate, which is the target product; The feed ratio of the sum of the mass of the modified product and the extracellular matrix microsheet to ultrapure water is (4-9) mg:10 mL, and the mass of genipin is 1-5% of the sum of the mass of the modified product and the extracellular matrix microsheet.

3. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, characterized in that: In step (1), the polyvinylidene fluoride trifluoroethylene solution is prepared by the following method: adding polyvinylidene fluoride trifluoroethylene solution powder to N,N-dimethylformamide solvent at a feeding ratio of (0.1~3) g: 25 mL, and stirring evenly to obtain the result; the cobalt ferrite dispersion is prepared by the following method: adding cobalt ferrite nanoparticles to ethanol at a feeding ratio of (2~60) mg: 15 mL, and ultrasonically dispersing evenly to obtain the result.

4. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, wherein: In step (1), the ultrasonic dispersion time is 0.5~2 h.

5. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, wherein: In step (1), the standing time for phase separation is 1 to 3 h.

6. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, wherein: In step (2), the feed ratio of the precipitate to ethanol is (10-500) mg:20 mL.

7. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, wherein: In step (3), the concentration of the ethanolamine aqueous solution is 14-16 mol / L, and the feed ratio of the hydrothermal product to the ethanolamine aqueous solution is (0.1-2) g:50 mL.

8. The method for preparing the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, wherein: In step (3), the cleaning is: cleaning with deionized water, or cleaning with 3% hydrogen peroxide solution followed by cleaning with deionized water.

9. Use of the injectable magnetoelectric composite anti-inflammatory microspheres according to claim 1 in the preparation of a drug for treating immune-mediated inflammatory diseases or in the preparation of an anti-inflammatory drug for wound repair.

10. Use of the injectable magnetoelectric composite anti-inflammatory microspheres obtained by the preparation method according to any one of claims 2 to 7 in the preparation of drugs for treating immune-mediated inflammatory diseases or in the preparation of anti-inflammatory drugs for wound repair.

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