Rhodium nano-enzyme compound and application thereof in preparation of medicine for preventing, relieving and / or treating medicine-induced liver injury

Abstract

The invention relates to a rhodium nano-enzyme compound and application thereof in preparation of drugs for preventing, relieving and / or treating drug-induced liver injury. The rhodium nano-enzyme compound comprises an extracellular vesicle simulant and rhodium nano-enzyme located in the extracellular vesicle simulant. The surface of the rhodium nano-enzyme is coated with the extracellular vesicle simulant, the extracellular vesicle simulant has good biocompatibility and low immunogenicity and can promote tissue repair and regeneration, and the extracellular vesicle simulant can be removed in a non-specific mode and then is enriched in the liver in a targeted mode, so that the rhodium nano-enzyme is enriched in the liver together with the extracellular vesicle simulant. The rhodium nano-enzyme has excellent simulation activity of superoxide dismutase (SOD) and catalase (CAT), can efficiently remove various active oxygen species, and can inhibit inflammatory reaction caused by APAP, so that the rhodium nano-enzyme has a targeted treatment effect on drug-induced liver injury.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and in particular to a rhodium nanozyme complex and its use in the preparation of drugs for the prevention, relief and / or treatment of drug-induced liver injury. Background Technology

[0002] Drug-induced liver injury (DILI) is a leading cause of acute liver failure worldwide, with acetaminophen (APAP) overdose being the most common cause. APAP is metabolized in hepatocytes to produce the toxic substance N-acetyl-p-benzoquinone imine (NAPQI), which depletes glutathione (GSH), triggering oxidative stress, inflammation, and hepatocyte necrosis, ultimately potentially leading to acute liver failure. Currently, N-acetylcysteine ​​(NAC), a first-line clinical drug, suffers from low bioavailability, poor stability, and a narrow therapeutic window. Furthermore, its mechanism of action is singular, failing to effectively address multiple pathological processes such as inflammation and regenerative disorders. Summary of the Invention

[0003] This application provides a rhodium nanozyme complex and its application in the preparation of drugs for the prevention, relief and / or treatment of drug-induced liver injury, which can effectively inhibit the inflammatory response induced by APAP, thereby having a targeted therapeutic effect on drug-induced liver injury.

[0004] In a first aspect, embodiments of this application provide a rhodium nanozyme complex comprising an extracellular vesicle mimic and a rhodium nanozyme located within the extracellular vesicle mimic.

[0005] In conjunction with the first aspect, in one embodiment, the extracellular vesicle mimic is an extracellular vesicle mimic derived from mesenchymal stem cells.

[0006] In conjunction with the first aspect, in one embodiment, the rhodium nanozyme has a particle size of less than 200 nm, and the rhodium nanozyme complex has a particle size of 100 nm to 1000 nm.

[0007] In conjunction with the first aspect, in one embodiment, the rhodium nanozyme has a particle size of 37.2 ± 1.2 nm; The rhodium nanozyme complex has a particle size of 436.4 ± 11.5 nm.

[0008] Secondly, embodiments of this application provide a method for preparing the rhodium nanozyme complex as described in the first aspect above, comprising: Mesenchymal stem cells were cultured, and the mesenchymal stem cells on the culture dish were scraped off. The obtained mesenchymal stem cell fragments were resuspended in PBS to obtain the first mixed solution. Rhodium nanozyme was added to the first mixed solution to obtain a second mixed solution; The second mixed solution was squeezed using an extrusion method to obtain a cell suspension; The cell suspension was subjected to gradient centrifugation to obtain the supernatant; Centrifuge the supernatant to obtain a rhodium nanozyme complex coated with extracellular vesicle mimicry.

[0009] In conjunction with the second aspect, in one embodiment, the concentration of rhodium nanozyme in the second mixed solution is 400–600 μg / mL; And / or, the second mixed solution is extruded using an extrusion method to obtain a cell suspension, which includes: extruding the cell suspension through a polycarbonate membrane with different pore sizes in descending order of pore size using an extruder, extruding it several times to obtain a cell suspension.

[0010] In conjunction with the second aspect, in one embodiment, the cell suspension is subjected to gradient centrifugation to obtain a supernatant, including: first centrifuging at 600g for 10 minutes, and then centrifuging at 1500g for 15 minutes to remove dead cells and debris, thereby obtaining a supernatant; And / or, centrifugation of the supernatant includes: centrifuging the resulting supernatant at 14000g for 60 minutes.

[0011] In conjunction with the second aspect, in one embodiment, the preparation of the rhodium nanozyme includes: Rhodium trichloride trihydrate RhCl3·3H2O and mercapto polyethylene glycol PEG-SH were dissolved in deionized water and stirred continuously. Subsequently, an aqueous solution of sodium borohydride was added dropwise with stirring, and the reaction was carried out at room temperature; Rhodium nanozymes were obtained by dialysis and purification in deionized water.

[0012] Thirdly, embodiments of this application provide the use of the rhodium nanozyme complex as described in the first aspect above in the preparation of medicaments for preventing, alleviating and / or treating drug-induced liver injury. Fourthly, embodiments of this application provide a medicament for preventing, alleviating, and / or treating drug-induced liver injury, the medicament containing the rhodium nanozyme complex as described in the first aspect above, or containing a rhodium nanozyme complex prepared using the preparation method of the rhodium nanozyme complex as described in the second aspect above.

[0013] The beneficial effects of the technical solution provided in this application include: This application provides a rhodium nanozyme complex and its application in the preparation of drugs for the prevention, relief, and / or treatment of drug-induced liver injury. The rhodium nanozyme is coated with extracellular vesicle mimics, which have good biocompatibility, low immunogenicity, and can promote tissue repair and regeneration. The extracellular vesicle mimics are non-specifically cleared and then targeted to the liver for enrichment. This allows the rhodium nanozyme to be enriched in the liver as well. The rhodium nanozyme has excellent superoxide dismutase (SOD) and catalase (CAT) mimicry activities, which can efficiently clear various reactive oxygen species and inhibit the inflammatory response induced by APAP, thereby providing a targeted therapeutic effect on drug-induced liver injury. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 Transmission electron microscope (TEM) images of Rhzymes, MEMs, and Rh@MEMs, and an HR-TEM image of Rhzymes, provided for Embodiment 1 of this application.

[0016] Figure 2 X-ray photoelectron spectroscopy (XPS) analysis of the composition and valence state of Rhzymes was provided in Example 1 of this application.

[0017] Figure 3 Hydrated particle size distribution diagrams of Rhzymes, MEMs, and Rh@MEMs provided in Embodiment 1 of this application.

[0018] Figure 4 The H2O2 removal effect of Rhzymes and Rh@MEMs provided in Embodiment 2 of this application.

[0019] Figure 5 The ability of Rhzymes and Rh@MEMs provided in Example 2 of this application to scavenge superoxide anion free radicals.

[0020] Figure 6 The ability of Rhzymes and Rh@MEMs to scavenge DPPH free radicals provided in Embodiment 2 of this application.

[0021] Figure 7 These are representative fluorescence images of EdU fluorescence staining on hepatocytes under different treatment conditions provided in Example 3 of this application.

[0022] Figure 8The images provided in Example 3 of this application are representative images of the levels of reactive oxygen species (ROS) in hepatocyte lines under different treatment conditions detected by DCFH-DA staining.

[0023] Figure 9 This is a statistical graph showing the cell viability of the L02 hepatocyte cell line after different treatments, as determined by the MTT assay in Example 3 of this application.

[0024] Figure 10 The statistical graph of in vitro DiR fluorescence intensity was analyzed from major organs collected from healthy mice and DILI model mice 24 hours after injection of MEMs or Rh@MEMs provided in Example 4 of this application.

[0025] Figure 11 The results of serum biochemical index detection related to liver function in healthy mice and DILI model mice receiving different treatments provided in Example 4 of this application are shown.

[0026] Figure 12 The representative H&E staining images provided in Example 4 of this application show the histopathological changes in the livers of healthy mice and DILI model mice after different treatments. The white dashed lines indicate necrotic areas and structurally disordered liver tissue.

[0027] Figure 13 This study provides a quantitative analysis of the necrotic liver regions in healthy mice and DILI mice treated with different methods, as provided in Example 4 of this application.

[0028] Figure 14 Quantitative analysis of the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum of healthy mice and DILI mice after different treatments provided in Example 4 of this application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] This application provides a rhodium nanozyme complex comprising extracellular vesicles and a rhodium nanozyme located within the extracellular vesicles.

[0031] Nanozymes, as nanomaterials with enzyme-like activity, have shown potential in antioxidant therapy. Among them, rhodium nanozymes (Rhzymes) possess excellent superoxide dismutase (SOD) and catalase (CAT) mimicry activities, and can efficiently scavenge various reactive oxygen species. However, their single antioxidant function is insufficient to address the complex pathological process of diabetic inflammatory disease (DILI).

[0032] Extracellular vesicles (EVs), especially those derived from mesenchymal stem cells, exhibit good biocompatibility, low immunogenicity, and the ability to promote tissue repair and regeneration. EVs are non-specifically cleared from circulation by the mononuclear phagocyte system, thus accumulating in the liver. Based on this characteristic, extracellular vesicles and their contained rhodium nanozymes can be enriched in the liver.

[0033] However, natural vesicles suffer from low yield and extraction difficulties, limiting their clinical application. Extracellular vesicle mimics (EMs) are prepared via physical extrusion, significantly increasing yield while retaining the core functions of natural EVs. EMs are typically non-specifically cleared from circulation by the mononuclear phagocyte system, thus accumulating in the liver. In DILI treatment, this limitation can be translated into a unique advantage, ensuring that a sufficient dose of EMs and their contents are delivered to the liver. Therefore, this application further provides a rhodium nanozyme complex comprising an extracellular vesicle mimic and a rhodium nanozyme located within the extracellular vesicle mimic, the extracellular vesicle mimic coating the surface of the rhodium nanozyme, and this rhodium nanozyme complex is designated Rh@MEMs.

[0034] The extracellular vesicle mimics are extracellular vesicle mimics derived from mesenchymal stem cells. Using extracellular vesicle mimics (MEMs) derived from mesenchymal stem cells to treat DILI can increase their production and exhibit liver tropism while retaining their ability to promote tissue repair and regeneration.

[0035] The rhodium nanozyme has a particle size of less than 200 nm, and the rhodium nanozyme complex has a particle size of 100 nm to 1000 nm. Preferably, the rhodium nanozyme has a particle size of 37.2 ± 1.2 nm, and the rhodium nanozyme complex has a particle size of 436.4 ± 11.5 nm.

[0036] The preparation method of the rhodium nanozyme complex includes the following steps: 101: Culture mesenchymal stem cells. Scrape the mesenchymal stem cells off the culture dish and resuspend the obtained mesenchymal stem cell fragments in PBS to obtain the first mixed solution.

[0037] 102: Add rhodium nanozyme to the first mixed solution to obtain a second mixed solution.

[0038] The concentration of rhodium nanozyme in the second mixed solution can be adjusted to 400-600 μg / mL, preferably, the concentration of rhodium nanozyme in the second mixed solution is 500 μg / mL.

[0039] 103: The second mixed solution is squeezed using an extrusion method to obtain a cell suspension.

[0040] In step 103, the second mixed solution is squeezed to obtain a cell suspension by squeezing: the cell suspension is obtained by squeezing the mixture through a polycarbonate membrane with different pore sizes in descending order of pore size using an extruder, and squeezing each membrane several times to obtain a cell suspension.

[0041] For example, as an example, a cell suspension can be obtained by extruding the cells through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm in sequence, 10 times for each membrane.

[0042] 104: The cell suspension was centrifuged using a gradient method to obtain the supernatant.

[0043] In step 104, the cell suspension is subjected to gradient centrifugation to obtain supernatant, including: first centrifuging at 600 g for 10 minutes, and then centrifuging at 1500 g for 15 minutes to remove dead cells and debris, thereby obtaining supernatant.

[0044] It is understandable that the above examples of centrifuging at 600 g for 10 minutes and centrifuging at 1500 g for 15 minutes are just examples, and the centrifugal force and centrifugation time can be adjusted according to the actual situation.

[0045] 105: Centrifuge the supernatant to obtain a rhodium nanozyme complex coated with extracellular vesicle mimicry.

[0046] In step 105, centrifuging the supernatant includes centrifuging the resulting supernatant at 14000 g for 60 minutes. It is understood that the above 14000 g centrifugation for 60 minutes is merely an example, and the centrifugal force and time can be adjusted according to actual conditions.

[0047] Furthermore, the preparation of the rhodium nanozyme includes: 201: Dissolve rhodium trichloride trihydrate (RhCl3·3H2O) and mercaptopolyethylene glycol (PEG-SH) in deionized water and stir continuously.

[0048] The amount of each substance added can be determined according to the actual preparation needs. For example, 60 mg of rhodium trichloride trihydrate RhCl3·3H2O and 40 mg of mercapto polyethylene glycol PEG-SH are dissolved in 100 mL of deionized water and stirred continuously.

[0049] Thiol-based polyethylene glycol can improve the solubility and stability of the modified molecule.

[0050] 202: Subsequently, sodium borohydride aqueous solution was added dropwise with stirring, and the reaction was carried out at room temperature.

[0051] For example, as an example, the concentration of sodium borohydride aqueous solution is 2 mg / mL, the amount added is 10 mL, and the reaction time is 5 min.

[0052] 203: Rhodium nanozyme was obtained by dialysis and purification in deionized water.

[0053] For example, purification may take 2 days, or the time may be adjusted depending on the actual purification situation.

[0054] This application also provides an application of a rhodium nanozyme complex in the preparation of medicaments for the prevention, relief, and / or treatment of drug-induced liver injury. This application also provides a medicament for preventing, alleviating and / or treating drug-induced liver injury, the medicament containing the rhodium nanozyme complex described above, or containing a rhodium nanozyme complex prepared using the method described above.

[0055] The present application will be described in detail below with reference to specific embodiments and accompanying drawings, but the present application is not limited to these embodiments.

[0056] Example 1: Fabrication and characterization of Rhzymes, MEMs and Rh@MEMs (1) Preparation of Rhzymes nanozymes Rhzymes were prepared by liquid-phase reduction.

[0057] Rhodium trichloride trihydrate (RhCl3·3H2O, 60 mg) and mercaptopolyethylene glycol (PEG-SH, 40 mg) were dissolved in deionized water (100 mL) with continuous stirring. Then, sodium borohydride aqueous solution (2 mg / mL, 10 mL) was added dropwise with continuous stirring. The reduction reaction was carried out at room temperature for 5 minutes. Subsequently, the solution was purified in deionized water for 2 days by dialysis (molecular weight cutoff: 3500) to obtain Rhzymes. The obtained Rhzymes could be lyophilized for further use.

[0058] (2) Preparation of extracellular vesicle mimics (MEMs) derived from mesenchymal stem cells Mesenchymal stem cells were cultured, and the mesenchymal stem cells were scraped off from the culture dish. The obtained mesenchymal stem cell fragments were then resuspended in PBS to obtain the first mixed solution.

[0059] The first mixed solution was extruded through a polycarbonate membrane with pore sizes of 10 μm, 5 μm, and 1 μm, 10 times for each, to obtain a cell suspension.

[0060] The cell suspension was subjected to gradient centrifugation, first at 600g for 10 minutes, then at 1500g for 15 minutes to remove dead cells and debris, and the supernatant was obtained.

[0061] The resulting supernatant was centrifuged at 14000g for 60 minutes to obtain mesenchymal stem cell-derived extracellular vesicle mimics (MEMs), which were then resuspended in PBS.

[0062] (3) Preparation of Rh@MEMs Rh nanozyme complex Mesenchymal stem cells were cultured, and the mesenchymal stem cells were scraped off from the culture dish. The obtained mesenchymal stem cell fragments were then resuspended in PBS to obtain the first mixed solution.

[0063] Rhodium nanozyme was added to the first mixed solution to obtain a second mixed solution with a concentration of 500 μg / mL of rhodium nanozyme.

[0064] The second mixed solution was extruded through a polycarbonate membrane with pore sizes of 10 μm, 5 μm, and 1 μm, 10 times for each, to obtain a cell suspension.

[0065] The cell suspension was subjected to gradient centrifugation, first at 600g for 10 minutes, then at 1500g for 15 minutes to remove dead cells and debris, and the supernatant was obtained.

[0066] The resulting supernatant was centrifuged at 14000g for 60 minutes to obtain the rhodium nanozyme complex Rh@MEMs coated with extracellular vesicle mimics, which was then resuspended in PBS.

[0067] It should be noted that the PBS is isotonic with the cells and has a concentration of 0.01M.

[0068] (4) Characterization results See Figure 1 As shown, transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM) images clearly reveal that the rhodium nanozymes have a near-spherical structure and are uniformly dispersed, while also exhibiting distinct lattice fringes. TEM imaging shows that the MEMs have a spherical membrane vesicle structure, and that the rhodium nanozymes are efficiently encapsulated within the MEMs.

[0069] See Figure 2 As shown, X-ray photoelectron spectroscopy analysis further confirmed the composition and valence state of the material. Its characteristic peaks at 305.7 eV and 310.4 eV correspond to Rh(0), and its characteristic peaks at 307.3 eV and 312.0 eV correspond to Rh(III).

[0070] See Figure 3As shown, dynamic light scattering analysis indicates that the average hydrated particle sizes of Rhzymes, MEMs, and Rh@MEMs are 37.2 ± 1.2 nm, 425.8 ± 10.3 nm, and 436.4 ± 11.5 nm, respectively.

[0071] Example 2: In vitro enzyme activity assessment of Rhzymes and Rh@MEMs (1) Catalase-like activity Dissolved oxygen was removed by purging ultrapure water with nitrogen for 30 minutes. Rhzymes or Rh@MEMs were then added to the nitrogen-deoxygenated ultrapure water to achieve a final Rhzymes concentration of 20 μg / mL. A layer of liquid paraffin was then applied to the solution surface as a protective layer, and H2O2 solution was injected using a syringe to a final concentration of 1 mM. O2 release was continuously measured using a portable dissolved oxygen meter (JPB-607A). See [link to relevant documentation]. Figure 4 As shown, an increase in dissolved oxygen concentration can be observed, confirming its catalase-like activity through the decomposition of H2O2.

[0072] (2) Superoxide dismutase-like activity The NBT photochemical reduction method was used to evaluate superoxide dismutase-like activity. The specific steps are as follows: The mixture was divided into two groups. In the first group, 50 μL of riboflavin (200 μM, Shanghai Aladdin Industrial Co., Ltd.), 50 μL of nitrotetrazole blue (NBT) (750 μM, Shanghai Aladdin Industrial Co., Ltd.), and 50 μL of methionine (130 mM, Shanghai Aladdin Industrial Co., Ltd.) were added to multiple 1.5 mL EP tubes. Then, different amounts of Rhzymes were added to each 1.5 mL EP tube, and PBS was added to bring the volume to 500 μL, so that the Rhzymes concentration in the mixed solution in each 1.5 mL EP tube was 5, 10, 20, 40, and 80 μg / mL, respectively.

[0073] In the second group, 50 μL of riboflavin (200 μM, Shanghai Aladdin Industrial Co., Ltd.), 50 μL of nitrotetrazole blue (NBT) (750 μM, Shanghai Aladdin Industrial Co., Ltd.), and 50 μL of methionine (130 mM, Shanghai Aladdin Industrial Co., Ltd.) were added to multiple 1.5 mL EP tubes. Then, different amounts of Rh@MEMs were added to each 1.5 mL EP tube, and PBS was added to bring the volume to 500 μL, so that the Rhzymes concentration in the mixed solution in each 1.5 mL EP tube was 5, 10, 20, 40, and 80 μg / mL, respectively.

[0074] After irradiation with white light for 5 minutes, the absorbance of the solution at 560 nm was measured. (See also...) Figure 5As shown, the results indicate that both Rhzymes and Rh@MEMs can effectively scavenge superoxide anion radicals in a dose-dependent manner.

[0075] (3) Reactive nitrogen species scavenging activity The scavenging activity of Rhzymes and Rh@MEMs for reactive nitrogen species was evaluated using DPPH radical scavenging experiments. The specific steps were as follows: 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH, THICA (Shanghai) Chemical Industry Development Co., Ltd.) was dissolved in ethanol, and the DPPH concentration was 200 μg / mL.

[0076] The mixture was divided into two groups. In the first group, 100 μL of DPPH and different amounts of Rhzymes were added to multiple 1.5 mL EP tubes to make the Rhzymes concentration in the mixed solution in each 1.5 mL EP tube 20, 40, 80, 160 and 320 μg / mL, respectively. After incubation for 20 min, the mixture was centrifuged at 10000 rpm for 5 min, and the absorbance of the supernatant was measured at 517 nm.

[0077] In the second group, 100 μL of DPPH and different amounts of Rh@MEMs were added to multiple 1.5 mL EP tubes to make the Rhzymes concentration in the mixed solution in each 1.5 mL EP tube 20, 40, 80, 160 and 320 μg / mL, respectively. After incubation for 20 min, the solution was centrifuged at 10000 rpm for 5 min, and the absorbance of the supernatant was measured at 517 nm.

[0078] See Figure 6 As shown, the results indicate that both Rhzymes and Rh@MEMs exhibit significant neutralization effects, demonstrating their excellent ability to scavenge reactive nitrogen (RNS) species.

[0079] Example 3: Evaluation of the in vitro regeneration-promoting effect of MEMs, the ROS scavenging ability of Rhzymes, and the cell-protective effect of Rh@MEMs 1. MEMs' ability to promote regeneration After 24 hours of adherent culture, L02 hepatocytes (Tico (Shanghai) Biotechnology Co., Ltd., hereinafter the same) were divided into two groups for treatment, each lasting 12 hours, as follows: Control group: Only complete culture medium was added.

[0080] APAP+MEMs group: Acetaminophen APAP (Shanghai Yuanye Biotechnology Co., Ltd.) and MEMs with a final concentration of 0.5 μg / mL were added to the complete culture medium and treated for 12 hours.

[0081] APAP group: treated with APAP alone at a final concentration of 10 mM for 12 hours.

[0082] Subsequently, each group was stained with EdU, and the intracellular EdU green fluorescence was observed using a fluorescence microscope. See also Figure 7 As shown, EdU staining results indicated that in the APAP-damaged hepatocyte line L02, the number of EdU-positive cells in the MEMs-treated group was greater than that in the PBS-treated group, suggesting that MEMs themselves have a significant ability to promote hepatocyte proliferation.

[0083] 2. Assessment of the intracellular ROS clearance capacity of Rhzymes and Rh@MEMs The intracellular reactive oxygen species (ROS) content was detected using the DCFH-DA probe (Shanghai Beyotime Biotechnology Co., Ltd.). The steps were as follows: Before the experiment, L02 hepatocytes were seeded in 24-well plates (5 × 10⁻⁶ cells / well). 4 Cells / well), cultured for 24 hours to allow for full adhesion. Subsequently, L02 hepatocytes were treated as follows: APAP group: treated with acetaminophen APAP at a final concentration of 10 mM for 1 hour.

[0084] APAP+MEMs group: Add acetaminophen APAP to a final concentration of 10 mM and MEMs to a final concentration of 5 μg / mL for 1 hour.

[0085] APAP+Rhzymes group: treated with acetaminophen APAP at a final concentration of 10 mM and Rhzymes at a final concentration of 50 µg / mL for 1 hour.

[0086] APAP+Rh@MEMs group: Add acetaminophen APAP and Rh@MEMs (with the final concentration of Rhzymes being 50 µg / mL) to a final concentration of 10 mM and treat for 1 hour.

[0087] Control group: No of the above substances were added.

[0088] Subsequently, each group was incubated with 0.5 mL of DCFH-DA (10 µM) for 30 minutes, and fluorescence images were acquired using a fluorescence microscope. Cells were then collected and quantitatively analyzed using a flow cytometer (Canto II, BD Biosciences, USA).

[0089] See Figure 8As shown, the results indicated that APAP treatment drastically increased ROS levels in L02 hepatocytes. Compared to the APAP group, APAP+Rhzymes or APAP+Rh@MEMs treatment effectively reduced fluorescence intensity.

[0090] 3. Cell-protective effects The protective effect of Rh@MEMs on hepatocytes was verified using the MTT assay. L02 hepatocytes were seeded in 96-well plates and cultured for 24 hours. Then, different concentrations of APAP (0-40 mM) were added, creating four groups: one group was designated as the APAP group, and the other three groups were further treated with MEMs (final concentration 5 μg / mL), Rhzymes (final concentration 50 µg / mL), or Rh@MEMs (with Rhzymes coated at a final concentration of 50 µg / mL), respectively, resulting in the APAP+MEMs group, APAP+Rhzymes group, and APAP+Rh@MEMs group.

[0091] Afterward, each group was incubated for another 24 hours, then washed with PBS, and 200 μL of MTT solution (5 mg / mL) was added to each well for 4 hours. To dissolve the generated formazan crystals, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was shaken for 15 minutes. Cells were then analyzed using a microplate reader (In...). The absorbance of each well in a 96-well plate was measured at a wavelength of 570 nm using an ite F50 (Tecan, Switzerland).

[0092] The results are as follows Figure 9 Rhzymes can improve cell viability after APAP treatment in a dose-dependent manner. In addition, due to the regenerative properties of MEMs, Rh@MEMs can further enhance cell viability compared to the Rhzymes treatment group alone.

[0093] Example 4: Evaluation of the liver tropism and therapeutic effect of the material 1. Distribution within the body C57 / BL6 mice (Hubei Beinte Biotechnology Co., Ltd.) were fasted (with free access to water) for 15 hours and then divided into four groups: MEMs group, Rh@MEMs group, APAP+MEMs group, and APAP+Rh@MEMs group. The treatments for the four groups of mice were as follows: MEMs group: C57 / BL6 mice were injected with DiR-labeled MEMs (MEMs dose of 0.5 mg / kg) via the tail vein and in vivo imaging was performed 24 hours later.

[0094] Rh@MEMs group: C57 / BL6 mice were injected with DiR-labeled Rh@MEMs (Rh@MEMs dose of 0.5 mg / kg) via the tail vein and in vivo imaging was performed 24 hours later.

[0095] APAP+MEMs group: C57 / BL6 mice were injected intraperitoneally with APAP solution at a dose of 300 mg / kg and then fed normally to induce DILI model mice. Then, DiR-labeled MEMs (MEMs dose of 0.5 mg / kg) were injected into the tail vein and in vivo imaging was performed 24 hours later.

[0096] APAP+Rh@MEMs group: C57 / BL6 mice were intraperitoneally injected with APAP solution at a dose of 300 mg / kg and then fed normally to induce DILI model mice. Then, DiR-labeled Rh@MEMs (Rh@MEMs dose of 0.5 mg / kg) were injected into the tail vein and in vivo imaging was performed 24 hours later.

[0097] APAP was administered via tail vein injection 12 hours after treatment.

[0098] See Figure 10 As shown, in vivo imaging revealed that both MEMs and Rh@MEMs were primarily enriched in the liver in both healthy and DILI model mice. Furthermore, in the same mouse model, the enrichment level of Rh@MEMs in the liver was comparable to that of MEMs, indicating that the liver tropism of MEMs was successfully conferred on Rh@MEMs. Additionally, the fluorescence signal in the liver region of DILI model mice was stronger than that in the liver of healthy mice, suggesting that liver injury enhanced the liver tropism of both MEMs and Rh@MEMs. This phenomenon may be due to the increased inflammatory response in mice caused by injury, leading to a more active mononuclear phagocytic system and exacerbated phagocytosis of materials, thereby increasing material accumulation in the liver.

[0099] 2. Treatment efficacy C57 / BL6 mice were fasted (with free access to water) for 15 hours and then randomly divided into five groups: a control group, an APAP group, an APAP+MEMs group (MEMs dose 1 mg / kg), an APAP+Rhzymes group (Rhzymes dose 10 mg / kg), and an APAP+Rh@MEMs group (where the coated Rhzymes dose was 10 mg / kg). The treatments for the five groups of mice were as follows: Control group: No treatment was given; healthy C57 / BL6 mice were included.

[0100] APAP group: C57 / BL6 mice were injected intraperitoneally with APAP solution at a dose of 300 mg / kg and then fed normally to induce DILI model mice.

[0101] APAP+MEMs group: C57 / BL6 mice were intraperitoneally injected with APAP solution at a dose of 300 mg / kg and then fed normally to induce DILI model mice. MEMs were then injected into the tail vein.

[0102] APAP+Rhzymes group: C57 / BL6 mice were injected intraperitoneally with APAP solution at a dose of 300 mg / kg and then fed normally to induce DILI model mice. Rhzymes were then injected into the tail vein.

[0103] APAP+Rh@MEMs group: C57 / BL6 mice were intraperitoneally injected with APAP solution at a dose of 300 mg / kg and then fed normally to induce DILI model mice. Rh@MEMs were then injected into the tail vein.

[0104] All formulations (MEMs, Rhzymes, and Rh@MEMs) were freshly prepared with PBS and administered via tail vein injection 12 hours after APAP treatment. Mice were sacrificed 24 hours after injection, and whole blood and major organs were collected for serum biochemical analysis, histological analysis, and ELISA detection. Cytokine levels were detected using a commercial ELISA kit (BioLegend, USA) according to the manufacturer's instructions.

[0105] (1) Liver function tests: To quantify the degree of liver damage, the levels of serum liver function markers—serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT)—were measured in mice. See [link to relevant documentation] Figure 11 As shown, ALT and AST levels in the APAP group mice increased dramatically (reaching 20.4 times and 14.1 times that of the control group, respectively). MEMs and Rhzymes could partially inhibit the increase of these indicators (MEMs inhibited ALT and AST by 16.5% and 23.4%, respectively; Rhzymes inhibited ALT and AST by 43.1% and 53.2%, respectively), while Rh@MEMs showed the strongest inhibitory effect, reducing ALT and AST levels by 74.8% and 78.9%, respectively.

[0106] (2) Histopathological analysis: Acute APAP administration induces widespread liver damage by promoting hepatocyte necrosis and apoptosis, with lesions primarily concentrated in the central region of the liver lobules. See also Figure 12 and Figure 13 As shown, although using MEMs and Rhzymes alone can reduce the APAP-induced necrosis area from 52.2% to 35.1% and 16.5% respectively, Rh@MEMs exhibits the strongest protective effect, further reducing the necrosis foci to 8.6%.

[0107] (3) Detection of pro-inflammatory factors: such as Figure 14As shown, Rhzymes and Rh@MEMs can downregulate the expression of key pro-inflammatory cytokines (TNF-α and IL-6) in DILI model mice, thus alleviating the inflammatory response induced by APAP.

[0108] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A rhodium nanozyme complex, characterized in that, It includes an extracellular vesicle mimicry and a rhodium nanozyme located within the extracellular vesicle mimicry.

2. The rhodium nanozyme complex according to claim 1, characterized in that: The extracellular vesicle mimicry is an extracellular vesicle mimicry derived from mesenchymal stem cells.

3. The rhodium nanozyme complex according to claim 1, characterized in that: The rhodium nanozyme has a particle size of less than 200 nm, and the rhodium nanozyme complex has a particle size of 100 nm to 1000 nm.

4. The rhodium nanozyme complex according to claim 3, characterized in that: The rhodium nanozyme has a particle size of 37.2 ± 1.2 nm; The rhodium nanozyme complex has a particle size of 436.4 ± 11.5 nm.

5. A method for preparing the rhodium nanozyme complex as described in claim 1, characterized in that, It includes: Mesenchymal stem cells were cultured, and the mesenchymal stem cells on the culture dish were scraped off. The obtained mesenchymal stem cell fragments were resuspended in PBS to obtain the first mixed solution. Rhodium nanozyme was added to the first mixed solution to obtain a second mixed solution; The second mixed solution was squeezed using an extrusion method to obtain a cell suspension; The cell suspension was subjected to gradient centrifugation to obtain the supernatant; Centrifuge the supernatant to obtain a rhodium nanozyme complex coated with extracellular vesicle mimicry.

6. The method for preparing the rhodium nanozyme complex as described in claim 5, characterized in that: The concentration of rhodium nanozyme in the second mixed solution is 400–600 μg / mL; And / or, the second mixed solution is extruded using an extrusion method to obtain a cell suspension, which includes: extruding the cell suspension through a polycarbonate membrane with different pore sizes in descending order of pore size using an extruder, extruding it several times to obtain a cell suspension.

7. The method for preparing the rhodium nanozyme complex as described in claim 5, characterized in that: The cell suspension was subjected to gradient centrifugation to obtain the supernatant, including: first centrifuging at 600g for 10 minutes, and then centrifuging at 1500g for 15 minutes to remove dead cells and debris, and obtain the supernatant. And / or, centrifugation of the supernatant includes: centrifuging the resulting supernatant at 14000g for 60 minutes.

8. The method for preparing the rhodium nanozyme complex as described in claim 5, characterized in that, The preparation of the rhodium nanozyme includes: Rhodium trichloride trihydrate RhCl3·3H2O and mercapto polyethylene glycol PEG-SH were dissolved in deionized water and stirred continuously. Subsequently, an aqueous solution of sodium borohydride was added dropwise with stirring, and the reaction was carried out at room temperature; Rhodium nanozymes were obtained by dialysis and purification in deionized water.

9. The use of a rhodium nanozyme complex as described in any one of claims 1 to 4 in the preparation of a medicament for the prevention, relief and / or treatment of drug-induced liver injury.

10. A medicament for preventing, alleviating, and / or treating drug-induced liver injury, characterized in that, The drug contains a rhodium nanozyme complex as described in any one of claims 1 to 4, or contains a rhodium nanozyme complex prepared by the method described in any one of claims 5 to 8.

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