M-dcn nano-enzyme, preparation method and application thereof

Abstract

The application belongs to the technical field of natural products, and discloses M-dCNs nano-enzyme as well as a preparation method and application thereof. The M-dCNs nano-enzyme comprises the following components in percentage by mass: hesperetin (HST) 84-87%, KPLH1130 5-8%, Fe 3+ 5-8%. The application constructs a metal-natural product complex, utilizes HST to complex with Fe 3+ , does not need to add a carrier or be modified through a covalent bond, can self-assemble to form nano-enzyme with uniform morphology, has small particle size, a drug loading capacity of 100%, and high stability, eliminates the dependence on a carrier and avoids the limitation of a drug loading rate, effectively improves the activity of HST by self-assembling to form nano-enzyme, exhibits liver aggregation targeting, greatly improves the treatment effect on liver damage, and can be applied to the preparation of a medicine for preventing and / or treating liver damage.

Description

Technical Field

[0001] This invention belongs to the field of natural product technology, and specifically relates to an M-dCNs nanozyme, its preparation method, and its application. Background Technology

[0002] Drug-induced liver injury (DILI) is liver damage caused by the drug itself or its metabolites, or by hypersensitivity or decreased tolerance to the drug due to specific individual conditions. Clinically, it can manifest as various acute and chronic liver diseases. Mild cases may resolve spontaneously after drug discontinuation, while severe cases can be life-threatening and require aggressive treatment and resuscitation. DILI can occur in healthy individuals with no prior history of liver disease or in patients with pre-existing serious conditions; it can occur with overdose or under normal dosage. In DILI, the regulation of inflammation plays a crucial role in promoting its therapeutic effect. Liver tissue contains a large number of macrophages. During DILI, pro-inflammatory macrophages promote the inflammatory response in the early stages of inflammation. Polarizing pro-inflammatory macrophages into anti-inflammatory macrophages is essential for reducing the inflammatory response in liver injury.

[0003] Hesperidin (HST) is a class of flavonoids with the molecular formula C2. 16 H 14 O6 is present in dried tangerine peel ( Citri Reticulatae Pericarpium Hesperidin derivatives of CRP (hesperidin, chloroform, and chloroform) have many potential biological benefits. They exert antioxidant and anti-inflammatory effects by downregulating NfkB and upregulating the expression of Nrf2 and AMPK. These pathways intersect and ultimately improve liver damage in terms of cell death, inflammation, and oxidative stress.

[0004] However, HST is poorly soluble in water, chloroform, and benzene at room temperature, but readily soluble in ethanol, limiting its effectiveness in the direct treatment of liver injury. Current technologies utilize carriers to load HST before application, leading to new problems such as carrier dependence and low drug loading rates. Therefore, further research is needed to effectively improve the therapeutic efficacy of the effective components of dried tangerine peel on liver injury.

[0005] In recent years, the effectiveness of metal-natural product complexes in the prevention and treatment of various diseases has attracted considerable attention from researchers. Metal ions can directly or indirectly activate the pharmacological effects of certain natural products. Furthermore, metal complexes can alter the properties of natural products, such as increasing water solubility and heat resistance, and enhancing drug activity. Based on the design of metal-natural product complexes, this invention provides a self-assembled M-dCNs nanozyme, thereby achieving both effective reduction of oxidative stress and synergistic anti-inflammatory effects. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an M-dCNs nanozyme.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned M-dCNs nanozyme.

[0008] Another object of the present invention is to provide the application of the above-mentioned M-dCNs nanozyme, particularly its application in the preparation of drugs for the prevention and / or treatment of liver injury.

[0009] The objective of this invention is achieved through the following solution:

[0010] An M-dCNs nanozyme comprises the following components by mass percentage: hesperidin (HST) 84–87%, KPLH1130 5–8%, Fe 3+ 5-8%.

[0011] Furthermore, the HST may be HST derived from dried tangerine peel.

[0012] This invention constructs metal-natural product complexes by utilizing HST and Fe 3+ Complexation allows for the self-assembly of uniformly morphological nanozymes without the need for carriers or covalent modifications. These nanozymes have small particle sizes, with an average particle size of less than or equal to 150 nm, 100% drug loading, and high stability. This not only eliminates dependence on carriers and avoids limitations on drug loading rates, but also effectively enhances the activity of HST through self-assembly, significantly improving the therapeutic effect on liver damage.

[0013] This invention also provides a method for preparing the above-mentioned M-dCNs nanozyme, comprising the following steps: adding hesperidin (HST) and Fe under stirring. 3+ The solution was stirred and mixed with KPLH1130, and then separated to obtain M-dCNs nanozyme.

[0014] Furthermore, in the reaction system, the concentration of HST can be 0.1-3 mg / mL, and Fe... 3+The concentration of [the substance] can be 0.1-2 mg / mL, and the concentration of KPLH1130 can be 0.1-3 mg / mL.

[0015] Furthermore, in the reaction system, Fe 3+ The mass ratio of HST to KPLH1130 can be (1-20): (1-30): (1-30).

[0016] Furthermore, in the reagents used in the method of the present invention, the Fe... 3+ Fe in solution 3+ The concentration can be 5-100 mg / mL.

[0017] Furthermore, the Fe 3+ The solution can be obtained by dissolving a soluble iron salt in water.

[0018] Furthermore, the soluble iron salt can be selected from iron salts such as ferric chloride and ferric sulfate; ferric chloride is preferred.

[0019] Furthermore, in the reagents used in the method of the present invention, the HST is soluble in DMSO to obtain an HST solution which is then used in the reaction.

[0020] Furthermore, the concentration of the HST solution can be 3-100 mg / mL.

[0021] Furthermore, in the reagents used in the method of the present invention, the KPLH1130 can be dissolved in DMSO to obtain a KPLH1130 solution which is then used in the reaction.

[0022] Furthermore, the concentration of the KPLH1130 solution can be 3-100 mg / mL.

[0023] Furthermore, the stirring time can be 5-15 minutes. Stirring is sufficient to ensure that all components are thoroughly mixed and reacted.

[0024] Furthermore, the separation can be achieved through conventional centrifugation, such as centrifuging at 10,000-20,000 rpm / min for 10-60 min, or centrifuging at 14,000 rpm / min for 30 min.

[0025] Furthermore, the separated precipitate can be washed with water.

[0026] Furthermore, the prepared M-dCNs nanozyme can be dispersed in water to obtain an M-dCNs nanozyme suspension.

[0027] The M-dCNs nanozyme of the present invention forms nanoparticles through self-assembly, with uniform and small particle size, and achieves 100% drug loading rate. It effectively solves the problems of low solubility, low bioavailability and poor antioxidant activity of HST. The obtained M-dCNs nanozyme has the advantages of stable functional substance activity, not easy to be oxidized, high bioavailability and good sustained release.

[0028] The M-dCNs nanozyme constructed in this invention utilizes HST and Fe 3+ Complexation allows for the self-assembly of uniformly morphological nanozymes without the need for carriers or covalent modifications. This not only eliminates dependence on carriers and avoids limitations on drug loading, but also effectively enhances the activity of HST through self-assembly of nanozymes. Furthermore, it exhibits liver aggregation and targeting, significantly improving the therapeutic effect on liver injury. This technology can be applied to the preparation of drugs for the prevention and / or treatment of liver injury. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A comparison of particle sizes for M-dCNs nanozymes with different proportions.

[0031] Figure 2 The image shows the physicochemical properties of M-dCNs nanozymes.

[0032] Figure 3 The graph shows the stability changes of M-dCNs nanozymes after storage at 25°C for 60 h.

[0033] Figure 4 The elemental analysis spectrum of M-dCNs nanozymes.

[0034] Figure 5 The ability of M-dCNs nanozymes to scavenge •DPPH in vitro.

[0035] Figure 6 The ability of M-dCNs nanozymes to scavenge •OH in vitro.

[0036] Figure 7 The distribution of M-dCNs in major organs of mice.

[0037] Figures 8-9 To demonstrate the early preventive effect of M-dCNs in DILI mice.

[0038] Figure 10 The late-stage therapeutic effect of M-dCNs in DILI mice.

[0039] Figures 11-12 To analyze the biosafety of M-dCNs in vivo. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0041] The hesperidin (HST) used in the following examples can be purchased from commercially available products. The amounts of each component are expressed in parts by weight and parts by volume, in mg and mL.

[0042] Example 1: Preparation of M-dCNs nanozymes

[0043] HST was dissolved in DMSO to obtain an HST solution, ferric chloride was prepared into an aqueous solution, and KPLH1130 was dissolved in DMSO to obtain a KPLH1130 solution.

[0044] According to the set Fe 3+ Mix the above solutions with HST:KPLH1130 in a stirring state, stir for 15 min, collect, centrifuge at 14000 rpm / min for 30 min, discard the supernatant, and resuspend the precipitate in distilled water to obtain the M-dCNs suspension.

[0045] Using different Fe 3+ M-dCNs nanozymes were prepared by mixing HST, ferric chloride, and KPLH1130 in a 2:3:2 ratio. Specifically, the concentrations of HST, ferric chloride, and KPLH1130 solutions were all prepared to 10 mg / mL. Then, 20 parts by volume of ferric chloride solution, 30 parts by volume of HST solution, and 20 parts by volume of KPLH1130 solution were added to 1000 parts by volume of water, respectively. The mixture was stirred and separated to obtain M-dCNs nanozymes with a mass ratio of 2:3:2. Different M-dCNs nanozymes were obtained by adjusting the amounts of each component. Note that, according to the dosage ratio, no self-assembly reaction occurred in the system when the iron ion concentration was less than 0.1 mg / mL.

[0046] The morphology of the prepared M-dCNs was observed using a transmission electron microscope at 80 kV. The particle size and PDI were measured using a Malvern particle size analyzer. The results are shown in the figure. Figure 1 As can be seen from the figure, the M-dCNs of this invention are particles with an average particle size of less than 150 nm and a uniform morphology, which are near-spherical particles. Figure 1 The paper lists particles prepared with some specific ratios. Particles prepared with other ratios have similar particle size and morphology, and will not be described in detail here.

[0047] Figure 2 The diagram shows the physicochemical properties of M-dCNs nanozymes. Figure 2 B- Figure 2 In C, Fe of M-dCNs 3+ The mass ratio of HST to KPLH1130 is 2:3:2. M-dCNs with different ratios have similar properties, which will not be elaborated upon here.

[0048] The stability of the above-mentioned M-dCNs suspension was tested by standing. The results showed that the M-dCNs nanozyme of the present invention can exist stably at room temperature. Figure 3 The graph shows the stability changes of M-dCNs nanozymes with a mass ratio of 2:3:2 after storage at 25°C for 60 h.

[0049] Elemental analysis of the M-dCNs nanozyme of this invention was performed using TEM-mapping, and the results are shown below. Figure 4 Fe in M-dCNs was determined using the o-phenanthroline spectrophotometric method. 3+ The contents of HST and KPLH1130 were determined by HPLC. The nanozyme synthesized at a mass ratio of 2:3:2 was centrifuged, the supernatant was discarded, and the nanozyme was resuspended in 1 mL of water. HPLC quantification showed that the concentration of HST dispersed in 1 mL of water was 138 mg / mL, the concentration of KPLH1130 was 11 mg / mL, and the Fe content was... 3+ The concentration was 12 mg / mL. For better comparison, the results listed in the following activity test examples are all for M-dCNs nanozymes prepared with a mass ratio of 2:3:2. M-dCNs nanozymes prepared with other ratios have similar activities, which will not be described in detail.

[0050] Example 2: •DPPH radical scavenging ability of M-dCNs nanozymes

[0051] (1) Grouping: M-dCNs nanozyme group, M-dCNs nanozyme was dispersed in methanol at a concentration of 138 mg / mL HST; and Fe was dispersed separately at the same concentration. 3+ For comparison, Fe solutions (solvent: methanol), HST solutions (solvent: methanol), and KPLH1130 solutions (solvent: methanol) were used, and Fe solutions of the same concentration were compared. 3+ The comparison was performed using HST + KPLH1130 solution (this comparison group is a simple physical mixture of the three reagents, and no self-assembly phenomenon occurred at this component ratio).

[0052] (2) Prepare a 0.1 mM DPPH solution by mixing DPPH with methanol and storing it in the dark. Then, mix different test solutions with DPPH or methanol, shake well, and let stand at room temperature in the dark for 30 min. Measure the absorbance at 517 nm and substitute it into the scavenging rate formula R%=[A0-(A X -A x0 )] / A0*100% (A0 is the absorbance of the blank control solution, A X A represents the absorbance after adding the test solution. X0 The scavenging rate was calculated using the background absorbance of the test solution. The results showed that the M-dCNs nanozyme of this invention exhibits significantly enhanced •DPPH radical scavenging ability. Figure 5 This demonstrates the ability of M-dCNs nanozymes to scavenge •DPPH in vitro. The left figure shows Fe... 3+ The right figure shows the results of particles with a ratio of HST:KPLH1130 = 2:3:2; the right figure shows the results of M-dCNs nanozymes prepared with different iron ions in vitro to scavenge •DPPH.

[0053] Example 3: Hydroxyl radical scavenging ability of M-dCNs nanozymes

[0054] (1) Grouping is the same as in Example 2;

[0055] (2) Add 400 mL of FeSO4 (9 mM), 400 mL of salicylic acid ethanol solution (2 mM), and 500 mL of sample solution sequentially to a colorimetric tube. Finally, add 400 mL of 0.03% H2O2, shake well, and heat in a water bath at 37℃ for 15 min. Using methanol as a reference solvent, measure the absorbance of the reaction solution at 510 nm. Substitute the values ​​into the clearance rate calculation formula to calculate the clearance rate. The clearance rate calculation formula is R% = [A0 - (A...] X -A X0 ) / A0*100% (where A0 is the absorbance value of the blank control, A X To increase the absorbance of the sample, A X0 (The absorbance is the value without the addition of 0.03% H2O2 as a colorimetric reagent). The results show that the M-dCNs nanozyme of the present invention significantly enhances the hydroxyl radical scavenging ability. Figure 6 In vitro M-dCNs nanozyme (Fe 3+ The ratio of HST to KPLH1130 (2:3:2) indicates the ability to scavenge •OH. Other ratios yielded similar results for nanozymes, which will not be detailed here.

[0056] Example 4: Cellular uptake of M-dCNs nanozymes

[0057] To assess the uptake capacity of M-dCNs nanozymes by cells, AML12 cells and Kupffer cells were incubated with Cy5.5-labeled M-dCNs (16.10 mg / L). After 2, 4, and 8 h of treatment, cells were washed with PBS and stained with Hoechst for 30 min. Intracellular fluorescence was observed using a confocal laser scanning microscope (CLSM). Cells were also collected and analyzed by flow cytometry. The results showed that the M-dCNs nanozymes of this invention could be taken up intracellularly by AML12 cells and Kupffer cells.

[0058] Example 5: Cytotoxicity assay of M-dCNs nanozymes (MTT assay)

[0059] (1) Grouping is the same as in Example 2;

[0060] (2) First, AML12 cells and Kupffer cells were seeded in 96-well plates. The solutions for each group were diluted by half, and then the AML12 cells and Kupffer cells were incubated in solutions of varying concentrations. After 24 h, 20 mL of MTT was added to each well. After 4 h, the culture medium in each well was replaced with 150 mL of DMSO. After shaking for 15 min, the absorbance of the DMSO solution in each well was measured using a microplate reader. Finally, cell viability was calculated by comparing the absorbance difference between the treated and untreated cells. The results showed that the cell viability of the treated group was similar to that of the untreated group, indicating that the M-dCNs nanozyme of the present invention has low cytotoxicity.

[0061] Example 6: ROS detection of M-dCNs nanozymes

[0062] (1) Grouping is the same as in Example 2;

[0063] (2) AML12 cells and Kupffer cells were incubated with their respective solutions for 2 h, and then co-cultured with 1 mL of APAP for 8 h. After culture, the cells were washed three times with PBS and then co-incubated with the ROS reactive oxygen species detection green probe DCFH-DA for 0.5 h. Intracellular fluorescence was captured and quantified using CLSM. The experimental results showed that the M-dCNs nanozyme of the present invention has ROS scavenging activity.

[0064] Example 7: In vivo distribution of M-dCNs nanozymes

[0065] To assess the biodistribution of M-dCNs nanozymes, an acute liver injury model was established in mice by intraperitoneal injection of APAP (350 mg / kg). DIR-labeled M-dCNs nanozymes (322 mg / L) were then injected into mice via tail vein injection. In vivo imaging was performed on the mice at 0 h, 4 h, 8 h, 12 h, and 24 h post-injection. Mice were sacrificed at 24 h, and major organs were harvested for fluorescence imaging. Liver tissue sections were stained with CD11B and Transferrin to observe the co-localization of DIR-labeled M-dCNs with CD11B and Transferrin. Figure 7 The distribution of M-dCNs in major organs of mice is shown. Fluorescence imaging results demonstrate that the M-dCNs nanozyme of this invention effectively accumulates in the liver, and in vitro organ imaging results show that M-dCNs accumulates most in the liver, 138.42, 9.43, 15.05, and 60.33 times that in the heart, spleen, lung, and kidney, respectively. This indicates that the M-dCNs nanozyme of this invention exhibits liver-specific aggregation targeting.

[0066] Example 8: Hepatoprotective effect of M-dCNs nanozymes on early acetaminophen-induced liver injury (AILI)

[0067] (1) Grouping is the same as in Example 2;

[0068] (2) C57BL / 6J mice were randomly divided into 7 groups (n = 4 / group). Female C57BL / 6J mice treated with APAP (350 mg / kg) were used as an acute liver injury (ALI) model. Two hours before APAP treatment, mice were injected via the tail vein with HST (10 mg / kg) and Fe. 3+ (0.87 mg / kg), KPLH1130 (0.80 mg / kg), HST (10 mg / kg) + Fe 3+ (0.87 mg / kg) + KPLH1130 (0.80 mg / kg) or M-dCNs nanozyme (11.67 mg / kg). The administration volume is 300 µL, and the concentration is HST (0.67 mg / mL) + Fe. 3+ (0.06 mg / mL), KPLH1130 (0.05 mg / mL), HST (0.67 mg / mL) + Fe 3+ (0.06 mg / mL) + KPLH1130 (0.05 mg / mL) or M-dCNs nanozyme (0.78 mg / mL). Four untreated mice were used as negative controls.

[0069] Mice were sacrificed 24 hours after APAP injection. Whole blood was collected from the heart, centrifuged at 5000 rpm for 15 min, and serum was collected. Serum ALT and AST levels were measured using a kit. Mouse liver tissue was collected, fixed in 4% paraformaldehyde, and stained with hepatocytes and liver cells (H&E). The presence of hepatocyte necrosis, infiltration, and inflammation in mouse H&E sections was estimated using ImageJ software, and the area of ​​pathological regions was calculated. The collected liver tissue was stained with immunofluorescence, and intracellular fluorescence was detected and quantitatively analyzed using CLSM. Figures 8-9 This study investigated the early preventive effect of M-dCNs on early-onset AILI in mice. Based on the ability of M-dCNs to accumulate in the liver of mice, this example examined the early preventive effect of M-dCNs on early-onset AILI in AILI mice. Figure 8 A). H&E staining results show the degree of liver damage in tissues after different treatment groups ( Figure 8 B). Notably, compared to the control group, AILI mice exhibited severe pathological areas in their liver tissue, with the pathological area reaching 26.85%, indicating the successful establishment of the APAP-induced liver injury model. Furthermore, the liver injury in pre-treated mice was alleviated to varying degrees. Importantly, the pathological area in the liver tissue of the M-dCNs group was significantly reduced to 6.99%, as shown in Figure 1. Figure 8 As shown in C. The plasma ALT and AST levels in each group of mice also showed similar trends. The ALT level in the APAP + M-dCNs group decreased by 43.74% compared to the APAP group, and the AST level in the APAP + M-dCNs group decreased by 34.78% compared to the APAP group. Figure 8 D, Figure 8 E). Notably, this invention used immunofluorescence staining to observe the expression of high-mobility group box 1 (HMGB1), a typical type of damage-associated molecular pattern (DAMP) released by necrotic hepatocytes, in liver tissues after treatment with different groups. The results showed that after APAP treatment, HMGB1 translocated from the nucleus to the cytoplasm, while in the presence of M-dCNs, HMGB1 efflux was inhibited. HMGB1 expression in the model group was mainly concentrated in the cytoplasm, rather than accumulating in the nucleus as in the control group. Surprisingly, similar to the control group, HMGB1 expression in the M-dCNs group appeared in the nucleus (…). Figure 9The results indicate that M-dCNs successfully inhibited APAP-induced HMGB1 secretion in hepatocytes. Tumor necrosis factor-alpha (TNF-α), IL-1β, and iNOS are phenotypic markers for macrophage M1, while IL-10 and CD206 are phenotypic markers for macrophage M2. Furthermore, APAP activates the cytochrome P450 enzyme Cyp2e1 during metabolism, leading to oxidative stress and inflammatory responses, accelerating liver injury. Nicotinamide adenine dinucleotide phosphate oxidase 2Nox2 can induce hepatic endocrine imbalance by promoting ROS production, leading to organelle dysfunction. Therefore, to evaluate the role of M-dCNs in regulating the inflammatory response in early AILI in vivo, this embodiment detected the inflammatory factor TNF-α (TNF-α) by immunofluorescence staining. Figure 8 F), the pro-oxidative stress gene Nox2 ( Figure 9 I), the pro-inflammatory cytokine interleukin-1β (IL-1β) Figure 9 H), anti-inflammatory cytokine interleukin-10 (IL-10) Figure 9 K), transforming growth factor-β1 (TGF-β1) Figure 9 L) and Mannose Receptor (CD206) Figure 9 The expression of TNF-α, Nox2, and IL-1β was reduced by 49.12%, 21.14%, and 44.90%, respectively, after M-dCNs treatment compared to the APAP treatment group (induced AILI). Furthermore, the expression levels of anti-inflammatory cytokines were significantly increased after M-dCNs treatment, with IL-10 and TGF-β1 expression levels being 1.91-fold and 2.34-fold higher than those in the APAP group, respectively. Compared to the control group, CD206 expression was downregulated by 60.01% in the model group, and the CD206 fluorescence intensity in the M-dCNs group recovered to a level similar to the control group compared to the APAP group. These results demonstrate the preventive effect of M-dCNs on early AILI.

[0070] Example 9: Therapeutic effect of M-dCNs nanozymes on advanced AILI

[0071] (1) Grouping is the same as in Example 2;

[0072] (2) Female C57BL / 6J mice treated with APAP (350 mg / kg) were used as an acute liver injury (ALI) model. The C57BL / 6 mice were randomly divided into 7 groups (n = 4 / group). The ALI model was established by intraperitoneal injection of APAP (350 mg / kg). Three hours after APAP induction, the mice were injected via the tail vein with HST (10 mg / kg) and Fe... 3+(0.87 mg / kg), KPLH1130 (0.80 mg / kg), HST (10 mg / kg) + Fe 3+ (0.87 mg / kg) + KPLH1130 (0.80 mg / kg) or M-dCNs nanozyme (11.67 mg / kg). The administration volume is 300 µL, and the dosage is HST (0.67 mg / mL) and Fe... 3+ (0.06mg / mL), KPLH1130 (0.05 mg / mL), HST (0.67 mg / mL) + Fe 3+ APAP (0.06 mg / mL) + KPLH1130 (0.05 mg / mL) or M-dCNs nanozyme (0.78 mg / mL). Four untreated mice served as negative controls. Mice were sacrificed 24 h after APAP injection. Whole blood was collected from the heart and centrifuged at 5000 rpm for 15 min to obtain serum. Serum ALT and AST were measured using a kit. Mouse liver tissue was collected, fixed with 4% paraformaldehyde, and stained with H&E. The degree of hepatocyte necrosis, infiltration, and inflammation in mouse H&E sections was estimated using ImageJ software, and the pathological area was calculated. The collected liver tissue was stained with immunofluorescence, and intracellular fluorescence was detected and calculated using CLSM. Figure 10 This illustrates the late-stage therapeutic effect of M-dCNs in DILI mice. The results show a schematic diagram of the M-dCNs treatment regimen in mice with late-stage liver injury. Figure 10 A). H&E staining images showed that severe liver lesions in mice with advanced liver injury were significantly reduced after treatment with M-dCNs (A). Figure 10 B and C). Compared with the APAP control group, M-dCNs treatment reduced ALT and AST levels by 63.05% and 58.05%, respectively. Figure 10 DE). In M-dCNs-treated mice with advanced liver injury, apap-induced HMGB1 cytoplasmic translocation was inhibited ( Figure 10 F). After treatment with M-dCNs, the expression of pro-inflammatory factors (TNF-α, IL-1β, Nox2) was downregulated and the expression of anti-inflammatory factors (CD206, IL-10, TGF-β1) was upregulated in mice with advanced liver injury. Figure 10 GL).

[0073] Example 10: Biosafety Assessment

[0074] (1) The grouping was the same as in Example 2, and the solvent was physiological saline;

[0075] (2) Female C57BL / 6 mice (18-20g) were randomly divided into 6 groups (n = 4 / group) and injected with HST (10mg / kg) and Fe via the tail vein, respectively.3+ (0.87 mg / kg), KPLH1130 (0.8 mg / kg), HST (10 mg / kg) + Fe 3+ (0.87 mg / kg) + KPLH1130 (0.8 mg / kg) or M-dCNs nanozyme (11.67 mg / kg). The administration volume was 300 µL, and the concentration was HST (0.67 mg / mL) + Fe 3+ (0.06 mg / mL), KPLH1130 (0.05 mg / mL), HST (0.67 mg / mL) + Fe 3+ (0.06 mg / mL) + KPLH1130 (0.05 mg / mL) or M-dCNs nanozyme (0.78 mg / mL). Mice were sacrificed 24 hours after injection, and whole blood was collected for biochemical analysis. Serum biochemical indicators ALT, AST, UREA, and UA levels were measured. H&E staining was used to observe damage to major organs such as the heart, liver, spleen, lungs, and kidneys. Figures 11-12 This study analyzed the biosafety of M-dCNs in vivo. In this example, H&E staining was used to evaluate the free drug HST (10 mg / kg) and Fe... 3+ (0.87 mg / kg), KPLH1130 (0.8 mg / kg), HST (10 mg / kg) + Fe 3+ Effects of (0.87 mg / kg) + KPLH1130 (0.8 mg / kg) or M-dCNs nanozyme (11.67 mg / kg) on ​​major organs such as heart, liver, spleen, lung, and kidney in AILI mice. Figure 11 Clearly, no pathological areas were observed in these major organs in any group of mice, indicating that these drugs have low systemic toxicity. More importantly, blood samples were collected from the mice, and blood biochemical indicators were tested (…). Figure 12 (AD). Hematological indicators remained at basically normal levels, indicating that M-dCNs nanozymes have good compatibility and great potential for in vivo application.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An M-dCNs nanozyme, characterized in that... The components include the following components by mass percentage: hesperidin 84–87%, KPLH1130 5–8%, Fe 3+ 5-8%; The M-dCNs nanozyme is prepared by the following steps: hesperidin is dissolved in DMSO to obtain a hesperidin solution, and soluble iron salt is dissolved in water to obtain Fe. 3+ KPLH1130 was dissolved in DMSO to obtain a KPLH1130 solution. The solution was stirred, mixed, and separated to obtain M-dCNs nanozyme. In the reaction system, the concentration of hesperidin is 0.1-3 mg / mL, and Fe... 3+ The concentration of [the substance] is 0.1-2 mg / mL, and the concentration of KPLH1130 is 0.1-3 mg / mL.

2. A method for preparing the M-dCNs nanoszyme of claim 1, characterized by comprising the steps of: mixing hesperetin, Fe 3+ solution, KPLH1130 under stirring, separating to obtain M-dCNs nanoszyme.

3. The method of claim 2, wherein: The concentration of hesperetin in the reaction system is 0.1-3 mg / mL, the concentration of Fe 3+ is 0.1-2 mg / mL, and the concentration of KPLH1130 is 0.1-3 mg / mL.

4. The preparation method according to claim 2, characterized in that: In the reaction system, Fe 3+ : Hesperitin: mass ratio of KPLH1130 is (1-20):(1-30):(1-30).

5. The method of claim 2, wherein: The Fe 3+ Fe in solution 3+ The concentration is 5-100 mg / mL.

6. The method of claim 2, wherein: The Fe 3+ The solution is obtained by dissolving a soluble iron salt in water.

7. The method of claim 6, wherein: The soluble iron salt is selected from at least one of ferric chloride and ferric sulfate.

8. The method of claim 2, wherein: The hesperidin is dissolved in DMSO to obtain a hesperidin solution, which is then used in the reaction; the KPLH1130 is dissolved in DMSO to obtain a KPLH1130 solution, which is then used in the reaction.

9. The method of claim 8, wherein: The concentration of the hesperidin solution is 3-100 mg / mL; the concentration of the KPLH1130 solution is 3-100 mg / mL.

10. The use of the M-dCNs nanozyme according to claim 1 in the preparation of drugs for the prevention and / or treatment of drug-induced liver injury.

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