Nano preparation for treating ischemia reperfusion injury as well as preparation method and application of nano preparation

Abstract

The invention discloses a nano preparation for treating ischemia reperfusion injury and a preparation method and application thereof, and belongs to the field of pharmaceutical preparations, the nano preparation comprises an active component, albumin, a liposome phospholipid bilayer and a target head, the active component is composed of an antioxidant and coenzyme Q10 and analogues thereof, and the target head is composed of a liposome phospholipid bilayer and a liposome phospholipid bilayer. The lipidosome phospholipid bilayer is composed of phospholipid, cholesterol and DSPE-mPEG2000, the nano preparation can remove active oxygen generated in cells to relieve intracellular oxidative stress, and can enter mitochondrial inner membranes to help mitochondrial respiratory chain electron transfer and repair damaged mitochondrial functions, generation of the active oxygen is reduced from the source, and the nano preparation can be used for treating the mitochondrial respiratory chain electron transfer and repairing the damaged mitochondrial function. Therefore, the effect of treating both symptoms and root causes is achieved. The invention provides a new strategy for treating ischemia-reperfusion injury related diseases, the oxidation resistance of cells is improved and oxidative injury caused by ferroptosis is reduced by removing active oxygen and effectively recovering a blocked electron transport chain, and the application has a relatively good practical application prospect.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to a nano preparation for treating ischemia-reperfusion injury, and a preparation method and application thereof. Background Art

[0002] As the center of energy metabolism in the body, mitochondria have a closed structure with double membranes. The inner membrane is concave inward to form ridges. It is the main place for cells to carry out oxidative phosphorylation and synthesize adenosine triphosphate (ATP). When mitochondrial function is impaired, the mitochondrial membrane potential decreases, ATP synthesis decreases, the activity of respiratory chain enzymes decreases, and oxidative stress products accumulate, eventually leading to cell apoptosis or death.

[0003] Ischemia reperfusion injury (IRI) refers to the situation in which the degree of tissue damage increases rapidly after the blood flow (reperfusion) is restored to tissue cells that have suffered a certain period of ischemia. It is more common 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 succinate during ischemia, oxygen enters the cells during reperfusion, which, on the one hand, leads to the enhancement of the reverse electron transport capacity of mitochondrial enzyme complex I and the outbreak of reactive oxygen species (ROS); on the other hand, it damages the mitochondrial membrane system, hinders the forward electron transport, and causes mitochondrial ATP synthesis disorders. The oxidative stress caused by mitochondrial damage will further induce protein and lipid peroxidation, impair the permeability of the mitochondrial membrane, activate the apoptotic pathway, and ultimately lead to cell apoptosis.

[0004] At present, the clinical treatments for IRI mainly include supportive therapy and replacement therapy, but the efficacy is not satisfactory. Some small molecule antioxidants have not yet been transformed into routine clinical practice due to their low delivery efficiency, poor targeting and easy toxic side effects. The main research direction for IRI is currently focusing on reducing oxidative stress or relieving inflammation, lacking attention to damaged mitochondrial respiratory chain and oxidative phosphorylation, so the efficacy is often poor and it is impossible to achieve the effect of treating both the symptoms and the root cause.

[0005] In order to achieve the treatment of IRI, it is necessary to design a nanoformulation that can effectively target the damaged site, relieve intracellular oxidative stress, and repair the mitochondrial respiratory chain. This nanoformulation can treat IRI by clearing the large amount of ROS that has been produced and repairing the damaged mitochondria that continue to produce ROS. Summary of the invention

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

[0007] In order to achieve the above object, the present invention adopts the following scheme:

[0008] In a first aspect, the present invention provides a nanoformulation for treating ischemia-reperfusion injury, comprising an active ingredient, albumin, a liposome phospholipid bilayer and a target head, wherein the active ingredient is composed of an antioxidant and coenzyme Q10 and its analogs, and the liposome phospholipid bilayer is composed of phospholipids, cholesterol and DSPE-mPEG. 2000 composition.

[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, mitochondriaquinone or decylubiquinone.

[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 lecithin (HSPC), egg yolk lecithin, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylcholine (DMPC).

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

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

[0016] In a second aspect, the present invention provides a method for preparing a nanoformulation for treating ischemia-reperfusion injury, the method comprising the following steps:

[0017] (1) Preparation of nanocores: The organic solution of the antioxidant and the organic solution of coenzyme Q10 and its analogs are fully mixed, and the mixed solution is added dropwise to the phosphate buffer solution of albumin under stirring at 300 rpm to spontaneously form a stable nanocore;

[0018] (2) Grafting of the target head: The prepared nanocore was added dropwise with a phosphate buffer solution of the target head under stirring conditions of 400 rpm to form a nanocore with specific targeting properties;

[0019] (3) Preparation of nanoformulations: Phospholipids, cholesterol and DSPE-mPEG 2000 The nanoparticles are dissolved in an organic solvent, rotary evaporated to form a film, vacuum dried overnight, and a nano-core with specific targeting is added to hydrate the lipid film to obtain a lipid suspension. The suspension is extruded through a liposome extruder and centrifuged to obtain the final nano-preparation.

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

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

[0022] Preferably, the concentration of the human serum albumin phosphate solution is controlled to be 0.5 to 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 present invention provides a use of the nanoformulation in preparing a drug for treating ischemia-reperfusion injury.

[0025] The present invention has the following beneficial effects: Different from single treatment methods such as relieving oxidative stress, inhibiting inflammatory environment or directly inhibiting ferroptosis, the present invention proposes for the first time a new dual-drug combination strategy for treating ischemia-reperfusion injury. On the one hand, it can relieve oxidative stress, and on the other hand, it can repair damaged mitochondrial function, restore the blocked electron transport chain, reduce ROS generation from the root, break the vicious cycle of ferroptosis caused by mitochondrial dysfunction, and thus achieve a symptomatic and root-cause treatment effect for the treatment of ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The particle size distribution, transmission electron microscope image (A) and stability (C) of the nanoformulation prepared in the example, and the potential comparison diagram of the nanoformulation prepared in the example and the comparative example (B).

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

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

[0029] Figure 4 After establishing a mouse model of renal ischemia-reperfusion injury, the H&E staining, EDU staining, and TUNEL staining of the ischemic-reperfused renal tissue of mice after tail vein injection of different nanoformulations (A is the tissue H&E staining result, B is the tissue EDU proliferation staining result, and C is the tissue TUNEL apoptosis staining result).

[0030] Figure 5 After establishing a mouse model of renal ischemia-reperfusion injury, the ATP content, lactate content, and MDA content in the ischemic-reperfused renal tissue of mice after tail vein injection of different nanoformulations (A is the quantitative result of ATP content in the tissue, B is the quantitative result of lactate 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. To compare the significant differences between experimental data, unpaired two-tailed t-tests were used for two-group comparisons, and one-way analysis of variance (ANOVA) with least significant difference (LSD) was used for multiple comparisons. All tests were two-sided, p > 0.05 indicates non-significance (ns), and a P value < 0.05 was considered statistically significant (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001; ****p < 0.0001). All data were analyzed using GraphPad Prism 8.0.1 software. Specific implementation manners

[0032] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood. The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0033] Example 1

[0034] Prepare 20 mg / mL FeCe6 dimethyl sulfoxide solution, 20 mg / mL IDE dimethyl sulfoxide solution, and 3 mg / mL human serum albumin phosphate buffer solution; fully mix the FeCe6 dimethyl sulfoxide solution and the IDE dimethyl sulfoxide solution, and the mass ratio of FeCe6 to IDE is 1.5:1. At 25°C and 300 rpm, the mixed solution is added dropwise to the phosphate buffer solution of human serum albumin, where the mass ratio of human serum albumin to active ingredient is 5:1, and IDFC NPs can be spontaneously formed during the dropwise addition process.

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

[0036] Weigh 9.8 mg dimyristoylphosphatidylcholine, 1.4 mg cholesterol, 1.4 mg DSPE-mPEG 2000 Dissolved in 1.33 mL of chloroform, added to a 25 mL round-bottom flask, the organic solvent was dried by rotary evaporator to form a uniform transparent film, HA-IDFC NPs were added and hydrated for 30 min, and then the liposome extruder was used to pass the membrane. The unencapsulated drug was removed by centrifugation at 5000 rpm / min for 5 min to obtain liposome nanoformulation 1 (IDFC@Lip-HA).

[0037] Comparative Example 1

[0038] FeCe6 was used as the drug ingredient. At 25°C and 300rpm, a FeCe6 dimethyl sulfoxide solution was added dropwise into a phosphate buffer solution of human serum albumin, wherein the mass ratio of human serum albumin to the active ingredient was 5:1. Nano-core FeCe6 NPs were spontaneously formed during the addition process.

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

[0040] Weigh 9.8 mg dimyristoylphosphatidylcholine, 1.4 mg cholesterol, 1.4 mg DSPE-mPEG 2000 Dissolved in 1.33 mL of chloroform, added to a 25 mL round-bottom flask, the organic solvent was dried by rotary evaporator to form a uniform transparent film, the targeted nanocore was added and hydrated for 30 min, and then the membrane was passed through a liposome extruder. The unencapsulated drug was removed by centrifugation at 5000 rpm / min for 5 min to obtain liposome nanoformulation 2 (FeCe6@Lip-HA).

[0041] Comparative Example 2

[0042] Using IDE as the drug ingredient, the IDE dimethyl sulfoxide solution was added dropwise into the phosphate buffer solution of human serum albumin at 25°C and 300rpm, where the mass ratio of human serum albumin to the active ingredient was 5:1. Nano-core IDE NPs could be spontaneously formed during the dropping process.

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

[0044] Weigh 9.8 mg dimyristoylphosphatidylcholine, 1.4 mg cholesterol, 1.4 mg DSPE-mPEG 2000 Dissolved in 1.33 mL of chloroform, added to a 25 mL round-bottom flask, the organic solvent was dried by rotary evaporator to form a uniform transparent film, the targeted nanocore was added and hydrated for 30 min, and then the liposome extruder was used to pass the membrane. The unencapsulated drug was removed by centrifugation at 5000 rpm / min for 5 min to obtain liposome nanoformulation 3 (IDE@Lip-HA).

[0045] Figure 1 The characterization results of the liposome nanoformulations prepared in Example 1 and Comparative Examples 1-2 are shown in FIG. Figure 1 As shown in A, the dynamic light scattering particle size of IDFC@Lip-HA is 181.97±3.10nm, and the transmission electron microscopy results show that it is a spherical particle with uniform size. Figure 1 C is the 7-day stability of IDFC@Lip-HA in glucose solution. 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 nanoformulations. The potentials of the three nanoformulations are all between -10 mV and 0 mV.

[0046] Experimental Example 1

[0047] Calcein AM is a living cell detection reagent. After entering the cell, it is hydrolyzed by esterases in the living cell to Calcein, which is retained in the cell and emits strong green fluorescence. PI is a commonly used nuclear fluorescent dye. Since it cannot penetrate the cell membrane of living cells, it can only stain dead cells with damaged cell membrane integrity. Calcein AM is usually used in combination with PI to perform dual fluorescent staining of living and dead cells at the same time, and the cell activity and cytotoxicity are detected according to their fluorescence intensity. DCFH-DA is a reactive oxygen detection reagent. It has no fluorescence itself, can freely pass through the cell membrane, and is hydrolyzed by esterases in the cell to DCFH and stored in the cell. It can be oxidized by intracellular reactive oxygen to a strong green fluorescent substance DCF. Therefore, the intracellular ROS level can be quantified according to its fluorescence intensity. Mitochondria are the main site of intracellular ROS production, and their function is closely related to the content of ROS in mitochondria. MitSOX Red is a dihydroethidium derivative with a cationic triphenylphosphate group. Dihydroethidium can be oxidized by superoxide to ethidium, and then bind to DNA to produce fluorescence. Therefore, the level of superoxide in mitochondria can be quantified according to its fluorescence intensity. Mitochondria are the main place where ATP is generated by the metabolic transformation of cellular substances. ATP is generally considered to be the most important energy metabolic substance in organisms. It can provide power for life activities and participate in various life activities. 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 progress of the mitochondrial respiratory chain, thereby simulating cell damage under ischemia-reperfusion conditions. Select human renal proximal tubular cells (HK2) as the research object, the cells are evenly spread in 6-well plates, placed in an incubator overnight, and when the density reaches 70-80%, the culture medium is discarded, and the groups are set as Control group, ROT group, ROT+IDE@Lip-HA group (hereinafter abbreviated as IDE@Lip-HA group), ROT+FeCe6@Lip-HA group (hereinafter abbreviated as FeCe6@Lip-HA group) and ROT+IDFC@Lip-HA group (hereinafter abbreviated as IDFC@Lip-HA group). Except for the Control group, the remaining groups are first diluted with serum-free MEM medium to 0.2 μM ROT dimethyl sulfoxide solution to damage mitochondrial function. After 24h, the liquid medicine is discarded, and the nanoformulations prepared in Example 1, Comparative Example 1 and Comparative Example 2 are respectively diluted to different concentrations using serum-free MEM medium, and 1 mL of the diluted liquid medicine is added to each well, and the culture is continued for 6h. Wherein the Control group is incubated with serum-free MEM medium. Subsequently, DCFH-DA and MitSOX Red staining were performed, and the fluorescence intensity of each group of cells after staining was observed using a laser confocal microscope and photographed and recorded.The above-mentioned administration operation was repeated, the cells were collected and fully lysed, and the ATP content in the cells was measured using an enhanced ATP content measurement kit.

[0048] Figure 2 The results of live-dead staining, intracellular reactive oxygen species, superoxide and ATP levels in mitochondria were detected in HK2 cells 6 hours after administration of rotenone under rotenone injury conditions. Figure 2 As shown in A, after rotenone damaged mitochondria, the number of dead cells increased significantly, and administration of IDFC@Lip-HA could effectively restore mitochondrial function and increase the number of living cells. Figure 2 As shown in B, after IDFC@Lip-HA intervened HK2 cells, the fluorescence intensity of intracellular DCF decreased significantly and the ROS level decreased. Figure 2 As shown in C, after IDFC@Lip-HA intervened in HK2 cells, the level of superoxide in mitochondria was significantly reduced, which can 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 in HK2 cells, ATP production decreased significantly. After IDFC@Lip-HA intervention, the intracellular ATP content increased significantly and mitochondrial function was effectively improved.

[0049] Experimental Example 2

[0050] BODIPY 581 / 591C11 is a fluorescent probe used to detect lipid peroxidation and antioxidant capacity in living cells. The maximum excitation wavelength of the reduced product of BODIPY 581 / 591C11 is 581nm, and the maximum emission wavelength is 591nm, which is mainly red fluorescence, and the red-green fluorescence ratio is large at this time; after oxidation by lipid peroxidation (Lipid Peroxidation, LPO), the maximum excitation and emission wavelengths shift to about 488 / 510nm, which is mainly green fluorescence, and the red-green fluorescence ratio decreases at this time. Malondialdehyde (MDA) is a degradation product of lipid peroxides. By detecting the level of MDA, the level of lipid peroxidation in cells can be effectively reflected. 4-Hydroxynonenal (4-HNE) is a metabolite of lipid peroxides, which can react with glutathione peroxidase 4 (GPX4) for glycosylation, promote the ubiquitination and degradation of GPX4, cause a vicious cycle, and aggravate cell damage. HK2 cells were selected as the research object, and the cells were evenly plated in a 6-well plate and placed in an incubator overnight. When the density reached 70-80%, the culture medium was discarded, and the groups were set as Control group, ROT group, IDE@Lip-HA group, FeCe6@Lip-HA group and IDFC@Lip-HA group. Except for the Control group, the ROT dimethyl sulfoxide solution was diluted to 0.2μM with serum-free MEM culture medium in the other groups, and then damaged for 24h. After discarding the drug solution, the nanoformulations prepared in Example 1, Comparative Example 1 and Comparative Example 2 were diluted to different concentrations using serum-free MEM culture medium, and 1 mL of the diluted drug solution was added to each well, and the culture was continued for 6 hours. The Control group was incubated with serum-free MEM culture medium. Subsequently, BODIPY 581 / 591C11 was used for staining, and the fluorescence intensity of each group of cells after staining was observed by laser confocal microscopy, and photographed and recorded. The above-mentioned drug administration operation was repeated, the cells were collected and fully lysed, and the intracellular MDA level was measured using an MDA detection kit. Repeat the above-mentioned drug administration operation, first wash twice with 1 mL of Hanks balanced solution, then fix with 1 mL of 4% paraformaldehyde at room temperature for 30 minutes, then permeabilize with 1 mL of 0.2% Triton X100 for 3 minutes, then block with 5% donkey serum at room temperature for 30 minutes, then incubate 4-HNE antibody overnight on a 4°C shaker, and finally incubate with fluorescently labeled secondary antibody (Alexa Fluor 647 labeled goat anti-mouse IgG) at 37°C in the dark. The fluorescence intensity of each group of cells after staining was observed by laser confocal microscopy and photographed and recorded.

[0051] Figure 3A is the result of intracellular BODIPY 581 / 591C11 staining. The results show that after rotenone-induced mitochondrial damage, the intracellular LPO level increased. IDFC@Lip-HA can effectively reduce the production of LPO and maintain normal lipid peroxidation levels. Figure 3 B is the quantitative result of intracellular MDA level. The results showed that compared with the single-drug nanoformulation group, the MDA level in the IDFC@Lip-HA group was reduced, which corresponded to the results of BODIPY 581 / 591C11. Figure 3 C is the quantitative result of intracellular 4-HNE level. As shown in the figure, after rotenone damaged mitochondria, unsaturated phospholipids were oxidized to generate a large amount of LPO, and the metabolite 4-HNE increased significantly. After administration of the final preparation, the LPO content in the damaged cells decreased, the 4-HNE content decreased, and the cell's own antioxidant capacity increased.

[0052] Experimental Example 3

[0053] In order to verify the therapeutic effect of IDFC@Lip-HA in treating renal ischemia-reperfusion in vivo, Balb / c male mice were randomly divided into 5 groups, 6 mice in each group, including Sham 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, surgical scissors are used to make an incision 1 cm away from the spine on the left and right sides. After the kidney is removed, the renal blood vessels are clamped with hemostatic forceps for 30 minutes, and then the kidney is put back into the abdominal cavity, and the muscle layer and skin layer are sutured. During reperfusion, different preparations were given through the tail vein. Three days after modeling, the mice were euthanized, and the kidney tissues of the mice were collected. After washing with saline, they were fixed with 4% paraformaldehyde. After fixation for 48 hours, they were embedded in paraffin and sliced. Hematoxylin and eosin were used for H&E staining to detect the morphological structure of the cells, EDU reagent was used for click chemical fluorescence staining to detect cell proliferation ability, and TUNEL reagent was used for TUNEL staining to detect cell apoptosis. Finally, the slices were scanned and analyzed using a digital slice scanner to comprehensively evaluate the overall therapeutic effect of the preparation.

[0054] Figure 4 Analysis of tissue staining results in mice with renal ischemia-reperfusion. Figure 4A is the result of tissue H&E staining, which shows that after ischemia-reperfusion injury, a large number of vacuolar degeneration (arrows) occurred in the renal tubular cells, and a large number of cell fragments of necrotic cells shed appeared in the renal tubular lumen, forming cell and granular casts (triangles), and there were also epithelial cells with condensed nuclei (squares) that shed, and even the entire renal tubular casts vacuolated, and a large amount of renal interstitial edema (circles). After the final preparation was given, renal interstitial edema was improved, vacuolation was alleviated, cell and granular casts were alleviated, and there was no interstitial edema, indicating that IDFC@Lip-HA can significantly improve the renal tubular cells and lumen damaged after renal ischemia-reperfusion and the renal microenvironment. Figure 4 B is the result of tissue EDU proliferation staining, which shows that after the kidney is injured by ischemia-reperfusion, a large amount of ROS damages the endogenous DNA of the cells, and the cell proliferation ability is significantly inhibited. After the final preparation is administered, the cell proliferation ability is greatly promoted, which plays a good role in promoting the repair of renal ischemia-reperfusion injury. Figure 4 C is the result of tissue TUNEL apoptosis staining. As shown in the figure, after the mice experienced renal ischemia-reperfusion, a large number of cells in the renal tissue underwent apoptosis, and the green fluorescence was significantly enhanced. After treatment with IDFC@Lip-HA, the oxidative stress of the cells was alleviated and the degree of cell apoptosis was significantly inhibited.

[0055] Experimental Example 4

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

[0057] Figure 5 To determine the tissue ATP content, lactic acid content and MDA content in mice with renal ischemia-reperfusion. Figure 5 A is the quantitative result of ATP content in tissues. As shown in the figure, after modeling, cells were damaged, mitochondrial function was impaired, and ATP production decreased. After treatment with the final preparation IDFC@Lip-HA, intracellular oxidative stress was relieved, mitochondrial function was significantly improved, and ATP content was significantly increased. Figure 5B is the quantitative result of the lactic acid content in the tissue. When the kidney experiences ischemia, the oxygen content in the tissue decreases, the oxygen required for oxidative phosphorylation of the cells is insufficient, and the cells convert part of the energy supply from the respiratory chain into glycolysis to produce lactic acid. During reperfusion, due to the damage of mitochondrial function in the cells, ATP cannot be produced through the respiratory chain when oxygen is full, resulting in the release of a large amount of ROS. The energy production of the cells depends on glycolysis, and lactic acid continues to accumulate. After the final preparation IDFC@Lip-HA is administered, mitochondrial function is restored, lactic acid content decreases significantly, and the respiratory chain returns to normal. Figure 5 C is the quantitative result of MDA content in tissues. After ischemia-reperfusion, the MDA content in tissues of mice increased. After treatment with the final preparation, the MDA content decreased significantly, effectively avoiding the occurrence of ferroptosis.

[0058] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A nanoparticle preparation for treating ischemia-reperfusion injury, characterized in that: The invention comprises an active ingredient, albumin, a liposome phospholipid bilayer and a target head, wherein the active ingredient is composed of an antioxidant and coenzyme Q10 and its analogs, and the liposome phospholipid bilayer is composed of phospholipids, cholesterol and DSPE-mPEG. 2000 composition.

2. The nanoformulation for treating ischemia-reperfusion injury according to claim 1, characterized in that: The antioxidant is selected from Fe(II)-Chlorin e6, Fe(III)-Chlorin e6, N-acetylcysteine, bilirubin, peroxidase or catalase.

3. The nano preparation for treating ischemia-reperfusion injury according to claim 1, characterized in that: The coenzyme Q10 and its analogs are selected from coenzyme Q10, idebenone, mitochondriaquinone or decylubiquinone.

4. The nanoformulation 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.

5. The nanoformulation 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.

6. The nanoformulation 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, distearoylphosphatidylcholine and dimyristoylphosphatidylcholine.

7. The nano preparation for treating ischemia-reperfusion injury according to claim 1, characterized in that: The mass ratio of the coenzyme Q10 and its analogues to the antioxidant is 1:0.5 to 1:10, and the phospholipid and DSPE-mPEG 2000 The mass ratio of the target head and the active ingredient is 1:1 to 1:100:1, the mass ratio of phospholipid and cholesterol is 10:1 to 50:1, the mass ratio of the albumin and the active ingredient is 1:1 to 20:1, and the mass ratio of the target head and the active ingredient is 1:1 to 1:

10.

8. The method for preparing the nanoformulation for treating ischemia-reperfusion injury according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) Preparation of nanocores: The organic solution of the antioxidant and the organic solution of coenzyme Q10 and its analogs are fully mixed, and the mixed solution is added dropwise to the phosphate buffer solution of albumin under stirring at 300 rpm to spontaneously form a stable nanocore; (2) Grafting of the target head: The prepared nanocore was added dropwise with a phosphate buffer solution of the target head under stirring conditions of 400 rpm to form a nanocore with specific targeting properties; (3) Preparation of nanoformulations: Phospholipids, cholesterol and DSPE-mPEG 2000 The nanoparticles are dissolved in an organic solvent, rotary evaporated to form a film, vacuum dried overnight, and a nano-core with specific targeting is added to hydrate the lipid film to obtain a lipid suspension. The suspension is extruded through a liposome extruder and centrifuged to obtain the final nano-preparation.

9. The preparation method according to claim 8, 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.

10. Use of the nanoformulation according to any one of claims 1 to 7 in the preparation of a drug for treating ischemia-reperfusion injury.

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