M-dCNs nano-enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of natural products, and discloses an M-dCNs nano-enzyme as well as a preparation method and application thereof. The M-dCNs nano enzyme disclosed by the invention is prepared from the following components in percentage by mass: 84 to 87 percent of hesperetin (HST), 5 to 8 percent of KPLH1130 and 5 to 8 percent of Fe < 3 + >. The metal-natural product complex is constructed, HST and Fe < 3 + > are complexed, a carrier does not need to be added or modification is carried out through a covalent bond, the nano-enzyme with uniform morphology can be formed through self-assembly, the particle size is small, the drug loading capacity reaches 100%, the nano-enzyme has high stability, dependence on the carrier is eliminated, limitation of the drug loading rate is avoided, the nano-enzyme is formed through self-assembly, and the drug loading efficiency is improved. According to the present invention, the HST activity is effectively improved, the aggregation targeting property of the liver is represented, the treatment effect on the liver injury is substantially improved, and the HST can be used for preparing the drugs for preventing and / or treating the liver injury.

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, metal-natural product complexes have attracted the attention of many researchers due to their effectiveness in preventing and treating various diseases. Metal ions can directly or indirectly activate the pharmacological effects of certain natural products. In addition, metal complexes can also change 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, the present application provides a M-dCNs nanoenzyme formed by self-assembly, thereby achieving the technical effects of effectively reducing oxidative stress and synergistically resisting inflammation. SUMMARY

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a M-dCNs nanoenzyme.

[0007] Another purpose of the present application is to provide a preparation method of the above-mentioned M-dCNs nanoenzyme.

[0008] Still another purpose of the present application is to provide the application of the above-mentioned M-dCNs nanoenzyme, particularly in the preparation of drugs for preventing and / or treating liver damage.

[0009] The purposes of the present application are achieved by the following solutions: A M-dCNs nanoenzyme, comprising the following components in mass percentage: 84-87 % of Hesperitin (HST), 5-8 % of KPLH1130, 5-8 % of Fe 3+ 5-8 %.

[0010] Further, the HST can be HST from dried tangerine or orange peel.

[0011] The present application constructs a metal-natural product complex, which is complexed with HST and Fe 3+ Without adding carriers or modifying by covalent bonds, the nanoenzyme with uniform morphology can be self-assembled, has a small particle size, an average particle size of less than or equal to 150 nm, a drug loading of 100 %, and high stability. Not only does it eliminate the dependence on carriers and avoid the limitation of drug loading rate, but also effectively improves the activity of HST and greatly improves the treatment effect on liver damage by self-assembling into a nanoenzyme.

[0012] The present application also provides a preparation method of the above-mentioned M-dCNs nanoenzyme, comprising the following steps: stirring Hesperitin (HST), Fe 3+ solution, and KPLH1130 to mix, separate, and obtain the M-dCNs nanoenzyme.

[0013] Further, in the reaction system, the concentration of HST can be 0.1-3 mg / mL, the concentration of Fe 3+The concentration of the Fe

[0014] Further, in the reaction system, the Fe 3+ The mass ratio of KPLH1130 to HST to Fe

[0015] Further, in the reagent used in the method, the Fe 3+ The concentration of the Fe 3+ may be 5-100 mg / mL.

[0016] Further, the Fe 3+ solution can be obtained by dissolving a soluble iron salt in water.

[0017] Further, the soluble iron salt can be selected from iron chloride, iron sulfate and the like; preferably iron chloride.

[0018] Further, in the reagent used in the method, the HST can be dissolved in DMSO to obtain an HST solution for use in the reaction.

[0019] Further, the concentration of the HST solution can be 3-100 mg / mL.

[0020] Further, in the reagent used in the method, the KPLH1130 can be dissolved in DMSO to obtain a KPLH1130 solution for use in the reaction.

[0021] Further, the concentration of the KPLH1130 solution can be 3-100 mg / mL.

[0022] Further, the stirring time can be 5-15 min. The stirring allows the components to be fully mixed and reacted.

[0023] Further, the separation can be achieved by conventional centrifugation and the like, for example, centrifugation at 10000-20000 rpm / min for 10-60 min, for example, centrifugation at 14000 rpm / min for 30 min.

[0024] Further, the obtained precipitate can be washed with water.

[0025] Further, the prepared M-dCNs nanoszyme can be dispersed in water to obtain an M-dCNs nanoszyme suspension.

[0026] 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.

[0027] 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

[0028] 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.

[0029] Figure 1 A comparison of particle sizes for M-dCNs nanozymes with different proportions.

[0030] Figure 2 The image shows the physicochemical properties of M-dCNs nanozymes.

[0031] Figure 3 The graph shows the stability changes of M-dCNs nanozymes after storage at 25°C for 60 h.

[0032] Figure 4 The elemental analysis spectrum of M-dCNs nanozymes.

[0033] Figure 5 The ability of M-dCNs nanozymes to scavenge •DPPH in vitro.

[0034] Figure 6 The ability of M-dCNs nanozymes to scavenge •OH in vitro.

[0035] Figure 7 The distribution of M-dCNs in major organs of mice.

[0036] Figures 8-9 To demonstrate the early preventive effect of M-dCNs in DILI mice.

[0037] Figure 10 To analyze the therapeutic effect of M-dCNs in DILI mice.

[0038] Figures 11-12 To analyze the biological safety of M-dCNs in vivo. DETAILED DESCRIPTION

[0039] The application will be further described in conjunction with the following examples, but the embodiments of the application are not limited thereto. The materials involved in the following examples can be obtained from commercial channels if no special instructions are given. The methods described are conventional methods if no special instructions are given.

[0040] The hesperetin (HST) used in the following examples can be purchased from a commercial product. The amount of each component is calculated in mass parts, volume parts, mg, and mL.

[0041] Example 1: Preparation of M-dCNs nanoscale enzyme HST was dissolved in DMSO to obtain a HST solution, ferric chloride was configured into an aqueous solution, and KPLH1130 was dissolved in DMSO to obtain a KPLH1130 solution; According to the set ratio of HST: KPLH1130, the above solutions were mixed under stirring, stirred for 15 min, collected, centrifuged at 14000 rpm / min for 30 min, the supernatant was discarded, and the precipitate was resuspended with distilled water to obtain a M-dCNs suspension. 3+ : HST: KPLH1130, the above solutions were mixed under stirring, stirred for 15 min, collected, centrifuged at 14000 rpm / min for 30 min, the supernatant was discarded, and the precipitate was resuspended with distilled water to obtain a M-dCNs suspension.

[0042] M-dCNs nanoscale enzymes were prepared with different ratios of Fe 3+ : HST: KPLH1130, the above solutions were mixed under stirring, stirred for 15 min, collected, centrifuged at 14000 rpm / min for 30 min, the supernatant was discarded, and the precipitate was resuspended with distilled water to obtain a M-dCNs suspension.

[0043] The morphology of the prepared M-dCNs was observed under a transmission electron microscope at 80 kv, and the particle size and PDI were determined using a Malvern particle size analyzer, and the results are shown in Figure 1 It can be seen from the figure that the M-dCNs of the application is a particle with an average particle size of less than 150 nm, and the morphology is uniform, which is a circular particle. Figure 1The 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 2: •DPPH radical scavenging ability of M-dCNs nanozymes (1) Grouping: M-dCNs nanozyme group, M-dCNs nanozyme was dispersed in methanol at a concentration of 138 mg / mL based on 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). (2) 0.1 mM DPPH solution was prepared by mixing DPPH with methanol and placed in the dark. Then, different test solutions were mixed with DPPH or methanol, shaken and placed in the dark at room temperature for 30 min. The absorbance was measured at 517 nm, and the clearance rate was calculated by substituting the clearance rate formula R% = [A0- (A X -A x0 )] / A0*100% (A0 is the absorbance of the blank control solution, A X is the absorbance after adding the test solution, and A X0 is the background absorption value of the test solution). The results show that the M-dCNs nanoscale enzyme has significantly improved DPPH free radical scavenging capacity. Figure 5 is the in vitro DPPH scavenging capacity of M-dCNs nanoscale enzyme. The left graph is the result of Fe 3+ : HST: KPLH1130 = 2:3:2 particles; the right graph is the in vitro DPPH scavenging capacity of M-dCNs nanoscale enzyme prepared by different iron ions.

[0048] Example 3: Hydroxyl radical scavenging capacity of M-dCNs nanoscale enzyme (1) Grouping is the same as in Example 2; (2) 400 mL FeSO4 (9 mM), 400 mL salicylic acid ethanol solution (2 mM), 500 mL sample solution were sequentially added in a colorimetric tube, and finally 400 mL 0.03% H2O2 was added, shaken, heated in a 37°C water bath for 15 min, and the absorbance of the reaction solution at 510 nm was measured with methanol as the reference. The clearance rate was calculated by substituting the clearance rate calculation formula. The clearance rate calculation formula is R% = [A0- (A X -A X0 )] / A0*100% (wherein A0 is the absorbance of the blank control, A X is the absorbance of the sample, and A X0 is the absorbance without the color developing agent 0.03% H2O2). The results show that the M-dCNs nanoscale enzyme has significantly improved hydroxyl radical scavenging capacity. Figure 6 is the in vitro OH scavenging capacity of M-dCNs nanoscale enzyme (Fe 3+ : HST: KPLH1130 = 2:3:2). The results of nanoscale enzymes with other proportions are similar and are not described here.

[0049] Example 4: Uptake of M-dCNs nanoscale enzyme by cells To evaluate the uptake ability of cells to M-dCNs nanoszymes, AML12 cells and Kupffer cells were incubated with Cy5.5-labeled M-dCNs (16.10 mg / L) respectively. After 2, 4, 8 h of treatment, the cells were washed with PBS and stained with Hoechst for 30 min, and the intracellular fluorescence was observed by confocal laser scanning microscopy (CLSM). In addition, the cells were collected for analysis by flow cytometry. The results showed that the M-dCNs nanoszymes of the application could be taken up into the cells by AML12 cells and Kupffer cells.

[0050] Example 5: Cell toxicity experiment (MTT experiment) of M-dCNs nanoszymes (1) Grouping is the same as in Example 2; (2) First, AML12 cells and Kupffer cells were seeded in a 96-well plate, and each group of solutions was diluted by half, then the AML12 cells and Kupffer cells were incubated in the gradient concentration of the solution. After 24 h, 20 mL of MTT was added to each well, and after 4 h, the culture medium in each well was replaced with 150 mL of DMSO, and after shaking for 15 min, the absorbance of the DMSO solution in the well was determined by an enzyme-labeled instrument. Finally, the cell viability was calculated by comparing the difference in absorbance between the dosed cells and the undosed cells. The results showed that the cell viability of the dosed group was similar to that of the undosed group, indicating that the M-dCNs nanoszymes of the application had low cytotoxicity.

[0051] Example 6: ROS detection of M-dCNs nanoszymes (1) Grouping is the same as in Example 2; (2) AML12 cells and Kupffer cells were incubated with each group of solutions for 2 h, and then co-cultured with 1 mL of APAP for 8 h. After the culture was completed, the cells were washed with PBS three times, and then co-incubated with the ROS active oxygen detection green probe DCFH-DA for 0.5 h. The intracellular fluorescence was photographed and quantified by CLSM. The experimental results showed that the M-dCNs nanoszymes of the application had ROS scavenging activity.

[0052] Example 7: In vivo distribution of M-dCNs nanoszymes To evaluate the biodistribution of M-dCNs nanosomes, the acute liver injury model of mice was established by intraperitoneal injection of APAP (350 mg / kg), and then the DIR-labeled M-dCNs nanosomes (322 mg / L) were injected into the mice through the tail vein. The small animals were imaged by a small animal living imager at 0 h, 4 h, 8 h, 12 h and 24 h after injection. The mice were sacrificed at 24 h, and the main organs were obtained for fluorescence imaging. The liver tissue sections were subjected to CD11B and Transferrin fluorescence staining to observe the colocalization of DIR-labeled M-dCNs with CD11B and Transferrin. Figure 7 The distribution of M-dCNs in the main organs of mice was shown in the fluorescence imaging results. The M-dCNs nanosomes of the application effectively accumulated in the liver, and the accumulation of M-dCNs in the liver was the most, which was 138.42, 9.43, 15.05 and 60.33 times of that in the heart, spleen, lung and kidney, respectively. That is, the M-dCNs nanosomes of the application showed liver aggregation targeting.

[0053] Example 8: Hepatoprotective effect of M-dCNs nanosomes on early acetaminophen-induced liver injury (AILI) (1) Grouping is the same as in Example 2; (2) C57BL / 6J mice were randomly divided into 7 groups (n = 4 / group), and C57BL / 6J female mice treated with APAP (350 mg / kg) were used as acute liver injury (ALI) models. Two hours before APAP treatment, mice were injected with HST (10 mg / kg), 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 nanosomes (11.67 mg / kg) through the tail vein. The volume of administration was 300 μL, and the concentration of administration 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 nanosomes (0.78 mg / mL). Four untreated mice were used as negative controls.

[0054] Mice were sacrificed 24 h after APAP injection. The whole blood was collected from the heart and centrifuged at 5000 rpm / min for 15 min to obtain serum. The serum ALT and AST levels were detected by kits. The liver tissues were collected and fixed with 4% paraformaldehyde for H&E staining. The necrosis, infiltration and inflammation of hepatocytes in the H&E sections of mice were estimated by using the software image J, and the pathological area was calculated. The collected liver tissues were subjected to immunofluorescence staining, and the intracellular fluorescence was detected and quantitatively analyzed by CLSM. Figures 8-9 To investigate the early prevention effect of M-dCNs on DILI in mice. Based on the ability of M-dCNs to accumulate in the liver of mice, this embodiment investigated the early prevention effect of M-dCNs on early AILI in AILI mice. Figure 8 A). The H&E staining results showed the degree of liver injury of the tissues after treatment in different groups Figure 8 B). It is worth noting that compared with the control group of mice, the liver tissues of AILI mice showed severe pathological areas, and the pathological area reached 26.85%, indicating that the APAP-induced liver injury model was successfully constructed. In addition, the liver injury of the pre-administration mice was relieved to varying degrees. Importantly, the pathological area of the liver tissues of the M-dCNs group mice was significantly reduced to 6.99%, as shown in Figure 8 C. The ALT and AST levels in the plasma of mice in each group also showed similar trends. The ALT level of the APAP + M-dCNs group decreased by 43.74% compared with the APAP group, and the AST level of the APAP + M-dCNs group decreased by 34.78% compared with the APAP group Figure 8 D, Figure 8 E). It is worth noting that the expression of high mobility group protein 1 (HMGB1), a typical type of damage-associated molecular pattern (DAMP) released by necrotic hepatocytes, in the liver tissues after treatment in different groups was observed by immunofluorescence staining. The results showed that after APAP treatment, HMGB1 translocated from the nucleus to the cytoplasm, while in the presence of M-dCNs, the efflux of HMGB1 was inhibited. The expression of HMGB1 in the model group was mainly concentrated in the cytoplasm, rather than in the nucleus as in the control group. Surprisingly, similar to the control group (control), the expression of HMGB1 in the M-dCNs group appeared in the nucleus Figure 9G), indicating that M-dCNs successfully inhibited the secretion of HMGB1 in liver tissue cells induced by APAP. Since Tumor necrosis factor-alpha (TNF-a), IL-1β and iNOS are markers of macrophage M1 phenotype, and IL-10 and CD206 are markers of macrophage M2 phenotype. In addition, APAP activates cytochrome P450 enzyme Cyp2e1 in the metabolic process, leading to oxidative stress and inflammatory response, accelerating the development of liver injury. Nicotinamide adenine dinucleotide phosphate oxidase 2 Nox2 can induce imbalance of liver internal environment by promoting the production of ROS, leading to dysfunction of organelles. Therefore, in order to evaluate the role of M-dCNs in regulating the inflammatory response produced by early AILI in vivo, this embodiment detects the expression of inflammatory factors TNF-a Figure 8 F), pro-oxidative stress gene Nox2 Figure 9 I), 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 J) by immunofluorescence staining. Compared with the APAP treatment group (inducing AILI), the levels of TNF-a, Nox2 and IL-1β were reduced by 49.12%, 21.14% and 44.90%, respectively, after M-dCNs treatment. In addition, the expression levels of anti-inflammatory cytokines were significantly increased after M-dCNs treatment, among which the expression levels of IL-10 and TGF-β1 were 1.91 times and 2.34 times that of the APAP group, respectively. Compared with the control group, the expression of CD206 in the model group was down-regulated by 60.01%, and compared with the APAP group, the fluorescence intensity of CD206 in the M-dCNs group was restored to the control group. These results reflect the preventive effect of M-dCNs on early AILI.

[0055] Example 9: Therapeutic effect of M-dCNs nanosomes on late AILI (1) The grouping is the same as that in Example 2; (2) Female C57BL / 6J mice treated with APAP (350 mg / kg) were used as an acute liver injury (ALI) model, and C57BL / 6 mice were randomly divided into 7 groups (n = 4 / group). The mice were intraperitoneally injected with APAP (350 mg / kg) to establish an acute liver injury (ALI) model. After 3 h of APAP induction, the mice were injected with HST (10 mg / kg), 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+ (0.06mg / 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 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).

[0056] Example 10: Biosafety Assessment (1) The grouping was the same as in Example 2, and the solvent was physiological saline; (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 nanoszyme (11.67 mg / kg). The administration volume was 300 μL, and the administration 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 nanoszyme (0.78 mg / mL). After 24 h of injection, the mice were sacrificed and whole blood was taken for biochemical detection. The serum biochemical indicators ALT, AST, UREA and UA levels were detected. The damage to the main organs of mice such as heart, liver, spleen, lung and kidney was observed by H&E staining. Figures 11-12 The bio-safety of M-dCNs in vivo was analyzed. In this embodiment, H&E staining method was used to evaluate the effects of free drug HST (10 mg / kg), Fe 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 nanoszyme (11.67 mg / kg) on the main organs of AILI mice such as heart, liver, spleen, lung and kidney Figure 11 ). Obviously, no pathological areas were observed in these main organs of mice in each group, indicating that the systemic toxicity of these drugs was low. More importantly, blood samples of mice were collected, and blood biochemical indicators were detected Figure 12 A-D). Hematology indicators basically maintained at normal levels, indicating that M-dCNs nanoszyme had good compatibility and great potential for in vivo application.

[0057] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. A M-dCNs nanozyme, characterized in that comprising the following components in percent by mass: hesperitin 84-87 %, KPLH1130 5-8 %, Fe 3+ 5-8 %.

2. A method for preparing the M-dCNs nanoszyme according to 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 method of claim 2, wherein: 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 at least one of ferric chloride and ferric sulfate.

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

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

10. Use of the M-dCNs nano-enzyme of claim 1 in the preparation of a drug for preventing and / or treating liver injury.

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