Injectable magnetoelectric composite anti-inflammatory microsphere and preparation and application thereof

Abstract

The invention discloses injectable magnetoelectric composite anti-inflammatory microspheres and preparation and application thereof, the microspheres can be recognized by a macrophage mode and phagocytized into macrophages, and meanwhile, a magnetic field applied externally can strengthen the phagocytic effect of the macrophages on the microspheres and promote the macrophages to be converted to the anti-inflammatory polarization direction. Therefore, the anti-inflammatory polarization effect of the macrophages can be regulated and controlled by regulating and controlling the magnetic field intensity from the outside, inflammation is eliminated, and tissue repair is promoted. Experiments of injecting an injection preparation prepared by dispersing the microspheres in normal saline into a mouse body prove that the injection preparation can effectively relieve rheumatoid arthritis. Therefore, the injectable magnetoelectric composite anti-inflammatory microsphere can be used for preparing drugs for treating inflammatory diseases and preparing anti-inflammatory drugs for wound repair. The preparation process is simple in method, easy to implement and suitable for popularization and application.

Description

Technical Field

[0001] The 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 is not controlled or chronically transformed, it often leads to disease and even induces systemic immune-inflammatory response disorders. For example, rheumatoid arthritis (RA) is one of the most common immune-mediated inflammatory diseases and is almost a persistent and difficult-to-treat disease. If effective treatment is not carried out in time, serious joint damage, disability and work disorders will occur due to chronic inflammation of the joint lining (synovium). One of the main characteristics of rheumatoid arthritis is that it can affect multiple joints at the same time, manifesting as joint symmetry and invasive arthritis of the limbs. In severe cases, it can even directly cause irreversible destruction and deformity of articular cartilage, reducing the patient's daily quality of life. The pathogenesis of rheumatoid arthritis involves a variety of cytokines and cells. In addition, in the process of trauma repair such as bone integration and bone repair or tissue regeneration, the presence of implant materials as foreign bodies in the body after implantation will inevitably cause the body to produce corresponding inflammatory responses, affecting the relationship between materials and bone tissue. Therefore, reducing the release of inflammatory factors and limiting inflammatory responses are of great significance in disease treatment and trauma repair.

[0003] At present, there are three main types of commonly used anti-inflammatory materials: one is materials with intrinsic anti-inflammatory properties, such as hyaluronic acid, chitosan, collagen, hydrogel, and metal nanoparticles; the second is materials used for implant coatings, such as glycosaminoglycans, hyaluronic acid, keratin, and magnesium ion coatings; the third is materials used for anti-inflammatory drug delivery, such as hydrogel scaffolds, hydroxymethylcellulose films, and chitosan-embedded microparticles. The first two are simple anti-inflammatory materials with a slow onset of action; the third type of anti-inflammatory drug delivery releases inaccurate amounts, and after these three materials enter the human body, they cannot be externally regulated for the time and effect of action, and cannot achieve a good anti-inflammatory effect. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an injectable magnetoelectric composite anti-inflammatory microsphere and its preparation and application, the microsphere can be recognized by macrophage pattern and engulfed into the macrophage, and at the same time, the magnetic field applied externally can strengthen the phagocytosis of the microsphere by macrophages and promote the transformation of macrophages to anti-inflammatory polarization. Therefore, the anti-inflammatory polarization effect of macrophages can be regulated by regulating the magnetic field intensity from the outside, 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 an extracellular matrix microsheet (ECM) coated on the outer layer of polyvinylidene fluoride trifluoroethylene (PVTF) / cobalt ferrite (CFO) composite microsphere.

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

[0008] In the 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) mixing 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, and uniformly dispersing by ultrasonication to obtain a PVTF / CFO mixed dispersion, wherein the mass ratio of polyvinylidene fluoride trifluoroethylene to cobalt ferrite is (20-100):1; placing deionized water with a volume ratio of (0.5-2):1 and the PVTF / CFO mixed dispersion in the lower and upper layers of the same container, respectively, and allowing to stand for phase separation to obtain a precipitate;

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

[0011] (3) placing the hydrothermal product obtained in step (2) in an aqueous solution of ethanolamine, 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 3-6h, 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 invention, in step (1), the ultrasonic dispersion time is 0.5 to 2 hours.

[0016] In some specific embodiments of the present invention, in step (1), the standing time for phase separation is 1 to 3 hours.

[0017] In some specific examples of the present invention, 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, the feed ratio of the two is (0.1-3) g: 25 mL, and stirring evenly; preferably, the stirring is magnetic stirring, and the stirring time is 2-6 hours;

[0018] The cobalt ferrite dispersion is prepared by the following method: adding cobalt ferrite nanoparticles to ethanol at a feed ratio of (2-60) mg:15 mL, and uniformly dispersing by ultrasonication; preferably, the ultrasonic dispersion time is 0.5-1 h.

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

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

[0021] In some specific examples of the present invention, 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.

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

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

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

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

[0026] In some specific examples of the present invention, the separation is centrifugal separation, and the rotation speed of the 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 recognized and phagocytosed by macrophage patterns, and have a magnetoelectric coupling effect under the condition of a low-frequency alternating magnetic field of 50Hz. The externally controlled magnetic field can strengthen 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 change in the direction of 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 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 proved that it can effectively reduce 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, and 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; when an external magnetic field is applied, the pattern recognition and phagocytosis of macrophages are further enhanced, and an electric potential response is generated on the surface of the microspheres, inducing the flow of calcium ions inside the macrophages, causing 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 regulating the magnetic field intensity from the outside, thereby achieving controllable anti-inflammation.

[0030] (2) In the present invention, a significant advantage is the easily operable regulation mechanism of the external magnetic field, which enables the anti-inflammatory effect inside the cell to be precisely controlled by external means. Specifically, by selectively turning on the external magnetic field to stimulate the function of macrophages at a specific time point, precise intervention in the inflammatory response is achieved. In addition, the present invention also allows the intensity of the magnetic field to be flexibly selected 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, easy to implement, and 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 to regulate the phagocytosis of macrophages and cell immune polarization in sequence under an alternating magnetic field through the three steps of "ECM recognition", "cell phagocytosis" and "magnetoelectric response" in vitro. 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 magnetoelectrically coupled bioactive microsphere brings an innovative method of anti-inflammatory injection in vivo. By adjusting the application time and intensity of the external magnetic field, the microspheres can accurately and efficiently exert anti-inflammatory effects inside the cells and regulate the cell 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 infrared spectra of the product obtained in step (1) and the product obtained in 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 of the microspheres obtained in step (2) of Example 1 and the product obtained in step (3).

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

[0039] Figure 6 It is the Fourier transform infrared spectrum of the target product obtained in step (5) in 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] Fig. 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] Fig.10 It 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] Fig.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] Fig.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 shall be 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 from the market. For example, PVTF powder is produced by Piezotech, France. FC30, the molar ratio of vinylidene fluoride monomer to trifluoroethylene monomer in vinylidene fluoride trifluoroethylene copolymer is 7:3. CFO nanoparticles were purchased from Sigma. The extracellular matrix sheet was derived from NIH3T3 cells, which were purchased from the cell bank of the Chinese Academy of Sciences. BMDM macrophages were derived from the bone marrow of SPF-grade C57BL / 6 mice, which were purchased from the Zhejiang Provincial Experimental Animal Center. BMDM macrophages can also be purchased from the market.

[0048] BMDM macrophage culture steps: Place mouse bone marrow cells in a centrifuge tube, add 3 mL of red blood cell lysis solution, place in an ice-water mixing bath for lysis 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, place in a constant temperature and humidity incubator at 37°C and a carbon dioxide concentration of 5%, wherein 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] Steps for preparing extracellular matrix sheets by repeated freeze-thaw method: Take 10 6 NIH3T3 cells were placed in a culture dish, 10 mL of DMEM high-glucose culture medium was added, and the cells were cultured in a constant temperature and humidity incubator at 37°C and a carbon dioxide concentration of 5%. The medium was changed every 2 days, and a cell sheet with complete growth was obtained after 7 days. After discarding the culture medium, the cells were washed twice with ultrapure water, each time (3-5) mL, frozen at -80°C for 1-5 hours, and then thawed at 25°C. After discarding the liquid, new ultrapure water was added, and the volume was still (3-5) mL. This freeze-thaw process was repeated more than 3 times to remove the cells. After that, an aqueous solution containing DNase and RNase (the concentration of both enzymes was 0.1 mg / mL) was added to remove the residual DNA and RNA on the sheet, and an extracellular matrix sheet was obtained.

[0050] Sample preparation and test plan for flow cytometry: At the mature stage of macrophage culture, the target product microspheres were added to the culture medium for phagocytosis, with an addition amount of 1 mg / 10 6 The cells were cultured in the incubator for a period of time according to the experimental conditions, then taken out and washed twice with PBS for 3 minutes each time to remove the unphagocytosed microspheres. They were incubated and digested with trypsin at 37°C twice, for 6 minutes each time, and collected by blowing into a special round-bottom centrifuge tube for flow cytometry and centrifuged at 1500rpm for 5 minutes each time. After collection, they were stained with dyes corresponding to the experimental conditions in turn, with each staining time of 30 minutes. After centrifugation, they were collected into centrifuge tubes, and 0.5mL of PBS was added to each tube of cells to suspend them evenly before they could be tested on the machine. Among them, in addition to the dyes required for the experimental conditions, macrophages were specifically stained with PE-F4 / 80. The target product microspheres have autofluorescence with a central wavelength of about 700nm and do not require additional staining. The detection parameters of the flow cytometer are the fluorescence intensity values ​​of a single cell in each fluorescence channel. In this experiment, each group of samples was tested three times, and the effective cell amount for each test was 10 5 indivual.

[0051] Example 1

[0052] (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 disperse ultrasonically 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 to disperse 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 / 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;

[0053] (2) The above 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 separated was the hydrothermal product;

[0054] (3) placing the hydrothermal product obtained in step (2) in 50 mL of 16 mol / L ethanolamine aqueous solution, heating in a 37° C. water bath for 30 min, then oxidizing and cleaning with a 3% mass concentration hydrogen peroxide solution, then cleaning with deionized water, and separating by centrifugation at 8000 rpm for 5 min; dispersing the separated solid phase product in a 75% volume concentration ethanol solution, soaking for 1 h for sterilization and disinfection, and centrifuging to obtain the solid phase as the modified product;

[0055] (4) Taking cells that have grown into complete sheets in a culture dish, preparing extracellular matrix sheets by -80°C / 25°C repeated freeze-thaw method, and breaking the complete sheets into micro-sheets by vibration during the thawing process. The size of the micro-sheets is 1 to 20 μm;

[0056] (5) Take 30 mg of the modified product obtained in step (3) and 5 mg of the extracellular matrix microsheets obtained in step (4), mix them in an ultrapure aqueous solution, adjust the pH to 6-8, add 1.05 mg of genipin for cross-linking, heat in a 37°C water bath for 5 h, then use ultrapure water for centrifugation and wash several times, and finally separate the precipitate to obtain 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 treated 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 the 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 (No treatment) obtained in step (1) and the product (Heated) obtained in step (2) of 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 crystal phase melting peak is in the range of 140-160°C. It can be seen that the product obtained in step (1) has no obvious ferroelectric phase, and the peak height and peak area of ​​the crystal phase melting peak are 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 morphology 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 are 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 this embodiment 1 were subjected to X-ray diffraction analysis, and the XRD pattern thereof is as follows: Figure 3 As shown in the spectrum curve. Among them, the peak of 19.1° corresponds to the (020) α phase crystal plane in PVTF, the peak of 19.6° corresponds to the (200) β phase crystal plane in PVTF, the peak of 35.0° corresponds to the strongest peak (311) crystal plane in CFO, and the peak of 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 Example 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 is increased, and the content of fluorine atoms is reduced.

[0063] The product obtained in step (5) was subjected to functional group analysis using Fourier transform infrared spectroscopy. The FTIR spectrum thereof is as follows: Figure 5 Among them, the peaks related to PVTF identification are marked in green, which are 1401cm -1 , 1176cm -1 , 1095cm -1 and 888cm -1 ; The peaks related to the PVTF ferroelectric phase are marked in blue, which are 1261cm -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 1638cm -1 Amide I band, 1550 cm -1 The amide II band, 1461 cm -1 CN stretching vibration peak, 1261cm -1 The 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 maintains the protein conformation of the extracellular matrix without being destroyed.

[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, it can be seen that in the target product, the extracellular matrix microsheets form 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 the extracellular matrix (ECM).

[0066] Performance Testing:

[0067] In an alternating magnetic field with a frequency of 50 Hz, the electrometer was used to test the magnetic and electric properties of the target product obtained in Example 1. The specific method is as follows: the target product obtained in Example 1 was cooled and condensed into a film with a thickness of about 130 nm in a hydrothermal process, and silver was plated in the 0.5 cm*0.5 cm area in the center of both sides of the film. The two poles of the voltage test were connected to the positive and negative poles of the electrometer respectively, and placed in a magnetic field for testing. The results are shown in FIG. Figure 7 As shown. Figure 7 According to the analysis, 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 is continuously increased, the corresponding surface potential fluctuation also increases and is almost linear, which shows 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: in the mature 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 The cells were cultured in the incubator for 6 hours before being taken out and washed twice with PBS to remove the unphagocytosed microspheres. The cytoskeleton was stained with FITC-phalloidin and the cell nucleus was stained with DAPI. The target product microspheres spontaneously fluoresced red and no additional staining was required. Figure 8 As shown. Figure 8 In the figure, the blue is the cell nucleus stained with DAPI, the green is the macrophage skeleton stained with FITC, and the red is the target product microsphere. It can be seen that the target product microsphere has passed the surface recognition mechanism of the macrophage and has been successfully engulfed into 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 disperse ultrasonically 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 to disperse 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 / 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 above 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 separated was the hydrothermal product;

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

[0076] (3) The hydrothermal product obtained in step (2) is placed in 50 mL of an aqueous solution of 16 mol / L ethanolamine, heated in a water bath at 37° C. for 30 min, then washed with deionized water, and centrifuged at 8000 rpm for 5 min; the separated solid phase product is dispersed in a 75% (volume concentration) ethanol solution, immersed for 1 h for sterilization and disinfection, and the solid phase obtained by centrifugation is 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 the PVTF solution to 15 mL of ethanol to obtain 40 mL of PVTF dispersion and mix by ultrasonic 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 above 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) placing the hydrothermal product obtained in step (2) in 50 mL of 16 mol / L ethanolamine aqueous solution, heating in a 37° C. water bath for 30 min, then washing with deionized water, and centrifuging at 8000 rpm for 5 min; dispersing the separated solid phase product in a 75% (volume concentration) ethanol solution, soaking for 1 h for sterilization, and centrifuging to obtain the solid phase as 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 disperse by ultrasonic method 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 to disperse by ultrasonic method 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 above precipitate was dispersed with 20 mL of ethanol and placed in a hydrothermal reactor, then heated in a muffle furnace at 120° C. for 0.5 h, centrifuged at 8000 rpm for 5 min, and the solid phase separated was the hydrothermal product;

[0084] (3) placing the hydrothermal product obtained in step (2) in 50 mL of 14 mol / L ethanolamine aqueous solution, heating in a 37° C. water bath for 10 min, then oxidizing and cleaning with a 3% mass concentration hydrogen peroxide solution, then cleaning with deionized water, and centrifuging at 8000 rpm for 5 min; dispersing the separated solid phase product in a 75% volume concentration ethanol solution, soaking for 0.5 h for sterilization and disinfection, and centrifuging to obtain the solid phase as the modified product;

[0085] (4) Taking cells grown into complete sheets in a culture dish, preparing extracellular matrix sheets by repeated freezing and thawing at -80°C to 25°C, and breaking the complete sheets into micro-sheets with a size of 1 to 20 μm by vibration during the thawing process;

[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 aqueous 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 for centrifugation and wash several times, and finally separate the precipitate to obtain the target product.

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

[0088] Example 3

[0089] (1) Add 3.0 g of PVTF powder to 25 mL of N,N-dimethylformamide (DMF) and stir magnetically for 3 h to obtain a PVTF solution; add 60 mg of CFO nanoparticles to 15 mL of ethanol and disperse ultrasonically for 2 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 to disperse ultrasonically for 1 h; take 20 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, stand for 3 h to separate the phases, centrifuge at 8000 rpm for 5 min, and retain the precipitate;

[0090] (2) The above 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 separated was the hydrothermal product;

[0091] (3) placing the hydrothermal product obtained in step (2) in 50 mL of 16 mol / L ethanolamine aqueous solution, heating in a 37° C. water bath for 60 min, then oxidizing and cleaning with a 3% mass concentration hydrogen peroxide solution, then cleaning with deionized water, and centrifuging at 8000 rpm for 5 min; dispersing the separated solid phase product in a 75% volume concentration ethanol solution, soaking for 2 h for sterilization and disinfection, and centrifuging to obtain the solid phase as the modified product;

[0092] (4) Taking cells grown into complete sheets in a culture dish, preparing extracellular matrix sheets by repeated freezing and thawing at -80°C to 25°C, and breaking the complete sheets into micro-sheets with a size of 1 to 20 μm by vibration during the thawing process;

[0093] (5) Take 40 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 aqueous solution, adjust the pH to 6-8, add 1.35 mg of genipin for cross-linking, heat in a 37°C water bath for 6 h, then use ultrapure water for centrifugation and wash several times, and finally separate the precipitate to obtain the target product.

[0094] The same method as in Example 1 was adopted for characterization, and the target product obtained in Example 2 was identified 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. Fig. 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 188.3 Gs, the applied frequency is 50 Hz, and the applied time is 10 min. In each group of bar graphs, the light color on the left indicates that no magnetic field is applied, and the dark color on the right indicates that a 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 the target product microspheres. From Fig. 9 It can be seen that in the comparison of each group, it can be found that: compared with the case without magnetic field, under the action of magnetic field, the phagocytosis of macrophages on each microsphere is improved, among which the EPC group modified with extracellular matrix surface has the most significant improvement. This shows that the external controlled magnetic field can enhance the phagocytosis of macrophages on microspheres, and the extracellular matrix can promote the pattern recognition and phagocytosis of macrophages on microspheres.

[0097] Flow cytometry was used to characterize the changes in macrophage polarization after BMDM macrophages engulfed different groups of microspheres under the influence of a magnetic field. Fig.10 As shown. APC-iNOS surface marker is used to represent M1 pro-inflammatory polarization, FITC-CD206 surface marker is used to represent M2 anti-inflammatory polarization, and the average fluorescence intensity of the fluorescence data of different channels measured is calculated and then divided to obtain the ratio of M2 / M1, which represents the degree of anti-inflammatory polarization of macrophages. The macrophages not treated with microspheres are set as the control group and recorded as 0, the group treated with the product obtained in comparative example 3 is recorded as P, and the group treated with the product in Example 1 is recorded as EPC. Fig.10 It can be seen that regardless of whether the magnetic field is applied or not, the M2 / M1 value of the EPC group is significantly higher than that of the P group and the control group 0; in the P group and the control group 0, the M2 / M1 value did not change significantly before and after the magnetic field is applied; in the EPC group, the M2 / M1 value increased significantly 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 macrophages, and at the same time, the external controlled magnetic field can act on the microspheres of the EPC group, further prompting the macrophages to shift towards the anti-inflammatory polarization direction.

[0098] Flow cytometry was used to characterize the changes in macrophage polarization after BMDM macrophages phagocytosed microspheres under the influence of magnetic fields of different intensities. Fig.11 As shown. Among them, the magnetic field strengths are 0Gs, 47.1Gs, 94.2Gs and 188.3Gs, the magnetic field frequency is 50Hz, and the application time is 10min. The results show that compared with the control group, after the macrophages phagocytize the target product microspheres obtained in Example 1, applying alternating magnetic fields of different intensities will significantly increase the anti-inflammatory polarization of the macrophages, among which the magnetic field strength of 94.2Gs is the best magnetic field strength to produce a pro-inflammatory effect.

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

[0100] In vivo experiments

[0101] The anti-inflammatory effect of the target product obtained in Example 1 in the animal body was observed by HE tissue staining of the feet of rheumatoid arthritis mice. Fig.12As shown. Among them, the rheumatoid arthritis mice adopted the CAIA induction model, and LPS was injected into the abdominal cavity on the 4th day after the tail vein injection of collagen combined with antibodies. This time was recorded as the 0th day of the inflammation onset time. Next, on the 2nd day, the microsphere preparation was injected into the ankle joint (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 the product of 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 the magnetic field on this basis was recorded as EPC+Mag. From Fig.12 It can be seen that starting from the RA group, from left to right, the number of dark inflammatory cells in the bone marrow gradually decreased, 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 recognized and phagocytosed by macrophage patterns, and have a magnetoelectric coupling effect under the condition of a low-frequency alternating magnetic field of 50Hz. 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 change in the direction of 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 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 they can effectively reduce 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, and in the preparation of anti-inflammatory drugs for wound repair.

[0103] It can be seen that the purpose of the present invention has been completely and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments. Without departing from the principles, the implementation methods can be modified arbitrarily. Therefore, the present invention includes all modified implementation methods based on 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) mixing 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, and uniformly dispersing by ultrasonication to obtain a PVTF / CFO mixed dispersion, wherein the mass ratio of polyvinylidene fluoride trifluoroethylene to cobalt ferrite is (20-100):1; taking deionized water with a volume ratio of (0.5-2):1 and the PVTF / CFO mixed dispersion, respectively, and placing them in the lower layer and the upper layer of the same container, standing for phase separation, and separating to obtain a precipitate; (2) dispersing the precipitate with ethanol and placing it in a hydrothermal reactor, followed by a hydrothermal reaction at (110-140)° C. for 0.5-2 h, separating, and obtaining a solid phase as a hydrothermal product; (3) placing the hydrothermal product obtained in step (2) in an aqueous solution of ethanolamine, 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, 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; (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 3-6h, washing, separating, and obtaining 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, the feeding ratio of the two is (0.1-3) g: 25 mL, and stirring to obtain uniformity; the cobalt ferrite dispersion is prepared by the following method: adding cobalt ferrite nanoparticles to ethanol, the feeding ratio of the two is (2-60) mg: 15 mL, and ultrasonically dispersing to obtain uniformity.

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

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

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

7. The method for preparing injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, characterized in that: 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 injectable magnetoelectric composite anti-inflammatory microspheres according to claim 2, characterized in that: 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 as claimed in claim 1.

10. Use of the injectable magnetoelectric composite anti-inflammatory microspheres obtained by the preparation method according to any one of claims 2 to 7.

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