Application of antioxidant lipidosome in preparation of medicine for preventing and / or treating inner ear injury caused by active oxygen accumulation

Abstract

The invention relates to application of an antioxidant liposome in preparation of a medicine for preventing and / or treating inner ear injury caused by active oxygen accumulation, and belongs to the field of medical technologies and biological medical materials. The antioxidant lipidosome is rutin-entrapped lipidosome, and the inner ear injury is preferably cis-platinum induced inner ear injury. The antioxidant liposome disclosed by the invention is used for inhibiting active oxygen accumulation, mitochondrial injury and cell apoptosis in cells; the compound is used for inhibiting auditory hair cell mitochondrial injury in cochlea. The antioxidant liposome disclosed by the invention can be used for effectively packaging medicines and improving the bioavailability and the stability of the medicines; the limitation of a tympanic membrane barrier and other biological barriers can be overcome, so that the medicine can effectively reach the inner ear part; precise targeted delivery of the medicine can be achieved, and it is ensured that the medicine is intensively released at the needed position.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and biomedical materials, and specifically relates to the use of antioxidant liposomes in the preparation of drugs for preventing and / or treating inner ear damage caused by accumulation of reactive oxygen species. Background Art

[0002] Cisplatin, as a commonly used chemotherapy drug in clinical practice, plays an important role in the treatment of various solid tumors such as lung cancer, head and neck cancer, testicular cancer, bladder cancer and ovarian cancer. However, the widespread use of cisplatin is also accompanied by a variety of adverse reactions, among which ototoxicity, nephrotoxicity and neurotoxicity are relatively common complications. Ototoxicity, as one of the main side effects of cisplatin treatment, has a high incidence and lacks effective treatment measures, which seriously affects the quality of life of patients. According to statistics, 40% to 60% of patients receiving cisplatin treatment will experience varying degrees of hearing loss during or after treatment. At present, there is no good method to prevent and treat ototoxicity caused by cisplatin. It is urgent to develop a more stable and effective treatment method to reduce the hearing damage caused by cisplatin and improve the quality of life of patients.

[0003] The specific mechanism of cisplatin-induced ototoxicity is not yet fully understood. A large number of studies have shown that it may be related to the generation and accumulation of reactive oxygen species (ROS) in the inner ear. As a strong oxidant, ROS can induce oxidative stress in cells, destroy membrane structures, DNA, and proteins, leading to cell damage and apoptosis. Under normal physiological conditions, cells neutralize ROS through their own antioxidant system to maintain redox homeostasis. However, when the body is affected by external damage such as chemotherapy drugs, ROS levels will increase significantly, exceeding the cell's own antioxidant capacity, leading to cell damage and dysfunction. In order to reduce or prevent ototoxicity caused by cisplatin treatment, researchers have been actively exploring various possible interventions. Among them, antioxidant therapy is considered to be an important approach. Antioxidants can neutralize ROS, reduce oxidative stress, and thus protect cells from damage.

[0004] Rutin is a flavonoid compound, also known as quercetin-3-O-rutinoside and vitamin P. It is found in a variety of plants and fruits, such as oranges, lemons, mulberries, buckwheat, cherries, grapes and plums. Rutin can effectively scavenge free radicals and has strong antioxidant properties. Studies have shown that rutin can effectively prevent ROS-related diseases, such as brain ischemia-related damage, nerve damage, diabetes and kidney damage. In these studies, rutin showed the effect of inhibiting oxidative stress response and reducing intracellular ROS, suggesting that it may have the potential value of alleviating cisplatin-induced ototoxicity. However, due to its poor water solubility, low permeability and diffusivity, rutin is difficult to pass through the multiple biological barriers of the inner ear, so it is rarely used in the treatment of inner ear diseases. Summary of the invention

[0005] The present invention solves the technical problems in the prior art that rutin has poor water solubility, low permeability and diffusivity, and is difficult to pass through multiple biological barriers of the inner ear, resulting in poor bioavailability and stability of the drug, and the inability to achieve precise targeted delivery. The present invention provides an application of an antioxidant liposome in the preparation of a drug for preventing and / or treating inner ear damage caused by accumulation of reactive oxygen species. The present invention uses liposomes as nanocarriers, which can effectively encapsulate drugs and improve their bioavailability and stability; and the penetration ability of liposomes helps to overcome the limitations of the tympanic membrane barrier and other biological barriers, so that the drug can effectively reach the inner ear and improve the targeting effect of the drug.

[0006] According to the purpose of the present invention, there is provided the use of antioxidant liposomes in preparing a drug for preventing and / or treating inner ear damage caused by accumulation of reactive oxygen species, wherein the antioxidant liposomes are liposomes encapsulating rutin.

[0007] Preferably, the inner ear injury is cisplatin-induced inner ear injury.

[0008] Preferably, the antioxidant liposomes are used to inhibit intracellular reactive oxygen accumulation, mitochondrial damage and cell apoptosis.

[0009] Preferably, the antioxidant liposomes are used to inhibit mitochondrial damage in auditory hair cells in the cochlea.

[0010] Preferably, the particle size of the antioxidant liposome is 122-190 nm.

[0011] Preferably, the antioxidant liposomes are prepared by the following steps:

[0012] (1) adding soybean lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol and rutin into an organic solvent and mixing thoroughly;

[0013] (2) removing the organic solvent from the mixed solution obtained in step (1) to obtain a lipid film;

[0014] (3) drying the lipid film obtained in step (2), then adding a buffer solution to hydrate the lipid film; and then filtering to obtain an antioxidant liposome nanoparticle solution.

[0015] Preferably, the mass ratio of the soybean lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol and rutin is 8:2:2:(1-2).

[0016] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0017] (1) The present invention utilizes liposomes as nano-delivery carriers to load rutin (Rutin) with antioxidant function to prepare nano-drug Lip-Rutin, which is used to reduce, prevent and alleviate ototoxic side effects (such as those caused by cisplatin). The nano-drug Lip-Rutin in the present invention can effectively protect auditory hair cells in the cochlea from damage caused by cisplatin, providing a new treatment strategy for the problem of ototoxicity. The nano-drug Lip-Rutin in the present invention verifies the potential of Lip-Rutin as a new antioxidant, especially its superiority in the ability to scavenge reactive oxygen species, and is suitable for the treatment of a variety of oxidative stress-related diseases. Due to its small size advantage, the nano-drug delivery system in the present invention, Lip-Rutin can be used as an effective nano-drug delivery device, which helps to improve the delivery efficiency and targeting of drugs in the body.

[0018] (2) Liposomes are a commonly used drug delivery carrier. They are spherical lipid bilayer structures with a diameter of 50 to 1000 nm. They can load both fat-soluble and water-soluble drugs and are amphiphilic nanomaterials. By improving the solubility, absorption rate and bioavailability of drugs, liposomes can effectively improve the therapeutic effect of drugs. Liposomes can carry drugs through the round window membrane into the inner ear and accurately deliver therapeutic drugs to the inner ear hair cells, thereby improving the local bioavailability of drugs in the inner ear. This provides a new technical approach for the treatment of inner ear diseases. Using nanoparticles as drug carriers and delivering them to the inner ear through intratympanic administration can significantly increase the concentration level of drugs in the inner ear. This is mainly attributed to the biocompatibility and nanoscale size of the nanoparticles themselves, the latter of which is conducive to improving the permeability through the round window membrane, thereby promoting the entry of drugs into the inner ear lymph and achieving higher tissue permeability and bioavailability. During the diffusion of nanoparticle preparations through the round window membrane into the inner ear lymph, they can protect the loaded drugs from accidental clearance and self-degradation, thereby regulating their release. Nanomaterial drug delivery systems have the ability to convert low water-soluble, poorly absorbed and unstable bioactive substances into drug forms that can be efficiently utilized by the body, opening up new ways to effectively achieve inner ear drug delivery. Therefore, the present invention develops a liposome loaded with rutin to enhance the lipid solubility and bioavailability of rutin, which is used to reduce cisplatin-induced ototoxicity.

[0019] (3) The present invention solves the problems of biological barrier limitation, bioavailability and stability of drugs and targeted drug delivery in the prior art:

[0020] Biological barriers: Biological barriers of the ear structures (such as the tympanic membrane, round window membrane, and blood-labyrinth barrier) limit the efficiency of drugs entering the inner ear through the cochlea. These barriers affect the delivery rate and efficacy of drugs, especially for those drugs with large molecules or poor water solubility.

[0021] Bioavailability and stability of drugs: Due to the limitations of their physical and chemical properties (such as poor water solubility, easy metabolic degradation, etc.), some drugs have low bioavailability before entering the inner ear, thereby reducing the therapeutic effect.

[0022] Precise targeted delivery: Traditional drug delivery systems have difficulty achieving precise delivery to specific parts of the inner ear, resulting in the inability to effectively concentrate the drug in the diseased area, increasing the difficulty of treatment and the risk of side effects.

[0023] The antioxidant liposomes provided by the present invention have the following technical effects:

[0024] Application of nanocarriers: Using liposomes as nanocarriers can effectively encapsulate drugs and improve their bioavailability and stability. Liposomes can protect drugs from biological degradation and prolong the half-life of drugs in the body, thereby increasing their therapeutic effects.

[0025] Overcoming biological barriers: Due to the nanoscale size and special surface properties of liposomes, they can easily pass through the RWM (round window membrane) and enter the cochlea. This penetration ability helps to overcome the limitations of the tympanic membrane barrier and other biological barriers, allowing drugs to effectively reach the inner ear.

[0026] Improve drug targeting: By adjusting the formulation and surface modification of liposomes, precise targeted delivery of drugs can be achieved, ensuring that the drugs are released in concentrated areas where they are needed, reducing the impact on healthy tissues, and enhancing the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Microstructure of Lip-Rutin, where A: particle size distribution of Lip-Rutin; B: microstructure of Lip-Rutin under transmission electron microscope (scale: 200 nm).

[0028] Figure 2 FTIR infrared spectrum of Lip-Rutin.

[0029] Figure 3 The stability of Lip-Rutin in different systems, where A: Particle size and zeta potential monitoring of Lip-Rutin in PBS; B: Particle size and zeta potential monitoring of Lip-Rutin in DMEM.

[0030] Figure 4 In vitro release of Lip-Rutin.

[0031] Figure 5DPPH inhibition efficiency of Lip-Rutin, where A: absorbance spectrum of DPPH after adding different concentrations of Lip-Rutin (2, 8, 32, 128 μM) at a wavelength of 450-600 nm; B: DPPH inhibition rate calculated based on absorbance after adding different concentrations of Lip-Rutin at a wavelength of 517 nm.

[0032] Figure 6 Analysis of Lip-Rutin-C6 uptake by OC-1 cells, where A: Fluorescence microscopy was used to observe the uptake of Lip-Rutin-C6 by OC-1 cells at 0.5, 1, 2, 4, 8 and 12 h (scale: 50 μm); B, C: Flow cytometry was used to detect the changes in the uptake of Lip-Rutin-C6 by OC-1 cells over time, and fluorescence intensity analysis was performed.

[0033] Figure 7 CCK-8 method was used to detect cell viability: OC-1 cells were pretreated with different concentrations of Lip-Rutin (6.3, 12.5, 25, 50, 100, 200 μM) for 4 h in advance, and then the cell viability was detected after 24 h of treatment with 10 μM CDDP.

[0034] Figure 8 Confocal microscopy and flow cytometry were used to detect DHE staining of OC-1 cells: the control group received no treatment, the Lip-Rutin group treated OC-1 cells with Lip-Rutin for 28 h, the CDDP group treated OC-1 cells with CDDP for 24 h, and the pretreatment group was pretreated with Lip-Rutin 4 h before the addition of CDDP, and then exposed to CDDP and Lip-Rutin for 24 h at the same time.

[0035] Fig. 9 Confocal microscopy was used to analyze the JC-1 staining of OC-1 cells, where A: JC-1 staining results of OC-1 cells under different treatments were observed under confocal microscopy (scale: 50 μm); B: The percentage of red and green fluorescence intensity of JC-1 staining of OC-1 cells in the total fluorescence intensity was calculated under different treatments.

[0036] Fig.10 AnnexinV / PI double staining method was used to detect apoptosis of OC-1 cells, where A: Flow cytometry was used to detect the results of AnnexinV / PI staining of OC-1 cells; Q2 quadrant was for AnnexinV and PI double-positive cells, indicating that the cells were in the late stage of apoptosis or dead; Q3 quadrant was for AnnexinV single-positive cells, indicating that the cells were in the early stage of apoptosis; B: Statistical analysis of the proportion of apoptotic cells in each group.

[0037] Fig.11The hair cells on the basilar membrane were observed under a confocal microscope. A: Taking the middle gyrus as an example, the hair cells on the basilar membrane of different groups were observed under a confocal microscope (scale: 50 μm); B: The number of surviving hair cells per 200 μm of basilar membrane under different treatments.

[0038] Fig.12 Mitosox staining of basement membrane hair cells was observed under confocal microscopy. A: Taking the middle gyrus as an example, Mitosox staining of basement membranes of different groups was observed under confocal microscopy; red fluorescence represents positive Mitosox staining; blue fluorescence represents Dapi-labeled cell nuclei, and green fluorescence represents positive phalloidin staining; (scale: 50 μm); B: The number of cells with positive Mitosox staining of basement membranes of different groups per 200 μm.

[0039] Fig.13 Zebrafish larvae pretreated with different concentrations of Lip-Rutin, where A: fluorescence intensity (scale: 200 μm) and hair cells in the ganglion (scale: 20 μm) of zebrafish larvae pretreated with different concentrations of Lip-Rutin, green fluorescence represents positive DASPEI staining; B: statistics of fluorescence intensity of zebrafish pretreated with different concentrations of Lip-Rutin; C: statistics of hair cell numbers of zebrafish pretreated with different concentrations of Lip-Rutin.

[0040] Fig.14 In vivo imaging of mice in different inner ear drug administration groups.

[0041] Fig.15 Measurement of hearing at D0 and D14: ABR tests were performed on mice one day before and 14 days after the start of CDDP administration, and the ABR thresholds of mice in each group were tested at 8, 16, 24, 28 and 32 Hz.

[0042] Fig.16 Counting of hair cells in the basement membrane of mice with different compositions.

[0043] Fig.17 Safety evaluation of Lip-Rutin, where A: HE-stained tissue sections of cochlea and important organs (scale: 50 μM); B: detection of biochemical indexes of liver function; C: detection of biochemical indexes of kidney function. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0045] The antioxidant liposomes prepared by the present invention have the following characteristics:

[0046] (1) The thin film hydration method was successfully used to prepare rutin-loaded liposomes Lip-Rutin. The liposome drug particle size was approximately 122-190 nm, with uniform distribution and size, and a potential of approximately -9.27 mV.

[0047] (2) The encapsulation efficiency of Lip-Rutin was about 83.4%, and the in vitro release characteristics were good, with about 67.8% of Rutin released within 72 hours and remaining stable in PBS and DMEM culture media within 7 days.

[0048] (3) Lip-Rutin exhibited significant free radical scavenging activity in vitro. At a concentration of 64 μM, the DPPH inhibition rate reached approximately 62.7%.

[0049] (4) Lip-Rutin nanoparticles can be internalized and taken up by OC-1 cells, and the uptake level remains at a high level after 4 hours.

[0050] (5) Treatment of OC-1 cells with cisplatin and CDDP caused a decrease in cell viability, while pretreatment with Lip-Rutin increased cell survival. Lip-Rutin could inhibit intracellular ROS accumulation, mitochondrial damage, and cell apoptosis caused by CDDP.

[0051] (6) In the cochlear explant model, pretreatment with Lip-Rutin (50 μM) reduced the proportion of CDDP-induced cochlear basal membrane hair cell damage and reduced CDDP-induced hair cell mitochondrial damage.

[0052] (7) In the zebrafish model, after pretreatment with Lip-Rutin (above 100 μM), the survival rate of hair cells in larval fish was significantly improved compared with that in the CDDP group.

[0053] (8) Lip-Rutin can antagonize cisplatin-induced hearing loss in mice at all frequency bands and reduce cisplatin-induced hair cell loss in mice.

[0054] (9) Lip-Rutin has good biocompatibility and minimal toxicity to major organs and the inner ear.

[0055] The following are specific embodiments

[0056] Example 1: Preparation of blank liposomes

[0057] The present invention adopts a thin film hydration method to prepare blank liposomes, using soybean lecithin (Lecitin), cholesterol (Cholesterol), distearoyl-phosphatidyl-ethanolamine-polyethylene glycol 2000 (Distearoyl-phosphatidyl-ethanolamine-polyethylene glycol 2000, DSPE-PEG 2000) as raw materials, and the specific preparation method is as follows:

[0058] (1) Weigh 20 mg of soybean lecithin, 5 mg of cholesterol, and 5 mg of DSPE-PEG 2000, respectively, add them to 8 ml of chloroform, and place them in an ultrasonic water bath to completely dissolve them;

[0059] (2) The mixed solution was transferred into a 100 ml round-bottom flask and chloroform was removed using a rotary evaporator at 42 °C;

[0060] (3) Use a vacuum drying oven to fully remove the residual chloroform in the round-bottom flask;

[0061] (4) Add 4 ml of PBS to the round-bottom flask and place it in an ultrasonic water bath to completely hydrate the thin film at the bottom of the flask;

[0062] (5) Filter using an aqueous filter with a pore size of 0.45 μm and collect the filtrate to obtain blank liposomes.

[0063] Example 2: Preparation of liposome nanoparticles

[0064] The liposome nanoparticles loaded with Rutin were prepared using soybean lecithin, cholesterol, DSPE-PEG 2000 and Rutin in a mass ratio of 4:1:1:1. The preparation method is as follows:

[0065] (1) Weigh 20 mg soybean lecithin, 5 mg cholesterol, 5 mg DSPE-PEG 2000 and 5 mg Rutin respectively, add them into 8 ml chloroform and mix well;

[0066] (2) Place in an ultrasonic water bath to completely dissolve all components;

[0067] (3) The mixed solution was transferred into a 100 ml round-bottom flask and chloroform was removed using a rotary evaporator at 42 °C to obtain a lipid film;

[0068] (4) Place the round-bottom flask in a vacuum drying oven to fully remove residual chloroform;

[0069] (5) Add 4 ml of PBS to the round-bottom flask, place it in an ultrasonic water bath to completely hydrate the thin film at the bottom of the flask, and collect the solution at the bottom of the flask;

[0070] (6) Filter using an aqueous filter with a pore size of 0.45 μm, collect the filtrate to obtain the Rutin-loaded liposome nanoparticle solution, and store it in a refrigerator at 4°C for later use.

[0071] Example 3

[0072] Particle size determination: Take 2 ml of the test solution containing nanoparticles and transfer it to the particle size sample cell. Place the sample cell in the DLS card slot, close the lid and check whether the instrument status is normal; set the measurement category, number of measurements and other parameters, and start the test; read and record the particle size and PDI information; perform three tests on each group of samples; after the measurement, clean the particle size sample cell and replace the sample for the next round of testing. Zeta potential is measured using a dedicated potential sample cell. After transferring the test solution to the potential sample cell, set the test parameters and start the test. The results are as follows: Figure 1 A in the figure shows that the average particle size of Lip-Rutin is 144.6 nm, the PDI is 0.267, and the average zeta potential is -9.27 mV.

[0073] Observe the morphology of Lip-Rutin by transmission electron microscopy: Take 10 μl of freshly prepared Lip-Rutin solution and carefully drop it on a special copper grid, and air-dry it overnight at room temperature. Place the prepared sample in a transmission electron microscope (TEM) to observe its microscopic morphology. The TEM shooting results are shown in Figure 2. Figure 1 B in the figure shows that the prepared Lip-Rutin has a uniform and complete spherical structure with a smooth surface, a diameter of about 150 nm, and is evenly distributed in the field of view.

[0074] Fourier transform infrared spectrometer (FTIR) was used to identify the structure of Lip-Rutin: 200 mg of potassium bromide was weighed and ground in a mortar; the ground potassium bromide powder was compressed and shaped using a film press to obtain a translucent round potassium bromide tablet; 20 μl of Rutin solution dissolved in DMSO, 20 μl of blank liposome solution, and 20 μl of Lip-Rutin solution were respectively taken and dropped on the potassium bromide disc; the disc was placed in a drying oven for drying; and FTIR was used for detection. The FTIR detection results are shown in the figure. Figure 2 It shows that the C=O bond (υC=O) in Rutin is at 1660 cm -1 The aromatic C=C bond (υC=C) is at 1504 cm -1 The phenolic CO bond (υC-O) is at 1362 cm -1 and 1297cm -1 The P=O bond (υP=O) in liposomes is at 1240cm -1 The POC bond (υP-OC) is at 1084cm -1 In addition, the 1362 cm-1 and 1297cm -1 and 1240 cm of P=O / POC in liposomes -1 / 1084cm -1 There are corresponding ones in Lip-Rutin, indicating that Rutin is successfully encapsulated in liposomes.

[0075] Evaluation of Lip-Rutin Stability: Freshly prepared Lip-Rutin was mixed with phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) at a ratio of 1:9, placed in a 37°C incubator, and the particle size and zeta potential of the mixed solution were measured using DLS for 7 consecutive days. Figure 3 The results showed that in both systems, the particle size and zeta potential of Lip-Rutin remained relatively stable, indicating that Lip-Rutin could remain stable in both PBS and DMEM culture media at 37°C.

[0076] Example 4

[0077] In vitro release study of Lip-Rutin: The in vitro release of Lip-Rutin was evaluated using a dialysis membrane and PBS as the release medium. The sample containing 0.2 mg of Rutin was diluted to 2 ml with PBS and transferred to a dialysis membrane bag with a molecular weight cutoff of 3000; the dialysis bag was sealed with a bag buckle and immersed in a beaker containing 50 ml of PBS; the flask was placed on a shaker at room temperature and shaken. After a specific time (0.5, 1, 2, 4, 10, 24, 48, 72, 96, 120 and 144 h), 5 ml of dialysate was extracted and replaced with an equal volume of PBS in the flask; the Rutin content in the dialysate extracted at each time point was detected, and the total amount of Rutin released in the corresponding time period was calculated. The results are as follows: Figure 4 The results show that in the first 10 hours, the release rate of Rutin was relatively fast, with about 24.6% of Rutin being released, showing a relatively rapid release effect. In the next 24 to 72 hours, the release rate of Rutin was relatively stable, with about 67.8% of Rutin continuously released. During this stage, the drug release was in a relatively stable period. Over time, the drug release rate gradually slowed down, and the final total release amount reached about 80.5%, and the release gradually tended to saturation.

[0078] Example 5

[0079] In vitro free radical scavenging properties of Lip-Rutin: Prepare Lip-Rutin solutions with concentrations of 40, 160, 640, 1280, and 2560 μM; draw 10 μL of each sample solution and add it to 190 μL DPPH free radical solution to make the final concentrations of Lip-Rutin 2, 8, 32, 64, and 128 μM, respectively; replace the sample solution with 10 μL of anhydrous ethanol in the control group and add it to 190 μL DPPH free radical solution; react the sample with the DPPH free radical solution at room temperature for 30 minutes; use an enzyme marker to measure the absorbance of the solution after each reaction at a wavelength of 450-600 nm, and draw a total absorbance spectrum; measure the absorbance value of the solution after each reaction at a wavelength of 517 nm; calculate the DPPH inhibition rate. The results are as follows: Figure 5 It shows that after 30 minutes of reaction, as the concentration of Lip-Rutin increases, the measured DPPH absorbance continues to decrease, showing the ability of Lip-Rutin to scavenge free radicals. At a wavelength of 517nm, low concentrations of Lip-Rutin have free radical scavenging activity. In addition, the inhibition rate of Lip-Rutin on DPPH gradually increases with the increase of concentration. At a concentration of 64μM, the DPPH inhibition rate reaches about 62.7%, and at a concentration of 128μM, the DPPH inhibition rate can reach about 73.2%.

[0080] Example 6

[0081] Study on the uptake of liposomal drugs by OC-1 cells: In order to detect the uptake of drugs by OC-1 cells, coumarin 6 (C6) was used to visualize and measure the uptake of OC-1 cells. First, the liposomal drug Lip-Rutin-C6 loaded with Rutin and C6 was prepared, and then fluorescence microscopy and flow cytometry were used to evaluate the details of Lip-Rutin-C6 uptake by OC-1 cells.

[0082] The preparation of Lip-Rutin-C6 is carried out according to the following method: accurately weigh 20 mg of lecithin, 5 mg of DSPE-PEG2000, 5 mg of cholesterol, 5 mg of Rutin and 2 mg of C6, dissolve them in 8 ml of chloroform, and fully dissolve and mix the components; transfer the mixed solution to a 100 ml round-bottom flask, and use a rotary evaporator to remove chloroform at 42°C; use a vacuum drying oven to fully remove the residual chloroform in the round-bottom flask; add 4 ml of PBS to the round-bottom flask, place it in an ultrasonic water bath to completely hydrate the thin film at the bottom of the flask; filter using an aqueous filter with a pore size of 0.45 μm to obtain Lip-Rutin-C6.

[0083] The method for detecting the uptake of Lip-Rutin-C6 by OC-1 cells using a fluorescence microscope is as follows: When the OC-1 cells grow to about 80%, wash, digest, centrifuge, resuspend and other operations are performed to obtain an OC-1 cell suspension; add 1 ml of culture medium to each well of a 12-well plate and inoculate an appropriate amount of cell suspension to make the cell density about 2.5×10 5 / ml, shake well and put into cell culture incubator; after cells are completely attached, remove the original culture medium in turn at 0.5, 1, 2, 4, 8 and 12h before termination of the experiment, and replace with culture medium supplemented with Lip-Rutin-C6; discard the old culture medium in all wells at the termination time point; rinse each well twice with PBS, add 250μl of 4% paraformaldehyde to each well, and fix at room temperature for 20min; remove the original liquid in each well, rinse twice with PBS, add 250μl Dapi dye solution, and stain at room temperature for 5min; remove the waste liquid in each well, rinse twice with PBS, and leave 200μl PBS in each well to cover the cells; observe the staining under a fluorescence microscope. The results are as follows Figure 6 A in the figure shows that OC-1 cells can be observed to have taken up a small amount of liposome drugs at 30 minutes. As time goes by, the green fluorescence at the corresponding position of the cells gradually increases, indicating that the cell uptake of liposomes gradually increases. After 4 hours, it is observed that the difference in green fluorescence intensity changes little over time, indicating that the cell uptake of liposomes gradually slows down or reaches saturation.

[0084] The flow cytometry method for detecting the uptake of Lip-Rutin-C6 by OC-1 cells is as follows: when the OC-1 cells grow to about 80%, wash, digest, centrifuge, resuspend and other operations are performed to obtain OC-1 cell suspension; add 2 ml of culture medium to each well of a 6-well plate and inoculate an appropriate amount of cell suspension to make the cell density about 1.2×10 6 / 2ml, shake well and put into cell culture box; after cells are completely attached, remove the original culture medium at 0.5, 1, 2, 4, 8 and 12h before termination, and replace with culture medium supplemented with Lip-Rutin-C6; remove the culture medium in all wells at the termination time point, wash twice with PBS; add 0.25% trypsin to each well for digestion, transfer cells to centrifuge tubes after digestion is terminated, mark them; centrifuge at 300g×5min, discard supernatant, add 200μl PBS to each tube, resuspend cells to make them evenly dispersed; detect and analyze by flow cytometry. The results are as follows Figure 6 B in Figure 6 C in the figure is consistent with the results of fluorescence microscopy, showing that the uptake of Lip-Rutin-C6 by OC-1 cells is time-dependent. Before 4 hours, the cell uptake increases with time and reaches the highest uptake level after 4 hours.

[0085] Example 7

[0086] Study on the protective effect of Lip-Rutin on CDDP-induced OC-1 cell damage: By detecting the changes in cell viability caused by pretreatment with different concentrations of Lip-Rutin and then adding CDDP. (1) OC-1 cells were seeded at an appropriate density in a 96-well plate and placed in a cell culture incubator; (2) After 20 hours, the original culture medium in each well was removed and 100 μl of fresh culture medium containing 0, 6.3, 12.5, 25, 50, 100, or 200 μM Lip-Rutin was added. Two groups without Lip-Rutin (i.e., a concentration of 0) were set up, with 5 replicate wells in each group, and the 96-well plate was placed in a cell culture incubator; After 4 hours, the original culture medium was removed and 100 μl of culture medium without Lip-Rutin and CDDP was added to each well of the control group; 100 μl of culture medium without Lip-Rutin and containing 10 μM CDDP was added to each well of the positive control group; and 100 μl of culture medium containing 10 μM CDDP and 6.3, 12.5, 25, 50, 100, or 200 μM CDDP was added to each well of the remaining groups. 100μl of Lip-Rutin culture medium; put the 96-well plate into the cell culture incubator; after 24 hours, take out the 96-well plate, remove the original culture medium, rinse each well with PBS twice, add 100μl pure DMEM and 10μl CCK-8 solution, mix well and put it back into the cell culture incubator; after 2 hours, use an ELISA reader to detect the absorbance of each well at a wavelength of 450nm and calculate the cell survival rate. The results are as follows Figure 7 It showed that low concentrations of Lip-Rutin were able to alleviate CDDP damage. When the concentration of Lip-Rutin exceeded 25 μM, it exhibited a stable and significant protective effect on OC-1 cells, indicating that Lip-Rutin pretreatment could reduce CDDP-induced cytotoxicity.

[0087] Example 8

[0088] DHE staining method was used to detect the generation of reactive oxygen species in OC-1 cells: By comparing the DHE staining of OC-1 cells treated with CDDP alone and pretreated with Lip-Rutin before CDDP treatment, the generation of reactive oxygen species in OC-1 cells caused by CDDP and the effect of Lip-Rutin pretreatment were evaluated. Confocal microscopy and flow cytometry were used for detection.

[0089] The experimental method of using confocal microscopy is as follows: spread cell slides on each well of the 12-well plate, then inoculate a cell suspension containing OC-1 cells of appropriate density, shake well and put into the cell culture incubator; discard the original culture medium after the cells are completely attached to the wall, and treat each group of cells differently, with 3 replicates for each group. After the treatment is completed, take out the 12-well plate, discard the waste liquid, and wash twice with PBS; add 500μl of DHE working solution prepared according to the instructions of the kit to each well, and incubate in the cell culture incubator in the dark for 30min; discard the waste liquid in each well, rinse twice with PBS, add 500μl of Hoechst dye solution prepared according to the instructions of the kit to each well, and incubate in the cell culture incubator in the dark for 30min; discard the waste liquid in each well, rinse twice with PBS; add a drop of anti-fluorescence quencher on the slide, carefully take out the cell slide in the 12-well plate, turn it upside down on the slide and seal it; observe with a confocal microscope; use Image J to analyze the fluorescence images of each group. The fluorescence intensity of each group of cells under the blue channel and red channel was counted respectively, and the relative fluorescence intensity of DHE in each group was calculated. Figure 8 Figure A shows that CDDP treatment significantly enhanced the fluorescence intensity of DHE staining in OC-1 cells, indicating that after CDDP treatment, the accumulation of ROS in OC-1 cells increased significantly, and the relative fluorescence intensity of DHE reached about 73.9%. When OC-1 cells were pretreated with Lip-Rutin and then treated with CDDP, although the fluorescence intensity of DHE staining did not fully recover to normal levels, the DHE fluorescence intensity still decreased significantly compared with OC-1 cells directly affected by CDDP, and the relative fluorescence intensity decreased to about 37.2%.

[0090] The experimental process of quantitatively analyzing the changes in DHE staining of OC-1 cells caused by different treatments by flow cytometry is as follows: inoculate a cell suspension containing appropriate density of OC-1 cells into each well of a 6-well plate, shake thoroughly and place in a cell culture incubator; discard the original culture medium after the cells are completely attached to the wall, and use different treatments for each group of cells, with 3 replicate wells for each group. After the treatment is completed, take out the 6-well plate, aspirate and discard the waste liquid, and wash twice with PBS; add 500μl of DHE working solution prepared according to the instructions of the kit to each well, put it in a cell culture incubator and incubate in the dark for 30 minutes; aspirate and discard the waste liquid from each well, and rinse twice with PBS. After digesting the cells with trypsin, collect the cells in a flow tube and mark them; centrifuge at 300g×5min and discard the supernatant, add 200ul PBS to each tube to resuspend the cells so that they are evenly dispersed; detect and analyze by flow cytometry. The results are as follows Figure 8 B in Figure 8C in the figure shows that the DHE staining of OC-1 cells was significantly enhanced after CDDP treatment. The fluorescence intensity of DHE staining of OC-1 cells pretreated with Lip-Rutin and then exposed to CDDP was significantly decreased compared with the fluorescence intensity of cells without pretreatment, and the fluorescence intensity was weakened by about 32.3%, indicating that the pretreatment of Lip-Rutin helps to reduce the accumulation of ROS in OC-1 cells caused by CDDP.

[0091] Confocal microscopy was used to observe the changes in mitochondrial membrane potential in OC-1 cells: In this experiment, the changes in JC-1 staining fluorescence were used to evaluate the changes in mitochondrial membrane potential in OC-1 cells. Cell slides were placed in each well of a 12-well plate, and then a cell suspension containing OC-1 cells of appropriate density was inoculated. After being fully shaken, the culture medium was discarded. Different treatments were applied to each group of cells, and three replicate wells were set up for each group. After adding CDDP, the 12-well plate was removed 24 hours later, the waste liquid was discarded, and it was washed twice with PBS. According to the instructions of the JC-1 kit, the JC-1 staining working solution and JC-1 staining buffer were prepared. 500 μl of the prepared JC was added to each well. -1 staining working solution, put it in a cell culture incubator and incubate it in the dark for 30 minutes; discard the waste liquid in each well, rinse it twice with JC-1 staining buffer, add 500μl Hoechst staining solution prepared according to the kit instructions to each well, put it in a cell culture incubator and incubate it in the dark for 30 minutes; discard the waste liquid in each well, rinse it twice with PBS; add a drop of anti-fluorescence quencher on the slide, carefully take out the cell slide in the 12-well plate, turn it upside down on the slide and seal it; observe with a confocal microscope. Statistical analysis was performed using ImageJ. For each group of pictures, the fluorescence intensity of the red and green channels was calculated respectively, and then the proportion of red and green fluorescence was calculated, representing the proportion of JC-1 polymer and JC-1 monomer, respectively. The results are as follows Fig. 9 The results showed that in the control group and Lip-Rutin group, the JC-1 staining reagent in OC-1 cells mainly existed in the form of polymers, producing red fluorescence. In the CDDP group, the green fluorescence signal of JC-1 staining was enhanced, accounting for about 85.8% of the total fluorescence intensity, and the red fluorescence signal was reduced to about 14.2%, indicating that the mitochondrial membrane potential in OC-1 cells was reduced, the JC-1 staining reagent was converted to monomeric form, and the mitochondria were depolarized. The red fluorescence intensity ratio of JC-1 staining in the Lip-Rutin pretreatment group was about 45.2%, which was less than 91.7% of the normal control group, but significantly higher than the CDDP group, indicating that Lip-Rutin pretreatment can help alleviate the changes in mitochondrial membrane potential caused by CDDP, thereby avoiding mitochondrial depolarization in OC-1 cells.

[0092] Flow cytometry was used to detect cell apoptosis: a cell suspension containing OC-1 cells of appropriate density was inoculated into each well of a 6-well plate, and the plate was placed in a cell culture incubator after being fully shaken; the original culture medium was discarded after the cells were completely attached to the wall, and different treatments were applied to each group of cells, with 3 replicate wells set up for each group. 24 hours after adding CDDP, the 6-well plate was removed, the waste liquid was discarded, and the plate was washed twice with PBS; after digesting the cells with trypsin, the cells were collected in a flow tube and marked; after centrifugation at 300g×5min, the supernatant was discarded, and 200μl 1×AnnexinV binding buffer was added to each tube to resuspend the cells; 10μl AnnexinV and 2μl PI were added to each tube, and the plates were gently mixed, and incubated at room temperature in the dark for 15min; flow cytometry was used for detection and analysis. The results are shown in Fig.10 The results showed that the apoptosis rate of OC-1 cells after CDDP treatment was 13.3%, which was significantly higher than that of the control group (5.5%). The total apoptosis rate of OC-1 cells pretreated with Lip-Rutin was about 8.0%, which was significantly lower than that of the CDDP group, but still higher than that of the control group, indicating that Lip-Rutin alleviated CDDP-induced apoptosis of OC-1 cells.

[0093] Example 9

[0094] Effect of Lip-Rutin on CDDP-induced damage of hair cells of basement membrane of neonatal mice: basement membrane of neonatal mice was removed by dissection, transferred to collagen gel in a well plate with culture medium, and cultured in an incubator overnight; basement membranes were grouped; culture was terminated 24 hours after adding CDDP-containing culture medium, the original culture medium was aspirated and washed; 4% paraformaldehyde solution was fixed at room temperature for 2 hours and then aspirated and washed 3 times with PBS, each time for 5 minutes; collagen gel with basement membrane of neonatal mice was peeled off, transferred to EP tube with blocking solution, and blocked on a shaker at room temperature for 1 hour; blocking solution was carefully aspirated and gently washed 3 times with PBS, each time for 5 minutes; 60 μl of rabbit anti-MYO7A primary antibody diluted 1:100 was added to each tube, and incubated on a shaker at 4°C overnight; primary antibody was recovered and washed 3 times with PTST, each time for 5 minutes; Alexa fluor diluted 1:300 was added to each tube 594 donkey anti-rabbit secondary antibody 60μl, incubate at room temperature on a shaker in the dark for 1h, then discard the secondary antibody, wash with PBST 3 times, 5min each time; add 60μL phalloidin to each tube, incubate at room temperature on a shaker in the dark for 20min, then recover phalloidin, wash with PBS 3 times, 5min each time; carefully remove the gel with basement membrane, spread on a slide with anti-fluorescence quencher in advance, pay attention to the side with basement membrane facing the slide, spread the cover glass and seal the slide; observe with a confocal microscope, and count the hair cells of each group of basement membrane. The results are as follows Fig.11The results showed that a large number of hair cells in the basement membrane of the neonatal mice in the CDDP group were lost, the cell morphology changed significantly, and the hair cells were arranged in a disordered manner. In the Lip-Rutin pretreatment group, the hair cells of the basement membrane were arranged more regularly and the morphology was more complete compared with the CDDP group, and the hair cell survival rate was significantly increased (apical gyrus: 87.7±1.7%, middle gyrus: 81.3%±2.6%, basal gyrus: 85.0%±4.2%). There was no significant difference in the damage of hair cells in the apical gyrus, middle gyrus, and basal gyrus of the basement membrane.

[0095] Example 10

[0096] Detection of ROS levels in basement membrane hair cells: Mitosox staining was used to evaluate the generation of ROS in basement membrane hair cells in this experiment. The number of hair cells with positive MitoSox staining per 200 μm basement membrane was counted to reflect the apoptosis of hair cells. After the grouping treatment, the waste liquid was discarded and washed 3 times with PBS; 1 ml of 4% paraformaldehyde solution was added to each well and fixed at room temperature for 2 hours; the paraformaldehyde solution was discarded and washed 3 times with PBS, each time for 5 minutes; in each 1.5 ml Add 60μl Mitosox working solution to the EP tube; gently peel off the collagen gel with the basement membrane of the newborn mouse, transfer it to the EP tube, and incubate it on a shaker at room temperature in the dark for 30 minutes; carefully discard the old solution, and gently wash it with PBS 3 times, 5 minutes each time; add 60μL of phalloidin to each tube, incubate it on a shaker at room temperature in the dark for 20 minutes, then recover the phalloidin, and wash it with PBS 3 times, 5 minutes each time; add 60μl of Dapi dye solution to each tube, incubate it on a shaker at room temperature in the dark for 15 minutes, then discard Dapi, and wash it with PBS 3 times, 5 minutes each time; carefully remove the gel with the basement membrane, and spread it on a slide with anti-fluorescence quencher in advance, with the side with the basement membrane facing the slide, spread the coverslip and seal the slide; observe with a confocal microscope. The results are as follows Fig.12 The results showed that in the CDDP-treated group, the number of hair cells with positive Mitosox staining per 200 μm of basement membrane was significantly higher than that in the control group (CDDP group: 55.0±4.08, control group 1.0±0.8). There were almost no hair cells with positive Mitosox staining in the Lip-Rutin group (1.3±0.5). The number of hair cells with positive Mitosox staining per 200 μm of basement membrane in the Lip-Rutin intervention group was lower than that in the CDDP group (24.3±4.2), indicating that Lip-Rutin pretreatment effectively reduced the accumulation of ROS caused by CDDP.

[0097] Embodiment 11

[0098] The effect of Lip-Rutin on CDDP-induced toxicity in zebrafish larvae was detected: zebrafish larvae 5 days after fertilization were used and grouped; DASPEI was used for staining for 30 minutes, and the larvae were washed twice with fresh embryo culture medium, each time for 5 minutes; the larvae were anesthetized with 0.4% tricaine for 5 minutes and mounted on a slide; the hair cells of the important neuromasts on the lateral line of zebrafish were counted and statistically observed under a fluorescence microscope. The results are shown in Fig.13 The results showed that compared with CDDP alone, when the concentration of Lip-Rutin reached 100 μM or more, it could significantly increase the DASPEI fluorescence intensity to more than 77.8%, and the survival rate of hair cells in young fish reached more than 78.8%, suggesting that Lip-Rutin intervention has a protective effect on zebrafish hair cells.

[0099] Example 12

[0100] Detection of the distribution of liposome nanoparticles in mice: Use small animal in vivo imaging technology with the help of cell membrane far-infrared probe DID to detect the distribution of drugs in mice. The specific method is as follows: Preparation of Lip-Rutin-DID: In the process of preparing Lip-Rutin, add 0.5 mg of DID powder to the organic solution when mixing the components, and inject 5 μL of saline, pure DID solution, and Lip-Rutin-DID solution into the bilateral tympanic cavity of each group of mice respectively; after 24 hours, the mice were anesthetized and imaged using a small animal in vivo imaging system, with the excitation wavelength set to 640 nm and the emission wavelength set to 700 nm. The mice were deeply anesthetized and killed, the inner ears of the mice were dissected out, and imaged using a small animal in vivo imaging system. The results are as follows: Fig.14 The results showed that after 24 hours, the fluorescence intensity in the ears of mice injected with Lip-Rutin-DID was significantly higher than that in the free DID solution group, indicating that Lip-Rutin-DID stayed in the ears of mice longer. The results of inner ear anatomy also showed that the fluorescence intensity of the liposome drug group was higher, suggesting that the drug in this group penetrated into the inner ear more effectively and maintained a higher drug concentration in the inner ear.

[0101] Example 13

[0102] Evaluation of hearing loss in mice: This study reflects the hearing condition of mice by testing the threshold changes of auditory brainstem response (ABR). The day of drug administration was taken as D1, and the ABR threshold of mice was tested on the day before drug administration, i.e. D0, and two weeks after drug administration, i.e. D14. The test method is as follows: After the mice are successfully anesthetized, they are placed on a constant temperature heating pad in a soundproof room. After disinfecting the electrodes with alcohol, the electrode to be tested is subcutaneously inserted into the mastoid area behind the ear of the mouse to be tested, the ground electrode is subcutaneously inserted into the mastoid area behind the ear of the contralateral ear, and the reference electrode is subcutaneously inserted in the center of the skull. The mouse is kept in a prone position, and the earphone is placed in the ear to be tested. Check whether the instrument connection is intact and whether the test can run normally; set the test frequency to 8kHz, 16kHz, 24kHz, 28kHz and 32kHz, and the sound field stimulation intensity is decreased from 90dB SPL, and the SPL is reduced by 5dB each time until it is judged that the hearing threshold is reached; record the hearing threshold corresponding to each test frequency for subsequent statistical analysis. The results are as follows Fig.15 The results showed that the ABR thresholds of mice treated with CDDP alone were significantly increased at all frequencies (8kHz: 55.0±3.5dB SPL, 16kHz: 57.5±2.5dB SPL, 24kHz: 61.3±5.4dB SPL, 28kHz: 65.0±5.0dB SPL, 32kHz: 67.5±2.5dB SPL). Compared with the control group, the ABR thresholds of mice treated with Lip-Rutin and CDDP were also increased, but significantly lower than those of mice treated with CDDP alone (8kHz: 33.8±4.1dB SPL, 16kHz: 36.3±6.5dB SPL, 24kHz: 32.5±2.5dB SPL, 28kHz: 38.8±2.2dB SPL, 32kHz: 40.0±5.0dB SPL). These results indicate that intraperitoneal injection of CDDP leads to hearing loss in mice, and intratympanic administration of Lip-Rutin can significantly alleviate this hearing loss.

[0103] Embodiment 14

[0104] Detection of hair cell damage in adult mice: After the last measurement of the ABR threshold, the mice were killed by cervical dislocation under deep anesthesia, the cochlea was dissected out, and the surrounding soft tissue was separated. A small hole was made at the top of the cochlea, and care was taken to avoid damaging the basement membrane, and the cochlea was transferred to 4% paraformaldehyde for immersion; after 24 hours, the cochlea was transferred from the paraformaldehyde to a rapid decalcification solution for immersion; after 24 hours, the cochlea was removed and the basement membrane was dissected out in sections under a microscope, and care was taken to avoid damaging the hair cells during the operation; the blocking solution was prepared: Triton-X100 was diluted with donkey serum to a concentration of 0.3%. The dissected basement membrane was placed in an EP tube containing blocking solution, blocked at room temperature for 1 hour, and carefully washed with PBS 3 times, 5 minutes each time; 1:100 diluted rabbit anti-MYO7A primary antibody was added to immerse the basement membrane, and incubated on a shaker at 4°C overnight; the primary antibody was recovered and washed with PBST 3 times, 5 minutes each time; 1:300 diluted Alexa fluor 594 donkey anti-rabbit secondary antibody was added to each tube to immerse the basement membrane, incubated on a shaker at room temperature in the dark for 1 hour, then the secondary antibody was discarded, and washed with PBST 3 times, 5 minutes each time; each tube added phalloidin, incubated on a shaker at room temperature in the dark for 20 minutes, then recovered phalloidin, and washed with PBS 3 times, 5 minutes each time; each tube added Dapi dye solution, incubated on a shaker at room temperature in the dark for 15 minutes, then Dapi was discarded, and washed with PBS 3 times, 5 minutes each time; the basement membrane was carefully clipped out and spread on a slide with anti-fluorescence quencher in advance, and a cover slip was laid and sealed; observed using a confocal microscope. The results are as follows Fig.16 The results showed that simple Lip-Rutin treatment did not cause changes in the number of hair cells in the basilar membrane (Lip-Rutin group: apical gyrus: 94.3±1.2, middle gyrus: 98.3±2.6, basal gyrus: 98.0±2.2; control group: apical gyrus: 95.3±1.2, middle gyrus: 98.3±2.0, basal gyrus: 99.3±1.0), suggesting that Lip-Rutin does not produce toxicity in the mouse cochlea. The number of hair cells per 200μm basilar membrane in mice treated with CDDP decreased (apical gyrus: 63.3±3.1, middle gyrus: 65.0±4.2, basal gyrus: 63.3±4.1), and there was no significant difference in the loss of hair cells in the apical gyrus, middle gyrus, and basal gyrus. The degree of hair cell damage in the basilar membrane of the Lip-Rutin intervention group was significantly reduced compared with the CDDP group alone.

[0105] Embodiment 15

[0106] Biosafety evaluation: After 7 days of intratympanic injection of normal saline and Lip-Rutin, about 0.5 ml of serum was taken from each mouse for biochemical examination. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CR) were tested to reflect liver and kidney function. The mice were deeply anesthetized and killed by cervical dislocation. The cochlea and heart, liver, kidney, spleen and lung were removed for hematoxylin-eosin (H&E) staining. The results are as follows: Fig.17 The results showed that the Lip-Rutin treated group showed no significant abnormalities compared with the saline group. In addition, the liver and kidney function parameters were within the normal reference range, indicating that Lip-Rutin did not cause significant liver or kidney toxicity. The results showed that Lip-Rutin has good biocompatibility and minimal toxicity to major organs and the inner ear.

[0107] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of antioxidant liposomes in the preparation of a drug for preventing and / or treating inner ear damage caused by accumulation of reactive oxygen species, characterized in that: The antioxidant liposome is a liposome encapsulating rutin.

2. The use according to claim 1, characterized in that The inner ear injury is cisplatin-induced inner ear injury.

3. The use according to claim 1, characterized in that The antioxidant liposome is used for inhibiting the accumulation of active oxygen in cells, mitochondrial damage and cell apoptosis.

4. The use according to claim 3, characterized in that The antioxidant liposomes are used to inhibit mitochondrial damage of auditory hair cells in the cochlea.

5. The use according to claim 1, characterized in that The particle size of the antioxidant liposome is 122-190 nm.

6. The use according to any one of claims 1 to 5, characterized in that: The antioxidant liposomes are prepared by the following steps: (1) adding soybean lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol and rutin into an organic solvent and mixing thoroughly; (2) removing the organic solvent from the mixed solution obtained in step (1) to obtain a lipid film; (3) drying the lipid film obtained in step (2), then adding a buffer solution to hydrate the lipid film; and then filtering to obtain an antioxidant liposome nanoparticle solution.

7. The use according to claim 6, characterized in that The mass ratio of soybean lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol and rutin is 8:2:2:(1-2).

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