Nano-robot CS-BT-MZ-NEs, preparation method thereof and application of nano-robot CS-BT-MZ-NEs in preparation of medicine for treating implant-related infection

Abstract

The invention relates to a nano-robot CS-BT-MZ-NEs preparation method, which comprises: mixing an ethanol solution of tetrabutyl titanate, an ammonia water solution and a clarified barium hydroxide water solution, and carrying out heat treatment to prepare nano-particles BaTiO3; mixing zinc nitrate hexahydrate, manganese nitrate, methanol and the nano-particles BaTiO3, stirring, dropwise adding a methanol solution of 2-methylimidazole, and continuously stirring, so as to prepare nano-particles BT-MZ; mixing an aqueous solution of chondroitin sulfate and the nanoparticles BT-MZ, and then stirring to prepare nanoparticles CS-BT-MZ; and incubating neutrophils with the nano-particles CS-BT (at) MZ, so as to prepare the nano-robot CS-BT (at) MZ (at) NEs. The nano-robot CS-BT-MZ-NEs provides an efficient and safe comprehensive solution for IAIs treatment by combining antibacterial activity, pH adjustment of a golf apparatus and immune memory activation, biological membrane related infection is directly eradicated, systemic immune response is activated and induced through an STING pathway, and lasting protection is provided for recurrent bacterial infection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and particularly relates to a nano-robot CS-BT@MZ@NEs, a preparation method thereof, and an application thereof in the preparation of a drug for treating implant-related infections. Background Art

[0002] Implant-related infections (IAIs) are a major challenge in modern medicine, especially in the fields of orthopedics, cardiology, and dentistry. These infections usually originate from the formation of bacterial biofilms. A biofilm is composed of a microbial community encapsulated in an extracellular polymeric matrix (EPS), which can protect bacteria from the host immune response and traditional antibiotics, thus resulting in high resistance to treatment. Even after surgical debridement and implant replacement, residual biofilm fragments often lead to recurrent infections or chronic inflammation, seriously affecting the prognosis of patients and increasing medical costs.

[0003] Traditional treatment methods for IAIs include systemic antibiotics and surgical intervention, but their efficacy is limited. Antibiotics are difficult to penetrate the dense biofilm structure, and the emergence of antibiotic-resistant pathogens further exacerbates the treatment challenge. In addition, the biofilm creates an immunosuppressive microenvironment (IME), which is characterized by the infiltration of myeloid-derived suppressor cells (MDSCs), reduced T cell activation, and impaired antigen presentation. These factors weaken the host's immune defense ability, making it difficult to clear the infection and prevent reinfection.

[0004] To address these limitations, researchers have turned to strategies based on advanced nanotechnology. Nano-robots in particular offer promising solutions because they can navigate in complex biological environments, precisely deliver therapeutic agents, and modulate the host immune response. However, effectively delivering nano-robots to deep infection sites (such as the bone marrow) remains a major obstacle. The bone marrow-blood barrier and low blood perfusion make it challenging to achieve sufficient therapeutic concentrations at these sites.

[0005] Neutrophils are the most abundant white blood cells in the body and play a key role in the innate immune response by crossing biological barriers and homing to infection or inflammation sites. Utilizing the natural migration properties of neutrophils can provide a unique method for nano-robots to cross the bone marrow-blood barrier and reach the infection site. This not only improves the delivery efficiency but also utilizes the inherent antibacterial function of neutrophils. Summary of the Invention

[0006] The purpose of the present invention is to provide, in view of the deficiencies in the prior art, a nano-robot CS-BT@MZ@NEs, a preparation method thereof, and an application thereof in the preparation of a drug for treating implant-related infections.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention is to provide a preparation method of a nano-robot CS-BT@MZ@NEs, and the steps include:

[0009] S1. Mix an ethanol solution of tetrabutyl titanate, an ammonia water solution, and a clarified barium hydroxide aqueous solution, and then perform heat treatment; then perform centrifugal separation, and successively wash, dry, calcine, and grind the obtained precipitate to obtain nano-particles BaTiO3;

[0010] S2. Mix zinc nitrate hexahydrate, manganese nitrate, methanol, and the nano-particles BaTiO3, and then stir; gradually add dropwise a methanol solution of 2-methylimidazole and continue stirring; then perform centrifugal separation, and wash the obtained precipitate to obtain nano-particles BT@MZ;

[0011] S3. Mix an aqueous solution of chondroitin sulfate and the nano-particles BT@MZ, and then stir; then perform centrifugal separation, and wash the obtained precipitate to obtain nano-particles CS-BT@MZ;

[0012] S4. Incubate neutrophils with the nano-particles CS-BT@MZ to obtain the nano-robot CS-BT@MZ@NEs.

[0013] Preferably, the S1 includes:

[0014] Mix tetrabutyl titanate with ethanol to obtain the ethanol solution of tetrabutyl titanate;

[0015] Add the ammonia water solution to the ethanol solution of tetrabutyl titanate to obtain a first mixed solution;

[0016] Dissolve hydrated barium hydroxide in deionized water to obtain the clarified barium hydroxide aqueous solution;

[0017] Add the clarified barium hydroxide aqueous solution to the first mixed solution to obtain a second mixed solution;

[0018] Perform heat treatment on the second mixed solution at 200 °C for 48 h; then perform centrifugal separation, and successively wash the obtained precipitate 2-3 times with acetic acid, 2-3 times with absolute ethanol, dry at 80 °C for 24 h, calcine in an air atmosphere at 800 °C for 10 h, naturally cool to room temperature, and grind to obtain the nano-particles BaTiO3.

[0019] Preferably, the S2 includes:

[0020] Dissolve the zinc nitrate hexahydrate and the manganese nitrate in the methanol to obtain a third mixed solution;

[0021] Add the nanoparticle BaTiO3 to the third mixed solution and stir for 20 min at room temperature to obtain a fourth mixed solution;

[0022] Dissolve 2-methylimidazole in methanol to obtain a methanol solution of 2-methylimidazole;

[0023] Dropwise add the methanol solution of 2-methylimidazole to the fourth mixed solution and continuously stir for 2 h;

[0024] Centrifuge at 13,000 rpm and wash the obtained precipitate with deionized water 2-3 times to obtain the nanoparticle BT@MZ.

[0025] Preferably, the step S3 includes:

[0026] Dissolve chondroitin sulfate in deionized water to obtain an aqueous solution of chondroitin sulfate;

[0027] Add the nanoparticle BT@MZ to the aqueous solution of chondroitin sulfate and stir at room temperature for 24 h;

[0028] Centrifuge at 13,000 rpm and wash the obtained precipitate with deionized water 2-3 times to obtain the nanoparticle CS-BT@MZ.

[0029] Preferably, the step S4 includes:

[0030] Under a sterile environment, culture the neutrophils in a serum-free medium for 1 h to obtain a fifth mixture;

[0031] Add the nanoparticle CS-BT@MZ to the fifth mixture and incubate at 37 °C for 1 h to obtain the nanorobot CS-BT@MZ@NEs.

[0032] Preferably, the neutrophils are bone marrow tropic neutrophils.

[0033] Preferably, the preparation steps of the bone marrow tropic neutrophils include:

[0034] Obtain a sterile bone sample;

[0035] Rinse the bone marrow with PBS, centrifuge at 250 g for 4 min, and then resuspend in PBS;

[0036] Prepare a density gradient centrifugation solution by sequentially layering 75% Percoll solution, 65% Percoll solution, and 55% Percoll solution, and add the single cell suspension on the top;

[0037] Centrifuge at 700 g for 25 min, and separate neutrophils at the interface of 65% Percoll solution and 75% Percoll solution;

[0038] Wash the neutrophils three times with ice-precooled PBS to remove the residual Percoll solution;

[0039] Culture in RPMI 1640 medium containing 1% penicillin / streptomycin in a humid atmosphere at 37 °C and 5% CO2, and the bone marrow tropic neutrophils are prepared after 6 h of in vitro culture.

[0040] The second aspect of the present invention is to provide a nanorobot CS-BT@MZ@NEs prepared by the aforementioned preparation method.

[0041] The third aspect of the present invention is to provide an application of the aforementioned nanorobot CS-BT@MZ@NEs in the preparation of a drug for treating implant-related infections.

[0042] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0043] The present invention provides an ultrasound-responsive nanorobot CS-BT@MZ@NEs, the core of which is barium titanate (BaTiO3, BT) with ultrasonic piezoelectric properties, encapsulated in a manganese-zinc metal-organic framework (Mn-Zif-8, MZ) to form a heterostructure, and chondroitin sulfate (CS) with Golgi targeting ability is coated on the surface to enhance targeting specificity and biocompatibility. By co-incubating with neutrophils, a CS-BT@MZ@NEs loading system is developed, and the natural migration characteristics of neutrophils are utilized to achieve targeted delivery to the infection site.

[0044] The nanorobot integrates the functions of targeting Golgi pH regulation, immune activation and biofilm disruption to address the challenges of implant-related infections (IAIs). Through the "hitchhiking" effect of neutrophils, the nanorobot achieves precise bone marrow-targeted delivery, overcoming the problem of low delivery efficiency of traditional therapies in deep infection sites. At the infection site, CS-BT@MZ@NEs destroys the bacterial biofilm structure through the ultrasonic driving effect, releases bacterial antigens, and at the same time its Golgi targeting ability regulates the Golgi pH, significantly enhancing the activity of the STING pathway. The acidified Golgi environment promotes STING oligomerization and TBK1 activation, drives the immune response centered on interferons and pro-inflammatory cytokines, enhances macrophage M1 polarization and dendritic cell (DCs) maturation, and amplifies the innate immune response. In addition, the nanorobot inhibits the infiltration of myeloid-derived suppressor cells (MDSCs) and increases CD4 + and CD8 +T cell infiltration achieved potent activation of adaptive immunity. CS-BT@MZ@NEs also exhibited excellent immune memory function, and the treated mice showed a higher proportion of memory T cells (CD4 + and CD8 + ) and significantly elevated IgG levels, demonstrating stronger immune defense ability upon re-infection with bacteria. Description of the Drawings

[0045] Figure 1 Schematic diagram and characterization of CS-BT@MZ; among them, Figure a is the schematic diagram of CS-BT@MZ construction, Figure b is the representative TEM and elemental mapping images of CS-BT@MZ, Figures c-d are the X-ray photoelectron spectra (XPS) of C1s, S2p, Ba 3d, Ti2p, Mn 2p and Zn2p of different samples, Figures e-f are the O1s XPS spectra of different samples and the change of OV / OL area ratio, Figure h is the XRD pattern of different samples;

[0046] Figure 2 Characterization of CS-BT@MZ nanorobot; among them, Figures a-d are the ESR spectra of CS-BT@MZ, Figure e is the output voltage of CS-BT@MZ under ultrasonic stimulation, Figure f is the PFM image of the surface of CS-BT@MZ, Figure g is the piezoelectric amplitude of CS-BT@MZ, Figure h is the phase angle of CS-BT@MZ, Figure i is the butterfly loop curve of CS-BT@MZ, Figure j is the piezoelectric switching curve of CS-BT@MZ, Figure k is the KPFM image of CS-BT@MZ and the corresponding surface potential distribution (with or without ultrasound), Figure l is the charge density distribution of BT / Mn-Zif heterostructure, Figure m is the electric field distribution of the multi-physics field simulation of BT / Mn-Zif heterojunction;

[0047] Figure 3Anti-biofilm activity of CS-BT@MZ+US; among them, Figure a shows live / dead biofilms treated with different samples. Figure b shows the damage to biofilms and the bacteria inside them by different treatments. Figures c-d show SPM images and quantitative analysis of bacteria in biofilms. Figure e shows representative images of the anti-biofilm effects of different treatments, evaluated using crystal violet staining. Figure f shows the absorbance measured at 595 nm after dissolving the crystal violet dye bound to the biofilm. Figures g-h show the yields of polysaccharides and eDNA after different treatments. Figures i-j show the sensitivities of bacteria treated with different systems and vancomycin to alkaline phosphatase activity and ONPG hydrolysis activity. Figure k shows the morphology of bacteria in biofilms of different treatment groups observed by transmission electron microscopy. Figures l-m show the evaluation of ROS generation using the DCFH-DA fluorescent probe by FCM and microplate reader. Figure n shows a heatmap of genes significantly upregulated and downregulated in the CS-BT@MZ+US group. Figure o shows KEGG pathway enrichment analysis. Figure p shows GO enrichment analysis. Figure q shows a schematic diagram of the enhanced biofilm antigen expression by CS-BT@MZ+US treatment;

[0048] Figure 4 Golgi targeting, pH regulation, and promotion of dendritic cell maturation in vitro by CS-BT@MZ; among them, Figure a shows confocal microscopy images showing the accumulation of different nanoparticles (NPs) in bone marrow-derived macrophages (BMDMs) after 2 hours or 12 hours. Figure b shows the co-localization of different nanoparticles in the Golgi of BMDMs. Figure c shows fluorescence microscopy used to detect the fluorescence changes of the probe in the Golgi of BMDMs after different treatments. Figure d shows the normalized fluorescence intensity. Figure e shows flow cytometry used to detect the fluorescence changes of the probe in the Golgi of BMDMs after different treatments. Figure f shows the expression levels of STING-related genes after different treatments. Figure g shows the expression levels of STING protein after different treatments. Figure h shows the preparation process of biofilm-conditioned medium (BCMs) and dendritic cell-conditioned medium (DCMs). Figure i shows the quantification of the proportion of mature dendritic cells (CD86 + CD80 + ) using flow cytometry (FCM). Figure j shows the relative expression levels of genes related to immune activation in dendritic cells treated with different BCMs. Figure k shows the concentrations of IFN-β and CXCL10 secreted into the medium after co-culturing dendritic cells with different BCMs. Figure l shows the expression levels of STING protein after different treatments. Figure m shows the expression levels of genes related to antigen presentation after co-culturing dendritic cells with BCMs. Figure n shows a schematic diagram of CS-BT@MZ targeting and regulating the pH of the Golgi to enhance STING activation and dendritic cell maturation, thereby promoting the immune response;

[0049] Figure 5The bone marrow targeting ability of CS-BT@MZ@NEs; among them, Figure a shows neutrophils isolated from the bone marrow taking up nanoparticles and returning to the bone marrow during aging, Figure b shows the purity of neutrophils analyzed by flow cytometry with dual staining (FITC-labeled mouse Ly-6G / Ly-6C (Gr1) and phycoerythrin (PE)-labeled mouse MAIR-IV (CLM-5) antibody), and Figure c shows DiD under different conditions + Representative flow cytometry plots of neutrophils, Figure d shows a schematic diagram of the Transwell migration experiment, cells were seeded in the upper chamber and CXCL12 was added to the lower chamber, Figure e shows fluorescence images of neutrophils or CS-BT@MZ@NEs (DiD-labeled) in the lower chamber under conditions with or without CXCL12 and AMD3100, Figures f-g show the quantification of the number of migrated neutrophils and CS-BT@MZ@NEs by ImageJ software, and Figure h shows fluorescence images of the trunk and major organs 12 hours after tail vein injection of CS-BT@MZ@NEs or DiR-CS-BT@MZ

[0050] Figure 6 CS-BT@MZ@NEs "in situ" inhibits bacterial growth and recruits and activates a large number of immune cells in IAIs; among them, Figure a shows a schematic diagram of the establishment of the IAIs model, Figure b shows a digital photograph of the infected bone 21 days after the primary surgery, Figure c shows X-ray imaging of the infected bone, Figure d shows MRI imaging of the infected bone, and the white circle indicates the infected site, Figure e shows a representative image of biofilm CLSM, Figures f-h show H&E staining, Giemsa staining, and MPO immunofluorescence images of the infected bone, and Figure i shows representative flow cytometry plots of mature DCs, CD4 + T cells, CD8 + T cells, plasmablasts, NK cells, and MDSCs in the bone marrow 21 days after treatment, and Figure j shows a schematic diagram of CS-BT@MZ@NEs promoting the immune process in vivo

[0051] Figure 7 The anti-biofilm immunotherapy mechanism of CS-BT@MZ@NEs based on transcriptome analysis; among them, Figure a shows a volcano plot of differentially expressed genes, Figure b shows GO enrichment analysis, Figure c shows KEGG pathway enrichment analysis, Figure d shows a heat map of significantly upregulated and downregulated genes, and Figures e-g show gene set enrichment analysis (GSEA) to evaluate representative immune signaling pathways after CS-BT@MZ@NEs treatment. The scale below the line represents the ranking of each gene, and NES refers to the normalized enrichment score;

[0052] Figure 8CS-BT@MZ@NEs triggers a systemic antibacterial immune response against contralateral IAIs; among them, Figure a shows the experimental design and timeline for evaluating the systemic immune response triggered by CS-BT@MZ@NEs, Figure b shows the digital photographs of the infected bone 14 days after the second surgery, Figure c shows the X-ray imaging of the infected bone, Figure d shows the MRI imaging of both sides of the infected bone, and the white and red circles respectively indicate the primary and secondary IAIs, Figure e shows the representative images of biofilm CLSM, Figures f-h show the H&E staining, Giemsa staining, and MPO immunofluorescence images of the infected bone, and Figures i-j show CD4 + T cells, CD8 + Representative flow cytometry plots and quantitative analysis of T cells, NK1.1 cells, and MDSCs in the spleen 14 days after secondary IAIs;

[0053] Figure 9 CS-BT@MZ@NEs triggers a long-term memory immune response to prevent the recurrence of IAIs; among them, Figure a shows the schematic diagram of CS-BT@MZ@NEs after surgery in the recurrent IAIs model; Figure b shows the digital photographs of the reinfected bone 21 days after reimplantation and MRSA injection; Figure c shows the X-ray imaging of the reinfected bone; Figure d shows the MRI imaging of the reinfected knee joint, and the white circle indicates the reinfected knee joint, Figure e shows the representative images of biofilm CLSM, Figures f-h show the H&E staining, Giemsa staining, and MPO immunofluorescence images of the reinfected bone, and Figures i-j show CD4 + memory T cells and CD8 + Representative flow cytometry plots and quantitative analysis of memory T cells, and Figures k-l show the IgG concentrations in the bone marrow and serum of the mice after re-challenge. Detailed Description of the Invention

[0054] The following will describe the specific embodiments of the present invention in detail.

[0055] Unless otherwise defined, the technical terms or scientific terms used in the claims and the specification should have the ordinary meanings understood by those of ordinary skill in the technical field to which the present invention belongs.

[0056] As used in the specification and claims of this patent application for the present invention, the words "comprising" or similar words are intended to mean that the items appearing before "comprising" cover the items listed after "comprising" or their equivalents, and do not exclude other items.

[0057] The numerical values mentioned in the present invention include all numerical values that increase unit by unit from low to high, assuming that there is an interval of at least two units between any lower value and higher value. For example, if it is said that a component amount or a physical quantity ranges from 1 to 100, preferably from 10 to 90, and most preferably from 20 to 80, it is intended to express that numerical values such as 5 to 95, 14 to 76, 23 to 67, 32 to 58, 41 to 49, etc. have been clearly listed in this specification; for numerical values less than 1, 0.0001, 0.001, 0.01 or 0.1 are considered as a suitable unit. The foregoing examples are for illustrative purposes only. In fact, all combinations of numerical values between the lowest value and the highest value listed are considered to be clearly listed in this specification in a similar manner.

[0058] All animal experiments in the present invention were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Naval Medical University (2023-564), and all operations and surgical procedures were carried out in accordance with the approved guidelines.

[0059] The bacteria used in the present invention were methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 43300), which was purchased from the American Type Culture Collection (ATCC) and cultured in TSB medium (Hopebio). Raw 264.7 cells were provided by the Shanghai Institute of Cell Biology and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Mouse bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs) were isolated and cultured according to the experimental protocol described below.

[0060] Unless otherwise specified, numerical data in the present invention are expressed as mean ± standard deviation. Two-group comparisons were performed using two-tailed Student's t-test, and multi-group comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. All calculations and statistical analyses were performed using Excel 2016 and GraphPad Prism 9. A P value less than 0.05 was considered statistically significant.

[0061] Preparation of Nano - robots CS - BT@MZ@NEs

[0062] 1) Mix 17.018 g (50 mmol) of tetrabutyl titanate (Ti[O(CH2)3CH3]4, 97%, Sigma-Aldrich) with 20 mL of ethanol (analytical grade) to obtain an ethanol solution of tetrabutyl titanate;

[0063] Add 7 mL of aqueous ammonia solution (25%, Sigma-Aldrich) to the ethanol solution of tetrabutyl titanate to obtain a first mixed solution;

[0064] Dissolve 14.204 g (75 mmol) of barium hydroxide hydrate (Ba(OH)₂·H₂O, 98%, Sigma - Aldrich) in 25 mL of deionized water to obtain a clear aqueous barium hydroxide solution;

[0065] Add the clear aqueous barium hydroxide solution to the first mixed solution to obtain a second mixed solution;

[0066] Transfer the second mixed solution to a 100 - mL stainless - steel autoclave with a Teflon liner, and perform heat treatment at 200 °C for 48 h; then perform centrifugal separation, and successively wash the obtained precipitate 2 - 3 times with acetic acid, 2 - 3 times with absolute ethanol, dry it in an oven at 80 °C for 24 h, transfer it to an alumina crucible, calcine it in an air atmosphere at 800 °C for 10 h, naturally cool it to room temperature, and finally grind it into a powder to obtain nanometer particles BaTiO₃;

[0067] 2) Dissolve 0.149 g of zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) and 0.125 g of manganese nitrate (Mn(NO₃)₂) in 11.3 mL of methanol to obtain a third mixed solution;

[0068] Add 0.1 g of the nanometer particles BaTiO₃ to the third mixed solution, and stir at room temperature for 20 min to obtain a fourth mixed solution;

[0069] Dissolve 0.656 g of 2 - methylimidazole in 11.3 mL of methanol to obtain a methanol solution of 2 - methylimidazole;

[0070] Dropwise add the methanol solution of 2 - methylimidazole to the fourth mixed solution, and continuously stir for 2 h;

[0071] Centrifuge at 13,000 rpm, and wash the obtained precipitate 2 - 3 times with deionized water to obtain nanometer particles BT@MZ.

[0072] 3) Dissolve 0.1 g of chondroitin sulfate (CS) in 20 mL of deionized water to obtain an aqueous solution of chondroitin sulfate;

[0073] Add 100 mg of the nanometer particles BT@MZ to the aqueous solution of chondroitin sulfate, and stir at room temperature for 24 h;

[0074] Centrifuge at 13,000 rpm, and wash the obtained precipitate 2 - 3 times with deionized water to obtain nanometer particles CS - BT@MZ;

[0075] 4) Collect sterile bone samples from euthanized mice on a sterile workbench;

[0076] The bone marrow was rinsed with PBS, centrifuged at 250 g for 4 min, and then resuspended in 1 mL of PBS;

[0077] A density gradient centrifugation solution was prepared by successively layering 2 mL of 75% (v / v) Percoll (Pharmacia) solution, 2 mL of 65% (v / v) Percoll solution, and 2 mL of 55% (v / v) Percoll solution, and 1 mL of the single-cell suspension was added on top;

[0078] Centrifuge at 700 g for 25 min, and neutrophils were separated at the interface between the 65% Percoll solution and the 75% Percoll solution;

[0079] The neutrophils were washed three times with ice-cold PBS to remove the residual Percoll solution;

[0080] Cultured in RPMI 1640 medium containing 1% penicillin / streptomycin in a humid atmosphere at 37 °C and 5% CO2, and bone marrow tropic neutrophils were obtained after 6 h of in vitro culture.

[0081] Note: Cells were stained with FITC-labeled anti-mouse Ly-6G / Ly-6C (Gr1) and PE-labeled anti-mouse MAIR-IV (CLM-5) antibodies (1:200 diluted in PBS), cell purity was evaluated by flow cytometry using NovoExpress software, and analyzed using FlowJo software.

[0082] In a sterile tube, the bone marrow tropic neutrophils were cultured in serum-free medium for 1 h to obtain a fifth mixture;

[0083] 1 mg / mL of the nanoparticle CS-BT@MZ was added to the fifth mixture and incubated at 37 °C for 1 h to obtain the nanorobot CS-BT@MZ@NEs.

[0084] Note: To quantify the amount of CS-BT@MZ in CS-BT@MZ@NEs, FITC-labeled CS-BT@MZ was co-incubated with neutrophils for 1 h according to the above steps, and then analyzed by flow cytometry. To evaluate the migratory ability of CS-BT@MZ@NEs to CXCL12 (SDF-1α), a Transwell experiment using a 3 μm pore size polyester membrane insert was performed. 10 6DiD-labeled neutrophils or CS-BT@MZ@NEs at a density of 0 cells / mL were seeded in the upper chamber, and CXCL12 (20 ng / mL) was added to the lower chamber. After 30 min, fluorescence images of the lower chamber were taken using an inverted fluorescence microscope and analyzed using ImageJ software. For the wells treated with AMD3100 (CXCR4 receptor antagonist), neutrophils were pre-incubated in 1 mg / mL AMD3100 for 15 min and then processed as described above.

[0085] Preparation of Other Nanoparticles

[0086] 1) Dissolve 0.1 g of chondroitin sulfate (CS) in 20 mL of deionized water to obtain an aqueous solution of chondroitin sulfate.

[0087] Add 100 mg of the nanoparticle BaTiO3 to the aqueous solution of chondroitin sulfate and stir at room temperature for 24 h.

[0088] Centrifuge at 13,000 rpm and wash the resulting precipitate with deionized water 2 - 3 times to obtain the nanoparticle CS-BT.

[0089] 2) Dissolve 0.149 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and 0.125 g of manganese nitrate (Mn(NO3)2) in 11.3 mL of methanol and stir at room temperature for 20 min to obtain a sixth mixed solution.

[0090] Dissolve 0.656 g of 2-methylimidazole in 11.3 mL of methanol to obtain a methanol solution of 2-methylimidazole.

[0091] Dropwise add the methanol solution of 2-methylimidazole to the sixth mixed solution and continue stirring for 2 h.

[0092] Centrifuge at 13,000 rpm and wash the resulting precipitate with deionized water 2 - 3 times to obtain the nanoparticle Mn-ZIF-8.

[0093] Then prepare the nanoparticle Mn-ZIF-8@CS using a preparation method similar to 1).

[0094] 3) Ultrasonically disperse 0.1 g of the nanoparticle CS-BT@MZ in 10 mL of deionized water and stir at room temperature for 5 min to obtain a seventh mixed solution.

[0095] Add 0.01 g of the FITC (DiD) fluorescent dye to the seventh mixed solution and stir for 8 h.

[0096] Centrifuge at 13,000 rpm, and wash the obtained precipitate with deionized water 2 - 3 times to obtain the nanoparticles BT@MZ@CS-FITC(DiD).

[0097] Subsequently, nanoparticles BT@CS-FITC(DiD) and MZ@CS-FITC(DiD) were prepared by a preparation method similar to 3).

[0098] Characterization

[0099] The structures and morphological properties of the synthesized various nanoparticles were analyzed by transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher, USA), and Fourier transform infrared spectroscopy (FTIR, Magna-IR750, Thermo Fisher, USA). To study the free radical mechanism in the ultrasonic Fenton piezoelectric system, electron paramagnetic resonance (EPR) free radical trapping experiments were carried out to determine the contributions of reactive species. TBA, Na2C2O4, BQ, and TEMP were used as quenchers for ·OH, h + , ·O2 - and 1 O2 respectively, and DMPO (10 mM) was used as a free radical trapping agent, and the concentrations were all 10 mM. Under ultrasonic triggering conditions, a digital oscilloscope (DHO1000, China) was used to measure the output voltage. The experimental parameters were set as follows: center frequency 1 MHz, pulse repetition frequency 1000 Hz, pressure intensity 30 mW / cm 2 , duty cycle 50%. Piezoresponse force microscopy (PFM) detects the piezoelectric response of materials using the inverse piezoelectric effect of piezoelectric materials. Before testing, the sample solution was dropped on a silicon wafer and characterized after drying. To study the free radical mechanism of the entire system under ultrasound, Kelvin probe force microscopy (KPFM, Bruker, Dimension Icon) was used to detect the surface potential distribution. Nano Scope Analysis software (1.40r1, Bruker, USA) was used to analyze and obtain the surface potential data, and then the surface potential data was fitted by Gaussian fitting, and finally the average surface potential was calculated according to the weighted average.

[0100] In Vitro Antibacterial Experiments

[0101] Thaw the frozen MRSA strains and shake-culture them overnight in TSB medium at 37°C. Subsequently, dilute the culture 1:10000 and continue culturing until the logarithmic growth phase. Take 500 μL of the bacterial solution in the logarithmic growth phase (concentration 1×10 7(CFU / mL) was added onto the sterilized titanium disks in 24-well plates and cultured at 37 °C for 24 h to form mature biofilms. Subsequently, different therapeutic agents (including PBS control, CS-BT, CS-MZ, CS-BT@MZ, and vancomycin) were added into the mature biofilms and cultured for 2 h. The biofilm formation ability was evaluated as follows: 360 μL of diluted MRSA solution (10 9 CFU / mL) was added onto the titanium disks in 24-well plates, 40 μL of the corresponding treatment system was added, and cultured for 2 h. Subsequently, these plates were irradiated with ultrasound for 5 min (1.5 W / cm 2 , 40 kHz) or not irradiated, and continued to be cultured for 24 h. The anti-biofilm effects of different treatments were evaluated using live / dead staining, crystal violet staining, and scanning electron microscopy: The LIVE / DEAD BacLight Bacterial Viability Kit was used according to the manufacturer's instructions. In addition, the biofilms were stained with 1% crystal violet for 15 min, the titanium disks were rinsed twice with deionized water, and dissolved with 96% ethanol. The optical density was measured at a wavelength of 595 nm using a microplate reader to evaluate the biofilm quality.

[0102] In addition, the biofilms were rinsed with sterile PBS and resuspended in 1 mL of physiological saline. Subsequently, Congo red was added to the solution to a final concentration of 40 μg / mL. The membranes were incubated at 37 °C and shaken at 200 rpm in a thermostatic shaker for 2 h. The absorbance of the supernatant was measured at a wavelength of 490 nm. When detecting, 1 mL of physiological saline containing Congo red was used as a control. The calculation formula for the Congo red binding rate is as follows:

[0103] Congo red binding rate (%) = 100 - [(OD of the test supernatant × 100) ÷ OD% of the control group]

[0104] In addition, dsDNA was extracted from the treated biofilms using a polymer-based circulating free DNA enrichment kit (Analytik Jena AG, Germany) and quantified according to the manufacturer's instructions. The dsDNA concentration was quantified using a NanoDrop spectrophotometer, and each experiment was repeated at least five times.

[0105] Subsequently, the biofilms were rinsed with sterile PBS, collected, and the bacterial viability was quantified by CFU assay. Meanwhile, the integrity of the bacterial membrane was evaluated by live / dead staining. Briefly, the bacterial suspension was washed three times with PBS, and then SYTO9 and PI double dyes were added. After co-incubation in the dark for 15 min, the samples were washed three times with PBS again and then analyzed by flow cytometry. The treated bacterial samples were incubated with propidium iodide (PI) for 30 min. The staining solution was also detected by flow cytometry. Subsequently, a microplate reader was used to measure the fluorescence intensity of these samples, and the excitation and emission wavelengths were set at λex = 535 nm and λem = 615 nm, respectively. The bacteria treated with different treatments were stained with DCFH-DA, and then the intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry and a microplate reader.

[0106] For TEM analysis, the bacterial samples were fixed overnight at 4 °C with 2.5% glutaraldehyde solution and then rinsed three times with PBS. The samples were then dehydrated through a series of gradient ethanol, infiltrated with the embedding medium and embedded. Ultrathin sections of the embedded samples were prepared on grids, stained with uranyl acetate, and finally observed by TEM. The effects of different treatments on bacterial disruption were evaluated using a hemolysin sensitivity assay, an alkaline phosphatase (AKP) kit, and an o-nitrophenyl-β-D-galactoside (ONPG) hydrolysis assay. First, the bacteria in the biofilms were treated differently. Subsequently, the culture medium was collected, and the supernatant was analyzed using an AKP detection kit. The absorbance (OD520) was measured at a wavelength of 520 nm using a microplate reader. Subsequently, the biofilms were collected by sonication and normalized to an optical density (OD600) of 0.1 at a wavelength of 600 nm. Finally, the absorbance was measured at wavelengths of 600 nm and 420 nm using a microplate reader. Each experiment was repeated five times.

[0107] In addition, the bacterial suspension was collected, and the well walls and sample surfaces were rinsed with 0.85% NaCl solution. The samples and all the bacterial suspensions were transferred to a 50 mL centrifuge tube and shaken vigorously for 5 min. Subsequently, the samples were taken out, and the remaining liquid was centrifuged at 6000 rpm for 10 min. The supernatant was discarded, and the precipitate was frozen with liquid nitrogen and temporarily stored in a -80 °C refrigerator. Subsequently, the samples were placed on dry ice and sent to Novogene Co., Ltd. in Beijing for analysis using an Illumina NovaSeq 6000. Gene function analysis was performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Differential gene expression analysis was performed using edgeR, and genes with P < 0.05 and log2FC > 1 were screened as differentially expressed genes.

[0108] In Vitro Cytocompatibility Experiments

[0109] After co - culturing cells (rBMSCs) with different systems for 24 h, they were incubated with a Calcein AM / PI Cell Viability / Cytotoxicity Assay Kit (Sigma) for 15 min. Subsequently, the cells were observed under a fluorescence microscope. To evaluate the distribution of cells within the designated materials, after co - culturing for 24 h, confocal microscopy images of cytoskeleton staining were taken.

[0110] Golgi Apparatus Targeting and pH Regulation Verification

[0111] BMDCs were extracted and induced according to the procedures of previous studies. The cells were seeded in 96 - well cell culture plates at 5×10 3 cells per well and cultured for 24 h. Subsequently, the cells were exposed to DMEM medium containing FITC - labeled nanoparticles for 1 h. After uptake, the medium containing the nanoparticles was collected, and the cells were gently washed twice with pre - warmed DMEM medium (37 °C), and then incubated in fresh cell medium for 2 h or 12 h for efflux. After efflux, the supernatant was collected, the cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and stained with 2-(4 - amidinophenyl)-6 - indolecarbamidine dihydrochloride (DAPI; ThermoFisher Scientific) for 10 min. Subsequently, the cells were observed using a confocal laser scanning microscope (CLSM). Cells cultured in 35 - mm glass - bottom dishes were incubated with nanoparticles in standard cell medium at 37 °C for 30 min. Subsequently, the medium was removed, and an appropriate amount of buffer (containing Ca 2+ and Mg 2+Wash the cells with Hanks' balanced salt solution. After removing the washing solution, add the pre-prepared Golgi-Tracker Red staining working solution and incubate at 4 °C for 30 min. Subsequently, recover the Golgi-Tracker Red staining solution, wash the cells with ice-precooled medium about three times, and then incubate in fresh medium at 37 °C for 30 min. Finally, wash the cells with fresh medium and observe using a fluorescence microscope or a laser confocal microscope typically. At this time, the Golgi apparatus shows bright and intense fluorescence staining, while other intracellular membrane systems show relatively weak fluorescence staining. To evaluate the ability of CS-BT@MZ to regulate the Golgi pH under ultrasound, the Golgi pH probe CPH (2-{4-(sulfamidophenylamino)-7-(1-ethyl-3,3-dimethyl(indol-2-ylidene)}-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl)-1-ethyl-3,3-dimethyl-3H-indolium) is used, whose fluorescence intensity increases with the increase of Golgi pH. Briefly, the cells cultured in 35 mm glass bottom dishes are incubated with nanoparticles or monensin (a Golgi oxidative stress inducer that neutralizes Golgi pH and reduces Golgi protein activity) in standard cell medium at 37 °C for 6 h. Subsequently, before CLSM and flow cytometry, the cells are incubated with the CPH probe for 25 min. In addition, PCR is used to evaluate the expression levels of genes related to STING activation after different treatments, and the activation status of STING protein is evaluated by immunofluorescence analysis.

[0112] In Vitro Dendritic Cell (DC) Maturation and Function Experiments

[0113] To prepare BCMs for culturing DCs, the supernatants of MRSA biofilms from different treatment groups were collected, centrifuged at 12,000 g for 10 min, and filtered using a 0.22 μm filter (Merck, Germany). The filtered biofilm supernatants were mixed with DMEM medium (Gibco, USA) at a ratio of 1:2, supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA). Notably, DCs cultured with BCMs prepared from untreated biofilm supernatants were used as the control group. For flow cytometry analysis, different DC subsets were isolated and incubated with fluorescently labeled flow cytometry antibodies (including CD45 (N418), CD80 (16-10-A1), and CD86 (clone GL-1)) for 30 min. After recovering the antibodies and washing the cells, the samples were analyzed using a flow cytometer, and the data were processed using FlowJo V10 software. Total RNA was extracted using the TRIzol method according to the above cell treatment protocol. Gene expression was normalized to the endogenous reference gene β-actin. In addition, the levels of IFN-β and CXCL10 in the culture supernatants were quantified using an enzyme-linked immunosorbent assay (ELISA) kit.

[0114] Biodistribution Studies

[0115] First, nanoparticles were labeled with DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Subsequently, DiR-NPs and DiR-NPs@NEs were intravenously injected into mice. After 12 h, in vivo imaging was performed, and then the mice were euthanized and the major organs (heart, liver, spleen, lung, kidney, and bone) were collected for ex vivo imaging.

[0116] Establishment of "In Situ" Implant Infection Model

[0117] To evaluate the direct therapeutic effect of the nanorobot, an "in situ" implant infection model was established. Sixty male C57 mice (6 weeks old) were randomly divided into six groups: control group, NEs group, CS-BT@NEs group, CS-MZ@NEs group, CS-BT@MZ@NEs group, and vancomycin group. After induction of anesthesia, the left leg of the mice was shaved and disinfected, the knee joint was exposed through a layered incision, and a bone channel was created on the tibial plateau using a medical drill. 50 μL of 10 7 CFU / mL MRSA was injected into the channel, and the wound was sutured layer by layer. Subsequently, the mice were closely observed for one day to monitor the progress of infection. Treatment began 24 h after infection, and the mice were intravenously injected with PBS (control group), CS-BT@NEs, CS-MZ@NEs, CS-BT@MZ@NEs, or vancomycin (10 mg / kg). After 24 h, the mice were ultrasonically treated (40 kHz, 1.5 W / cm2 , 1 min per cycle, for a total of 5 cycles). To evaluate the effects of different treatments, general observations, clinical imaging evaluations, and bacterial viability tests were performed at predetermined time points. The knee joint area was recorded using calipers and quantified using the formula:

[0118] Knee joint area = π × a × b

[0119] where a and b are the lengths of the semi-major axis and semi-minor axis, respectively.

[0120] On the 20th day after surgery, the implants were removed and stained, and bacteria were detected by CLSM and SEM. The removed femurs were fixed and decalcified in EDTA for two weeks, and then pathological sections were prepared for HE and Giemsa staining. Immunofluorescence staining of MPO, iNOS, and CD206 was also performed.

[0121] The tibial end was incised, and bone marrow aspirates were collected using a syringe. The harvested bone marrow suspension was digested at 37 °C for 30 min in digestion buffer (100 μg / mL DNase I and 1 mg / mL collagenase IV). Subsequently, the cell suspension was filtered through a 70-μm filter to obtain a single-cell suspension. Red blood cells were lysed using ACK lysis buffer (Gibco) and washed three times with ice-cold PBS. For immunophenotypic analysis, the cell suspension was incubated with CD16 / 32 for 10 min, and then stained with antibodies against CD45 (S18009D), CD3 (17A2), CD4 (RM4-5), CD8a (53-6.7), CD11b (M1 / 70), CD11c (N418), CD80 (16-10-A1), CD86 (GL-1), Gr-1 (RB6-8C5), NK1.1 (BM8), CD19 (6DS), and CD138 (281-2). Cytokines (CXCL10, IL-10, IL-12, and IFN-β) and antibodies (IgM and IgG) in the bone marrow were quantified using an ELISA kit according to the manufacturer's instructions. In addition, mice carrying biofilms were injected with PBS and CS-BT@MZ@NEs, respectively. After the treatment was completed, the infected tissues were collected and sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for high-throughput sequencing. The data were analyzed using an interactive analysis platform, and differentially expressed genes (DEGs) with an adjusted P value less than 0.05 and [log2FoldChange] greater than 1 were selected for enrichment analysis.

[0122] Monitoring of Systemic Immune Responses in Vivo

[0123] To evaluate the systemic antibacterial immune response, a contralateral murine endophyte infection model was established. First, an "in situ" endophyte infection model was established in the left knee joint and treated with the above different methods. After 14 days, an endophyte infection model was established in the right femur using the same method. The isolation and staining procedures of spleen-related immune cells were similar to those of bone marrow. Briefly, the spleen was dissected and cut into small pieces, enzymatically digested and mechanically disrupted through a 70 μm cell strainer. After removing red blood cells using ACK lysis buffer, the cell suspension was co-incubated with CD16 / 32 for 10 min, and then stained with antibodies against CD45 (S18009D), CD3 (17A2), CD4 (RM4-5), CD8a (53-6.7), NK1.1 (BM8), CD11b (M1 / 70), CD11c (N418) and Gr-1 (RB6-8C5) on ice according to the manufacturer's instructions. Serum cytokines (CXCL10 and IFN-β) and antibody levels (IgG and IgM) were measured using ELISA kits according to the manufacturer's instructions.

[0124] Results

[0125] Multiple nanocomposites were characterized by scanning electron microscopy (SEM), and the results showed that BT@MZ was successfully synthesized with relatively uniform size and an average particle size of about 100 nm. Transmission electron microscopy (TEM) images clearly showed the CS coating, and the elemental analysis spectrum indicated the presence of C and S elements in the coating ( Figure 1 b). These results jointly verified the successful synthesis of CS-BT@MZ, providing a solid structural basis and characterization support for subsequent studies.

[0126] X-ray photoelectron spectroscopy (XPS) analysis showed that during the coating process, the binding energies of C, Ti, Ba, Mn, and Zn elements remained basically unchanged ( Figure 1 c-d), indicating that the structural integrity and chemical states of these elements were well maintained during the synthesis process. The stability of the binding energy indicated that the functionalization process did not change the core properties of the material, thus ensuring the stability and activity of the key components of the nanorobot. This stability is crucial for the performance of the material in biological systems and its response ability to external stimuli such as ultrasound. The oxygen vacancy content of different nanoparticles was analyzed by XPS, and the results showed that CS-MZ had the highest proportion of oxygen vacancies, while CS-BT had the lowest proportion of oxygen vacancies, and CS-BT@MZ was between the two ( Figure 1e - f). This trend indicates that the formation of oxygen vacancies in different nanoparticles is affected by compositional regulation: manganese and zinc ions promote the generation of oxygen vacancies, while the BT structure exhibits higher lattice stability, thus reducing the formation of oxygen vacancies. It is worth noting that the oxygen vacancy content significantly affects the piezoelectric effect of nanoparticles. Excessive oxygen vacancies will destroy the piezoelectric properties of the crystal, leading to uneven charge distribution and thus weakening the piezoelectric effect. The moderate proportion of oxygen vacancies in CS - BT@MZ indicates that while maintaining a certain activity, it effectively balances the influence of oxygen vacancies, ensuring the piezoelectric performance of the nanoparticles. This optimized oxygen vacancy ratio provides crucial material support for the applications of CS - BT@MZ in ultrasonic response - driven and immunomodulation.

[0127] Figure 1 Figure g shows the Fourier transform infrared spectroscopy (FTIR) peaks of CS - BT@MZ, elucidating the molecular structure and compositional details of the material. The weak peak at 1731 cm -1 corresponds to the stretching vibration of C=O, indicating that there may be carbonyl functional groups on the surface of the nanoparticles. The peak at 1639 cm -1 is also attributed to the stretching vibration of C=O, further confirming the existence of carbonyl groups. The characteristic peak at 1421 cm -1 is related to the bending vibration of Ba - Ti - O, indicating the presence of the barium titanate core. The doublet peaks at 1236 cm -1 and 1227 cm -1 correspond to the stretching vibration of C - N, which may originate from the amino modification on the surface of the nanoparticles or organic molecular components. The characteristic peak at 422 cm -1 is attributed to the Ti - O vibration, further confirming the structural integrity of barium titanate. Figure 1 Figure h shows the X - ray diffraction (XRD) patterns of different nanocomposites, clearly demonstrating the crystal structure characteristics of each material. The XRD pattern of CS - BT shows a typical perovskite - type crystal structure, with characteristic diffraction peaks at 2θ = 22.8°, 31.7°, and 45.5°, corresponding to the (100), (110), and (200) crystal planes of BT, respectively. These characteristic peaks indicate that the core structure of barium titanate remains intact during the functionalization process. The XRD pattern of CS - MZ shows significantly different diffraction characteristics, and the distribution of characteristic peaks reflects the influence of manganese - zinc (Mn - Zn) ion modification on the crystal structure of the material. This structure formation may endow the material with unique functional properties, such as enhanced chemical reactivity or improved surface characteristics. In the XRD pattern of CS - BT@MZ, the diffraction characteristics of both CS - BT and CS - MZ are observed simultaneously, although the overall peak intensity changes slightly. This change indicates the successful synthesis of CS - BT@MZ, and the surface modification has limited influence on the core crystal structure, mainly affecting the surface characteristics. In addition, the coated CS does not show obvious diffraction peaks, which is consistent with its amorphous nature.

[0128] Further study the ability of the nanorobot CS-BT@MZ to generate reactive oxygen species (ROS) under ultrasonic stimulation, including hydroxyl radicals (·OH), singlet oxygen ( 1 O2), and superoxide radicals (·O2 - ), while monitoring the generation of holes (h + ). As Figure 2 shown in a-d, the electron spin resonance (ESR) spectra clearly show the characteristic peaks of ·OH, ·O2 - , 1 O2, and h + under ultrasonic conditions, indicating that the nanorobot can efficiently generate various ROS under ultrasonic action. It is worth noting that the peak intensities of ·OH, ·O2 - , and 1 O2 of CS-BT@MZ are significantly higher than those of other control groups, indicating its excellent ROS generation ability under ultrasonic stimulation. This enhanced ROS generation ability may be attributed to the material design of CS-BT@MZ, which effectively promotes electron transfer and ROS generation through the ultrasonic-induced piezoelectric effect. In addition, for holes (h + ), the peak intensity of CS-BT@MZ is the lowest, indicating that its material design is conducive to the effective separation of electrons and holes, thus preventing electron-hole recombination and further improving the ROS generation efficiency.

[0129] To further verify the piezoelectric conversion ability of CS-BT@MZ, detect its output voltage response under ultrasonic pressure. As Figure 2 shown in e, CS-BT@MZ exhibits a significant electrical signal output under dynamic ultrasonic pressure, and the signal intensity increases significantly with the increase of ultrasonic power. This linear relationship indicates that CS-BT@MZ can efficiently convert the mechanical energy of ultrasound into electrical signals, highlighting its excellent piezoelectric properties. Through the piezoresponse force microscopy (PFM) of atomic force microscopy (AFM), study the local point-to-point piezoelectric response of CS-BT@MZ materials. After applying a tip bias of 10 V to the sample surface, a piezoelectric response amplitude of 66.5 pm and a phase angle of 143° ( Figure 2 g-h) are observed, indicating a significant piezoelectric response on the material surface. These findings confirm the excellent piezoelectric properties of CS-BT@MZ. Further analysis shows that the non-zero remanent polarization or ferroelectric properties of the sample are clearly defined by the PFM amplitude butterfly hysteresis loop and phase hysteresis loop ( Figure 2 i-j). CS-BT@MZ exhibits a typical hysteresis loop and a significant butterfly curve, indicating that the material can undergo spontaneous polarization under the action of a switching voltage.

[0130] To study the spatial distribution of sonogenerated charge transfer in CS-BT@MZ, in-situ ultrasonic irradiation Kelvin probe force microscopy (KPFM) was used for detection. As Figure 2 shown in k, under ultrasonic irradiation, the average surface potential of CS-BT@MZ increased significantly to 423.44 mV, while it was 412 mV without ultrasound, with a difference of 11.44 mV. This result indicates that ultrasonic irradiation effectively promotes the charge transfer process in CS-BT@MZ, further verifying the piezoelectric effect and charge separation ability of the nanorobot under ultrasonic stimulation. These results further verify the piezoelectric properties and ferroelectric characteristics of CS-BT@MZ, laying a solid foundation for its multifunctional applications in ultrasonic-driven environments. The efficient piezoelectric response not only supports its stable operation in the ultrasonic environment but also provides a reliable energy basis for ROS generation, local acidic environment regulation, and precise regulation of Golgi function.

[0131] To more intuitively clarify the charge transfer mechanism of CS-BT@MZ, its charge density distribution ( Figure 2 m) was simulated. The results show that electrons transfer from BT to Mn-Zif through the heterostructure, theoretically confirming that this heterostructure effectively promotes the separation and transfer of carriers. This efficient charge transfer mechanism provides a theoretical basis for the enhanced sonodynamic effect of the nanorobot under ultrasonic irradiation. In addition, the COMSOL Multiphysics software was used to simulate the spatial potential distribution and electric field direction of the CS-BT@MZ piezoelectric heterojunction ( Figure 2 n). The simulation results show that at the BT / Mn-Zif interface, the direction of the built-in electric field (IEF) is from BT to Mn-Zif. This interfacial electric field helps to effectively separate the electron-hole pairs generated under ultrasonic stimulation and reduce electron-hole recombination. Under ultrasonic irradiation, due to the synergistic effect of the heterojunction energy band structure and functional interface, the CS-BT@MZ heterojunction significantly enhances the sonodynamic effect, thereby improving the ROS generation efficiency.

[0132] In summary, the simulation results of the charge density distribution and piezoelectric heterojunction show that the heterostructure of CS-BT@MZ significantly enhances the sonodynamic effect and ROS generation ability by promoting electron transfer and carrier separation, providing a solid material basis for its efficient application in antibacterial therapy.

[0133] Biofilm formation is a key factor for bacteria to evade host immune defenses, further increasing the complexity of treating IAIs by traditional methods. Studies have shown that the biofilm formed during the infection process not only serves as a protective barrier for immune cells but also functions as a predatory mechanism to effectively eliminate immune cells. Bacterial biofilms have evolved various strategies to evade detection by the immune system, including mimicking host cell structures, regulating antigen expression, and secreting EPS. These mechanisms provide a physical barrier for bacteria and interfere with immune signal transduction and suppress effector functions. In addition, the structural complexity and heterogeneity of biofilms create a microenvironment that inhibits the infiltration and activity of immune cells, enabling the bacteria within the biofilms to survive and proliferate for a longer time. Given these complex defense mechanisms, inducing the release of biofilm antigens has become a crucial step in developing effective biofilm-targeted immunotherapies. By exposing the antigens hidden in the biofilm matrix, these strategies can enhance the immune system's recognition ability, thereby activating innate and adaptive immune responses and ultimately destroying the biofilm and clearing the bacteria within it. Therefore, understanding and exploiting the processes that trigger biofilm antigen release and immune activation are crucial for advancing novel treatment methods against IAIs.

[0134] As Figure 3 shown in a-b, the CS-BT@MZ+US treatment significantly disrupted the structure of mature biofilms and effectively reduced the number of viable bacteria within them. This indicates that the sound-driven nanorobots can effectively disrupt the integrity of the biofilm matrix through mechanical forces and chemical reactivity, significantly weakening its function as a bacterial protective barrier. The study emphasizes the key role of EPS degradation in releasing bacterial antigens and enhancing immune cell recognition. The degradation of EPS not only disrupts the stability of the biofilm but also exposes hidden bacterial antigens, thereby enhancing the host immune system's ability to detect pathogens. Further quantitative analysis ( Figure 3 c-d) showed that compared with other groups, the number of viable bacteria within the biofilms in the CS-BT@MZ+US treatment group was significantly reduced, indicating its excellent antibacterial effect. In addition, the biofilms were stained using the 1% crystal violet staining method, and the absorbance of the treated samples was measured using a spectrophotometer after incubation at 37 °C for 24 hours ( Figure 3 e-f). The results showed that the CS-BT@MZ+US treatment significantly reduced the adhesion of bacteria to the implant surface and significantly weakened the structural integrity of the biofilms. In addition, as Figure 3As shown in g-h, the CS-BT@MZ+US treatment demonstrated a remarkable ability to disrupt key components of the biofilm matrix, such as polysaccharides and extracellular DNA (eDNA). These matrix components are crucial for maintaining the structural integrity and stability of the biofilm, and their disruption plays a key role in inhibiting biofilm formation. Among all the evaluated treatments, the CS-BT@MZ+US showed the most significant inhibitory effect on polysaccharides and eDNA. Specifically, compared with the control group cultured with CS-BT@MZ alone, the CS-BT@MZ+US treatment significantly reduced the eDNA biomass to 122.3±2.5 μg / mL after 5 minutes of ultrasound treatment, indicating that ultrasound-assisted nanorobots can effectively degrade the core components of the biofilm matrix.

[0135] The effects of the CS-BT@MZ+US treatment on MRSA embedded in mature biofilms were further investigated by detecting extracellular alkaline phosphatase (AKP) levels and performing o-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis experiments. The results showed that the extracellular AKP concentration increased significantly after the CS-BT@MZ+US treatment ( Figure 3 i), and the release of AKP is a key indicator of bacterial cell wall damage. This indicates that the treatment significantly damaged the bacterial cell wall. The ONPG hydrolysis experiment further showed that ONPG molecules can penetrate the bacterial cytoplasm and be cleaved by intracellular β-galactosidase to generate o-nitrophenol, indicating a significant increase in bacterial cell membrane permeability. Notably, the absorbance value at 420 nm increased significantly after the treatment ( Figure 3 j), confirming the enhanced bacterial cell membrane permeability. In addition, transmission electron microscopy (TEM) analysis showed that the CS-BT@MZ+US treatment had a profound impact on the bacterial structure. The treated bacteria showed significant cell membrane shrinkage, blurred boundaries, and a corrugated morphology, while the bacteria in the control group PBS had a smooth, intact, and undamaged cell membrane ( Figure 3 k). These observations indicate that the CS-BT@MZ+US treatment effectively cleared the bacteria embedded in the biofilm by disrupting the integrity of the bacterial cell wall and cell membrane, thus promoting further immune system responses. Importantly, the residues of apoptotic and damaged bacteria are important sources of bacterial antigens. Studies have shown that these antigens can be efficiently internalized by DCs, promoting antigen cross-presentation and stimulating downstream adaptive immune responses. These findings highlight the potential of CS-BT@MZ+US in disrupting biofilms, enhancing immune recognition, and bactericidal activity, providing new insights into immunocombinatorial therapies for IAIs.

[0136] Subsequently, the bactericidal mechanism mediated by GMBC+US was further explored. In previous studies, it was confirmed that the CS-BT@MZ+US treatment produced a large amount of ROS ( Figure 2 a-d), indicating that its antibacterial properties may be attributed to the activity of ROS. AsFigure 3 As shown in l-m, the generation of ROS in bacteria after CS-BT@MZ+US treatment increased significantly. Further analysis showed that this treatment method might penetrate the biofilm surface layer by layer through the synergistic effect of ROS, effectively destroying the integrity of the bacterial cell membrane, thereby achieving powerful biofilm disruption. Notably, in addition to the EPS barrier of the biofilm, the persistent viable bacteria within the biofilm are another major challenge in the treatment of MRSA. The research results showed that CS-BT@MZ+US could not only effectively penetrate the MRSA biofilm, but also produce a variety of synergistic active substances under the action of ultrasound. These active substances not only enhanced the antibacterial effect of ROS, but also further promoted the degradation of the biofilm and the clearance of bacteria.

[0137] To further explore the potential mechanism of CS-BT@MZ+US in eradicating MRSA biofilms, RNA sequencing analysis was performed on the CS-BT@MZ+US treatment group and the control group, with three replicates in each group. The results showed that CS-BT@MZ+US treatment significantly changed the gene expression pattern of bacteria. Heat map analysis of differentially expressed genes (DEGs) Figure 3 n) showed that genes related to the quorum sensing (QS) system and bacterial biofilm formation (such as AgrA, AgrB, AgrD, and secA2) were significantly downregulated in the CS-BT@MZ+US treatment group. In addition, genes related to cell membrane integrity (such as kdpA and kdpB) also showed downregulated expression in the CS-BT@MZ+US group. Notably, CS-BT@MZ+US treatment significantly affected the manganese ion homeostasis of bacteria. Specifically, the manganese ion uptake genes (mend and menH) were upregulated, while the manganese ion efflux gene (deoB) was downregulated. This may be attributed to the synergistic effect of sonodynamic therapy (SDT) and chemodynamic therapy (CDT). The large amount of ROS generated by CS-BT@MZ+US destroyed the integrity of the bacterial cell wall and cell membrane, affecting the proton transport channels, thereby increasing the influx of metal ions into the bacterial cytoplasm. To further explore the anti-biofilm mechanism of CS-BT@MZ+US, enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Figure 3 o) to systematically study the biochemical metabolism and signal transduction pathways. The analysis showed that the metabolic pathways related to ribosome function were significantly disrupted, which might be due to ROS interfering with the protein transporters on the bacterial membrane. The action of ROS reduced the efficiency of the protein transporters, significantly increased the permeability of the bacterial membrane, thereby promoting the entry of harmful substances into the bacteria and further destroying the ribosome structure. At the same time, the results of gene ontology (GO) enrichment analysis further verified these observations Figure 3p). The results showed that molecular functions such as ribosomal subunit, rRNA binding, protein-containing complex, ribonucleoprotein complex, and structural molecule activity were significantly affected, indicating that the metabolic and biosynthetic processes of bacteria were severely disrupted. These changes were closely related to a significant decrease in the viability of bacteria.

[0138] In summary, CS-BT@MZ+US eradicates bacteria by disrupting bacterial biofilms, inducing bacterial DNA damage and protein leakage using ROS-related oxidative stress toxicity, and inhibiting the tricarboxylic acid (TCA) cycle ( Figure 3 q). These findings provide crucial experimental evidence for the further development of ROS-based anti-biofilm treatment strategies and highlight the potential application value of CS-BT@MZ+US as an anti-infective therapy.

[0139] To determine whether various nanoparticles (NPs) could effectively penetrate into the interior of cells, their phagocytosis efficiency in BMDMs was evaluated. The research results showed that these NPs had a uniform size distribution and excellent stability. In addition, fluorescently labeled NPs (FITC-NPs) exhibited rapid and efficient internalization ability in BMDMs, as Figure 4 shown in a, rather than just attaching to the cell surface. These results indicated that NPs had a strong intracellular entry ability, laying a foundation for further functional studies. To further verify whether the CS-coated nanoparticles could target the Golgi apparatus for co-localization experiments. As Figure 4 shown in b, after co-culturing FITC-labeled nanoparticles (FITC-NPs) with BMDMs for 2 hours, the fluorescence signal (green) of the uncoated CS nanoparticles was significantly separated from the Golgi-selective dye (red), indicating that these particles failed to localize to the Golgi apparatus. In contrast, the FITC-labeled CS-coated nanoparticles (CS-NPs) showed significant Golgi localization signals, indicating that CS-NPs were effectively transported to the Golgi apparatus after endocytosis. The Golgi apparatus is rich in glycosyltransferases (GTs), glycosidases, and nucleotide sugars, and these components work together to promote carbohydrate synthesis and covalently link sugar chains to proteins. Among them, N-acetylgalactosaminyltransferase (GalNAc-Ts) specifically recognizes polypeptide chains and transfers N-acetylgalactosamine (GalNAc) to them. Since GalNAc is the main component of CS, the affinity of CS-NPs for the Golgi apparatus can be attributed to the specific interaction between GalNAc-Ts and CS. This finding further confirmed the Golgi targeting ability of CS-NPs and provided a theoretical basis for its application in the regulation of Golgi function.

[0140] Subsequently, the Golgi pH fluorescent probe CPH was used to study the effects of different treatments on Golgi pH. Monensin was used as a positive control (as it induces Golgi oxidative stress and significantly increases its pH), while PBS was used as a negative control. BMDMs were pretreated with PBS, CS-BT@MZ, and monensin for 7 hours, followed by the addition of the fluorescent probe CPH. After incubation for 25 minutes, confocal microscopy imaging was performed. Fluorescence emission wavelengths of 700 nm - 760 nm were collected under 633 nm laser excitation for analysis ( Figure 4 c-d). The results showed that the fluorescence intensity of the probe in the monensin-treated group was significantly enhanced (about 2.3 times that of the control group), indicating that monensin treatment significantly increased Golgi pH. In contrast, the fluorescence intensity of the CS-BT@MZ+US-treated group was significantly lower than that of the PBS control group, indicating that CS-BT@MZ+US effectively reduced Golgi pH. These findings confirmed the role of CS-BT@MZ+US in regulating the acidic environment of the Golgi. In further experiments, cells treated with trypsin digestion and incubated with the probe were rinsed with PBS and collected into 1.5 mL EP tubes for flow cytometry analysis. The experimental results were consistent with the CLSM results, further demonstrating that CS-BT@MZ+US treatment significantly reduced Golgi pH( Figure 4 e).

[0141] The mechanism by which CS-BT@MZ+US reduces Golgi pH may involve the following processes: i) Ultrasound-driven effect promotes the formation of a local acidic environment: CS-BT@MZ is an ultrasound-responsive nanoparticle, and its piezoelectric effect under ultrasound can initiate the ion transport process. Specifically, under ultrasound activation, the nanoparticles generate mechanical vibrations and local thermal effects, which can trigger the release of H + ions. These H + ions rapidly diffuse within the cell, especially accumulate in the Golgi region, thereby reducing the local pH. ii) Golgi targeting: CS is a key component of the CS-BT@MZ nanoparticles and has a high affinity for specific glycosyltransferases in the Golgi (such as GalNAc-Ts). This targeting effect enables the nanoparticles to be directed to the Golgi after endocytosis, ensuring that the released H + ions are mainly concentrated within the Golgi, enhancing the local acidification effect. iii) ROS synergy: Under ultrasound, CS-BT@MZ generates a large amount of ROS. These ROS not only directly damage cell membranes and intracellular structures but may also alter the ion channel function on the Golgi membrane, further promoting the influx of H + ions or restricting the efflux of H +efflux, thereby exacerbating Golgi acidification. iv) Regulation of proton transport channels: The combined action of ROS and nanoparticles may affect the activity of proton pumps (such as V-ATPase) on the Golgi membrane. Ultrasound stimulation may enhance the activity of proton pumps by changing membrane fluidity or protein structure, further increasing the H + concentration in the Golgi lumen and decreasing its pH. In summary, CS-BT@MZ+US effectively reduces the Golgi pH through multiple mechanisms such as ultrasound-driven processes, targeted delivery, ROS generation, and regulation of proton transport. This multi-level acidification effect not only directly changes the functional state of the Golgi but may also indirectly affect cell signaling and immune responses, providing a new strategy for regulating the intracellular acidic microenvironment and treating IAIs.

[0142] Within the Golgi apparatus, the molecular structure of STING undergoes a conformational change to form a functional oligomeric complex, which is crucial for downstream signal transduction (such as the recruitment and phosphorylation of TBK1). Studies have shown that the acidic pH environment of the Golgi is essential for maintaining the conformation and function of the STING protein. The optimal acidic environment not only promotes STING oligomerization but also significantly enhances the recruitment efficiency of the TBK1 kinase, thereby amplifying the signal transduction effect. Notably, CS-BT@MZ+US effectively regulates the Golgi pH through its ultrasound-driven mechanism, promoting the formation of an acidic environment. To further investigate the specific effect of the Golgi acidic environment induced by CS-BT@MZ+US on STING activation, subsequent verification experiments were conducted. First, the PCR technique was used to evaluate the effect of CS-BT@MZ+US treatment on the expression of genes related to the STING pathway ( Figure 4 f). The results showed that compared with the control group, CS-BT@MZ+US significantly upregulated the expression levels of STING, IRF3, TBK1, and IFN-β, indicating that this treatment can activate key components of the STING signaling pathway. In addition, immunofluorescence analysis further verified these findings, showing a significant increase in the STING expression level in the CS-BT@MZ+US treatment group ( Figure 4 g). These findings suggest that CS-BT@MZ+US promotes the activation of the STING signaling pathway by regulating the Golgi pH, thereby providing a molecular basis for enhancing the host immune response.

[0143] Subsequently, supernatants were extracted from MRSA biofilms treated with different methods and prepared into biofilm-conditioned media (BCMs) for culturing bone marrow-derived dendritic cells (BMDCs) ( Figure 4 h). The results showed that after treatment with CS-BT@MZ+US, the proportion of mature dendritic cells (CD80 + CD86 + ) increased significantly from 4.59% to 33.8% (Figure 4 i). The maturation of dendritic cells further leads to a significant increase in the expression levels of inflammatory cytokines and chemokines ( Figure 4 j - k), thus enhancing the intensity of the innate immune response. Immunofluorescence experiments also confirmed that CS - BT@MZ+US could effectively activate the expression of STING ( Figure 4 l). In addition, in the CS - BT@MZ+US treatment group, the activation of the cGAS - STING signaling pathway led to a significant up - regulation of the gene expression related to antigen presentation and T - cell migration in dendritic cells ( Figure 4 m).

[0144] In summary, CS - BT@MZ+US effectively activated the cGAS - STING signaling pathway by significantly enhancing the release of bacterial antigens and promoting Golgi acidification. In addition, this treatment significantly promoted the maturation of dendritic cells, thereby enhancing the activation and effector functions of adaptive immunity ( Figure 4 n). Finally, this dual - action mechanism synergistically enhanced the overall immune defense ability of the host, providing new strategies and possibilities for the immunotherapy of biofilm infections.

[0145] The low blood perfusion characteristics of the bone marrow and the existence of the bone marrow - blood barrier pose significant challenges for the treatment of bone - related diseases such as osteomyelitis, implant - associated infections (IAIs), and osteoporosis. Despite significant progress in medical research over the past few decades, the problem of drugs having difficulty penetrating deep into the bone marrow remains unresolved. This limitation mainly stems from the inefficiency of drug delivery systems in the bone marrow environment. The bone marrow - blood barrier hinders the penetration of therapeutic molecules and drugs, while low blood perfusion further reduces the distribution efficiency of drugs in the bone marrow. To address this problem, researchers have developed various nanocarriers and functional molecules (such as bisphosphonates, aptamers, and peptides) for encapsulating and targeting therapeutic drugs. However, the bone marrow targeting ability of these nanoparticles is still limited, mainly because it is difficult for them to efficiently cross vascular endothelial cells and enter bone marrow tissue. In addition, the low perfusion rate in the blood further reduces the chance of drugs reaching the bone marrow. Therefore, there is an urgent need to develop novel and efficient drug delivery strategies to overcome this challenge.

[0146] Neutrophils are the most common type of white blood cells in the body, and a large number of their precursor cells differentiate into neutrophils in the bone marrow. Neutrophils have unique life cycle and migration characteristics, making them a potential drug delivery vehicle. Notably, neutrophils can cross the bone marrow-blood barrier and directly deliver therapeutic drugs to the bone marrow, significantly improving the therapeutic effect. In addition, studies have shown that neutrophils cultured in vitro for 6 hours can significantly upregulate the expression of CXCR4. The high expression of CXCR4 promotes neutrophil homing to the bone marrow because CXCR4 can specifically bind to CXCL12 (SDF-1) secreted in the bone marrow environment. This property makes neutrophils an ideal vehicle for delivering therapeutic molecules to the bone marrow.

[0147] Based on this, the present invention proposes to use neutrophils cultured in vitro for 6 hours as a drug delivery tool. By co-incubating nanoparticles (NPs) with these neutrophils highly expressing CXCR4, it aims to achieve effective loading of the nanoparticles and utilize the natural migration characteristics of neutrophils to precisely deliver the drug deep into the bone marrow, thereby overcoming the limitations of traditional drug delivery strategies and providing a new method for the treatment of IAIs ( Figure 5 a). Neutrophils were isolated from the bone marrow by density gradient centrifugation and their purity was identified by flow cytometry, and the results showed that the purity was about 91% ( Figure 5 b). Subsequently, DiD-labeled nanoparticles (nNPs) were co-cultured with neutrophils for 1 hour to evaluate the internalization degree of the nanoparticles. The results showed that the concentration of the nanoparticles significantly affected the uptake efficiency of neutrophils. As the concentration of the nanoparticles increased, the fluorescence signal detected in neutrophils gradually increased. Specifically, the proportion of neutrophils (DiD-NPs@NEs) + increased from 33.1% at 50 μg / mL to 96% at 500 μg / mL. ( Figure 5 c). This finding indicates that the concentration of the nanoparticles directly affects their internalization efficiency in neutrophils, providing an important basis for optimizing the nanoparticle delivery system.

[0148] To evaluate the chemotactic response of neutrophils to CXCL12, a Transwell assay was used ( Figure 5 d). Neutrophils (CS-BT@MZ@NEs loaded with nanoparticles) isolated from the bone marrow and cultured for 6 hours were seeded in the upper chamber of the Transwell, and CXCL12 was added to the lower chamber. The experimental results ( Figure 5e-g) showed that CS-BT@MZ@NEs exhibited significant chemotaxis towards CXCL12, capable of efficiently crossing a 3-μm pore size membrane and migrating to the lower chamber. After 0.5 h, compared with the control group without CXCL12, the number of neutrophils in the lower chamber with CXCL12 was significantly increased, while almost no cell migration was observed in the control group. In addition, the addition of the CXCR4 receptor antagonist AMD3100 significantly reduced the chemotactic ability of neutrophils towards CXCL12, further confirming the crucial role of the CXCR4-CXCL12 axis in neutrophil chemotaxis. These results indicated that CS-BT@MZ@NEs had high sensitivity and chemotactic ability towards CXCL12, providing a theoretical support and experimental basis for its efficient bone marrow delivery in vivo. To further investigate the in vivo distribution of CS-BT@MZ@NEs, DiR-labeled nanoparticles (DiR-CS-BT@MZ) and nanoparticles loaded with neutrophils (DiR-CS-BT@MZ@NEs) were injected via the tail vein, and detected using an in vivo imaging system. The results showed that the fluorescence signal of DiR-NPs was mainly concentrated in the liver, indicating that it was easily captured by the reticuloendothelial system (RES). In contrast, the signal of DiR-CS-BT@MZ@NEs was significantly reduced in the liver, while significantly increased in the spleen, lung, and bone marrow ( Figure 5 h).

[0149] These findings indicated that the addition of neutrophil carriers could effectively change the biodistribution pattern of nanoparticles, reduce liver accumulation, and enhance targeted delivery to the bone marrow. This finding further confirmed the potential of CS-BT@MZ@NEs in bone marrow targeted therapy, providing an important experimental basis for its application in the treatment of IAIs.

[0150] In vitro studies showed that CS-BT@MZ@NEs could activate the STING pathway and enhance antigen presentation mediated by dendritic cells (DCs). Based on these findings, it was speculated that CS-BT@MZ@NEs might induce local inflammation and enhance the host's immune defense mechanism in immunosuppressive IAIs. To verify this hypothesis, the knee abscess size, microbial changes, and imaging features were evaluated in the IAIs model ( Figure 6 a). The experimental results showed that the mice in the control group (PBS) exhibited severe knee swelling and suppuration, while the knee abscess in the CS-BT@MZ@NEs group of mice was significantly reduced, and there were no obvious signs of infection ( Figure 6 b). X-ray analysis further showed that chronic biofilm infection led to significant periosteal reaction and osteolysis, while the bone destruction in the CS-BT@MZ@NEs treatment group was minimal and the bone structure was preserved ( Figure 6c). Micro-CT analysis showed that the CS-BT@MZ@NEs group significantly reduced osteolysis around the implant. In addition, MRI showed a significant decrease in T2 signal in the CS-BT@MZ@NEs group, reflecting a substantial decline in the severity of infection ( Figure 6 d). To further verify the antibacterial effect, confocal microscopy (CLSM) observation was performed on the harvested implants. The results showed that the bacterial colonies in the CS-BT@MZ@NEs treatment group were significantly reduced ( Figure 6 e). Histological analysis further showed that neutrophil and bacterial infiltration in this treatment group were significantly reduced ( Figure 6 f-h).

[0151] To further clarify the intrinsic antibacterial mechanism of CS-BT@MZ@NEs, a comprehensive analysis of the immune cell composition in the bone marrow was performed, focusing on the phenotypes of infection-related macrophages (IAMs) and the dynamic changes of immune cells. IAMs with the M1 phenotype are key effector cells for clearing bacteria through phagocytosis and antigen-dependent mechanisms. The results showed that CS-BT@MZ@NEs treatment significantly polarized IAMs in the bone marrow of mice towards the M1 phenotype. Further analysis of the bone marrow immune cell composition showed that CS-BT@MZ@NEs treatment significantly enhanced the infiltration of key driving cells for antibacterial immunity ( Figure 6 i). Notably, the proportion of mature DCs (CD80 + CD86 + ) in the CS-BT@MZ@NEs group was significantly increased. Previous studies have shown that a higher proportion of mature DCs can significantly enhance the adaptive immune response in cancer treatment. Similarly, it is speculated that as a bacterial therapeutic agent, the increase in DCs may significantly improve antigen presentation efficiency, thereby enhancing the bacteria-specific immune response. Further flow cytometry analysis showed a significant increase in T cell infiltration in the CS-BT@MZ@NEs group, including CD4 + helper T cells (CD45 + CD3 + CD4 + ) and CD8 + cytotoxic T cells (CD45 + CD3 + CD8 + ). The increase in these T cells is crucial for the adaptive immune response of infected hosts. CD4 + helper T cells amplify the antibacterial effect by regulating the functions of other immune cells, while CD8 + T cells directly mediate the clearance of the bacterial infection site.

[0152] To trigger a protective humoral immune response, B cells must differentiate into long-lived plasma cells capable of producing antibodies or memory B cells capable of rapidly responding to re-exposure to pathogens. The results showed that CS-BT@MZ@NEs significantly increased the proportion of plasma cells (CD45 + CD19 + CD138 + ) in infected bone tissue, nearly tripling compared to the untreated group. This indicates enhanced generation of long-lived plasma cells, providing an important source of lifelong protective antibodies, which is crucial for the treatment of IAIs. Additionally, a common immune evasion strategy of invading pathogens is to hide inside host cells. Natural killer (NK) cells and CD8 + T cells are the main innate and adaptive lymphocytes that recognize and eliminate infected host cells. Notably, CS-BT@MZ@NEs treatment significantly enhanced the recruitment of NK cells (CD45 + NK1.1 + ) to the infected bone marrow. This treatment also significantly promoted the infiltration of NK cells and CD8 + T cells, indicating its ability to effectively recognize and eliminate bacteria within host cells. These results further highlight the potential value of CS-BT@MZ@NEs in enhancing host immune defense and clearing persistent infections.

[0153] A hallmark of IAIs is the significant infiltration of MDSCs in the bone marrow. To evaluate the regulatory effect of CS-BT@MZ@NEs on the immunosuppressive IAIs microenvironment (IME), the infiltration of MDSCs was analyzed. The results showed that the proportion of CD45 + Gr1 + MDSCs was significantly reduced in the CS-BT@MZ@NEs treatment group. Given that the extensive infiltration of MDSCs can lead to T cell dysfunction and hinder the effector functions of immune cells, the depletion of MDSCs induced by CS-BT@MZ@NEs is considered a key mechanism for enhancing the efficacy of bacterial immunotherapy. These results further confirm that CS-BT@MZ@NEs can trigger a strong antibacterial immune response in low-immunogenic IAIs. Inducing a protective antibody response is a key goal in the development of therapeutic agents for infectious diseases.

[0154] In summary, CS-BT@MZ@NEs can trigger strong innate and adaptive antibacterial immune responses in IAIs. The innate immune response is achieved through the STING pathway activated by CS-BT@MZ@NEs, including macrophage M1 polarization and DCs maturation; the adaptive immune response is jointly mediated by sonodynamic therapy (SDT) assisted by CS-BT@MZ@NEs, humoral immunity driven by protective antibodies induced by CS-BT@MZ@NEs, and T cell immunity activated by antigen-presenting cells (APCs). These immune responses indicate that CS-BT@MZ@NEs can effectively combat bacterial infections.

[0155] To comprehensively elucidate the anti-biofilm mechanism of CS-BT@MZ@NEs, transcriptome analysis was performed on the infected tissues of mice treated with PBS or CS-BT@MZ@NEs. The results showed that compared with the biofilm group, CS-BT@MZ@NEs treatment led to upregulation of 1056 genes and downregulation of 1138 genes ( Figure 7 a). Gene Ontology (GO) analysis indicated that immune system-related pathways were significantly enriched, especially immune system processes and innate immune responses ( Figure 7 b), suggesting that CS-BT@MZ@NEs is closely related to immune-related pathways, thus triggering a strong anti-biofilm immune response. In addition, KEGG pathway analysis further verified these findings ( Figure 7 c). CS-BT@MZ@NEs activated the STING pathway, triggering a series of immune responses, including upregulation of interferon-stimulated genes (such as Ifng), pro-inflammatory cytokines (such as Il1b and Il17a), leukocyte recruitment chemokines (such as Cxcr2, Cxcr5, Cxcl3), and activation markers of T cells and DCs (such as CD4, H2-Q10) ( Figure 7 d). The upregulation of these immune effector molecules indicates that CS-BT@MZ@NEs effectively promotes the recruitment and activation of various immune cells. In addition, gene set enrichment analysis (GSEA) showed that the activities of the IL-17 signaling pathway, Th1 and Th2 cell differentiation pathways, and cytoplasmic DNA sensing pathway were significantly enhanced in the CS-BT@MZ@NEs treatment group ( Figure 7 e-g). These changes indicate that key co-stimulatory signaling pathways are effectively activated, further amplifying the anti-biofilm immune response.

[0156] Therefore, the CS-BT@MZ@NEs nanorobot can efficiently activate the STING pathway in vivo, induce the expression of interferon-stimulated genes, amplify pro-inflammatory signals, and promote the recruitment and activation of various immune cells, ultimately achieving a strong anti-biofilm effect. This finding provides important theoretical and practical guidance for the immunotherapy of IAIs.

[0157] Uncontrolled local bacterial infections can gradually lead to fatal bacteremia and distant organ damage. Additionally, infection-related immunosuppression increases the host's susceptibility to secondary infections. To evaluate whether CS-BT@MZ@NEs nanorobots can prevent the formation of distant infection foci by inducing an effective systemic antibacterial immune response, a murine contralateral IAIs model ( Figure 8 a) was established. The experimental procedure involved first establishing an in situ IAIs model and immunizing mice with CS-BT@MZ@NEs, followed by establishing another IAIs model in the contralateral knee joint. Fourteen days after model establishment, multidimensional analyses of the contralateral infected knee joint were performed, including knee joint abscess size, microbial changes, imaging features, and Giemsa staining. The results showed that the severity of infection in mice treated with CS-BT@MZ@NEs was significantly reduced, and knee joint swelling was significantly alleviated ( Figure 8 b). X-ray and MRI results showed that diffuse edema and liquefactive necrosis occurred in the joint cavity and surrounding muscles of control mice, while the infectious lesions in the CS-BT@MZ@NEs treatment group were significantly alleviated ( Figure 8 c-d). CLSM images showed that the bacterial load in immunized mice was significantly reduced ( Figure 8 e). Pathological sections further supported these findings: control mice showed extensive neutrophil and bacterial infiltration, while the tissue morphology in the CS-BT@MZ@NEs treatment group was close to normal ( Figure 8 f-h). These results indicate that in situ inoculation with CS-BT@MZ@NEs can significantly reduce the severity of infection in the contralateral IAIs model.

[0158] To further explore the mechanism of this phenomenon, the immune response in the spleens of mice was analyzed. As the main lymphoid organ, the spleen plays a central role in the host's systemic pathogen defense. After antigen capture, a large number of APCs migrate to the spleen and interact with immune cells to trigger a systemic antibacterial immune response. The results showed that the spleens of IAIs mice exhibited significant immune activation characteristics, including dual responses of innate and adaptive immunity ( Figure 8 i-j). These immune effects characterized by increased pro-inflammatory cytokines and activation of adaptive immune cells provide strong systemic immune support for infection control. Further analysis showed that the infiltration of CD4 + and CD8 + T cells, as well as NK cells, reached a peak in the spleen tissue of CS-BT@MZ@NEs-immunized mice. CD8 +The expansion of T cells in lymphoid organs is a hallmark of systemic immune enhancement, representing a significant boost in cellular immunity. Meanwhile, CS-BT@MZ@NEs effectively inhibited the influx of regulatory immune cells such as MDSCs, thereby alleviating the immunosuppressive effect induced by bacterial infection. This immune cell profile collectively verified that CS-BT@MZ@NEs could induce a strong systemic humoral and cellular immunity to prevent bacterial dissemination. To comprehensively characterize the host immune response, ELISA analysis was performed on cytokine and antibody levels in serum ( Figure 8 k). The results showed that the levels of pro-inflammatory cytokines were significantly increased, while the levels of anti-inflammatory cytokines were significantly decreased in the CS-BT@MZ@NEs treatment group, indicating that CS-BT@MZ@NEs could trigger a pro-inflammatory cytokine storm, thereby amplifying the antibacterial immune response and achieving the therapeutic effect. In addition, the IgM and IgG responses of CS-BT@MZ@NEs-immunized mice were significantly enhanced, exceeding those of other experimental groups, further demonstrating a strong humoral immune response.

[0159] In short, the CS-BT@MZ@NEs nanorobot not only effectively controlled local infection through in-situ immunity but also significantly alleviated the severity of distant infection foci by enhancing the systemic immune response. This finding provides important support for the application of CS-BT@MZ@NEs in immunotherapy and demonstrates its great potential in the treatment of IAIs.

[0160] To explore the potential of the CS-BT@MZ@NEs nanorobot to trigger an antibacterial immune recall response in a prophylactic setting, its ability to prevent infection recurrence after primary immune clearance was further investigated. To simulate a clinically relevant scenario, first, CS-BT@MZ@NEs immunotherapy was used to clear IAIs in mice, and then these mice were challenged with MRSA again ( Figure 9 a). The results showed that mice pre-immunized with CS-BT@MZ@NEs exhibited significant anti-infection ability during the secondary bacterial challenge, with mild infection symptoms and a significant reduction in bacterial load ( Figure 9 b-h). Notably, the high antigen variability and microbiome heterogeneity in bacterial infections usually limit the immune memory response of current antibacterial therapies to specific bacteria. However, flow cytometry analysis showed that CS-BT@MZ@NEs treatment significantly expanded the populations of CD4 + memory T cells (CD4 + CD44highCD62Llow) and CD8 + memory T cells (CD8 + CD44highCD62Llow) in the spleen ( Figure 9(i-j). In addition, to evaluate the antibody generation potential of CS-BT@MZ@NEs-pretreated mice upon re-challenge with bacterial antigens, IgG levels in their sera and bone marrows were analyzed. The results showed that the IgG levels in the CS-BT@MZ@NEs treatment group were significantly higher than those in other groups( Figure 9 (k-l). These findings indicate that the CS-BT@MZ@NEs nanorobot can effectively induce a strong immune memory response, providing long-term protective immunity against recurrent infections.

[0161] In summary, CS-BT@MZ@NEs not only showed significant therapeutic effects in the primary infection but also enhanced the host's resistance to recurrent infections by inducing long-lasting memory T cell responses and antibody generation. These findings highlight the potential application of CS-BT@MZ@NEs in combating recurrent infections and maintaining immune defense.

[0162] In conclusion, the nanorobot CS-BT@MZ@NEs provides an efficient and safe comprehensive solution for the treatment of IAIs by combining antibacterial activity, Golgi pH regulation, and immune memory activation. It not only directly eradicates biofilm-related infections but also induces a systemic immune response through STING pathway activation, providing durable protection against recurrent bacterial infections. This invention opens up a new path for the deep integration of immunotherapy and nanotechnology and lays a solid foundation for future clinical translation.

[0163] The above are only the preferred embodiments of the present invention, and thus do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all equivalent replacements and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a nanorobot CS-BT@MZ@NEs, characterized in that the steps include: S1, mixing an ethanol solution of tetrabutyl titanate, an aqueous ammonia solution, and a clarified aqueous barium hydroxide solution and then performing heat treatment; Then centrifuge and sequentially wash, dry, calcine and grind the precipitate to obtain nano-particles of BaTiO3; S2, mixing zinc nitrate hexahydrate, manganese nitrate, methanol, and the nanoparticles BaTiO3 and stirring; then adding a methanol solution of 2-methylimidazole dropwise and stirring continuously; Then centrifuge and wash the precipitate to obtain nanoparticles BT@MZ; S3, mixing the aqueous solution of chondroitin sulfate and the nanoparticles BT@MZ and stirring; Then centrifuge and wash the precipitate to obtain nanoparticles CS-BT@MZ; S4. Incubate the neutrophils with the nanoparticles CS-BT@MZ to obtain the nanorobots CS-BT@MZ@NEs.

2. The preparation method according to claim 1, characterized in that: The S1 includes: Mixing tetrabutyl titanate with ethanol to obtain an ethanol solution of the tetrabutyl titanate; adding the aqueous ammonia solution to the ethanol solution of tetrabutyl titanate to obtain a first mixed solution; dissolving hydrated barium hydroxide in deionized water to obtain the clear barium hydroxide aqueous solution; adding the clear barium hydroxide aqueous solution to the first mixed solution to obtain a second mixed solution; The second mixed solution is heat-treated at 200°C for 48 hours; then centrifuged and the resulting precipitate is washed with acetic acid 2-3 times, washed with anhydrous ethanol 2-3 times, dried at 80°C for 24 hours, calcined in an air atmosphere at 800°C for 10 hours, naturally cooled to room temperature, and ground to obtain the nanoparticles BaTiO3.

3. The preparation method according to claim 1, characterized in that: The S2 includes: dissolving the zinc nitrate hexahydrate and the manganese nitrate in the methanol to obtain a third mixed solution; Adding the nanoparticles BaTiO3 to the third mixed solution and stirring at room temperature for 20 minutes to obtain a fourth mixed solution; Dissolving 2-methylimidazole in methanol to obtain a methanol solution of the 2-methylimidazole; Add the methanol solution of 2-methylimidazole dropwise into the fourth mixed solution, and continue stirring for 2 hours; The mixture was centrifuged at 13,000 rpm, and the obtained precipitate was washed with deionized water 2-3 times to obtain the nanoparticles BT@MZ.

4. The preparation method according to claim 1, characterized in that: The S3 includes: Dissolving chondroitin sulfate in deionized water to obtain an aqueous solution of chondroitin sulfate; The nanoparticles BT@MZ were added to the aqueous solution of chondroitin sulfate and stirred at room temperature for 24 h; The mixture was centrifuged at 13,000 rpm, and the obtained precipitate was washed with deionized water 2-3 times to obtain the nanoparticles CS-BT@MZ.

5. The preparation method according to claim 1, characterized in that: The S4 includes: Under a sterile environment, culturing the neutrophils in a serum-free medium for 1 hour to obtain a fifth mixture; The nanoparticles CS-BT@MZ were added to the fifth mixture and incubated at 37° C. for 1 h to obtain the nanorobots CS-BT@MZ@NEs.

6. The preparation method according to claim 1 or 5, characterized in that: The neutrophils are bone marrow-tropic neutrophils.

7. The preparation method according to claim 6, characterized in that: The preparation steps of the bone marrow tropistic neutrophils include: Obtain a sterile bone sample; The bone marrow was washed with PBS, centrifuged at 250 g for 4 min, and then resuspended in PBS; Density gradient centrifugation was prepared by sequentially layering 75% Percoll solution, 65% Percoll solution, and 55% Percoll solution, and adding the single cell suspension on top; Neutrophils were separated at the interface of 65% Percoll solution and 75% Percoll solution by centrifugation at 700 g for 25 min; Neutrophils were washed three times with ice-cold PBS to remove residual Percoll solution; The cells were cultured in a humidified atmosphere of 37° C. and 5% CO 2 using RPMI 1640 medium containing 1% penicillin / streptomycin. After 6 hours of in vitro culture, the bone marrow-tropic neutrophils were obtained.

8. A nanorobot CS-BT@MZ@NEs prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the nanorobot CS-BT@MZ@NEs as claimed in claim 8 in the preparation of a drug for treating implant-related infection.

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