Ultrasound-responsive targeted nanozyme, preparation method thereof and application thereof in diagnosis and treatment of liver fibrosis

Abstract

The application discloses an ultrasonic response type targeted nano-enzyme, a preparation method thereof and application of the nano-enzyme in diagnosis and treatment of liver fibrosis, and belongs to the technical field of biomedical nanomaterials. The preparation method comprises the following steps: (1) mixing Fe-MnO2 nanoparticles, mesoporous silica and an organic solvent, drying, and calcining under an inert atmosphere to obtain MS; (2) performing amino treatment on the MS to obtain MS-NH2; mixing the MS-NH2, a PBS buffer, EDC, NHS and L-arginine, and performing reaction to obtain MSL; and (3) mixing the MSL, a PBS buffer, EDC, NHS and a collagen targeting peptide, and performing reaction to obtain the product. By introducing the collagen targeting peptide CBP, the material can specifically recognize the excessive deposition of type I collagen in the lesion area of liver fibrosis, so that the enrichment efficiency of the material in the lesion part is improved, and the invalid distribution in non-lesion tissues is reduced, and the material has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to an ultrasound-responsive targeted nanozyme, its preparation method, and its application in the diagnosis and treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological stage in the progression of various chronic liver injuries. Essentially, it is an abnormal repair response of the liver under the stimulation of persistent inflammation, oxidative stress, and tissue damage. This is mainly manifested as abnormal activation of hepatic stellate cells, excessive deposition of extracellular matrix such as type I and type III collagen, and remodeling of liver tissue structure. Current research indicates that oxidative stress plays a crucial role in the development of liver fibrosis. Excessive reactive oxygen species (ROS) can not only directly damage hepatocytes but also promote hepatic stellate cell activation, amplify inflammation, and activate pro-fibrotic signaling pathways, thereby driving fibrosis progression. Meanwhile, nitric oxide (NO) also plays an important role in regulating hepatic sinusoidal microcirculation, maintaining sinusoidal endothelial cell function, and inhibiting hepatic stellate cell activation. However, the biological effects of NO are closely related to the release site, concentration, and duration.

[0003] In the diagnosis of liver fibrosis, liver biopsy is still widely regarded as an important basis for pathological staging. However, this method has drawbacks such as being highly invasive, carrying risks of complications such as bleeding, having limited sample representativeness, and being difficult to dynamically repeat assessments. To overcome these shortcomings, non-invasive detection technologies such as ultrasound elastography, transient elastography, and magnetic resonance elastography (MRE) have been developed. Among them, MRE, as a non-invasive quantitative assessment method based on magnetic resonance imaging, has shown high accuracy in staging liver fibrosis. However, it mainly reflects changes in tissue mechanical properties and lacks direct feedback capability on oxidative stress, inflammation, and molecular pathological changes in the fibrotic microenvironment. Therefore, it still cannot meet the needs of early molecular diagnosis and simultaneous monitoring of treatment.

[0004] In recent years, the development of nanomedicine has provided new insights into the diagnosis and treatment of liver fibrosis. Nanoenzyme materials, in particular, with catalytic activity similar to natural enzymes, have attracted attention in the treatment of oxidative stress-related diseases due to their good stability, ease of modification, and ability to be integrated into multiple functions. Manganese-based oxide nanomaterials, especially MnO2-based nanomaterials, can participate in redox reactions and regulate hydrogen peroxide and reactive oxygen species levels, and can also release Mn²⁺ under specific microenvironments. + This results in a T1-weighted magnetic resonance signal enhancement effect, thus possessing the potential for integrated therapeutic and imaging applications. Furthermore, research on manganese oxide nanoparticles as a T1 MRI contrast material indicates their promising potential as an alternative to traditional gadolinium-based contrast agents.

[0005] On the other hand, exogenous physical stimulus-responsive nanomaterials have gradually become a research hotspot. Ultrasound, as an external stimulus with good tissue penetration, non-invasiveness, and spatiotemporal controllability, has been widely used in drug delivery and disease treatment. Existing research shows that ultrasound can change the electronic behavior of nanomaterial interfaces through mechanical vibration, cavitation effects, and energy conversion, and can enhance the catalytic activity of stimulus-responsive nanozymes, thereby improving their ability to regulate the lesion microenvironment. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide an ultrasound-responsive targeted nanozyme and its application.

[0007] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes a method for preparing ultrasound-responsive targeted nanoenzyme materials, comprising the following steps: (1) Fe-MnO2 nanoparticles, mesoporous silica and organic solvent were mixed, dried and calcined under an inert atmosphere to obtain Fe-MnO2@mesoporous silica composite material (denoted as MS); (2) Amination of MS to obtain MS-NH2; MS-NH2, PBS buffer, EDC, NHS and L-arginine are mixed and reacted to obtain MS loaded with L-arginine (denoted as MSL). (3) Mix MSL, PBS buffer, EDC, NHS and collagen-targeting peptide and react to obtain MSL (denoted as MSLC) coupled with collagen-targeting peptide.

[0008] PBS buffer: also known as phosphate buffer saline or PBS solution.

[0009] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, CAS No. 1892-57-5.

[0010] NHS: N-hydroxysuccinimide, CAS number 6066-82-6.

[0011] L-arginine: English name L-arginine, CAS number 74-79-3.

[0012] Collagen-targeting peptide: English name Collagen binding peptide, CAS number 94040-53-6.

[0013] The Fe-MnO2 nanoparticles are composite nanoparticles obtained by doping Fe element into MnO2 nanomaterials. Preparation methods include, but are not limited to, any one of the following: hydrothermal method, solvothermal method, redox precipitation method, and chemical bath deposition method.

[0014] Preferably, in (1), the Fe-MnO2 nanoparticles are prepared by the following process: dispersing MnO2 nanomaterials in deionized water, and adding Fe 3+ The solution is obtained through a hydrothermal reaction.

[0015] More preferably, the molar ratio of Fe to Mn is 1:(1~3). The hydrothermal reaction conditions are: 100~140℃, 6~10 h.

[0016] Preferably, in (1), the mass ratio of Fe-MnO2 nanoparticles to mesoporous silica is (10~30):(70~100); more preferably, it is 20:80.

[0017] More preferably, in (1), the ratio of Fe-MnO2 nanoparticles, mesoporous silica, and organic solvent is (10~30) mg: (70~100) mg: (30~60) mL; more preferably, it is 20 mg: 80 mg: 40 mL.

[0018] More preferably, the organic solvent includes, but is not limited to, one or more of anhydrous ethanol, methanol, propanol, and acetone.

[0019] Preferably, in (1), the calcination conditions are: 400~500℃, 0.5~2h; further preferably 420~480℃, 0.8~1.2h; and even more preferably 450℃, 1h.

[0020] Preferably, in (2), the amination treatment of MS is performed by mixing MS, 3-aminopropyltriethoxysilane and anhydrous ethanol, heating and refluxing, centrifuging, washing and drying to obtain amination MS (MS-NH2).

[0021] 3-Aminopropyltriethoxysilane: Abbreviated as APTES, CAS number 919-30-2.

[0022] Further preferred, the ratio of MS to 3-aminopropyltriethoxysilane is (10~30) mg: (150~250) μL; even more preferred is 20 mg: 200 μL.

[0023] Further preferred, the heating and reflux reaction conditions are 60~100℃ for 4~8h; even more preferred: 80℃ for 6h.

[0024] Preferably, in (2), the mass ratio of MS-NH2 to L-arginine is (10~30):(30~60); more preferably, it is 20:40.

[0025] More preferably, in (2), the ratio of MS-NH2, PBS buffer, EDC, NHS and L-arginine is (10~30) mg: (10~40) mL: (10~30) mg: (8~20) mg: (30~60) mg; and even more preferably, it is 20 mg: 20 mL: 20 mg: 12 mg: 40 mg.

[0026] Preferably, in (2), the reaction conditions are: reacting at room temperature for 10~14 h.

[0027] Preferably, in (3), the mass ratio of MSL to collagen-targeting peptide is (10~30):(1~5); more preferably, it is 20:2.

[0028] Further preferably, in (3), the ratio of MSL, PBS buffer, EDC, NHS and collagen-targeting peptide is (10~30) mg: (10~40) mL: (10~25) mg: (5~20) mg: (1~8) mg; and even more preferably, it is 20 mg: 20 mL: 15 mg: 10 mg: 5 mg.

[0029] Preferably, in (3), the reaction conditions are: 3~5℃, 20~28h; more preferably 4℃, 24h.

[0030] This invention also proposes an ultrasonic-responsive targeted nanozyme material prepared by the above preparation method.

[0031] This invention also proposes the application of the above-mentioned ultrasound-responsive targeted nanozyme material in the preparation of drugs for the diagnosis and / or treatment of liver fibrosis.

[0032] After being administered in vivo, nanozyme materials (MSLC) specifically bind to the excessively deposited type I collagen in the liver fibrosis lesion area via CBP, thereby targeting and enriching the lesion site.

[0033] The beneficial effects of this invention are as follows: 1. This invention provides an ultrasound-responsive targeted nanozyme platform, comprising a Fe-MnO2 bimetallic nanozyme, a mesoporous silica carrier, L-arginine (L-Arg), and a collagen-targeting peptide (CBP). The Fe-MnO2 bimetallic nanozyme serves as the catalytic active center, regulating the level of reactive oxygen species in the liver fibrosis microenvironment; the mesoporous silica serves as the carrier, improving the structural stability, dispersibility, and loading capacity of the material; L-Arg serves as a nitric oxide precursor, generating NO in the lesion area; and the CBP identifies excessively deposited type I collagen in the liver fibrosis area, thereby enhancing the material's enrichment capacity in the lesion area.

[0034] 2. This invention provides an ultrasound-responsive targeted nanozyme platform and its application in the diagnosis and treatment of liver fibrosis. The platform uses Fe-MnO2 bimetallic nanozymes as the functional core and mesoporous silica as the carrier. By loading L-arginine (L-Arg) and further coupling it with collagen-targeting peptides (CBP), a composite nanosystem with lesion targeting, ultrasound response, reactive oxygen species scavenging, nitric oxide generation, and magnetic resonance imaging functions is constructed, denoted as MSLC.

[0035] 3. By introducing collagen-targeting peptides (CBP), the nanoplatform can specifically recognize excessively deposited type I collagen in liver fibrosis lesions, thereby improving the enrichment efficiency of materials at the lesion site and reducing ineffective distribution in non-lesion tissues. Using Fe-MnO2 bimetallic nanozymes as the catalytic active center, the electron transfer process can be enhanced through multivalent cycling between Fe and Mn, thereby improving the ability to regulate reactive oxygen species and exhibiting good antioxidant microenvironment regulation effects. 4. Loading L-arginine (L-Arg) as a NO precursor into the nanoplatform is beneficial for improving the local NO generation efficiency in the lesion area, thereby improving local microcirculation, reducing inflammatory response, and inhibiting hepatic stellate cell activation. Introducing low-intensity ultrasound as an exogenous activation method can further enhance the catalytic activity of nanozymes after the material is enriched in the lesion area, and promote L-Arg release and NO generation, thereby improving the local therapeutic effect. 5. The manganese-related components in this application can enhance T1-weighted magnetic resonance imaging signals under the influence of the lesion microenvironment, thereby achieving visual monitoring of the lesion area and possessing the characteristics of integrated treatment and imaging. By organically combining targeted delivery, ultrasound response, ROS regulation, NO intervention, and MRI feedback, a technical path of "targeted enrichment—local activation—synergistic treatment—image monitoring" is formed, demonstrating good comprehensive application effects. It can effectively solve the problems of insufficient diagnostic specificity for liver fibrosis, poor targeting of antioxidant therapy, uncontrollable NO release, and separation of diagnostic and therapeutic functions in existing technologies, showing promising application prospects.

[0036] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0037] Figure 1 The preparation process (A) of the ultrasound-responsive targeted nanozyme platform in Example 1 of the present invention and its overall application in the diagnosis and treatment of liver fibrosis are shown in Figure (B).

[0038] Figure 2 The figures show the structural characterization results of the Fe–MnO2, Fe–MnO2@MS (MS), Fe–MnO2@MS@L-Arg (MSL), and Fe–MnO2@MS@L-Arg@CBP (MSLC) nanomaterials prepared in Example 1 of this invention. In the figures, A is a transmission electron microscope image and a high-resolution transmission electron microscope image; B is a scanning electron microscope image and an elemental mapping image; C is an X-ray diffraction pattern; D is an X-ray photoelectron spectrum; E and F are the particle size and zeta potential detection results, respectively; G and H are the ultraviolet-visible absorption spectrum and ultraviolet photoelectron spectrum, respectively; and I is an atomic force microscope image.

[0039] Figure 3 The figures shown are characterization results of the catalytic performance and reactive oxygen species regulation ability of the materials in Example 1 of this invention under ultrasonic treatment. A: Response release results of the materials under different ultrasonic treatment times; B: NO generation of different materials under ultrasonic stimulation; C and D are RhB degradation curves under conditions without US and US, respectively; E and F are MB degradation curves under conditions without US and US, respectively; G, H, and I are ·OH and O2, respectively. - , 1 The ESR spectrum of O2; J and M represent the DPPH detection results under US-free and US-free conditions, respectively; K and N represent the NBT detection results under US-free and US-free conditions, respectively; L and O represent the DPBF detection results under US-free and US-free conditions, respectively. US in the figures represents ultrasound.

[0040] Figure 4 The figures show the results of macrophage transcriptome sequencing and NF-κB signaling pathway verification after US+MSLC treatment in Example 1 of this invention. A: Heatmap of differentially expressed genes in two groups; B: Volcano plot of differentially expressed genes; C: GO functional enrichment analysis of differentially expressed genes; D: KEGG pathway enrichment analysis of the top 20 signaling pathways; E: GSEA analysis showing the enrichment of differentially expressed genes in the NF-κB signaling pathway; F: WB detection results of NF-κB signaling pathway-related proteins in different treatment groups.

[0041] Figure 5The images show the results of MSLC nanomaterials promoting intracellular nitric oxide generation and scavenging reactive oxygen species under ultrasound in Example 1 of this invention. A and B: confocal laser scanning microscopy fluorescence images and semi-quantitative analysis results of NO generation in cells of different treatment groups; C and D: flow cytometry detection results of NO generation in LX-2 cells of different treatment groups and quantitative analysis of average NO fluorescence intensity; E and F: confocal laser scanning microscopy fluorescence images and semi-quantitative analysis results of ROS in cells of different treatment groups; G and H: flow cytometry detection results of ROS in LX-2 cells of different treatment groups and quantitative analysis of average ROS fluorescence intensity.

[0042] Figure 6 The images show the results of MSLC nanomaterials inhibiting the activation and migration of hepatic stellate cells and regulating macrophage polarization under ultrasound in Example 1 of this invention. A: Immunofluorescence images of α-SMA and COL1A1 in LX-2 cells from different treatment groups; B and C: Quantitative analysis of mRNA expression levels of α-SMA and COL1A1; D: Representative microscopic images of LX-2 cells from different treatment groups at 0 h and 24 h after scratching; E: Confocal fluorescence staining images of CD86 and CD206 in cells from the same treatment group; F: Flow cytometry detection results of CD86 and CD206 in cells from different treatment groups.

[0043] Figure 7 The diagram shows the in vivo targeted distribution of MSLC nanomaterials in Example 1 of this invention and the results of co-localization with type I collagen in liver fibrosis tissue; where A and B are in vivo fluorescence imaging and quantitative analysis diagrams; C and D are in vitro organ fluorescence imaging and quantitative analysis diagrams; and E is the immunofluorescence co-localization analysis diagram of each treatment group.

[0044] Figure 8 The images show the T1-weighted magnetic resonance imaging performance and in vivo enhanced imaging results of the MSLC nanomaterials in Example 1 of this invention; where A: in vitro T1-weighted MRI images of Ga and MSLC at different concentrations; B: linear relationship curve between longitudinal relaxation rate (1 / T1) and concentration; C: T1-weighted MRI images (sagittal and axial) of liver fibrosis mice before and after administration, with the dashed line representing the liver region; D: quantitative analysis of liver ΔCNR in each group.

[0045] Figure 9This is a diagram showing the MRI evaluation, pathological and serum index analysis of MSLC in a mouse model of liver fibrosis in Example 1 of this invention; where A: Schematic diagram of the establishment and treatment process of CCl4-induced liver fibrosis model, blue indicates intraperitoneal injection of CCl4, yellow indicates tail vein injection of nanomaterials or ultrasound treatment, green indicates MRI scan, and red indicates sacrifice and tissue collection; where B: T1-weighted MRI images of the liver of mice in each group (before and after drug administration), with the liver region outlined by dashed lines; where C: Quantitative analysis of ΔCNR in the liver of each group; D, E, F: Expression level analysis of pro-inflammatory factors IL-6, IL-1β and TNF-α in each group; G, H: Expression level analysis of anti-inflammatory factors IL-10 and IL-4 in each group; I, ​​J: Serum ALT and AST level analysis diagram.

[0046] Figure 10 The figures show the evaluation results of the therapeutic effect of MSLC nanomaterials on liver fibrosis in mice under ultrasound in Example 1 of this invention; where A: gross morphology of liver in each group of mice, HE staining of liver tissue, Masson staining, immunohistochemistry of α-SMA, and immunofluorescence staining results of α-SMA and Collagen I; B: quantitative analysis of Masson staining positive area; C: quantitative analysis of α-SMA immunohistochemistry positive area; D, E: quantitative analysis of α-SMA and Collagen I immunofluorescence positive area; F, G: analysis of mRNA expression levels of α-SMA and COL1A1 in liver tissue.

[0047] Figure 11 Figure 1 shows the in vitro and in vivo biocompatibility evaluation results of MSLC nanomaterials in Example 1 of this invention; A, B: Detection of ALT and AST levels in mouse serum; C, D: Hemolysis experiments after MSLC and US+MSLC treatment; E: HE staining and Masson staining of major organ tissues. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0049] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0050] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0051] In the following examples, Fe-MnO2 nanoparticles were prepared using the following process: Potassium permanganate and manganese sulfate were dissolved in deionized water at a molar ratio of 2:3. After stirring for 30 minutes, the solution was transferred to a polytetrafluoroethylene (PTFE) hydrothermal reactor and hydrothermally reacted at 180°C for 12 hours. After the reaction, the solution was allowed to cool to room temperature, and the black precipitate was collected by centrifugation. The precipitate was washed three times each with deionized water and anhydrous ethanol, and then dried at 60°C for 12 hours to obtain pure-phase MnO2 nanomaterials. The MnO2 nanomaterials were ultrasonically dispersed in deionized water, and FeCl3·6H2O was added at a Fe to Mn molar ratio of 1:2. After stirring for 1 hour, the solution was transferred to a PTFE hydrothermal reactor and hydrothermally reacted at 120°C for 8 hours. After the reaction, the solution was allowed to cool to room temperature, and the black precipitate was collected by centrifugation. The precipitate was washed three times each with deionized water and anhydrous ethanol, and then vacuum dried at 60°C for 12 hours to obtain Fe-MnO2 nanoparticles.

[0052] Example 1: A method for preparing an ultrasound-responsive targeted nanozyme material includes the following steps: (1) Synthesis of MS 20 mg of Fe-MnO2 nanoparticles were dispersed in 40 mL of anhydrous ethanol, and 80 mg of mesoporous silica powder was added. After ultrasonic dispersion for 30 min, the mixture was magnetically stirred (300 rpm) at room temperature for 12 h. The resulting product was then vacuum dried at 60 °C for 12 h and calcined at 450 °C for 1 h under a nitrogen atmosphere to uniformly anchor the Fe-MnO2 nanoparticles within the mesoporous silica channels. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 min and washed three times each with anhydrous ethanol and deionized water to obtain the Fe-MnO2@mesoporous silica composite material (MS).

[0053] (2) Synthesis of MSL 20 mg MS was dispersed in 40 mL of anhydrous ethanol, and 200 μL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 80 °C for 6 h. After the reaction, the mixture was centrifuged at 10,000 rpm for 10 min, washed three times with anhydrous ethanol and deionized water, and dried under vacuum at 60 °C for 12 h to obtain aminated MS-NH2. Subsequently, 20 mg of the obtained MS-NH2 was dispersed in 20 mL of PBS buffer (0.01 mol / L, pH 7.4), and 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 12 mg of N-hydroxysuccinimide (NHS) were added. The mixture was activated at room temperature for 30 min, and then 40 mg of L-arginine (L-Arg) was added. The mixture was then magnetically stirred (300 rpm) at room temperature for 12 h. After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 10 min, washed thoroughly with deionized water three times to remove unreacted L-Arg, and dried under vacuum at 60 °C for 12 h to obtain MSL.

[0054] (3) Synthesis of MSLC 20 mg MSL was dispersed in 20 mL of PBS buffer (0.01 mol / L, pH 7.4), and 15 mg EDC and 10 mg NHS were added. The mixture was activated at room temperature for 30 min. Then, 2 mg of collagen-targeting peptide (CBP) was added, and the reaction was carried out at 4 °C with slow stirring (200 rpm) for 24 h. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against deionized water for 48 h, with the deionized water changed every 6 h to ensure complete removal of uncoupled peptide molecules. Finally, the obtained product was... After pre-freezing at 80 °C for 12 h, Fe-MnO2@MS@L-Arg@CBP (MSLC) was obtained by freeze-drying for 48 h.

[0055] The overall schematic diagram of the preparation process of the ultrasound-responsive targeted nanozyme material in Example 1 and its application in the diagnosis and treatment of liver fibrosis is shown below. Figure 1 As shown. Figure 1 In section A: First, Fe-MnO2 bimetallic nanozymes were prepared and loaded onto a mesoporous silica support to form a composite framework; then, L-arginine (L-Arg) was loaded and collagen-targeting peptides (CBP) were coupled to the material surface to obtain the ultrasound-responsive targeted nanozyme platform MSLC. Figure 1After in vivo administration, MSLC specifically binds to the excessively deposited type I collagen in the liver fibrosis lesion area via CBP, thereby targeting and enriching the lesion site. Under ultrasound stimulation, the platform enhances the catalytic activity of Fe-MnO2 bimetallic nanozymes, promotes the scavenging of reactive oxygen species and the generation of NO, and achieves lesion imaging and efficacy monitoring through the T1-weighted magnetic resonance signal enhancement effect of manganese-related components.

[0056] The material obtained in Example 1 was tested and characterized: (1) Structural characterization of materials: The specific steps are as follows: TEM characterization: The microstructure, particle size and dispersion state of the samples were characterized by transmission electron microscopy, and the lattice fringes and interplanar spacing were analyzed by high-resolution transmission electron microscopy.

[0057] SEM characterization: The surface morphology and dispersion state of the samples were observed using scanning electron microscopy, and the elemental composition and distribution of the samples were analyzed in conjunction with EDS.

[0058] XRD analysis: X-ray diffraction was used to analyze the crystal phase composition, crystallinity and structural characteristics of the sample.

[0059] XPS analysis: X-ray photoelectron spectroscopy is used to analyze the elemental composition and surface chemical state of the sample.

[0060] UV-DRS analysis: The diffuse reflectance spectrum of the sample was measured using ultraviolet-visible diffuse reflectance spectroscopy to analyze its optical absorption characteristics.

[0061] Particle size and zeta potential analysis: The hydrated particle size distribution and zeta potential of the samples were determined by dynamic light scattering to evaluate their dispersibility and colloidal stability.

[0062] AFM testing: The surface morphology and piezoelectric response characteristics of the samples were tested using an atomic force microscope and its PFM module.

[0063] Results: The structural characterization results of the Fe–MnO2, Fe–MnO2@MS (MS), Fe–MnO2@MS@L-Arg (MSL) and Fe–MnO2@MS@L-Arg@CBP (MSLC) nanomaterials prepared in Example 1 are shown in the figure below. Figure 2 As shown. By Figure 2 As shown in Figure A, Fe–MnO2 retains a relatively complete particle morphology after being coated with mesoporous silica and subsequently modified, and the corresponding lattice fringes of Fe–MnO2 can be observed. Figure 2 As shown in section B, Fe, Mn, O, Si, C, and N elements are uniformly distributed in the MSLC, indicating that each component was successfully constructed. Figure 2As shown in C and D, the material retains the characteristic diffraction peaks of Fe–MnO2 and contains elements such as Fe, Mn, O, Si, C, and N. From... Figure 2 As shown in equations E and F, with the progress of coating and modification, the particle size of the material gradually increases, and the surface potential changes accordingly. Figure 2 As can be seen from G, H, and I, MSLC has a stable optical response, electronic structure, and nanoscale surface morphology.

[0064] (2) Determination of material properties: The specific steps are as follows: L-Arg release assay: MSLCs were placed in dialysis bags and immersed in a PBS release system. Samples were taken at different time points with and without sonication. The L-Arg concentration at each time point was calculated based on the standard curve, and the cumulative release amount and release rate were further analyzed.

[0065] NO release detection: Fe-MnO2, MS, MSL, and MSLC were dispersed in PBS, and after sonication, the supernatant was reacted with Griess reagent. The NO release capacity of different materials was evaluated by measuring the absorbance at 540 nm.

[0066] RhB ultrasonic catalytic degradation experiment: Using 5 mg / L RhB aqueous solution as a model pollutant, after adding MSLC, the mixture was first stirred in the dark for 30 min to reach adsorption-desorption equilibrium, and then the reaction was carried out with or without ultrasound. During the reaction, samples were taken at regular intervals for centrifugation, and the absorption spectrum was scanned using an ELISA reader to evaluate the ultrasonic catalytic degradation performance of the material.

[0067] MB ultrasonic catalytic degradation experiment: Using 2 mg / L MB aqueous solution as the model pollutant, Fe-MnO2, MS, MSL, and MSLC were added respectively. After stirring in the dark for 30 min, each group was reacted with or without ultrasound. During the reaction, samples were taken and centrifuged at regular intervals, and the absorbance changes were detected by an ELISA reader to compare the ultrasonic catalytic degradation ability of different materials.

[0068] ESR analysis: X-band ESR was used to detect the material's ability to modulate ROS, with DMPO capturing ·OH and O2· - ¹O2 was captured using TEMP. The material's ability to generate or scavenge reactive oxygen species was analyzed by comparing the changes in the intensity of characteristic signal peaks in different treatment groups.

[0069] DPPH radical scavenging experiment: Using DPPH ethanol solution as a free radical model system, different materials were added and reacted in the dark followed by ultrasonic treatment. The scavenging ability of each material for DPPH free radicals was evaluated by detecting the change in absorbance over time and calculating the scavenging rate.

[0070] NBT superoxide anion scavenging experiment: using NBT solution as O2· - The detection system was tested, and H2O2 was added as a reaction substrate to investigate the regulatory effects of different materials under ultrasonic conditions. The scavenging ability of the materials for superoxide anions was evaluated by measuring the absorbance change and calculating the scavenging rate.

[0071] DPBF Singlet Oxygen Detection: Using DPBF ethanol solution as the¹O₂ probe system, different materials were added and treated under ultrasonic conditions. The ability of the materials to scavenge or generate singlet oxygen was analyzed by detecting the absorbance attenuation.

[0072] Results: The characterization results of the catalytic performance and reactive oxygen species regulation ability of each material under ultrasonic irradiation in Example 1 are shown in the figure below. Figure 3 As shown. Among them, Figure 3 Figure A shows the response release results of the material under different ultrasonic treatment times, indicating that as the ultrasonic treatment time increases, the release of active components gradually increases and tends to stabilize; Figure 3 B~F are colorimetric photographs and UV-Vis absorption spectra of different reaction systems. The results show that MSLC can significantly promote the oxidation or degradation of the substrate under ultrasonic treatment, and its catalytic effect is better than that of the material group that has not been ultrasonically treated. Figure 3 G~I is the electron paramagnetic resonance spectrum, which shows that MSLC can generate reactive oxygen species such as hydroxyl radicals, superoxide anion radicals and singlet oxygen under ultrasonic excitation conditions. Figure 3 J~O represent the colorimetric results, absorption spectra, and quantitative analysis results of the catalytic reaction of the material under different conditions, indicating that MSLC has good catalytic activity and stable reactive oxygen species regulation ability under ultrasonic irradiation. (3) RNA sequencing analysis of RAW246.7 cells The specific steps are as follows: RNA transcriptome sequencing: To perform RNA transcriptome sequencing analysis, two models were used: an H2O2-induced LX-2 cell oxidative stress model and an LPS-induced RAW264.7 cell inflammation model. Three biological replicates were set up for each group, and the cells were treated with either MSLC or PBS. The US+MSLC group was further subjected to ultrasound irradiation. After treatment, total RNA was extracted from each group, and after passing quality checks, it was used for library construction and high-throughput sequencing on the Illumina NovaSeq 6000 platform. After quality control, the sequencing data were used to screen for differentially expressed genes using DESeq2, and further GO functional annotation, KEGG pathway enrichment, and GSEA analysis were performed to systematically evaluate the transcriptome changes under different treatment conditions.

[0073] Western blot analysis: After cell treatment, total protein was extracted using RIPA lysis buffer, and protein concentration was determined by BCA method. The protein samples were then denatured, separated by SDS-PAGE electrophoresis, and transferred to a PVDF membrane. Sequential blocking, primary antibody incubation, secondary antibody incubation, and chemiluminescence imaging were performed. The expression level of the target protein was evaluated by band grayscale analysis.

[0074] Results: The results of macrophage transcriptome sequencing and NF-κB signaling pathway verification after ultrasound (US) + MSLC treatment in Example 1 are shown in the figure below. Figure 4 As shown. Among them, by Figure 4 The results from the study showed significant differences in gene expression profiles between the control group and the US+MSLC group; Figure 4 As shown in B, multiple inflammation-related genes were significantly downregulated after US+MSLC treatment; Figure 4 Figures C and D show that differentially expressed genes are mainly enriched in cytokine receptor interactions, the TNF signaling pathway, the NF-κB signaling pathway, and inflammatory response-related biological processes; Figure 4 The results from the study indicate that US+MSLC treatment can significantly inhibit the activity of the NF-κB signaling pathway; Figure 4 The results showed that the expression of p-NF-κB and p-IκBα proteins was reduced in the US+MSLC group, further indicating that the MSLC nanomaterials prepared in this embodiment can inhibit the activation of the NF-κB signaling pathway under ultrasound, thereby exerting an anti-inflammatory effect.

[0075] (4) NO conversion rate and ROS removal efficiency The specific steps are as follows: NO confocal fluorescence microscopy observation: Intracellular nitric oxide (NO) levels were labeled and detected using a DAF-FM DA fluorescent probe, and cell nuclei were counterstained with Hoechst 33342. Fluorescence images of cells in each treatment group were then acquired using confocal fluorescence microscopy. Quantitative analysis of fluorescence intensity was performed to evaluate changes in intracellular NO levels under different treatment conditions.

[0076] NO flow cytometry analysis: DAF-FM DA fluorescent probes were used to stain treated cells, and single-cell suspensions were prepared. Fluorescence signals in the FITC channels of each group of cells were detected using flow cytometry. Intracellular NO levels under different treatment conditions were quantitatively analyzed by comparing average fluorescence intensity.

[0077] ROS confocal fluorescence microscopy observation: Intracellular reactive oxygen species (ROS) levels were labeled and detected using the DCFH-DA fluorescent probe, and cell nuclei were counterstained using Hoechst 33342. Fluorescence images of cells in each treatment group were then acquired using confocal fluorescence microscopy. Quantitative analysis of fluorescence intensity was performed to evaluate changes in intracellular ROS levels under different treatment conditions.

[0078] ROS flow cytometry analysis: Cells were stained with the DCFH-DA fluorescent probe, and single-cell suspensions were prepared. Fluorescence signals in the FITC channels of each cell group were detected using flow cytometry. Intracellular ROS levels under different treatment conditions were quantitatively analyzed by comparing the average fluorescence intensity.

[0079] Results: The results of MSLC nanomaterials in Example 1 promoting intracellular nitric oxide production and scavenging reactive oxygen species under ultrasound are shown in the figure below. Figure 5 As shown. Among them, by Figure 5 The results from the A-group showed that the US+MSLC group exhibited the strongest green fluorescence, indicating that this group had the highest intracellular nitric oxide level; Figure 5 As shown in Figure B, this further confirms that ultrasound can promote the release of L-arginine-related nitric oxide in MSLC. Figure 5 The results showed that the level of reactive oxygen species (ROS) in the model group cells was significantly increased, while the level of ROS decreased after MSLC treatment, with the most significant decrease observed in the US+MSLC group; Figure 5 The results from the DH assay showed that the levels of reactive oxygen species (ROS) and the quantitative analysis were consistent with the results of fluorescence staining. These results indicate that the MSLC nanomaterials prepared in this embodiment can effectively promote intracellular nitric oxide production and significantly scavenge excess ROS under ultrasonic treatment.

[0080] (5) Determination of in vitro antifibrotic and anti-inflammatory effects The specific steps are as follows: Immunofluorescence staining of α-SMA and COL1A1: Except for the Control group, all other groups were incubated with TGF-β1 (10 ng / mL) for 24 h to induce a pro-fibrotic phenotype. Subsequently, under continuous TGF-β1 stimulation, the cells were further divided into groups: the PBS group and the US+PBS group received an equal volume of PBS, while the Fe-MnO2 group, MSL group, MSLC group, and US+MSLC group received 100 μg / mL of the corresponding material. The US+PBS group and the US+MSLC group were simultaneously sonicated (1.0 MHz, 0.5 W / cm², 50% duty, 5 min) for 24 h. After treatment, the cells were fixed, permeabilized, and blocked. They were then incubated overnight at 4 ℃ with COL1A1 and α-SMA primary antibodies, followed by nucleus staining with the corresponding fluorescent secondary antibodies and DAPI. After mounting, images were acquired using a confocal microscope, and the fluorescence intensity of α-SMA and COL1A1 was quantitatively analyzed to evaluate the in vitro anti-fibrotic effect of each treatment group.

[0081] RT-qPCR analysis of cells: After cell treatment, the culture medium was discarded and the cells were gently washed twice with PBS. Total RNA was extracted with pre-chilled TRIzol lysis buffer, followed by chloroform separation, isopropanol precipitation, and washing with 75% ethanol. RNA was then dissolved in RNase-free water, and its concentration and purity were determined. A suitable amount of total RNA was used to prepare a 10 μL reverse transcription reaction system according to the kit instructions for cDNA synthesis. Subsequently, using cDNA as a template, real-time quantitative PCR was performed using the SYBR Green method. The total reaction volume was 10 μL, including 2× qPCR Mix, forward and reverse primers, cDNA template, and RNase-free water. After amplification, melting curve analysis was performed to verify product specificity, and β-actin was used as an internal control. The relative mRNA expression levels of target genes such as α-SMA and COL1A1 were calculated using the ΔΔCt method to evaluate the changes in the expression of fibrosis-related genes under different treatment conditions.

[0082] Cell scratch assay: Logarithmic growth phase cells were seeded in 6-well plates and cultured until confluence reached 90%–100% the next day. A sterile 200 μL pipette tip was used to scratch the cells in a straight line, followed by gentle washing with PBS 2–3 times to remove floating cells and debris. Subsequently, TGF-β1 (10 ng / mL) was used to induce a fibrotic phenotype in the cells. Simultaneous grouping was performed under low serum medium (1% FBS): the PBS group and the US+PBS group received an equal volume of PBS; the Fe-MnO2 group, MSL group, MSLC group, and US+MSLC group received 100 μg / mL of the corresponding material. The US+PBS group and the US+MSLC group were immediately sonicated after drug administration (1.0 MHz, 0.5 W / cm², 50% duty, 5 min). The Control group received no TGF-β1 and no treatment. After incubation for another 24 hours, scratch images were acquired at 0h and 24h. Three fixed fields of view were selected from each well. The scratch area was measured using ImageJ software, and the wound closure rate (%) was calculated as follows: [(A0)] A t ) / A0] × 100% was used for quantitative analysis to assess the effects of different treatment conditions on cell migration ability.

[0083] Immunofluorescence staining of CD86 and CD206: Except for the Control group, all other groups were stimulated for 12 h with complete medium containing LPS (100 ng / mL) to induce macrophage inflammatory phenotype. After induction, the medium was discarded and the cells were washed twice with PBS. Subsequently, the cells were treated under continuous LPS stimulation: the PBS group was treated with fresh complete medium containing LPS and an equal volume of PBS; the MSLC group was treated with fresh complete medium containing LPS and MSLC (100 μg / mL); the US+MSLC group was treated with fresh complete medium containing LPS and MSLC (100 μg / mL) and immediately sonicated at the following parameters: 1.0 MHz, 0.5 W / cm², 50% duty cycle, 5 min; the Control group was not treated. After incubation for another 24 h, the medium was discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA for 1 h. Subsequently, CD86 and CD206 primary antibodies were added, and the mixture was incubated overnight at 4 °C. After washing with PBS, the corresponding fluorescent secondary antibodies were added, and the mixture was incubated at room temperature in the dark for 1 h. After counterstaining the cell nuclei with DAPI and mounting the slides, images were observed and acquired using a confocal microscope to analyze the expression of the macrophage phenotypic markers CD86 and CD206 in each group.

[0084] Flow cytometry analysis of CD86 and CD206: Except for the Control group, all other groups were stimulated for 12 h with complete medium containing LPS (100 ng / mL). After induction, the medium was discarded and the cells were washed twice with PBS. Under continuous LPS stimulation, the cells were further divided into groups: the PBS group was given fresh complete medium containing LPS and an equal volume of PBS; the MSLC group was given fresh complete medium containing LPS and MSLC (100 μg / mL); the US+MSLC group was given fresh complete medium containing LPS and MSLC (100 μg / mL) and sonicated at 1.0 MHz, 0.5 W / cm², 50% duty cycle, and 5 min; the Control group received no treatment. After 24 h of incubation, cells were collected and single-cell suspensions were prepared. After Fc receptor blocking, APC-labeled CD86 antibody and FITC-labeled CD206 antibody were added, and the cells were incubated at 4 ℃ in the dark for 30 min. After staining, the cells were washed and resuspended, and the expression levels of CD86 and CD206 in each group of cells were detected by flow cytometry to evaluate the regulatory effect of different treatment conditions on macrophage phenotypic transformation.

[0085] Results: The results of MSLC nanomaterials inhibiting hepatic stellate cell activation and migration and regulating macrophage polarization under ultrasound in Example 1 are shown in the figure below. Figure 6 As shown. Among them, by Figure 6 The results from the US+MSLC study showed that the expression of α-SMA and COL1A1 was significantly increased in the model group, while it decreased after MSLC treatment, with the most significant decrease observed in the US+MSLC group. Figure 6 The results from the B study showed that the cell migration ability of the US+MSLC group was significantly reduced; Figure 6 The results from the CF study showed that after treatment with US+MSLC, CD86 expression decreased and CD206 expression increased, indicating that MSLC under ultrasound can inhibit the activation and migration of hepatic stellate cells and promote the transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype. (6) In vivo targeting assessment The specific steps are as follows: Preparation of Cy5.5-labeled MSLCs: MSL and MSLCs were dispersed separately in PBS and mixed with Cy5.5-COOH at a predetermined ratio. The mixture was incubated at room temperature for 24 h in the dark to load Cy5.5 onto the surface of the nanoparticles. After incubation, the nanoparticles were separated by centrifugation, and the supernatant was discarded. The nanoparticles were then resuspended in PBS and washed 2–3 times to remove unbound free Cy5.5, ultimately yielding Cy5.5-MSL and Cy5.5-MSLCs for subsequent in vitro and in vivo fluorescence imaging experiments.

[0086] Small animal in vivo imaging: MSL and MSLC were incubated with Cy5.5-COOH in the dark for 24 h, respectively. After centrifugation and washing to remove free dye, Cy5.5-MSL and Cy5.5-MSLC were prepared. Six weeks after CCl4-induced liver fibrosis, mice were randomly divided into FreeCy5.5, Cy5.5-MSL, and Cy5.5-MSLC groups. After tail vein injection of the corresponding preparation (5 mg / kg), small animal in vivo fluorescence imaging was performed at 1, 6, and 24 h, and ROI quantitative analysis of the fluorescence signal in the liver area was performed. Mice were sacrificed 24 h later, and major organs such as liver, spleen, kidney, lung, and heart were collected for in vivo fluorescence imaging to evaluate the distribution characteristics of different preparations in vivo and the liver accumulation capacity.

[0087] Immunolocalization: Mice were sacrificed 12 h after drug administration, and liver tissue was rapidly harvested to prepare frozen sections. After fixation, permeabilization, and blocking, COL1A1 primary antibody and corresponding FITC-labeled secondary antibody were added for immunofluorescence staining, and cell nuclei were counterstained with DAPI. Subsequently, Cy5.5, FITC, and DAPI channel images were acquired, and background subtraction and colocalization analysis were performed using ImageJ software to evaluate the targeting enrichment ability of MSLC for type I collagen in fibrotic liver tissue.

[0088] Results: The in vivo targeted distribution of MSLC nanomaterials in Example 1 and their co-localization with type I collagen in liver fibrosis tissue are shown in the figure below. Figure 7 As shown. Among them, by Figure 7 Images A and B show that the Cy5.5-MSLC group exhibited stronger fluorescence signals and longer retention times in the liver region; Figure 7 C and D indicate that Cy5.5-MSLCs were more enriched in fibrotic livers than Cy5.5-MSLs and free Cy5.5. Figure 7 The image shows a significant overlap between the MSLC signal and the type I collagen signal. These results indicate that the MSLC nanomaterials prepared in this embodiment can be effectively enriched in fibrotic livers in vivo and possess good collagen-targeting ability.

[0089] (7) T1-weighted MRI imaging performance and in vivo enhancement effect of MSLC: The specific steps are as follows: Relaxation rate: Gd-DTPA and MSLC were prepared at concentrations of 0, 0.07, 0.13, 0.25, 0.5 and 1.0 mM, and placed in 2 mL round-bottom centrifuge tubes. Spin-echo T1WI scanning was performed on 3.0T MRI using a flexible coil with an echo time of 8.8 ms and repetition times of 300, 500, 600, 800 and 1000 ms. After acquiring magnetic resonance images, the signal intensity was measured and the longitudinal relaxation time (T1) was calculated. The ratio 1 / T1 was plotted, and the slope of the straight line was the r1 relaxation rate of the contrast agent.

[0090] Establishment of a CCl4-induced mouse liver fibrosis model: Male C57BL / 6 mice (6–8 weeks old) were randomly divided into groups and a liver fibrosis model was established by intraperitoneal injection of CCl4. CCl4 was diluted with olive oil to prepare a 10% (v / v) working solution, which was administered intraperitoneally at a rate of 1 mL / kg body weight twice a week for 4, 6, or 8 weeks as required for subsequent experiments. The control group received the same volume of olive oil. Body weight and general condition were monitored regularly during the modeling period.

[0091] MR Imaging: Before scanning, model mice were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 1 mL / 100 g body weight. After adequate anesthesia, triplanar localization scanning was performed first, followed by magnetic resonance imaging (MRI) of the mouse abdomen. Axial and coronal T1-weighted imaging (T1WI) scans and axial T1 mapping imaging were performed first. Subsequently, mice in the Gd-DTPA group were injected with 40 μmol / kg Gd-DTPA solution via the tail vein, while the MSLC and MSL groups were injected with the same dose of MSLC and MSL. Axial and coronal enhanced T1WI scans were performed 15 min after contrast agent injection. The MRI parameters were set as follows: matrix 252 × 248, field of view (FOV) 5.0 × 5.0 cm, slice thickness 1.5 mm, interslice spacing 0.5 mm, and number of excitations (NEX) 6–8. T1WI uses a fast spin echo (FSE) sequence with parameters TR / TE = 200 / 10 ms.

[0092] MR image data processing: A region of interest (ROI) containing liver parenchyma was manually traced, avoiding major blood vessels. A second ROI was placed on the dorsal muscle visible in the same image slice to quantify the signal intensity in the muscle for comparison. Subsequently, all three ROIs were placed in a field of view without any tissue air to measure changes in background signal. The same analysis was performed on pre- and post-injection sequence images. Image visualization and quantification were performed in RadiAntViewer software. The contrast-to-noise ratio (CNR) was calculated by subtracting the SI in the muscle from the signal intensity (SI) in the liver and normalizing it to the SD of the signal in the animal's external air. The CNR was then used to calculate the contrast-to-noise ratio. post Subtract CNR pre The change in ΔCNR is calculated using the following formula: CNR = (SI) liver SI muscle ) / SD air ΔCNR = CNR post CNR pre .

[0093] Results: The T1-weighted magnetic resonance imaging performance and in vivo enhanced imaging results of the MSLC nanomaterials in Example 1 are shown in the figure below. Figure 8 As shown. Among them, by Figure 8 As shown in section A, the sample signal gradually increases with increasing Fe+Mn concentration, indicating that MSLC has a concentration-dependent T1 enhancement capability; Figure 8 From B, we know that the r1 value of MSLC is 7.88 mM. -1 ·s -1 4.11 mM higher than Gd-DTPA -1 ·s -1 This indicates that MSLC has good T1 contrast performance; Figure 8 In Figures C and D, the results showed that the liver signal enhancement was more significant after injection in the MSLC group, indicating that the MSLC nanomaterials prepared in this embodiment can be used as a T1-weighted magnetic resonance contrast agent for liver enhancement imaging.

[0094] (8) MRI evaluation and pathological and serum marker analysis of MSLC in a mouse model of liver fibrosis The specific steps are as follows: Mice were randomly divided into four groups (n=5): Normal, PBS, US+PBS, MSL, US+MSL, MSLC, and US+MSLC. Except for the Normal group, all other groups underwent intraperitoneal injection of CCl4 for 6 consecutive weeks to establish a liver fibrosis model. The Normal group received an equal volume of olive oil. From the third week of modeling, each group received PBS, MSL, or MSLC (5 mg / kg) via tail vein injection twice weekly. The US+PBS, US+MSL, and US+MSLC groups underwent ultrasound treatment (1.0 MHz, 1.0 W / cm², 50% duty, 5 min) 30 min after each administration. Enhanced MRI was performed after the last treatment. T1-weighted images of the liver region were acquired first, followed by enhanced T1-weighted images acquired within a preset time window after MSLC injection via tail vein. The CNR and ΔCNR before and after enhancement were calculated by delineating the liver region and background noise ROI to quantitatively evaluate the treatment effect of each group. Whole blood was collected 24 hours after imaging to separate serum. The levels of inflammatory factors such as IL-6, IL-1β, TNF-α, IL-10 and IL-4 were detected by ELISA, and the levels of ALT and AST were detected by a fully automated biochemical analyzer to comprehensively evaluate the effects of different treatments on liver fibrosis, inflammatory state and liver function.

[0095] Results: The MRI evaluation, pathological findings, and serum marker analysis of MSLC in a mouse model of liver fibrosis in Example 1 are shown in the figure below. Figure 9 As shown. Among them, by Figure 9 Results A and B showed that the liver enhancement signal was more obvious in the PBS group and the US+PBS group, while the enhancement signal was weakened in the MSLC and US+MSLC groups, with the most significant changes observed in the US+MSLC group. Figure 9 The results showed that MSLC, under ultrasound stimulation, could improve the degree of liver lesions and reduce fibrosis-related imaging indicators. These results indicate that the MSLC nanomaterials prepared in this embodiment can exert anti-liver fibrosis effects under ultrasound stimulation, and the therapeutic effect can be assessed via magnetic resonance imaging.

[0096] (9) Evaluation of the therapeutic effect in mice with liver fibrosis: The specific steps are as follows: Mice were sacrificed 24 hours after the last treatment, and their livers were harvested for gross observation and photographic recording of liver color, surface morphology, and nodular changes. Liver tissues were then used for paraffin embedding, cryopreservation, or RNA extraction. Paraffin sections were dewaxed and hydrated before HE and Masson staining to observe the pathological structure and collagen deposition in the liver tissue. Immunohistochemical and immunofluorescence staining were also performed to detect the expression of fibrosis-related markers such as α-SMA and COL1A1. Total RNA was extracted from a portion of the liver tissue, and cDNA was synthesized via reverse transcription. RT-qPCR was used to detect the mRNA expression levels of target genes such as α-SMA and COL1A1, with β-actin as an internal control. The ΔΔCt method was used for quantitative analysis to comprehensively evaluate the effects of different treatments on liver fibrosis from histological, protein, and gene levels.

[0097] Results: The evaluation results of the therapeutic effect of MSLC nanomaterials on liver fibrosis in mice under ultrasound in Example 1 are shown in the figure below. Figure 10 As shown in the figure, the liver tissues of mice in the PBS group and the US+PBS group exhibited significant inflammatory damage, collagen deposition, and fibrosis, with significantly increased expression of α-SMA and COL1A1. After MSLC treatment, all of the above pathological changes were improved, with the US+MSLC group showing more intact liver tissue structure and the most significant reduction in collagen deposition and α-SMA and COL1A1 expression. These results indicate that the MSLC nanomaterials prepared in this embodiment can effectively reduce liver inflammation and collagen deposition under ultrasound, thereby improving the degree of liver fibrosis.

[0098] (10) Biosafety assessment of MSLC in mice: The specific steps are as follows: Healthy mice were randomly divided into a Control group and a US+MSLC group. The US+MSLC group received MSLC via tail vein injection at a dose of 5 mg / kg twice a week for 4 consecutive weeks, and underwent sonication after each administration (1.0 MHz, 1.0 W / cm², 50% duty, 5 min). The Control group received an equal volume of PBS concurrently. Mouse weight, mental status, and general physiological condition were continuously monitored during the administration period. After the last treatment, whole blood was collected, serum was separated, and ALT and AST levels were measured using an automated biochemical analyzer to evaluate liver function safety. Separately, fresh anticoagulated blood was used to prepare red blood cell suspensions, which were incubated with different concentrations of MSLC or US+MSLC, and the absorbance at 540 nm was measured. Blood compatibility was calculated using the hemolysis rate formula. Mice were then sacrificed, and major organs such as heart, lungs, liver, spleen, and kidneys were harvested. After fixation with 4% paraformaldehyde, dehydration, embedding, and sectioning, the tissues were stained with hematoxylin and eosin (HE) and Masson's trichrome staining, respectively. The pathological morphology and collagen deposition of each tissue were observed under a microscope to systematically evaluate the biosafety of MSLC in mice.

[0099] Results: The in vitro and in vivo biosafety evaluation results of the MSLC nanomaterials in Example 1 are shown in the figure below. Figure 11 As shown. Among them, by Figure 11 In the case of AD, the results showed no obvious hemolysis within the tested concentration range, indicating that MSLC has good blood compatibility; Figure 11 The results showed no obvious pathological damage to the major organs. These results indicate that the MSLC nanomaterials prepared in this embodiment have good biocompatibility.

[0100] Targeted delivery solution In one embodiment of the present invention, the ultrasound-responsive targeted nanoenzyme material, through surface-modified collagen-targeting peptides (CBP), can actively identify and enrich liver fibrosis lesions. Due to the abnormal deposition of type I collagen in liver fibrosis tissue, the CBP can specifically bind to the overexpressed type I collagen in the lesion area. This allows the nanoplatform to preferentially remain in the fibrosis area after reaching the liver via in vivo circulation, increasing the local concentration of the material at the lesion site, reducing ineffective distribution in non-lesion tissues, and providing a targeted delivery basis for subsequent ultrasound-responsive activation, ROS regulation, NO generation, and magnetic resonance imaging.

[0101] • Synergistic regulation scheme for ROS scavenging and NO generation In one embodiment of the present invention, the targeted nanozyme platform achieves synergistic regulation in the liver fibrosis lesion area through two pathways: ROS scavenging and NO generation. The Fe-MnO2 bimetallic nanozyme participates in electron transfer reactions through the cyclic conversion between the multiple valence states of Fe and Mn, thereby reducing excess H2O2 and O2· in the lesion area.- The platform removes reactive oxygen species such as ·OH and ¹O2, thereby reducing oxidative stress damage and improving the local microenvironment. At the same time, L-Arg loaded on the platform is released under the action of ultrasound stimulation and local catalytic conditions, promoting NO production. The NO can further improve local microcirculation, alleviate inflammatory response, inhibit hepatic stellate cell activation and reduce collagen deposition, thereby achieving a combined intervention on the pathological process of "oxidative stress-inflammation-fibrosis".

[0102] • Ultrasonic response activation scheme In one embodiment of the present invention, after the targeted nanozyme platform is enriched in the liver fibrosis lesion area, low-intensity ultrasound stimulation is applied to the lesion site. The mechanical vibration, cavitation effect, and local energy conversion generated by ultrasound enhance the interfacial electron migration ability of the Fe-MnO2 bimetallic nanozyme, improve electron-hole separation efficiency, and accelerate the redox cycle between Fe and Mn, thereby enhancing the material's catalytic regulation ability of reactive oxygen species in the lesion microenvironment. Simultaneously, ultrasound stimulation can also promote the release of L-Arg loaded in the material and improve its conversion efficiency to NO, causing the nanoplatform to further transform from a static enrichment state in the lesion area to a dynamically activated state, thereby enhancing its comprehensive therapeutic effect.

[0103] Integrated Magnetic Resonance Imaging Diagnosis and Treatment Solution In one embodiment of the present invention, the ultrasound-responsive targeted nanozyme platform also has magnetic resonance imaging capabilities, specifically, the manganese-related components in the platform can release Mn²⁺ under the influence of the liver fibrosis lesion microenvironment. + The Mn² + Exhibiting paramagnetism, it can enhance T1-weighted magnetic resonance imaging signals, thereby enabling visual monitoring of lesion areas. By comparing changes in T1 signal intensity, T1 value, or ΔCNR before and after drug administration or treatment, the enrichment of the material in liver fibrosis lesions, the degree of lesion, and the treatment response can be evaluated. Therefore, the nanoplatform can not only achieve targeted therapy of lesion areas but also provide simultaneous imaging feedback, thereby constructing an integrated diagnostic and therapeutic technical solution for liver fibrosis.

[0104] The working principle of this invention is as follows: Liver fibrosis lesions are characterized by massive deposition of type I collagen, enhanced oxidative stress, sustained amplification of inflammation, and microcirculatory disturbances. This invention endows a nanoplatform with the ability to recognize type I collagen through CBP, enabling MSLC (microenzyme-dependent fibrotic lesions) to preferentially accumulate in liver fibrosis areas. Under ultrasound stimulation, the activity of the enriched MSLC, specifically the Fe-MnO2 bimetallic nanozyme, is further enhanced. The multivalent cycling between Fe and Mn accelerates electron transfer and enhances enzyme-like catalytic reactions, thereby efficiently clearing excess ROS in the lesion area and restoring local redox homeostasis. Simultaneously, L-Arg loaded in the material is released and promotes NO (nitrogenous oxygen) generation under ultrasound stimulation and the local catalytic microenvironment. The generated NO improves local hemodynamics, reduces inflammation, inhibits hepatic stellate cell activation, and lowers collagen deposition levels. Furthermore, the Mn-related components in the material can produce an MRI signal enhancement effect in the lesion microenvironment, enabling lesion imaging and treatment monitoring. Thus, this invention achieves a synergistic diagnostic and therapeutic process of "lesion-targeted delivery—ultrasound-responsive activation—ROS clearance—NO generation—MRI feedback."

[0105] Example 2: The difference between this embodiment and Embodiment 1 is that: In the preparation method of ultrasound-responsive targeted nanozyme materials, In step (1), the amounts of Fe-MnO2 nanoparticles, mesoporous silica, and anhydrous ethanol were 10 mg, 70 mg, and 30 mL, respectively; the calcination conditions were 400 °C for 2 h. In step (2), the amounts of MS and 3-aminopropyltriethoxysilane were 30 mg and 250 μL, respectively; the reflux reaction conditions were 100 °C for 4 h; the amounts of MS-NH2, PBS buffer, EDC, NHS, and L-arginine were 30 mg, 40 mL, 30 mg, 20 mg, and 60 mg, respectively; and the reaction conditions were: 14 h at room temperature. In step (3), the amounts of MSL, PBS buffer, EDC, NHS, and collagen-targeting peptide are 10 mg, 10 mL, 10 mg, 5 mg, and 1 mg, respectively; the reaction conditions are: 5℃, 20 h. The rest is the same as in Example 1.

[0106] Example 3: The difference between this embodiment and Embodiment 1 is that: In the preparation method of ultrasound-responsive targeted nanozyme materials, In step (1), the amounts of Fe-MnO2 nanoparticles, mesoporous silica, and anhydrous ethanol were 30 mg, 100 mg, and 60 mL, respectively; the calcination conditions were 500 °C for 0.5 h. In step (2), the amounts of MS and 3-aminopropyltriethoxysilane were 10 mg and 150 μL, respectively; the reflux reaction conditions were 60 °C for 8 h; the amounts of MS-NH2, PBS buffer, EDC, NHS, and L-arginine were 10 mg, 10 mL, 10 mg, 8 mg, and 30 mg, respectively; and the reaction conditions were: 10 h at room temperature. In step (3), the ratio of MSL, PBS buffer, EDC, NHS, and collagen-targeting peptide is 30 mg, 40 mL, 10 mg, 5 mg, and 1 mg; the reaction conditions are: 3℃, 28 h. The rest is the same as in Example 1.

[0107] The ultrasonic-responsive targeted nanoenzyme materials prepared in Examples 2 and 3 have similar properties to those in Example 1.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an ultrasound-responsive targeted nanoenzyme material, characterized in that, Includes the following steps: (1) Fe-MnO2 nanoparticles, mesoporous silica and organic solvent were mixed, dried and calcined under an inert atmosphere to obtain Fe-MnO2@mesoporous silica composite material, denoted as MS; (2) Amination of MS to obtain MS-NH2; MS-NH2, PBS buffer, EDC, NHS and L-arginine are mixed and reacted to obtain MS loaded with L-arginine, denoted as MSL; (3) Mix MSL, PBS buffer, EDC, NHS and collagen-targeting peptide, and react to obtain the product.

2. The preparation method according to claim 1, characterized in that, In (1), the mass ratio of Fe-MnO2 nanoparticles to mesoporous silica is (10~30):(70~100); the calcination conditions are: 400~500℃, 0.5~2h.

3. The preparation method according to claim 1, characterized in that, (2) The amination treatment of MS is as follows: MS, 3-aminopropyltriethoxysilane and anhydrous ethanol are mixed, heated and refluxed, centrifuged, washed and dried to obtain the product.

4. The preparation method according to claim 1, characterized in that, In (2), the mass ratio of MS-NH2 to L-arginine is (10~30): (30~60); the reaction conditions are: 10~14 h at room temperature.

5. The preparation method according to claim 1, characterized in that, In (2), the ratio of MS-NH2, PBS buffer, EDC, NHS and L-arginine is (10~30) mg: (10~40) mL: (10~30) mg: (8~20) mg: (30~60) mg.

6. The preparation method according to claim 1, characterized in that, In (3), the mass ratio of MSL to collagen-targeting peptide is (10~30):(1~5).

7. The preparation method according to claim 1, characterized in that, In (3), the ratio of MSL, PBS buffer, EDC, NHS and collagen-targeting peptide is (10~30) mg: (10~40) mL: (10~25) mg: (5~20) mg: (1~8) mg.

8. The preparation method according to claim 1, characterized in that, (3) The reaction conditions are: 3~5℃, 20~28h.

9. The ultrasonically responsive targeted nanozyme material prepared by the preparation method according to any one of claims 1-7.

10. The use of the ultrasound-responsive targeted nanozyme material of claim 8 in the preparation of medicaments for diagnosing and / or treating liver fibrosis.

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