Osteomyelitis-related macrophage targeting engineered biological function 2D nano-drug, preparation method and application thereof
By designing 2D nano drugs targeting osteomyelitis macrophages, the problem of impaired macrophage activity in osteomyelitis treatment is solved, efficient digestion and activation of MRSA, and a brand new metal immunotherapy is provided.
Patent Information
- Application Number
- CN202510230104.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-30
AI Technical Summary
The treatment of osteomyelitis is difficult to effectively eradicate bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). Due to the impaired phagocytic and bactericidal activity of macrophages, it leads to immune paralysis and persistent infection.
Design an engineered 2D nanopharmaceutical targeting of osteomyelitis-associated macrophages to modify 2D ternary transition metal phosphorus chalcogen nanosheets through surface functionalization to achieve targeted reprogramming of macrophages in osteomyelitis lesions, promote highly reactive ROS outbreaks, digest bacteria in cells and activate immune responses.
Accurate treatment of osteomyelitis lesions is achieved, and a new metal immunotherapy is provided by enhancing the bactericidal activity and immune response of macrophages, terminating persistent infection and re-establishing the bactericidal effect of the host.
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Figure CN120053689A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a macrophage-targeted engineered biofunctional 2D nano-drug related to osteomyelitis, a preparation method thereof and an application thereof, belonging to the technical field of nano-drugs. Background Art
[0002] Osteomyelitis is a particularly severe complication after orthopedic surgery or bacteremia and is a major clinical challenge. The treatment of osteomyelitis requires complex treatment strategies, including long-term antibiotic treatment and extensive surgical debridement. However, due to the persistent presence of bacteria, limited clinical progress has been made in eradicating osteomyelitis. Among the various pathogens associated with osteomyelitis, Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), is the most common and destructive pathogen in osteomyelitis. Since Staphylococcus aureus causing osteomyelitis can cause host immune paralysis and evade antibiotic treatment, reactivating bactericidal immunity in the bone infection microenvironment is crucial for eradicating osteomyelitis. However, there is still a lack of effective treatment means currently.
[0003] Macrophages (MΦs) play a key role in coordinating the bactericidal immune response against Staphylococcus aureus, mainly relying on the burst of reactive oxygen species (ROS) after internalizing pathogens and continuously initiating adaptive immunity through antigen presentation. However, postoperative local tissue reactions and progressive infections lead to immune paralysis, impairing the phagocytic and bactericidal activities of MΦs. In addition, when Staphylococcus aureus infects MΦs, the ROS level is insufficient, weakening phagolysosome killing, thus allowing the pathogen to survive within the cell and allowing persistent infection.
[0004] Arming infected MΦs with nano-drugs to trigger a highly reactive ROS burst in phagosomes is crucial for effectively eliminating intracellular bacteria. Generally, ROS-catalyzed metal-based enzymatic nano-drugs exhibit a powerful Fenton / Fenton-like reaction, which can generate highly toxic hydroxyl radicals (·OH). In addition, transition metal ions, especially manganese ions, are effective immune adjuvants for metal immunotherapy. Two-dimensional (2D) ternary transition metal phosphochalcogenide nanomaterials, such as MnPSe 3 and MnPS 3 , have become emerging biomaterials due to their unique planar topological structure, inherent enzyme mimicking and potential immune-stimulating properties. However, biological barriers and non-specific distribution are still huge challenges for achieving precise treatment with 2D ternary transition metal phosphochalcogenide nanosheets. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide a biofunctional 2D nanomedicine for targeting osteomyelitis-related macrophages, a preparation method thereof, and an application thereof. A nanomedicine designed to precisely target and reprogram MΦs in osteomyelitis lesions using 2D ternary transition metal phosphochalcogenide nanosheets can in-situ engineer MΦs in osteomyelitis lesions to digest intracellular multidrug-resistant bacteria and reactivate antibacterial immunity, thereby realizing a host-directed bactericidal strategy and providing a new metal immunotherapy for the precise eradication of osteomyelitis.
[0006] To achieve the above object, the technical solution of the present invention is as follows.
[0007] A biofunctional 2D nanomedicine for targeting osteomyelitis-related macrophages, wherein the biofunctional 2D nanomedicine has a sheet-like core-shell structure, the core layer is a 2D ternary transition metal phosphochalcogenide nanosheet, and the biochemical functional modification material on the surface of the core layer is the shell layer. The biochemical functional modification material is phosphatidylserine capped with senescent neutrophil membrane, CXCR4-MΦs bifunctional targeting peptide, or bone targeting peptide;
[0008] The mass ratio of the biochemical functional modification material to the 2D ternary transition metal phosphochalcogenide nanosheet is 1:0.5 - 2.
[0009] Preferably, the thickness of the core-shell structure is 6 - 25 nm, and the average hydrated particle size is 210 - 230 nm; the thickness of the core layer is 3 - 8 nm, and the average hydrated particle size is 180 - 190 nm; the thickness of the shell layer is 3 - 17 nm.
[0010] Preferably, the mass ratio of the biochemical functional modification material to the 2D ternary transition metal phosphochalcogenide nanosheet is 1:0.8 - 1.5.
[0011] Preferably, the transition metal in the 2D ternary transition metal phosphochalcogenide nanosheet is Mn, Zn, or Co. More preferably, the 2D ternary transition metal phosphochalcogenide nanosheet is a 2D MnPSe 3 nanosheet.
[0012] Preferably, the senescent neutrophil membrane is prepared from a senescent neutrophil population with a neutrophil proportion of 20% - 40%. high
[0013] Preferably, the senescent neutrophil population is obtained by the following method: Neutrophils are in vitro aged and cultured at a cell density of 1×10 6 - 3×10 6 / ml for 6 - 10 h.
[0014] A preparation method of a biofunctional 2D nanomedicine for targeting osteomyelitis-related macrophages, the method steps include:
[0015] Disperse the aqueous dispersion of 2D ternary transition metal phosphosulfide nanosheets and the biochemical functional modification material in a glass sample container, and then place it in a water bath at 4-8 °C. Continuously ultrasonicate for 2-5 min at 120-150 W and 20-40 kHz. After the ultrasonication, centrifuge at 4-6 °C and 10,000-14,000 rpm for 5-10 min to collect a biofunctional 2D nano-drug targeted at osteomyelitis-related macrophages.
[0016] Preferably, the 2D ternary transition metal phosphosulfide nanosheets are prepared by the following method: Under the condition of an ice-water bath, disperse the 2D layered ternary transition metal phosphosulfide bulk solution with a concentration of 1-5 mg / ml, and intermittently ultrasonicate at 600-800 W for 2-6 h, turning off for 2-3 s every 3-5 s of ultrasonication to obtain a suspension; then centrifuge at 4000-5000 rpm for 5-15 min, collect the supernatant, and centrifuge at 12,000-14,000 rpm for 5-10 min to collect the 2D ternary transition metal phosphosulfide nanosheets.
[0017] Use of a biofunctional 2D nano-drug targeted at osteomyelitis-related macrophages in the preparation of a drug for treating methicillin-resistant Staphylococcus aureus (MRSA) infectious osteomyelitis.
[0018] Preferably, the MRSA infectious osteomyelitis includes implant-related osteomyelitis, fracture-related infections, hematogenous osteomyelitis, diabetic foot infections, septic arthritis, and primary spinal osteomyelitis.
[0019] Beneficial effects
[0020] The present invention provides a biofunctional 2D nano-drug targeted at osteomyelitis-related macrophages, which realizes bone marrow lesion homing and sequential targeting engineering of related macrophages through surface functional modification of 2D ternary transition metal phosphosulfide nanosheets. The above biofunctional 2D nano-drug digests intracellular bacteria by generating highly toxic ·OH, and mediates immune activation through the included transition metal ions to reprogram bactericidal immunity, thereby synergistically terminating the pathological cascade reaction caused by persistent infection and re-establishing host-directed bactericidal efficacy, achieving the purpose of developing a new metal immunotherapy for osteomyelitis.
[0021] The present invention provides a preparation method of a biofunctional 2D nano-drug targeted at osteomyelitis-related macrophages, which ultrasonicates the aqueous dispersion of 2D ternary transition metal phosphosulfide nanosheets and the biochemical functional modification material under specific conditions to prepare a core-shell structure material with high shell integrity. Description of the drawings
[0022] Figure 1Schematic diagram for the extraction of senescent neutrophil membranes and the preparation of the biomimetic 2D aNM@MPS nanomedicine in Example 1.
[0023] Figure 2 Transmission electron microscopy image of the 2D aNM@MPS nanomedicine in Example 1.
[0024] Figure 3 Fluorescence image of the 2D aNM@MPS nanomedicine in Example 1.
[0025] Figure 4 Quantitative migration result graph of the 2D aNM@MPS nanomedicine in the Transwell model in Example 1.
[0026] Figure 5 Fluorescence image of MΦs uptake of the 2D aNM@MPS nanomedicine in Example 2;
[0027] Figure 6 Co-localization fluorescence image of the 2D aNM@MPS nanomedicine and MRSA in Example 2.
[0028] Figure 7 Quantitative result graph of ·OH generation in MΦs induced by the 2D aNM@MPS nanomedicine in MRSA infection in Example 2.
[0029] Figure 8 Result graph of the 2D aNM@MPS nanomedicine for clearing intracellular bacteria in Example 2.
[0030] Figure 9 Result graph of gene expression of TNF-α, IFN-β, and IL-10 detected by real-time fluorescence quantitative PCR in Example 2.
[0031] Figure 10 Result graph of in vivo bone marrow homing of the 2D aNM@MPS nanomedicine in Example 3.
[0032] Figure 11 Gram staining images of the implanted femurs under different treatment regimens in Example 4.
[0033] Figure 12 Result graph of biofilm formation on the implant surface under different treatment regimens in Example 4.
[0034] Figure 13 For 2D aNM in Comparative Example 1 8h @MPS and aNM 16h Comparative fluorescence result graph of the enrichment of @MPS nanomedicine in the bone marrow.
[0035] Figure 14 For aNM@MPS in Comparative Example 2 3Fluorescence images of nano-drugs.
[0036] Figure 15 For the 2D aNM@MPS in Comparative Example 2 3 and the comparative fluorescence images of the MΦs uptake of aNM@MPS nano-drugs. Detailed implementation manners
[0037] The present invention will be further described in detail below in conjunction with specific embodiments.
[0038] Example 1 Preparation and characterization of 2D aNM@MPS nano-drugs with sequential targeting to bone marrow-MΦs
[0039] As Figure 1 shown, (1) 4 ml of 69% (vol / vol) Percoll and 4 ml of 78% (vol / vol) Percoll were stacked by density gradient centrifugation, and fresh neutrophils with an average purity of 96.5% were extracted by centrifugation at 1600 g (5 up and 0 down) for 35 min. They were cultured in vitro for aging at a cell density of 2×10 6 ~3×10 6 / ml. Senescent neutrophils with an average CXCR4 high proportion of 36% in neutrophils after 8 h of in vitro aging were selected to obtain senescent neutrophil membranes (aNM).
[0040] (2) A 4 mg / ml MnPSe 3 bulk was intermittently ultrasonicated (ultrasonic for 3 s, off for 2 s) at 800 W for 6 h in an ice bath. The suspension was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Then it was centrifuged at 14000 rpm for 10 min to collect 2D MPS NSs with a hydrodynamic diameter of 190 nm.
[0041] (3) The aqueous dispersions of aNM and 2D MPS NSs were blended at a mass ratio of 1:1 in a 5 ml glass sample bottle, placed in a water bath at 4 - 8 °C, and continuously ultrasonicated at a power of 120 W and 40 kHz for 5 min. At 4 °C, it was centrifuged at 12000 rpm for 10 min to collect 2D aNM@MPS nano-drugs with sequential targeting to bone marrow-MΦs.
[0042] The morphological characteristics of aNM biomimetic 2D MPS NSs were investigated by transmission electron microscopy, and the encapsulation of MPS NSs by aNM was verified by fluorescence co-localization experiments; the migration ability of 2D aNM@MPS nano-drugs mediated by the CXCR4 / CXCL12 signaling axis was evaluated using a Transwell model.
[0043] The transmission electron microscopy image of 2D aNM@MPS nano-drugs shows a typical sheet-like core-shell structure asFigure 2 As shown; the visualization image of fluorescence colocalization proves that the 2D MPS NSs labeled with the red dye RhB and the aNM labeled with the green dye CFSE are successfully combined, and shows a relatively high cell membrane coating integrity, as Figure 3 shown; the evaluation by the Transwell model shows that aNM@MPS has responsiveness to CXCL12, proving that the 2D aNM@MPS nanomedicine has the property of bone marrow homing, as Figure 4 shown.
[0044] Example 2 Biological evaluation of 2D aNM@MPS nanomedicine
[0045] 1. Targeting of 2D aNM@MPS nanomedicine to MΦs
[0046] Bone marrow-derived MΦs were cultured in vitro, and then incubated with 2D aNM@MPS and 2D MPS NS (equivalent dose of 20 μg / ml of 2D MPS NSs) respectively. After incubation for 2 h, the free nanomedicine was washed away. After labeling the cell nuclei, it was placed under a laser confocal microscope for observation. The results are as Figure 5 shown. The 2D aNM@MPS treatment group showed more significant MΦs uptake compared with the 2D MPS NSs treatment group, proving the excellent MΦs targeting and high efficient cell internalization ability of the 2D aNM@MPS nanomedicine.
[0047] 2. Colocalization of 2D aNM@MPS nanomedicine with intracellular bacteria and generation of ·OH
[0048] In this example, MRSA was taken as an example to construct an MΦs intracellular infection model based on fluorescently labeled MRSA. Then, the infected MΦs were treated with RhB-labeled 2D aNM@MPS and co-cultured for 2 h. Then, the cell nuclei of the infected MΦs were stained with 10 nM DAPI for 15 min, and then fluorescence images were captured by CLSM.
[0049] The hydroxy-phenyl fluorescein fluorescent probe was used to evaluate the generation of Mn 2+ -mediated intracellular ·OH in MRSA-infected MΦs after internalizing the 2D aNM@MPS nanomedicine, and the ·OH signal was detected by flow cytometry.
[0050] As Figure 6 shown, RhB-labeled 2D aNM@MPS showed good colocalization with MRSA, indicating that aNM@MPS has the ability to selectively kill bacteria in phagosomes on the basis of achieving MΦs targeting and high efficient uptake.
[0051] Detection of the generation of ·OH in MRSA-infected MΦs induced by the 2D aNM@MPS nanomedicine, as Figure 7As shown, the 2D aNM@MPS nanomedicine treatment group can effectively catalyze the generation of ·OH in infected cells due to its excellent cell internalization effect, so as to achieve efficient sterilization.
[0052] 3. 2D aNM@MPS nanomedicine clears intracellular bacteria
[0053] An intracellular infection model of MΦs based on MRSA was established. The infected MΦs were incubated with PBS, Vancomycin (Van, 20 μg / ml), MPS NSs (20 μg / ml), and aNM@MPS (equivalent dose of 20 μg / ml 2D MPS NSs), respectively. After incubation for 12 h, the cells treated differently were washed with PBS and lysed with 0.1% Triton X-100. The obtained lysates were cultured on TSA plates to quantify the bacterial colony-forming units. As Figure 8 shown, the aNM@MPS nanomedicine treatment group showed the lowest bacterial load, demonstrating that the aNM@MPS nanomedicine can significantly enhance bacterial killing and prevent the survival of bacteria in cells.
[0054] 4. 2D aNM@MPS nanomedicine mediates MΦs immune activation
[0055] MΦs were seeded in 6-well plates. After culturing for 24 h, PBS, Van, MPS NSs, and aNM@MPS were added respectively. Subsequently, the MΦs were exposed to different treatments overnight and then incubated in fresh medium for 24 h. The gene expressions of TNF-α, IFN-β, and IL-10 were detected by real-time fluorescence quantitative PCR, and β-actin was used as an internal reference gene, and quantified by the 2 -ΔΔCt method. As Figure 9 shown, the expressions of the pro-inflammatory biomarker TNF-α and the type I interferon biomarker IFN-β increased in MΦs treated with the aNM@MPS nanomedicine, while the expression of the anti-inflammatory biomarker IL-10 decreased. These results indicate that the aNM@MPS nanomedicine-mediated reprogramming of MΦs can enhance the polarization into a pro-inflammatory phenotype, which is beneficial to enhancing the phagocytosis and bactericidal activities of MΦs.
[0056] Example 3 In vivo bone marrow homing of 2D aNM@MPS nanomedicine
[0057] In this example, an in-situ osteomyelitis mouse model was taken as an example. Fluorescently labeled MPS NSs and aNM@MPS nanomedicine were injected into the osteomyelitis mice through the tail vein. At 8 h and 24 h after injection, the mice were sacrificed, and the hind limb long bones were taken and analyzed using an IVIS system (Ex / Em: 748 / 780 nm).
[0058] As Figure 10As shown, aNM@MPS nanoparticles are superior to MPS NSs in terms of bone accumulation, indicating that the 2D aNM@MPS nanoparticles have specific bone marrow homing ability.
[0059] Example 4 In Vivo Anti-Osteomyelitis Study of 2D aNM@MPS Nanoparticles
[0060] In this example, the blood-borne osteomyelitis induced by MRSA was taken as an example to verify the feasibility of eradicating osteomyelitis with the 2D aNM@MPS nanoparticles prepared by the present invention. First, a mouse femoral implant model was established. 21 days after the operation, 100 μl (2×10 5 CFUs / ml) of MRSA was inoculated by retro-orbital injection. On the 2nd day after the injection of bacteria, each group of mice was injected with PBS (100 μl), Van (20 mg / kg), MPS NSs (10 mg / kg), and aNM@MPS (equivalent dose of 10 mg / kg MPS NSs) via the tail vein, and the drug was administered 3 times within 7 days. On the 7th day after the third administration, the treated mice were euthanized, and antibacterial activity evaluation and biofilm detection were performed.
[0061] Figure 11 Shown are Gram-stained images of the implanted femurs under different treatment regimens at the study endpoint. The results show that there is no obvious bacterial infiltration in the aNM@MPS nanoparticle treatment group, demonstrating its strong antibacterial activity.
[0062] An important pathogenesis of Staphylococcus aureus-induced osteomyelitis is the formation of biofilms. Figure 12 The treatment with aNM@MPS nanoparticles reduced the overall tendency of biofilm formation, while the implants from mice in other treatment groups, especially the control group and the vancomycin treatment group, showed dense biofilm aggregates.
[0063] Comparative Example 1
[0064] Select aged neutrophils-derived aNM that have been aged in vitro for 16 h and have an average proportion of CXCR4 high neutrophils of 50%. Then, 1 ml of aNM and 1 ml of MPS (average hydrated particle size of 190 nm) were blended at a mass ratio of 1:1 in a 5-ml glass sample bottle, placed in a water bath at 4-8°C, and continuously sonicated for 5 min at a power of 120 W and a frequency of 40 kHz. The 2D nanoparticles were collected by centrifugation at 12,000 rpm for 10 min at 4°C and denoted as aNM 16h @MPS. Fluorescently labeled aNM 8h @MPS and aNM 16h @MPS nanoparticles were injected into the osteomyelitis mice via the tail vein. At 24 h after the injection, the mice were sacrificed, the implanted femurs were removed, and after tissue sectioning, the enrichment of the 2D nanoparticles in the femurs was observed by fluorescence. The results are asFigure 13 As shown, although aNM 16h @MPS nano-drug membrane surface CXCR4 expression relative to aNM 8h @MPS is high, but because apoptosis-related molecules on the membrane surface are also upregulated, aNM 16h @MPS nanomedicines are cleared from the systemic circulation, impairing the ability of 2D nanomedicines to home to bone marrow lesions and affecting drug concentration in lesions.
[0065] Comparative Example 2
[0066] 1 ml aNM and 1 ml MPS were mixed in a 5 ml glass sample bottle at a mass ratio of 1:3, placed in a 4-8 °C water bath, and continuously sonicated for 5 min at a power of 120 W and 40 kHz. 2D aNM@MPS nanomedicine was collected by centrifugation at 4 °C and 12000 rpm for 10 min, and recorded as aNM@MPS 3 The integrity of aNM-encapsulated MPS NSs was verified by fluorescence co-localization experiments. Figure 14 As shown, the fluorescence co-localization visualization image shows that the 2D MPS NSs labeled with the red dye RhB are only partially coated by the aNM labeled with the green dye CFSE. 3 Insufficient coating integrity; further investigation of aNM@MPS using bone marrow-derived MΦs cultured in vitro 3 Cellular uptake of aNM@MPS and aNM@MPS 3 After 2 hours, the free nanomedicine was washed away, and after labeling the cell nucleus, the cells were observed under a laser confocal microscope. Figure 15 As shown, aNM@MPS 3 The treated group showed lower MΦs uptake compared with the aNM@MPS treated group, indicating that the insufficient integrity of the cell membrane coating hindered the targeted uptake of MΦs by aNM@MPS, affecting the drug efficacy.
[0067] In summary, the invention includes but is not limited to the above embodiments. Any equivalent substitution or partial improvement made under the spirit and principle of the invention shall be deemed to be within the protection scope of the invention.
Claims
1. A biofunctional 2D nanomedicine targeted to osteomyelitis-related macrophages, characterized by: The biofunctional 2D nanomedicine is a sheet-like core-shell structure, the core layer is a 2D ternary transition metal phosphorus-sulfide nanosheet, the biochemical functional modification material on the surface of the core layer is a shell layer, and the biochemical functional modification material is phosphatidylserine capped with an aged neutrophil membrane, a CXCR4-MΦs bifunctional targeting peptide, or a bone targeting peptide; The mass ratio of the biochemical functional modification material to the 2D ternary transition metal phosphorus-sulfur nanosheets is 1:0.5-2.
2. The biofunctional 2D nanomedicine targeted to osteomyelitis-associated macrophages according to claim 1, characterized in that: The core-shell structure has a thickness of 6 to 25 nm, and an average hydrated particle size of 210 to 230 nm; the core layer has a thickness of 3 to 8 nm, and an average hydrated particle size of 180 to 190 nm; and the shell layer has a thickness of 3 to 17 nm.
3. The biofunctional 2D nanomedicine targeted to osteomyelitis-associated macrophages according to claim 1, characterized in that: The mass ratio of the biochemical functional modification material to the 2D ternary transition metal phosphorus-sulfur nanosheets is 1:0.8-1.
5.
4. The biofunctional 2D nanomedicine targeted to osteomyelitis-associated macrophages according to claim 1, characterized in that: The transition metal in the 2D ternary transition metal phosphorus sulfide nanosheet is Mn, Zn or Co; preferably, the 2D ternary transition metal phosphorus sulfide nanosheet is a 2D MnPSe3 nanosheet.
5. The biofunctional 2D nanomedicine targeted to osteomyelitis-associated macrophages according to claim 1, characterized in that: The senescent neutrophil membrane expresses CXCR4 high A population of aged neutrophils in which the neutrophils account for 20% to 40% is prepared.
6. The osteomyelitis-associated macrophage-targeted biofunctional 2D nanomedicine according to claim 1 or 5, characterized in that: The senescent neutrophil population is obtained by the following method: neutrophils are divided into 1×10 6 ~3×10 6 / ml cell density for in vitro aging culture, the culture time is 6 to 10 hours.
7. A method for preparing the osteomyelitis-associated macrophage-targeted engineered biofunctional 2D nanomedicine according to any one of claims 1 to 6, characterized in that: The method steps include: An aqueous dispersion of 2D ternary transition metal phosphorus sulfide nanosheets and a biochemical functional modification material are mixed in a glass sample container, then placed in a water bath at 4-8°C, and continuously ultrasonicated for 2-5 minutes at 120-150W and 20-40kHz. After the ultrasonication, the mixture is centrifuged at 4-6°C and 10000-14000rpm for 5-10 minutes to collect a biofunctional 2D nanomedicine targeted to osteomyelitis-related macrophages.
8. The method for preparing a biofunctional 2D nanomedicine targeted to osteomyelitis-related macrophages according to claim 7, characterized in that: The 2D ternary transition metal phosphorus sulfide nanosheets are prepared by the following method: under ice-water bath conditions, a 2D layered ternary transition metal phosphorus sulfide block dispersion with a concentration of 1 to 5 mg / ml is subjected to intermittent ultrasonication at 600 to 800 W for 2 to 6 hours, with ultrasonication turned off for 2 to 3 seconds every 3 to 5 seconds to obtain a suspension; then centrifuged at 4000 to 5000 rpm for 5 to 15 minutes, the supernatant is collected, and the 2D ternary transition metal phosphorus sulfide nanosheets are collected by centrifugation at 12000 to 14000 rpm for 5 to 10 minutes.
9. Use of the osteomyelitis-associated macrophage-targeted engineered biofunctional 2D nanomedicine according to any one of claims 1 to 6 in the preparation of a drug for treating MRSA-infected osteomyelitis.
10. The use according to claim 9, characterized in that: The MRSA infectious osteomyelitis includes implant-related osteomyelitis, fracture-related infection, hematogenous osteomyelitis, diabetic foot infection, septic arthritis and primary spinal osteomyelitis.