A nano-preparation for treating ischemia-reperfusion injury and a preparation method and application thereof

Abstract

The application discloses a kind of nano preparation for treating ischemia-reperfusion injury and its preparation method and application, belong to the field of pharmaceutical preparation, the nano preparation includes active ingredient, albumin, liposome phospholipid bilayer and target head, active ingredient is composed of antioxidant and coenzyme Q10 and its analog, liposome phospholipid bilayer is composed of phospholipid, cholesterol and DSPE-mPEG 2000 Composition, the nano preparation can both remove active oxygen produced in cell and relieve intracellular oxidative stress, also can enter mitochondrial inner membrane and help mitochondrial respiratory chain electron transfer, repair damaged mitochondrial function, reduce the generation of active oxygen from root, so as to play the effect of treating both symptoms and root cause.The application proposes a kind of new strategy for treating ischemia-reperfusion injury related diseases, by removing active oxygen, effectively restoring blocked electron transport chain, improving the antioxidant capacity of cell itself, reducing oxidative damage caused by ferroptosis, with good practical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to a nano-preparation for treating ischemia reperfusion injury and a preparation method and application thereof. BACKGROUND

[0002] As the center of energy metabolism of the body, mitochondria have a closed structure of double-membrane nesting, and the inner membrane is concave inward to form a ridge, which is the main place for cells to perform oxidative phosphorylation and synthesize adenosine triphosphate (ATP). When the function of mitochondria is impaired, the mitochondrial membrane potential decreases, ATP synthesis decreases, respiratory chain enzyme activity decreases, and oxidative stress products accumulate, ultimately leading to cell apoptosis or death.

[0003] Ischemia reperfusion injury (IRI) refers to the condition that the degree of tissue damage rapidly increases after the blood flow (reperfusion) of the tissue cells suffering from ischemia for a certain period of time, which is often seen after organ transplantation and vascular recanalization. Many studies have shown that ischemia reperfusion injury is closely related to mitochondrial dysfunction. Due to the accumulation of a large amount of succinic acid during ischemia, oxygen enters the cell during reperfusion, which on the one hand leads to the enhancement of the reverse electron transport capacity of mitochondrial enzyme complex I, and the burst of reactive oxygen species (ROS); on the other hand, it damages the membrane system of mitochondria, hinders the forward electron transport, and causes mitochondrial ATP synthesis disorder. Oxidative stress caused by mitochondrial damage can further induce protein and lipid peroxidation, damage the permeability of the mitochondrial membrane, activate the apoptosis pathway, and ultimately lead to cell apoptosis.

[0004] Currently, the main clinical treatment methods for IRI are supportive therapy and replacement therapy, but the efficacy is not satisfactory. Some small molecule antioxidants have not been converted into routine clinical practice due to their low delivery efficiency, poor targeting and easy production of toxic side effects. The current main research direction for IRI is to reduce oxidative stress or relieve inflammation alone, and there is a lack of attention to damaged mitochondrial respiratory chain and oxidative phosphorylation, so the effect is often poor and it is difficult to achieve the effect of treating both the symptoms and the root cause.

[0005] In order to achieve the treatment of IRI, a nano-preparation capable of effectively targeting the damaged site, relieving intracellular oxidative stress, and repairing the mitochondrial respiratory chain is needed. The nano-preparation can treat IRI by removing a large amount of ROS that has been generated and repairing damaged mitochondria that continuously produce ROS. SUMMARY

[0006] The present application provides a nano-preparation for treating ischemia reperfusion injury and a preparation method and application thereof to solve the problems in the prior art.

[0007] To achieve the above object, the present application adopts the following scheme:

[0008] In the first aspect, the present application provides a nano-preparation for treating ischemia-reperfusion injury, comprising active ingredients, albumin, liposome phospholipid bilayer and target head, wherein the active ingredients are composed of antioxidants and coenzyme Q10 and its analogs, the liposome phospholipid bilayer is composed of phospholipids, cholesterol and DSPE-mPEG 2000 .

[0009] Preferably, the antioxidant is selected from Fe(II)-Chlorin e6 (FeCe6), Fe(III)-Chlorin e6, N-acetylcysteine, bilirubin, peroxidase or catalase.

[0010] Preferably, the coenzyme Q10 and its analogs are selected from coenzyme Q10 (CoQ10), idebenone, ubiquinone or decyl ubiquinone.

[0011] Preferably, the albumin is selected from human serum albumin, bovine serum albumin, ovalbumin or mouse serum albumin.

[0012] Preferably, the target head is selected from DSPE-PEG-Hyaluronic Acid (DSPE-PEG-HA), DSPE-PEG-Folic Acid (DSPE-PEG-FA), DSPE-PEG-Heparin or DSPE-PEG-Biotin.

[0013] Preferably, the phospholipid is selected from one or more of soybean lecithin, hydrogenated soybean phospholipid (HSPC), egg yolk lecithin, dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) and dimyristoyl phosphatidylcholine (DMPC).

[0014] Preferably, the mass ratio of the coenzyme Q10 and its analogs to the antioxidant is 1:0.5-1:10, the mass ratio of the phospholipid to DSPE-mPEG 2000 is 1:1-100:1, the mass ratio of the phospholipid to cholesterol is 10:1-50:1, the mass ratio of the albumin to the active ingredients is 1:1-20:1, and the mass ratio of the target head to the active ingredients is 1:1-1:10.

[0015] Preferably, the preparation method of the liposome phospholipid bilayer is any one or several of the thin film dispersion method, the solvent injection method, the direct titration method or the solvent exchange method.

[0016] In the second aspect, the present application provides a preparation method of a nano-preparation for treating ischemia-reperfusion injury, comprising the following steps:

[0017] (1) Preparation of the nanocore: the organic solution of the antioxidant and the organic solution of coenzyme Q10 and its analogues are mixed thoroughly, and the mixed solution is added dropwise into the phosphate buffer solution of albumin under the condition of stirring at 300 rpm, and a stable nanocore is formed spontaneously;

[0018] (2) Grafting of the target head: the prepared nanocore is added dropwise into the phosphate buffer solution of the target head under the condition of stirring at 400 rpm, and a nanocore with specific targeting is formed;

[0019] (3) Preparation of the nanofomulation: phospholipid, cholesterol and DSPE-mPEG 2000 are dissolved in an organic solvent, rotary evaporation is performed to form a film, vacuum drying is performed overnight, the nanocore with specific targeting is added to the hydrated lipid film, a lipid suspension is obtained, and the final nanofomulation is obtained after centrifugation of the lipid suspension after extrusion by a liposome extruder.

[0020] Preferably, the concentration of the organic solution of the antioxidant is controlled to be 1-25 mg / mL.

[0021] Preferably, the concentration of the organic solution of coenzyme Q10 and its analogues is controlled to be 1-40 mg / mL.

[0022] Preferably, the concentration of the phosphate solution of human serum albumin is controlled to be 0.5-30 mg / mL.

[0023] Preferably, the organic solvent is selected from any one or more of dimethyl sulfoxide, N,N-dimethylformamide, chloroform, dichloromethane, methanol, ethanol, acetonitrile, and acetone.

[0024] In a third aspect, the application provides a use of the nanofomulation in the preparation of a drug for treating ischemia-reperfusion injury.

[0025] The application has the following beneficial effects: unlike single treatment methods such as relieving oxidative stress, inhibiting inflammatory environment, or directly inhibiting ferroptosis, the application first proposes a new strategy of double-drug combination for treating ischemia-reperfusion injury. On the one hand, oxidative stress can be relieved, and on the other hand, damaged mitochondrial function can be repaired, and the blocked electron transport chain can be restored, thereby reducing the generation of ROS from the root, breaking the vicious cycle of ferroptosis caused by mitochondrial dysfunction, and thus achieving a cure-all effect on ischemia-reperfusion injury. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The particle size distribution and the image under transmission electron microscope (A) and the stability (C) of the nanofomulation prepared in the examples are shown, and the potential comparison diagram (B) of the nanofomulation prepared in the examples and the comparative examples is shown.

[0027] Figure 2 The survival of cells after co-incubation with different nano-preparations after rotenone-induced mitochondrial damage in HK2 cells, intracellular and mitochondrial reactive oxygen species, and intracellular ATP production (A is the cell Calcein AM / PI staining result graph, B is the intracellular DCFH-DA staining quantitative result, C is the intracellular MitoSOX Red staining quantitative result, D is the intracellular ATP level quantitative result).

[0028] Figure 3 The accumulation of lipid peroxides in cells and 4-HNE content determination results after co-incubation with different nano-preparations after rotenone-induced mitochondrial damage in HK2 cells (A is the cell BODIPY 581 / 591C11 staining result graph, B is the intracellular MDA level quantitative result, C is the intracellular 4-HNE level quantitative result).

[0029] Figure 4 H&E staining, EDU staining and TUNEL staining of ischemia-reperfusion kidney tissues of mice after tail vein injection of different nano-preparations after the construction of mouse renal ischemia-reperfusion injury model (A is the tissue H&E staining result, B is the tissue EDU proliferation staining result, C is the tissue TUNEL apoptosis staining result).

[0030] Figure 5 ATP content, lactic acid content and MDA content in ischemia-reperfusion kidney tissues of mice after tail vein injection of different nano-preparations after the construction of mouse renal ischemia-reperfusion injury model (A is the quantitative result of ATP content in the tissue, B is the quantitative result of lactic acid content in the tissue, Figure 5 C is the quantitative result of MDA content in the tissue).

[0031] All data are shown as mean ± standard deviation. In order to compare the significant differences between experimental data, unpaired two-tailed t test was used for two-group comparison, and least significant difference (LSD) one-way analysis of variance (ANOVA) was used for multiple comparisons. All tests are bilateral, p>0.05 indicates not significant (ns), P value <0.05 is considered statistically significant (*0.01 DETAILED DESCRIPTION

[0032] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention. The invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] Prepare a 20 mg / mL FeCe6 dimethyl sulfoxide solution, a 20 mg / mL IDE dimethyl sulfoxide solution, and a 3 mg / mL human serum albumin phosphate buffer solution. Thoroughly mix the FeCe6 dimethyl sulfoxide solution and the IDE dimethyl sulfoxide solution, with a FeCe6 to IDE mass ratio of 1.5:1. At 25°C and 300 rpm, add the mixed solution dropwise to the human serum albumin phosphate buffer solution, where the human serum albumin to active ingredient mass ratio is 5:1. During the dropwise addition, IDFC NPs spontaneously form.

[0035] A DSPE-PEG-HA PBS solution with a concentration of 1 mg / mL was prepared and added dropwise to IDFC NPs at 25 °C and 400 rpm. The mass ratio of DSPE-PEG-HA to the active ingredient was 1:6.25, thus obtaining targeted nanocore HA-IDFC NPs.

[0036] Weigh out 9.8 mg of myristoyl phosphatidylcholine, 1.4 mg of cholesterol, and 1.4 mg of DSPE-mPEG. 2000 Dissolved in 1.33 mL of chloroform, the solution was added to a 25 mL round-bottom flask. The organic solvent was evaporated to dryness using a rotary evaporator until a uniform and transparent film was formed. HA-IDFC NPs were added and hydrated for 30 min. After hydration, the film was extruded using a liposome extruder. The unencapsulated drug was removed by centrifugation at 5000 rpm for 5 min, yielding liposome nanoformulation 1 (IDFC@Lip-HA).

[0037] Comparative Example 1

[0038] Using FeCe6 as the drug component, FeCe6 dimethyl sulfoxide solution was added dropwise to a phosphate buffer solution of human serum albumin at 25°C and 300 rpm, with the mass ratio of human serum albumin to active ingredient being 5:1. During the dropwise addition process, FeCe6 nano-cores (NPs) could be spontaneously formed.

[0039] A solution of DSPE-PEG-HA PBS with a concentration of 1 mg / mL was prepared, and the solution was added dropwise to the nanocore at 25°C and 400 rpm, with the mass ratio of DSPE-PEG-HA to active ingredient being 1:6.25, to obtain the targeted nanocore HA-FeCe6 NPs;

[0040] 9.8 mg of dimyristoyl phosphatidylcholine, 1.4 mg of cholesterol, and 1.4 mg of DSPE-mPEG 2000 were weighed, dissolved in 1.33 mL of chloroform, added to a 25 mL round-bottom flask, and the organic solvent was spun dry by a rotary evaporator to form a uniform transparent film. The targeted nanocore was hydrated for 30 min, and after hydration, the film was passed through a liposome extruder. Unwrapped drugs were removed by centrifugation at 5000 rpm / min for 5 min to obtain the liposome nanoscale preparation 2 (FeCe6@Lip-HA).

[0041] Comparative Example 2

[0042] IDE dimethyl sulfoxide solution was added dropwise to a human serum albumin phosphate buffer solution at 25°C and 300 rpm, with the mass ratio of human serum albumin to active ingredient being 5:1, and the nanocore IDE NPs were spontaneously formed during the addition process.

[0043] A solution of DSPE-PEG-HA PBS with a concentration of 1 mg / mL was prepared, and the solution was added dropwise to the nanocore at 25°C and 400 rpm, with the mass ratio of DSPE-PEG-HA to active ingredient being 1:6.25, to obtain the targeted nanocore HA-IDE NPs;

[0044] 9.8 mg of dimyristoyl phosphatidylcholine, 1.4 mg of cholesterol, and 1.4 mg of DSPE-mPEG 2000 were weighed, dissolved in 1.33 mL of chloroform, added to a 25 mL round-bottom flask, and the organic solvent was spun dry by a rotary evaporator to form a uniform transparent film. The targeted nanocore was hydrated for 30 min, and after hydration, the film was passed through a liposome extruder. Unwrapped drugs were removed by centrifugation at 5000 rpm / min for 5 min to obtain the liposome nanoscale preparation 3 (IDE@Lip-HA).

[0045] Figure 1 The characterization results of the liposome nanoscale preparations prepared in Example 1 and Comparative Examples 1-2 are shown in Table 1. Figure 1 As shown in Table 1 and FIG. A, the dynamic light scattering particle size of IDFC@Lip-HA was 181.97 ± 3.10 nm, and the transmission electron microscopy result was a uniform spherical shape.Figure 1 C is the 7-day stability of IDFC@Lip-HA in glucose solution, and the results show that it can exist stably in glucose solution for 7 days, indicating that IDFC@Lip-HA still has good stability under physiological conditions. Figure 1 B is the potential results of the three nano-preparations, and the potentials of the three nano-preparations are between -10 mV and 0 mV.

[0046] Experimental Example 1

[0047] Calcein AM as a kind of live cell detection reagent, after entering the cell, it is hydrolyzed into Calcein by esterase in the live cell and stays in the cell, emitting strong green fluorescence. PI is a commonly used nuclear fluorescent dye, which cannot penetrate the cell membrane of the live cell, so it can only stain dead cells with damaged cell membranes. Calcein AM is usually used in combination with PI to simultaneously perform double fluorescence staining on live cells and dead cells, and the cell activity and cytotoxicity are detected according to the fluorescence intensity. DCFH-DA as a kind of active oxygen detection reagent, has no fluorescence itself, can freely penetrate the cell membrane, and is hydrolyzed into DCFH by esterase in the cell and stored in the cell, which can be oxidized to strong green fluorescent substance DCF by intracellular active oxygen. Therefore, the intracellular ROS level can be quantified according to the fluorescence intensity. Mitochondria is the main place of ROS generation in cells, and its function is closely related to the content of ROS in mitochondria. MitSOX Red is a dihydroethidium derivative with a cationic triphenyl phosphate group. Dihydroethidium can be oxidized to ethidium by superoxide, and then combined with DNA to produce fluorescence. Therefore, the superoxide level in mitochondria can be quantified according to the fluorescence intensity. Mitochondria is the main place of ATP generation in cell material metabolism. ATP is generally considered to be the most important energy metabolism substance in organisms, which can provide power for life activities and participate in various life processes. Detecting the content of intracellular ATP can effectively measure the function of mitochondria. Rotenone (ROT) as an inhibitor of mitochondrial enzyme complex I can effectively block the normal operation of the mitochondrial respiratory chain, thereby simulating the damage to cells under the condition of ischemia-reperfusion. Human kidney proximal tubular cells (HK2) were selected as the research object, and the cells were evenly spread in a 6-well plate and placed in a culture box overnight. When the density reached 70-80%, the culture medium was discarded, and the groups were set as Control group, ROT group, ROT+IDE@Lip-HA group (hereinafter referred to as IDE@Lip-HA group), ROT+FeCe6@Lip-HA group (hereinafter referred to as FeCe6@Lip-HA group) and ROT+IDFC@Lip-HA group (hereinafter referred to as IDFC@Lip-HA group). Except for the Control group, the rest of the groups were first diluted with ROT dimethyl sulfoxide solution to 0.2 μM in serum-free MEM medium to damage the function of mitochondria. After 24 h, the drug solution was discarded, and the nano-preparation prepared in Example 1, Comparative Example 1 and Comparative Example 2 was diluted with serum-free MEM medium to different concentrations, 1 mL of the diluted drug solution was added to each well, and the culture was continued for 6 h. The Control group was incubated with serum-free MEM medium. Then DCFH-DA and MitSOX Red staining were performed, and the fluorescence intensity of the cells after staining in each group was observed by laser confocal microscope and photographed.The above administration operation was repeated, the cells were collected and fully lysed, and the ATP content in the cells was determined using an enhanced ATP content determination kit.

[0048] Figure 2 The results of intracellular live and dead staining, intracellular reactive oxygen species level, mitochondrial superoxide level and ATP level detection of HK2 cells after 6 hours of administration under rotenone injury condition are shown in Figures A-D. Figure 2 As shown in Figure A, after rotenone damaged mitochondria, the number of dead cells increased significantly, and the administration of IDFC@Lip-HA could effectively restore mitochondrial function and increase the number of live cells. Figure 2 As shown in Figure B, after the HK2 cells were intervened by IDFC@Lip-HA, the fluorescence intensity of intracellular DCF decreased significantly, and the ROS level decreased. Figure 2 As shown in Figure C, after the HK2 cells were intervened by IDFC@Lip-HA, the mitochondrial superoxide level decreased significantly, which could effectively alleviate mitochondrial oxidative stress. Figure 2 D is the quantitative result of intracellular ATP level. After rotenone induced the decrease of mitochondrial enzyme complex I activity of HK2 cells, the ATP generation decreased significantly. After the intervention of IDFC@Lip-HA, the intracellular ATP content increased significantly, and the mitochondrial function was effectively improved.

[0049] Experimental Example 2

[0050] BODIPY 581 / 591C11 is a fluorescent probe for detecting lipid peroxidation and antioxidant capacity in living cells. The reduced product of BODIPY 581 / 591C11 has a maximum excitation wavelength of 581 nm and a maximum emission wavelength of 591 nm, mainly red fluorescence, at this time the red-green fluorescence ratio is large; after oxidation by lipid peroxidation (Lipid Peroxidation, LPO), the maximum excitation and emission wavelength shifts to about 488 / 510 nm, mainly green fluorescence, at this time the red-green fluorescence ratio decreases. Malondialdehyde (Malondialdehyde, MDA) as a degradation product of lipid peroxides, by detecting the level of MDA can effectively reflect the level of lipid peroxidation in cells. 4-Hydroxynonenal (4-Hydroxynonenal, 4-HNE) is a metabolite of lipid peroxides, which can undergo glycosylation with glutathione peroxidase 4 (Glutathione Peroxidase 4, GPX4), promote the ubiquitination and degradation of GPX4, and cause the occurrence of vicious cycle, aggravate cell damage. Select HK2 cells as the research object, evenly spread the cells in a 6-well plate, and place it in the incubator overnight. When the density reaches 70-80%, discard the culture medium, set up groups as Control group, ROT group, IDE@Lip-HA group, FeCe6@Lip-HA group and IDFC@Lip-HA group. Except for the Control group, the rest of the groups are first diluted with serum-free MEM medium ROT dimethyl sulfoxide solution to 0.2 μM, and damaged for 24 hours. After discarding the drug solution, use serum-free MEM medium to dilute the nanofactories prepared in Example 1, Comparative Example 1 and Comparative Example 2 to different concentrations, add 1 mL of diluted drug solution to each well, and continue to culture for 6 hours. The Control group is incubated with serum-free MEM medium. Then use BODIPY 581 / 591C11 for staining, and observe the fluorescence intensity of each group of cells after staining by laser confocal microscope, and take pictures. Repeat the above dosing operation, collect the cells and perform thorough lysis, and use the MDA detection kit to determine the MDA level in the cells. Repeat the above dosing operation, first wash twice with 1 mL of Hanks' balanced solution, then fix with 1 mL of 4% paraformaldehyde at room temperature for 30 min, then permeabilize with 1 mL of 0.2% Triton X100 for 3 min, then block with 5% horse serum at room temperature for 30 min, then incubate the 4-HNE antibody at 4°C on a shaker overnight, and finally incubate the fluorescently labeled secondary antibody (Alexa Fluor 647-labeled goat anti-mouse IgG) at 37°C in the dark. Observe the fluorescence intensity of each group of cells after staining by laser confocal microscope, and take pictures.

[0051] Figure 3A is the intracellular BODIPY 581 / 591C11 staining result map, the results show that after rotenone induces mitochondrial damage, the intracellular LPO level increases, and IDFC@Lip-HA can effectively reduce the production of LPO and maintain the normal level of lipid peroxidation. Figure 3 B is the quantitative result of intracellular MDA level, the results show that compared with the single drug nano preparation group, the MDA level in the IDFC@Lip-HA group is reduced, which corresponds to the BODIPY 581 / 591C11 result. Figure 3 C is the quantitative result of intracellular 4-HNE level, as shown in the figure, after rotenone damages mitochondria, a large amount of LPO is generated by the oxidation of unsaturated phospholipids, and the metabolic product 4-HNE is significantly increased. After giving the final preparation, the LPO content in the damaged cells is reduced, the 4-HNE content is reduced, and the antioxidant capacity of the cells is improved.

[0052] Experimental Example 3

[0053] In order to verify the treatment effect of IDFC@Lip-HA on renal ischemia-reperfusion in vivo, Balb / c male mice were randomly divided into 5 groups, 6 in each group, and the groups included Sham (sham operation) group, IRI group, IRI+IDE@Lip-HA group (hereinafter referred to as IDE@Lip-HA group), IRI+FeCe6@Lip-HA group (hereinafter referred to as FeCe6@Lip-HA group) and IRI+IDFC@Lip-HA group (hereinafter referred to as IDFC@Lip-HA group). The specific modeling method of IRI is as follows: the skin of the back of the mouse is prepared, and after the mouse is anesthetized, the surgical scissors are used to make an incision at 1 cm from the spine on the left and right sides, the kidney is taken out, and the hemostatic forceps is used to clamp the renal blood vessels for 30 min, then the kidney is put back into the abdominal cavity, and the muscle layer and skin layer are sutured. When reperfusion, different preparations are given through the tail vein, and three days after modeling, the mice are euthanized, the kidney tissues of the mice are collected, washed with physiological saline, and fixed with 4% paraformaldehyde, and after 48 h of fixation, paraffin embedding and tissue sectioning are performed, hematoxylin and eosin are used for H&E staining to detect the morphological structure of cells, EDU reagent is used for click chemistry fluorescent staining to detect the cell proliferation ability, and TUNEL reagent is used for TUNEL staining to detect the cell apoptosis. Finally, the digital slice scanner is used to scan and analyze the slices, and the overall treatment effect of the preparation is comprehensively evaluated.

[0054] Figure 4 The results of tissue staining in vivo of renal ischemia-reperfusion mice are analyzed. Figure 4A is the result of H&E staining of the tissue, which shows that after ischemia-reperfusion injury, a large number of vacuolar degeneration of renal tubular cells (arrow), a large number of necrotic cell shedding cell fragments in the renal tubular lumen, forming cell and granular casts (triangle), and pyknosis of the epithelial cells (square) also appear. After treatment with the final preparation, the renal interstitial edema is improved, the vacuolization is reduced, the cell and granular casts are alleviated, and there is no interstitial edema, indicating that IDFC@Lip-HA can significantly improve the renal tubular cells and lumen and the microenvironment of the kidney after ischemia-reperfusion injury. Figure 4 B is the result of EDU proliferation staining of the tissue, which shows that after the kidney is subjected to ischemia-reperfusion injury, a large number of ROS destroy the endogenous DNA of the cells, and the cell proliferation ability is significantly inhibited. After the final preparation is given, the cell proliferation ability is greatly promoted, and the repair of the kidney ischemia-reperfusion injury is well promoted. Figure 4 C is the result of TUNEL apoptosis staining of the tissue, as shown in the figure, after the mouse experiences renal ischemia-reperfusion, a large number of cells in the kidney tissue undergo apoptosis, and the green fluorescence is significantly enhanced. After treatment with IDFC@Lip-HA, the oxidative stress of the cells is alleviated, and the degree of cell apoptosis is significantly inhibited.

[0055] Experimental Example 4

[0056] The mouse was subjected to renal ischemia-reperfusion injury model construction, and after grouping and treatment according to the above treatment scheme, the mouse was euthanized, the kidney tissue of the mouse was dissected, washed with physiological saline, and then frozen with liquid nitrogen, and stored at -80°C. An appropriate amount of animal tissue was fully lysed with lysis buffer, ground, and then the supernatant was taken for use. The ATP content, lactate content and MDA content of the tissue were detected according to the experimental scheme of the enhanced ATP detection kit, lactate detection kit and MDA detection kit.

[0057] Figure 5 The ATP content, lactate content and MDA content of the tissue in the kidney ischemia-reperfusion mouse in vivo were determined. Figure 5 A is the quantitative result of the ATP content in the tissue, as shown in the figure, after modeling, the cells are damaged, the mitochondrial function is impaired, and the ATP generation is decreased. After treatment with the final preparation IDFC@Lip-HA, the oxidative stress in the cells is alleviated, the mitochondrial function is significantly improved, and the ATP content is significantly increased. Figure 5B is the quantitative result of lactic acid content in the tissue. When the kidney experiences ischemia, the oxygen content in the tissue decreases, the oxygen required for cell oxidative phosphorylation is insufficient, the cell is partially converted from respiratory chain energy supply to glycolysis, and lactic acid is produced. During reperfusion, due to the damaged mitochondria in the cell, oxygen cannot produce ATP through the respiratory chain when oxygen is filled, resulting in the release of a large amount of ROS, and the energy production of the cell depends on glycolysis, and lactic acid continues to accumulate. After the final preparation IDFC@Lip-HA is given, the mitochondrial function is restored, and the lactic acid content is significantly reduced, and the respiratory chain is restored to normal. Figure 5 C is the quantitative result of MDA content in the tissue. After the mouse experiences ischemia-reperfusion, the MDA content in the tissue increases, and after treatment with the final preparation, the MDA content is significantly reduced, effectively avoiding the occurrence of ferroptosis.

[0058] The above uses specific examples to illustrate the present application, which is only used to help understand the present application and does not limit the present application. For those skilled in the art to which the present application belongs, according to the idea of the present application, a number of simple deductions, deformations or substitutions can be made.

Claims

1. A nano-formulation for treating ischemia-reperfusion injury, characterized in that, Comprising an active ingredient consisting of an antioxidant and coenzyme Q10 and its analogues, an albumin, a liposomal phospholipid bilayer consisting of phospholipids, cholesterol and DSPE-mPEG 2000 consisting of an antioxidant and coenzyme Q10 and its analogues, an albumin, a liposomal phospholipid bilayer consisting of phospholipids, cholesterol and DSPE-mPEG The antioxidant is selected from Fe(II)-Chlorin e6, Fe(III)-Chlorin e6, N-acetylcysteine, bilirubin, peroxidase or catalase; The coenzyme Q10 and its analogues are selected from coenzyme Q10, idebenone, mitomone, or decyl ubiquinone; The mass ratio of coenzyme Q10 and its analogues to antioxidants is 1:0.5 to 1:

10.

2. The nano-formulation for treating ischemia-reperfusion injury according to claim 1, characterized in that, The albumin is selected from human serum albumin, bovine serum albumin, ovalbumin or mouse serum albumin.

3. The nano-formulation for treating ischemia-reperfusion injury according to claim 1, characterized in that, The target head is selected from DSPE-PEG-Hyaluronic Acid, DSPE-PEG-Folic Acid, DSPE-PEG-Heparin, or DSPE-PEG-Biotin.

4. The nano-formulation for treating ischemia-reperfusion injury according to claim 1, characterized in that, The phospholipid is selected from one or more of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, and dimyristoylphosphatidylcholine.

5. The nano-formulation for treating ischemia-reperfusion injury according to claim 1, characterized in that, The phospholipid and the DSPE-mPEG 2000 The mass ratio of the phospholipid and the DSPE-mPEG is 1:1-100:1, the mass ratio of the phospholipid and the cholesterol is 10:1-50:1, the mass ratio of the albumin and the active ingredient is 1:1-20:1, and the mass ratio of the targeting head and the active ingredient is 1:1-1:

10.

6. The method for preparing the nano-formulation for treating ischemia-reperfusion injury according to any one of claims 1-5, characterized in that, The method includes the following steps: (1) Preparation of nanocores: The organic solution of antioxidant and the organic solution of coenzyme Q10 and its analogues are thoroughly mixed. The mixed solution is added dropwise to the albumin phosphate buffer solution under stirring at 300 rpm to spontaneously form stable nanocores. (2) Grafting of the target: The prepared nanocore is added dropwise with the phosphate buffer solution of the target under stirring at 400 rpm to form a nanocore with specific targeting properties; (3) Preparation of nano-formulations: Phospholipids, cholesterol and DSPE-mPEG are combined. 2000 Dissolved in an organic solvent, the lipid film is formed by rotary evaporation and vacuum dried overnight. A hydrated lipid film with a specific targeting nanocore is added to obtain a lipid suspension. The suspension is extruded through a liposome extruder and centrifuged to obtain the final nanoformulation.

7. The preparation method according to claim 6, characterized in that, The organic solvent is selected from any one or more of dimethyl sulfoxide, N,N-dimethylformamide, chloroform, dichloromethane, methanol, ethanol, acetonitrile, and acetone.

8. The use of the nanoformulation according to any one of claims 1-5 in the preparation of drugs for treating ischemia-reperfusion injury.

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