Method for regulating and controlling in-vitro programmed polarization of macrophages

Abstract

The invention discloses a method for regulating and controlling in-vitro programmed polarization of macrophages. Carrying out ultrasonic treatment on MXene to obtain MXene nanosheets, and adding the MXene nanosheets into a DMEM (Dulbecco Modified Eagle Medium) to obtain a regulation culture medium; the method comprises the following steps: culturing macrophages by using a DMEM culture medium until the macrophages are adhered to the wall, replacing the DMEM culture medium with a regulation culture medium, placing the macrophages in a rotating magnetic field generated by a rotating permanent magnet after the macrophages endocytose MXene nanosheets, enabling the MXene nanosheets to polarize the macrophages into M1-type macrophages through electromagnetic induction, and after the rotating magnetic field is removed, enabling the M1-type macrophages to become M2-type macrophages, the in-vitro program polarization of the macrophages is realized. The invention provides a simple method for establishing the macrophage polarization model, reduces the cost of establishing the model, improves the repeatability of a model experiment, and promotes related immune mechanism research.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for regulating programmed polarization of macrophages in vitro. Background Art

[0002] Macrophages are essential components of the body's innate immune system, possessing a high degree of functional diversity and plasticity. They play a crucial role in immune regulation and pathogen clearance. They are also a key cell type in the immune system, a type of white blood cell distributed in tissues that originates from monocytes. Their primary functions in the body include phagocytosis of pathogens, clearance of dying cells, regulation of inflammatory responses, and involvement in tissue repair. In response to various environmental cues, macrophages can polarize into distinct subtypes, the most prominent of which are the M1 and M2 subtypes. M1 macrophages (classically activated macrophages) are typically activated by pathogen components such as bacteria and viruses (such as LPS) or cytokines (such as IFN-γ). They produce high levels of proinflammatory cytokines such as TNF-α, IL-6, and IL-12, possess potent bactericidal and tumor cell-killing abilities, promote Th1 immune responses, and contribute to cellular immunity. M2 macrophages (alternatively activated macrophages) are typically activated by anti-inflammatory cytokines (such as IL-4 and IL-13) or immune complexes; they produce anti-inflammatory cytokines such as IL-10 and TGF-β, which participate in tissue repair, promote angiogenesis, and inhibit inflammation. In short, M1 macrophages are pro-inflammatory cells, while M2 macrophages are anti-inflammatory cells.

[0003] Macrophages play an essential role in infection resolution and tissue repair by dynamically regulating their polarization between M1 and M2 phenotypes. They not only eliminate microorganisms, dead cells, and debris through phagocytosis but also modulate immune responses through the secretion of cytokines (such as IL-1, TNF-α, and IL-10) and chemokines, participating in tissue repair, remodeling, and inflammatory responses. In vitro manipulation of macrophage polarization facilitates the development of macrophage polarization models for biomedical research. However, certain pathogens, such as drug-resistant bacteria, viruses, or fungi, can evade macrophage phagocytosis and killing through various mechanisms, rendering macrophages unable to effectively respond to these pathogens and contributing to the persistence or worsening of infection. Therefore, a programmed two-stage regulatory strategy should be adopted: first, promoting macrophage differentiation toward the M1 phenotype to enhance bactericidal efficacy, and then regulating their conversion to the M2 phenotype to accelerate tissue regeneration. This approach leverages the immunomodulatory function of macrophages for the efficient treatment of infected wounds. Manipulating macrophage polarization in vitro can provide a cellular model for biopharmaceutical research. This is not only of great significance in basic research on inflammatory diseases, tumor immune regulation, and tissue regeneration, but also allows for faster mechanistic studies and in-depth understanding of macrophage-mediated immune mechanisms, such as regulation of inflammatory responses, changes in phagocytic function, and functional differentiation of tumor-associated macrophages (TAMs). It can also be used for disease simulation, establishing in vitro models of inflammatory diseases, the tumor microenvironment, fibrosis, and other related diseases to support the analysis of pathological processes; and can be used to evaluate the immunomodulatory effects and mechanisms of action of anti-inflammatory drugs, immunomodulators, and tumor therapeutics. In particular, research on the immune mechanisms involved in macrophages can facilitate the development of new anti-tumor and antibacterial drugs and provide new entry points for the development of relevant clinical treatment strategies.

[0004] Currently, drugs that regulate macrophage polarization primarily include small molecule inhibitors (such as dexamethasone) and cytokines (such as IL-4 / IL-10). However, these drugs can only unilaterally modulate macrophage phenotypes (inhibiting M1 polarization or promoting M2 polarization). These methods suffer from low induction efficiency, unstable polarization phenotype conversion, and significant impact on cell viability, making them difficult to meet the application requirements of high-throughput screening, precise immune regulation, and clinical translation. Patent CN118141988A discloses a component for regulating the osteogenic immune microenvironment, its preparation method, and its application. Titanium dioxide nanotubes are coated on titanium sheets to form BTTs (BTTs), which are then cultured on the surface of the BTTs to regulate macrophage polarization with or without magnetic field intervention. However, this method requires not only coating the titanium sheets with carbon dioxide nanotubes but also culturing RAW264.7 cells in the BTTs for three days, resulting in a long and costly regulation process. Therefore, there is an urgent need for a more efficient and inexpensive method for programmed macrophage polarization that can quickly obtain the required cell models to improve experimental reproducibility, promote mechanism research, and provide more promising technical means for immune regulation of clinically relevant diseases. SUMMARY

[0005] In view of the prior art, the present application aims to provide a method for regulating in vitro programmed polarization of macrophages. After the MXene nanosheet is endocytosed by the macrophages, it is placed in a switching rotating magnetic field, and wireless radio stimulation is generated under the driving of the rotating magnetic field to promote the sequential regulation of M1 to M2 phenotype transformation of the macrophages. The present application provides a simple method for establishing a macrophage polarization model, reduces the cost of establishing the model, improves the repeatability of the model experiment, and promotes the research on related immune mechanisms.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application provides a method for regulating in vitro programmed polarization of macrophages, wherein the method comprises the following steps: MXene is subjected to ultrasonic treatment to obtain MXene nanosheets, which are added to DMEM culture medium to obtain a regulation culture medium; macrophages are cultured in the DMEM culture medium until they adhere to the wall, the DMEM culture medium is replaced with the regulation culture medium, and after the macrophages endocytose the MXene nanosheets, they are placed in a rotating magnetic field generated by a permanent magnet, the MXene nanosheets cause the macrophages to polarize into M1 type macrophages through electromagnetic induction, and after the rotating magnetic field is removed, the M1 type macrophages transform into M2 type macrophages, thereby realizing in vitro programmed polarization of the macrophages.

[0007] Preferably, the ultrasonic treatment is ice bath ultrasonic treatment of MXene under nitrogen protection; the power of the ultrasonic treatment is 500w, and the time is 10min.

[0008] Preferably, the MXene nanosheets are single-layer nanosheets with a size of 200-300nm.

[0009] Preferably, the concentration of the MXene nanosheets in the regulation culture medium is 5 μg / mL.

[0010] Preferably, the time for the macrophages to endocytose the MXene nanosheets is 2-4h.

[0011] Preferably, the electromagnetic induction refers to that the rotating magnetic field generates magnetic induction lines, and the MXene nanosheets cut the magnetic induction lines in the rotating magnetic field to generate a micro-current in the macrophages.

[0012] Preferably, the rotating speed of the rotating magnetic field is 500rpm; the application time of the rotating magnetic field is 15min-24h; and the magnetic field strength of the rotating magnetic field is 1.0 Tesla.

[0013] Preferably, the macrophages are located below the permanent magnet at a distance of 1.0-2.0cm from the permanent magnet.

[0014] Preferably, the time for the M1 macrophages to become M2 macrophages is 15 minutes to 24 hours.

[0015] In addition to in vitro regulation, MXene nanosheets can also be used in vivo.

[0016] In a second aspect, the present invention provides the use of MXene nanosheets in the preparation of a drug that promotes wound healing. The MXene nanosheets are dispersed on the wound, allowed to stand, and then the wound is placed under a rotating permanent magnet. The permanent magnet is then removed to promote wound healing. The MXene nanosheets are single-layer nanosheets with a size of 200-300 nm.

[0017] When MXene nanosheets are used as medicine: MXene nanosheets are dispersed on the wound, allowed to stand, and then the wound is placed under a rotating permanent magnet, and then the permanent magnet is removed to promote wound healing.

[0018] After being engulfed by macrophages, MXene nanosheets are placed in a rotating magnetic field, where they polarize to the M1 type and produce high levels of pro-inflammatory cytokines, which contribute to cellular immunity. When the rotating magnetic field is turned off, the macrophages transition from the M1 to the M2 phenotype. M2 macrophages produce anti-inflammatory cytokines, which participate in tissue repair, promote angiogenesis, and inhibit inflammation. This allows the development of drugs that promote wound healing and other related effects.

[0019] Beneficial effects of the present invention: (1) This invention involves endocytosing MXene nanosheets into macrophages and then placing them in a switching rotating magnetic field. The changing magnetic field generates radio stimulation to promote the sequential regulation of macrophage phenotype transition from M1 to M2. This provides a simple method for establishing a macrophage polarization model, reduces the cost of model establishment, improves the reproducibility of model experiments, and promotes research on related immune mechanisms.

[0020] (2) The MXene nanosheets of the present invention sequentially regulate the phenotypic transition of macrophages from M1 to M2 in the presence or absence of a rotating magnetic field, thereby enhancing the bactericidal ability of macrophage lysosomes and removing excess reactive oxygen species (ROS) in macrophages to alleviate oxidative stress and reduce excessive inflammation, thereby promoting tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :(a) TEM image of MXene nanosheets, scale bar is 100 nm; (b) XRD image of MXene nanosheets; (c) XPS image of MXene nanosheets; (d) current diagram of MXene nanosheets at different rotation speeds; (e) electrokinetic potential diagram of MXene nanosheets.

[0022] Figure 2:(a) Live / dead assay of RAW264.7 after culturing with different concentrations of MXene nanosheets for 2 days, with live and dead cells stained green and red, respectively; (b) Viability of RAW264.7 after culturing with different concentrations of MXene nanosheets for 1, 2, and 3 days; (c) Survival rate of RAW264.7 after culturing for 3 days; (d) Representative fluorescence images of RAW264.7 after incubation with 5 μg / mL MXene nanosheets (100-200 nm) for 4 h; (e) Representative fluorescence images of RAW264.7 after incubation with 5 μg / mL MXene nanosheets (100-500 μm) for 4 h; Figure 3 : (a) RT-qPCR analysis results of RAW264.7-related marker mRNA under magnetic field driving, (b) RT-qPCR analysis results of RAW264.7-related marker mRNA after the magnetic field is removed; Figure 4 :(a) Analysis results of IL-6 and CD206 secretion factors of RAW264.7 cells after magnetic field drive; (b) Analysis results of IL-6 and CD206 secretion factors of RAW264.7 cells after magnetic field removal; Figure 5 : (a) Quantitative analysis of the co-localized fluorescence images and mean fluorescence intensity of RAW264.7 and Staphylococcus aureus in each group after 15 min of treatment; (b) Quantitative analysis of the co-localized fluorescence images and mean fluorescence intensity of RAW264.7 and Staphylococcus aureus in each group after 40 min of treatment; Figure 6 : Analysis of plate coating and colony count results after co-culture of RAW264.7 and Staphylococcus aureus in each group; Figure 7 : Quantitative analysis of ROS fluorescence images and mean fluorescence intensity of RAW264.7 cells after 24 h of treatment in each group; Figure 8 : Quantitative analysis results of WB bands and gray values ​​of signaling pathways in RAW264.7 treated by each group. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0024] As introduced in the background section, macrophage polarization can not only provide cell models for the research and development of biological medicine, but also treat immune-related diseases. There are reports that macrophages are placed on the surface of BTTs for culture, and the polarization of macrophages is regulated under the intervention of a magnetic field or without the intervention of a magnetic field, but the regulation time is long, and the cells need to be cultured for more than 3 days.

[0025] Based on this, the purpose of the present application is to provide a method for regulating the in vitro polarization of macrophages. After the MXene nanosheet of the present application is endocytosed by macrophages, it is placed in an on-off rotating magnetic field, and radio stimulation is generated under the changing magnetic field to promote the sequential regulation of M1 to M2 phenotype transformation of macrophages. Macrophages endocytose MXene, and through radio stimulation, MXene generates an induced current in the macrophage in vivo, promoting the phenotype transformation of macrophages.

[0026] However, when macrophages are placed on the surface of MXene two-dimensional material for culture and then placed in a rotating magnetic field, the macrophages polarize to M2, and after the magnetic field is removed, there is no effect on the macrophages. Therefore, it can be seen that although MXene and BTTs are both Ti-containing materials, due to their specific composition and morphology, etc., they will all affect the polarization of macrophages.

[0027] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific embodiments.

[0028] The test materials used in the embodiments of the present application are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1: Regulation of macrophage polarization (1) Preparation of MXene nanosheet: MXene two-dimensional material (purchased from Shandong Enyan New Material Technology Co., Ltd.) was ultrasonicated (220v, 500w) under nitrogen protection for 10 min in an ice bath to obtain MXene nanosheet (200-300 nm).

[0030] (2) MXene nanosheet was added to fresh DMEM culture medium to make its concentration 5 μg / mL to obtain a regulation culture medium.

[0031] (3) Regulation of macrophage polarization: RAW264.7 cells (mouse mononuclear macrophage leukemia cells) were cultured in a T25 flask containing DMEM with 10% FBS and placed in a 37°C, 5% CO2 incubator to observe the cell growth status. When the cell growth density reached 90%, the old culture medium was aspirated with a 1 mL pipette, 2-3 mL of sterile phosphate buffered saline (PBS) solution was added, and the bottle was gently shaken to rinse. This action was repeated twice and then poured out. Since the RAW264.7 cell line is a semi-adherent cell, trypsin is not required. 2 mL of fresh DMEM culture medium was added to the T25 flask, and a 1 mL pipette was used to gently blow off the RAW264.7 cells at the bottom of the bottle and mix to obtain a cell suspension. RAW264.7 cells were plated at a concentration of 1×10 6 cells / mL in a 12-well plate. The culture medium was then replaced with conditioned medium. After 4 hours, after the RAW264.7 cells had engulfed the MXene nanosheets, a rotating magnetic field was applied. Culture was continued under a permanent magnet, directly below the magnet, at a distance of 1.0 cm and a magnetic field strength of 1.0 Tesla at 500 rpm. After 15 minutes of incubation, the RAW264.7 cells began to polarize to the M1 phenotype; after 24 hours of incubation, most RAW264.7 cells were polarized to the M1 phenotype. After removing the magnetic field, the cells were incubated under standard culture conditions. After 15 minutes of incubation, the M1 macrophages began to transition to the M2 phenotype; after 24 hours of incubation, most M1 macrophages had transitioned to the M2 phenotype.

[0032] Comparative Example 1 The difference from Example 1 is that in step (2), MXene nanosheets are replaced with MXene two-dimensional materials (with a size of 100-500 μm) and added to fresh DMEM culture medium to a concentration of 5 μg / mL to obtain a regulated culture medium.

[0033] In the presence or absence of a magnetic field, RAW264.7 cells are basically polarized to the M2 type.

[0034] Comparative Example 2 The difference from Example 1 is that in step (2), LPS (lipopolysaccharide) is used to replace MXene nanosheets and added to fresh DMEM culture medium. Because LPS stimulation at a concentration of 5 μg / mL for 24 h will cause significant cell apoptosis, its concentration is selected to be 1 μg / mL to obtain a regulated culture medium.

[0035] In the presence or absence of a magnetic field, RAW264.7 cells are basically polarized to the M1 type.

[0036] Example 2: Characterization pass Figure 1The transmission microscopy (TEM) image in (a) shows that the MXene nanosheets prepared in Example 1 are 200-300 nm in size, have a single-layer structure and a smooth surface. Figure 1 From the X-ray diffraction (XRD) pattern in (b), it can be seen that the diffraction peak at 6.6° is attributed to the crystal plane around (002) of the crystal structure, confirming the existence of a layered structure in the MXene two-dimensional material. Subsequently, in order to detect the elemental composition and chemical state of the surface of the MXene nanosheet, the X-ray photoelectron spectroscopy (XPS) pattern was further observed, as shown in Figure 2. Figure 1 As can be seen in (c), there are four forms of titanium in MXene nanosheets, namely titanium carbide, Ti 2+ and Ti 4+ (TiO2). The C1s spectrum shows four characteristic peaks at 281.5 eV, 284.7 eV, 285.5 eV, and 288.5 eV, attributed to Ti-C, C-C, CO, and C=O bonds, respectively. This confirms that ultrasonic treatment has no effect on the other properties of the MXene material, other than converting it into single-layer MXene nanosheets.

[0037] To further investigate the magnetoelectric responsiveness of MXene nanosheets, a current meter was used to evaluate the strength of the electrical signals generated by the MXene nanosheets in different rotating magnetic fields. The rotating magnetic field is provided by a magnet driven by a motor, which can control different speeds. The electrical signals are generated by electromagnetic induction. Figure 1 (d) shows that when the magnet rotates at different rotation speeds of 300rpm, 400rpm and 500rpm, the corresponding induced currents generated by the MXene nanosheets are 0.001μA, 0.015μA and 0.078μA respectively. The periodic changes in the rotating magnetic field produce different induced current signals, and the induced current signals at this time are consistent with the size of the rotating magnetic field. The above results show that the induced current signal and frequency generated by the MXene nanosheets can be controlled by rotating magnetic fields at different speeds. In addition, the zeta potential value of the MXene nanosheets is as follows Figure 1 As shown in (e), the negatively charged MXene nanosheets have good dispersibility in ultrapure water, ethanol, PBS and DMEM. Especially when dispersed in DMEM, the MXene nanosheets show good stability and a potential value of -125.07mV.

[0038] Example 3: Biocompatibility To evaluate the optimal concentration of MXene nanosheets in cells, the compatibility of cells with different concentrations (0-40 μg / mL) of MXene nanosheets was explored by evaluating cytotoxicity using live / dead staining and cell counting kit 8 (CCK-8 assay). Considering the rapid proliferation of macrophages, the compatibility was evaluated for 3 days. RAW264.7 cells were cultured at a rate of 5×10 5 cells / mL were seeded in a 24-well plate and co-cultured with MXene nanosheets for 48 h. After the incubation, live / dead cell double staining reagent (C2015M, Biyuntian) was added and incubated at 37°C in the dark for 15 min. Fluorescence imaging was performed using a laser scanning confocal microscope. For CCK-8 detection, RAW264.7 cells were plated at 1×10 5 Cells were seeded at a density of 10 cells / mL in 96-well plates and co-cultured with different concentrations of MXene nanosheets for 24, 48, and 72 hours. After adding 100 μL of CCK-8 working solution to each well and incubating at 37°C for another 2 hours, the absorbance (OD) of each well was measured at 450 nm using a multi-function microplate reader.

[0039] The results are as follows Figure 2 As shown in (a), when stained with calcein green-AM and propidium iodide (PI), live cells appear green, while dead cells appear red. When the concentration of MXene nanosheets is less than 20 μg / mL, no obvious dead cells are observed, and there is no significant difference from the control group; when the concentration rises to 40 μg / mL, the proportion of dead cells increases significantly, showing obvious red fluorescence aggregation. In addition, according to Figure 2 (b) CCK-8 results show that the absorbance value (OD450) of different concentrations of MXene nanosheets changes in the same trend as the live / dead staining results. It is worth noting that Figure 2 Panel (c) shows that a 5 μg / mL concentration of MXene nanosheets exhibited significantly higher cell viability on day 3 than other concentrations, demonstrating that this concentration not only exhibits excellent cytocompatibility but also actively enhances cell proliferation. In the presence of a 500 rpm rotating magnetic field (where MXene nanosheet-mediated electrical signals are most pronounced), exposure to a magnetic field was observed to have no effect on cell viability. Based on these cytocompatibility results, the combination of 5 μg / mL MXene nanosheets and a 500 rpm magnetic field was selected as the experimental condition for subsequent experiments.

[0040] Example 4: Endocytosis Detection A 200 μg / mL MXene nanosheet solution was dispersed in a 10 mL ethanol / water (9 / 1, v / v) mixture and reacted with 100 μL APTES under vigorous stirring for 2 h. Ammonia was then added to adjust the pH to 10. The nanosheets were collected by centrifugation and washed with ethanol several times to obtain amino-modified MXene nanosheets. 1 mg / mL FITC was then diluted 500 times and mixed with the amino-modified MXene nanosheets (volume ratio 1:1) and incubated at 4 °C in the dark for 24 h. Finally, centrifugation and ethanol washing were used to ensure the removal of any unbound fluorescent dye to obtain fluorescently labeled nanosheets (FITC-MXene nanosheets). FITC-MXene nanosheets were added to DMEM culture medium to obtain a regulated medium with a concentration of 5 μg / mL. 5×10 5 Cells at a density of cells / mL were cultured in a 24-well plate using a regulated medium for 4 h, and then washed slowly with PBS buffer three times, and then stained with lysosomal tracer (LysoTracker TM The samples were stained with DAPI (red, 1:1000 dilution) in the dark for 30 min, and then the nuclei were stained with DAPI (blue) in the dark for 10 min. Finally, the samples were imaged using a laser scanning confocal microscope. Figure 2 (d) shows the co-localization image of the MXene nanosheets from Example 1 with lysosomes, which were successfully internalized by RAW264.7 cells. As can be seen in Figure 2(e), the MXene two-dimensional material from the comparative example was not internalized by RAW264.7 cells.

[0041] Example 5: Marker Detection The experiment was divided into four groups: Example 1, Comparative Examples 1-2, and a blank control group. The blank control group differed from Example 1 in that no MXene nanosheets were added.

[0042] (1) RT-qPCR detection Cells were collected from the blank control group, Example 1 group, and Comparative Examples 1-2 groups after and after the rotating magnetic field was removed. Total RNA was extracted using an RNA extraction kit (Nanjing Novozymes Biotechnology Co., Ltd.). RNA absorbance at 260 nm and 280 nm was measured using a Q-5000 spectrophotometer to assess concentration (A260) and purity (A260 / A280 ratio). Subsequently, the extracted RNA was reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Kit (Accor Biotech). Using a LightCycler 96 Real-Time PCR System, mRNA expression levels of IL-1β and IL-6 (M1-type markers) and Arg-1 and CD206 (M2-type markers) were measured using β-actin as an internal reference gene. Reaction conditions included pre-denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Primer sequences for the genes involved were synthesized by Shanghai Bioengineering and are shown in Table 1.

[0043] Table 1 Related sequences RT-qPCR analysis showed that the use of MXene two-dimensional materials in Comparative Example 1 significantly upregulated the mRNA expression levels of M2 phenotype markers (Arg-1 and CD206) in the presence or absence of a magnetic field, and the mRNA expression levels were higher in the presence of a magnetic field. Comparative Example 2 used LPS to significantly upregulated the mRNA expression levels of M1 phenotype markers (IL-1β and IL-6) in RAW264.7 cells in the presence or absence of a magnetic field, confirming that LPS can only induce RAW264.7 cells to polarize toward M1 in the presence or absence of a magnetic field. Figure 3 As shown in (a), compared to the blank control, MXene nanosheets combined with magnetic field treatment for 24 hours increased the expression of these markers, indicating that MXene nanosheets under magnetic field conditions promote the polarization of macrophages toward the M1 phenotype. Conversely, in the presence of a magnetic field, the expression of anti-inflammatory genes (Arg-1 and CD206) in cells treated with MXene nanosheets was significantly reduced compared to the blank control.

[0044] like Figure 3 As shown in (b), 24 hours after the magnetic field was removed, the expression of pro-inflammatory genes (IL-1β and IL-6) decreased significantly compared to the blank control and LPS groups, while the expression of anti-inflammatory genes (Arg-1 and CD206) was significantly higher than in the other groups. These results indicate that MXene nanosheets can modulate the transition of macrophages from the M1 to the M2 phenotype in the absence of a magnetic field. RT-qPCR results indicate that MXene nanosheets promote the polarization of macrophages toward the M1 phenotype under magnetic field exposure and promote the transition from the M1 to the M2 phenotype after the magnetic field is removed.

[0045] According to Comparative Example 1 and Example 1, it can be seen that if the cells do not internalize MXene, the cells can only polarize to M2 in the presence or absence of a magnetic field; after the cells internalize MXene, in the presence or absence of a magnetic field, the cells will first polarize to M1 and then to M2, achieving sequential polarization.

[0046] (2) ELISA test The cell supernatants of the blank control group, Example 1 group, and Comparative Examples 1-2 groups were collected before and after the rotating magnetic field was removed, and the secretion levels of IL-6 (pro-inflammatory factor) and CD206 (anti-inflammatory factor) in the supernatants were detected by enzyme-linked immunosorbent assay (ELISA) kits (Shanghai ELISA Biotechnology).

[0047] After the cells were treated with Comparative Example 1, the secretion of M2 cytokine CD206 increased, and the expression level was higher under magnetic field conditions. Figure 4 As shown in (a), after 24 h of magnetic field exposure, the secretion of IL-6 (pro-inflammatory cytokine) by RAW264.7 cells treated with MXene nanosheets increased significantly compared with the blank control group, while there was no difference in the secretion of CD206 (anti-inflammatory cytokine). After removing the magnetic field for 48 h, the secretion of CD206 by RAW264.7 cells treated with MXene nanosheets increased significantly compared with the control group, while there was no significant difference in the secretion of IL-6 by the cells ( Figure 4 (b)). These findings provide evidence at the cytokine level that MXene nanosheets mediate a bidirectional regulatory mechanism during macrophage polarization, initially promoting M1 phenotype activation and subsequently promoting the M1 to M2 transition.

[0048] ELISA testing also revealed that if cells do not internalize MXene, they can only polarize toward M2 in the presence or absence of a magnetic field. However, after internalizing MXene, the cells first polarize toward M1 and then toward M2 in the presence or absence of a magnetic field, achieving sequential polarization. The ELISA results were consistent with those of RT-qPCR.

[0049] Example 6: Antibacterial ability detection (1) Visual detection of bacterial phagocytosis: Staphylococcus aureus was cultured in LB liquid medium at 37 °C with shaking (200 rpm) for 6 h, washed with PBS and resuspended to 1.0 × 10 5 CFU / mL. Take 1 mL of bacterial solution and mix with pHrodo TMGreen STP ester (10 μM, green) was labeled at room temperature in the dark for 30 min, and the cells were centrifuged to remove the free dye and then resuspended in PBS. The experiment was divided into two groups: Example 1 group and blank control group. The difference between the blank control group and Example 1 group was that no MXene nanosheets were added; MXene+magnetic field drive and MXene respectively represented MXene treatment with or without a magnetic field. They were Example 1 groups at different times, and were tested under magnetic field drive and magnetic field removal conditions to illustrate the different effects of magnetic field on cells. The labeled bacteria were added to the culture plate of RAW264.7 cells after 24 hours of treatment with magnetic field-driven MXene nanosheets and 24 hours of treatment without magnetic field removal, and co-cultured at 37 ° C and 5% CO2 for 15 min and 40 min. The culture medium was discarded and the cells were washed with PBS containing 5% double antibody solution. Using LysoTracker TM Lysosomes were labeled with Red DND-99 (red), and the samples were incubated at 37°C for 30 minutes. Finally, the samples were imaged using a laser scanning confocal microscope. Fluorescence signals were semi-quantitatively analyzed using Image J software.

[0050] like Figure 5 As shown, phagocytosis of bacteria by macrophages was detected after just 15 minutes of co-incubation, with colocalization of bacteria with lysosomes confirming the occurrence of phagocytosis. Phagocytic activity increased in a time-dependent manner, with a significant increase in the efficiency of bacterial internalization into lysosomes observed at the 40-minute time point. Notably, the MXene nanosheets combined with magnetic field significantly enhanced macrophage phagocytosis compared to the other groups, indicating that MXene nanosheets effectively enhance bacterial capture efficiency by promoting M1-type polarization.

[0051] (2) Bactericidal efficacy test of RAW264.7: The test grouping was the same as in Example 6 (1). Staphylococcus aureus (1.0×10 5 CFU / mL) were co-cultured with RAW264.7 cells treated with magnetic field-driven MXene nanosheets for 24 hours and then removed from the magnetic field for another 24 hours. After incubation, the cells were gently washed three times with PBS to remove extracellular bacteria. Intracellular bacteria were lysed by adding 0.1% Triton-X 100 for 15 minutes. The cells were then diluted and plated onto LB solid medium and incubated at 37°C for 24 hours. Colony forming units (CFU) were then counted to quantify the antibacterial activity of RAW264.7 cells.

[0052] To quantitatively evaluate the bactericidal efficacy of macrophages, intracellular bacteria were extracted from macrophages and plated for colony forming unit (CFU) counting. Figure 6The results show that different groups have different bactericidal abilities. The MXene group showed limited antibacterial activity, while the MXene group showed significant antibacterial effects under magnetic field drive, with an inhibition rate of over 50% against Staphylococcus aureus. This result confirms that MXene nanosheets can significantly enhance the bactericidal ability of macrophages by enhancing the M1 polarization of macrophages under magnetic field conditions.

[0053] (3) ROS detection The experimental grouping was the same as in Example 6 (1). After 24 hours of magnetic field-driven MXene nanosheet treatment and 24 hours of magnetic field removal, RAW264.7 cells were washed three times with PBS (5 min / time) and then incubated at 37°C in the dark for 30 minutes to detect intracellular ROS levels. Subsequently, the samples were imaged using a laser scanning confocal microscope. The fluorescence signal was semi-quantitatively analyzed using Image J software.

[0054] Fluorescence imaging analysis showed that the green fluorescence intensity of the MXene-MF group was significantly enhanced compared with the other groups, indicating that MXene nanosheets exposed to magnetic fields can significantly promote the generation of ROS in cells ( Figure 7 This phenomenon is consistent with the polarization characteristics of M1 macrophages and is a key molecular mechanism by which MXene nanosheets enhance their bactericidal activity. Taken together, these findings demonstrate that magnetic fields combined with MXene nanosheets significantly enhance bacterial clearance in M1 macrophages through multiple mechanisms, including increased phagocytic activity and ROS production. This establishes the potential of MXene nanosheets as a novel nanomaterial for regulating macrophage antimicrobial immunity.

[0055] In summary, MXene nanosheets internalized by cells can polarize toward M1 under magnetic field conditions, producing high levels of pro-inflammatory cytokines, which contribute to cellular immunity. After the magnetic field is removed, they sequentially polarize to M2, producing anti-inflammatory cytokines that participate in wound repair, promote angiogenesis, and inhibit inflammation, thereby promoting rapid and efficient wound healing.

[0056] Example 7: mRNA Sequencing The experiment was divided into two groups: Example 1 group and a blank control group. The difference between the blank control group and Example 1 group was that no MXene nanosheets were added.

[0057] Cells were collected from the blank control group and Example 1 group after the rotating magnetic field and after the rotating magnetic field was removed. Total RNA was extracted using the Trizol method. After quality control and quantification, mRNA sequencing was performed by Shanghai Boshi Biotechnology Co., Ltd. The differentially expressed genes were functionally characterized using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Key signaling pathway proteins selected based on the sequencing results were verified by Western blotting ( Figure 8 ).

[0058] MXene nanosheets significantly upregulated the expression of phosphorylated p65 under magnetic field exposure, a core transcription factor in the NF-κB signaling pathway. Conversely, after magnetic field removal, MXene nanosheets promoted the expression of phosphorylated JNK2, activating the pathway through a JNK2-mediated JAK / STAT protein phosphorylation cascade. This differential regulation of signaling pathways promotes the sequential polarization of macrophages from M1 to M2 phenotypes, providing a molecular basis for maintaining immune balance and tissue homeostasis.

[0059] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for regulating programmed polarization of macrophages in vitro, characterized in that: The method is: MXene is ultrasonically treated to obtain MXene nanosheets, which are then added to DMEM culture medium to obtain a regulated culture medium. Macrophages are cultured with DMEM culture medium until they adhere to the wall, and the DMEM culture medium is replaced with a regulated culture medium. After the macrophages internalize the MXene nanosheets, they are placed in a rotating magnetic field generated by a rotating permanent magnet. The MXene nanosheets polarize the macrophages into M1 macrophages through electromagnetic induction. After the rotating magnetic field is removed, the M1 macrophages become M2 macrophages, realizing the programmed polarization of macrophages in vitro.

2. The method according to claim 1, characterized in that The ultrasonic treatment is performed by ultrasonicating MXene in an ice bath under nitrogen protection; the ultrasonic power is 500w and the time is 10min.

3. The method according to claim 1, characterized in that The MXene nanosheets are single-layer nanosheets with a size of 200-300 nm.

4. The method according to claim 1, wherein The concentration of MXene nanosheets in the regulation culture medium is 5 μg / mL.

5. The method according to claim 1, wherein The time for the macrophages to internalize the MXene nanosheets is 2 to 4 hours.

6. The method according to claim 1, characterized in that The electromagnetic induction refers to the generation of magnetic flux lines in a rotating magnetic field. MXene nanosheets cut the magnetic flux lines in the rotating magnetic field, generating microcurrents in macrophages.

7. The method according to claim 1, characterized in that The rotation speed of the rotating magnetic field is 500 rpm; the application time of the rotating magnetic field is 15 minutes to 24 hours; and the magnetic field strength of the rotating magnetic field is 1.0 Tesla.

8. The method according to claim 1, characterized in that The macrophages are located below the permanent magnet, with a distance from the permanent magnet of 1.0 to 2.0 cm.

9. The method according to claim 1, characterized in that The time it takes for the M1 macrophages to become M2 macrophages is 15 minutes to 24 hours.

10. Application of MXene nanosheets in the preparation of drugs that promote wound healing, characterized in that: MXene nanosheets are dispersed on the wound, allowed to stand, and then the wound is placed under a rotating permanent magnet, which is then removed to promote wound healing. The MXene nanosheets are single-layer nanosheets with a size of 200-300 nm.

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