Selenium composite SOD / CAT cascade catalytic nano material and preparation method and application thereof

Abstract

The invention relates to the technical field of nano-medicines, and discloses a selenium composite SOD / CAT cascade catalytic nano-material as well as a preparation method and application thereof. The selenium composite SOD / CAT cascade catalytic nano-material is prepared from catalase, superoxide dismutase, human serum albumin, glutaraldehyde and a diselenide cross-linking agent through a self-assembly and cross-linking method, ROS in the inner ear environment can be effectively removed, auditory cells are protected against oxidative damage, and the selenium composite SOD / CAT cascade catalytic nano-material has the advantages of being safe and reliable. The composite has the effect of treating hearing loss caused by oxidative stress induced by noise, has good biocompatibility, and can be used for systemic administration to treat hearing loss. In addition, the selenium composite SOD / CAT cascade catalytic nano material is simple in preparation method and mild in reaction condition, and large-scale production can be realized. The invention provides a novel method for treating noise-induced hearing loss by utilizing a nano system for cascade catalysis of a selenium biological material and natural enzyme.

Description

Technical Field

[0001] The invention relates to the fields of nanomaterials and biomedicine, and in particular to a method for preparing a selenium composite SOD / CAT cascade catalytic nanomaterial and an application of the selenium composite SOD / CAT cascade catalytic nanomaterial in preparing a medicine for treating noise-induced hearing loss. Background Art

[0002] Noise-induced hearing loss (NIHL) is a prevalent sensory disorder characterized by decreased auditory sensitivity and speech comprehension, often accompanied by symptoms such as dizziness, headache, hypertension, and tinnitus. Notably, excessive noise exposure is associated with impaired neurogenesis in the hippocampus, which may affect learning and cognitive function and increase the risk of dementia. The use of hearing restoration devices can reduce the risk of cognitive decline in patients with hearing loss by 19%. This link between NIHL and dementia highlights the urgency of addressing NIHL. Despite trials of antioxidants such as vitamins A, C, and E, minerals such as magnesium, and compounds such as N-acetylcysteine ​​and glutathione, effective clinical treatments for NIHL remain elusive, highlighting the need for advanced antioxidant, biocompatible nanomaterials specifically tailored for NIHL treatment.

[0003] In noise-induced hearing loss, high levels of ROS and excess iron deposition are considered to be important factors causing cell death and damage. Noise exposure triggers excessive production of ROS in the cochlea, including superoxide anions (O 2 · - ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (·OH), which affect mitochondria-rich areas such as hair cells, spiral ganglion cells, and vascular striae, causing damage and death of cochlear cells due to oxidative stress. Excessive ROS can also cause the accumulation of iron-dependent lipid peroxides in cells, leading to lipid peroxidation and causing ferroptosis of cells.

[0004] Clinically, the use of hearing aids and cochlear implants are potential approaches to improve hearing loss. However, both hearing aids and cochlear implants prefer to improve hearing in a quiet rather than a noisy environment. Preclinical researchers have mainly focused on the regeneration of damaged sensory cells or neurons, with the help of gene therapy to replace mutated or defective genes, and cell therapy to transplant stem cells (embryonic or adult), in addition to delivering potential otoprotectants to rescue hearing loss. However, due to the existing bony labyrinth (rigid outer wall) and blood labyrinth barriers, the intrinsic defects of systemic administration, such as low permeability, limited distribution, and insufficient bioavailability, limit their clinical efficacy. In addition, most drugs with rapid physical and chemical degradation characteristics require intratympanic / intracochlear intervention, which poses a major challenge to long-term treatment and induces secondary damage. Therefore, there is an urgent need to explore better alternatives to combat noise-induced hearing loss. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a selenium composite SOD / CAT cascade catalytic nanomaterial and a preparation method thereof and an application thereof in the preparation of a drug for treating noise-induced hearing loss. The selenium composite SOD / CAT cascade catalytic nanomaterial can be used for treatment by systemic administration. The preparation method of the selenium composite SOD / CAT cascade catalytic nanomaterial is simple and convenient, and the conditions are mild, and it can be used for large-scale production.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a selenium-composite SOD / CAT cascade catalytic nanomaterial, which is a multifunctional nanocatalytic material with a cascade catalytic effect prepared by self-assembly and cross-linking of catalase, superoxide dismutase, human serum albumin, glutaraldehyde and a diselenide cross-linking agent; wherein human serum albumin is used as a carrier material, glutaraldehyde is a preliminary curing cross-linking agent, and the diselenide cross-linking agent is 3,3-diselenodiprolic acid di(N-succinimide) ester, which has ROS responsiveness.

[0008] The selenium composite SOD / CAT cascade catalytic nanomaterial of the present invention has the following properties: (1) possessing natural catalase and superoxide dismutase activities; and (2) possessing the ability to scavenge hydroxyl radicals, superoxide anions and 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt free radicals.

[0009] The selenium composite SOD / CAT cascade catalytic nano-platform of the present invention has the effects of reducing the intracellular ROS level, alleviating oxidative stress and inhibiting ferroptosis, thereby achieving the effect of treating noise-induced hearing loss.

[0010] Specifically, the selenium composite SOD / CAT cascade catalytic nanomaterial is a hybrid catalyst with a spherical particle structure and a particle size of 50-200 nm.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned selenium composite SOD / CAT cascade catalytic nanomaterial, comprising the following steps:

[0012] S1, dissolving catalase, superoxide dismutase and human serum albumin in ultrapure water, adding glutaraldehyde solution and stirring to solidify at room temperature to obtain a mixed solution;

[0013] S2. Add 3,3-diselenodipronate (N-succinimide) to the mixed solution and continue stirring, collect the material, and obtain the selenium composite SOD / CAT cascade catalytic nanomaterial after dialysis.

[0014] Specifically, in step S1, the mass ratio of catalase, superoxide dismutase and human serum albumin added is (0.5-2):(0.5-2):10, the concentration of the glutaraldehyde solution is 20wt%-25wt%, the stirring time is 12-24 hours, and the stirring speed is 1000-1200rpm.

[0015] Specifically, in step S1, the mass ratio of glutaraldehyde to human serum albumin is (2-3):10, and the concentration of glutaraldehyde in the mixed solution is 0.05-0.1 mg / ml.

[0016] Specifically, the mass ratio of 3,3-diselenedipropionic acid bis(N-succinimide) added in step S2 to human serum albumin added in step S1 is (10-20):1, the concentration of 3,3-diselenedipropionic acid bis(N-succinimide) in the mixed solution is 8-12 mg / ml, the stirring time in step S2 is 12-24 hours, and the stirring speed is 1000-1200 rpm.

[0017] In a third aspect, the present invention provides the use of the above-mentioned selenium composite SOD / CAT cascade catalytic nanomaterial in the preparation of a drug for treating noise-induced hearing loss.

[0018] The noise-induced hearing loss includes an increase in the hearing threshold.

[0019] The selenium composite SOD / CAT cascade catalytic nanomaterial is used in the preparation of a medicine for treating noise-induced hearing loss. The medicine for treating noise-induced hearing loss is a systemically administrable preparation.

[0020] Beneficial effects achieved by the technical solution of the present invention:

[0021] The experiments show that the selenium composite SOD / CAT cascade catalytic nanomaterial has natural catalase (CAT) and superoxide dismutase (SOD) activities, and has the ability to produce hydroxyl radicals (·OH) and superoxide anions (O 2 · - ) and 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt free radical (ABTS· + ) scavenging ability, can effectively remove ROS in the inner ear environment, thereby reducing the damage caused by oxidative stress to sensory hair cells, including alleviating lipid peroxidation, ferroptosis and reducing DNA and protein damage, and the selenium composite SOD / CAT cascade catalytic nanomaterial is composed of natural enzymes, and selenium is also an essential element in the human body, so it has good biocompatibility. The present invention uses the selenium composite SOD / CAT cascade catalytic nanomaterial to prepare a drug for treating noise-induced hearing loss, which can achieve a better noise-induced hearing loss drug effect, and the selenium composite SOD / CAT cascade catalytic nanomaterial has stable performance and is a safe, convenient and ideal drug. In addition, the preparation process of the selenium composite SOD / CAT cascade catalytic nanomaterial is simple and easy to operate, the preparation conditions are mild, and it is suitable for mass production. The present invention provides a new method for treating noise-induced hearing loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscope (TEM) photograph of the selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1 of the present invention;

[0023] Figure 2 This is a diagram showing the hydrodynamic size detection results of the selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1 of the present invention;

[0024] Figure 3 This is a result diagram of the catalase (CAT) simulation activity test of the selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1;

[0025] Figure 4 This is a result diagram of the superoxide dismutase (SOD) simulation activity test of the selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1;

[0026] Figure 5 This is a result diagram of the test of the ability of the selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1 to remove hydroxyl radicals (·OH);

[0027] Figure 6 The selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1 removes 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt free radicals (ABTS·+ ) Ability test result graph;

[0028] Figure 7 This is a result diagram of the cytotoxicity of different concentrations of selenium-complexed SOD / CAT cascade catalytic nanomaterials to HEI-OC1 cells using CCK-8 assay;

[0029] Figure 8 In order to detect the inhibition of H by different concentrations of selenium-composite SOD / CAT cascade catalytic nanomaterials using CCK-8 assay, the 2 O 2 Results of the killing of the Otitis Institute-Corti 1 (HEI-OC1) cells capacity;

[0030] Fig. 9 This is a graph showing the results of flow cytometry detection of selenium-complexed SOD / CAT cascade catalytic nanomaterials reducing ROS levels in HEI-OC1 cells;

[0031] Fig.10 This is a graph showing the results of flow cytometry detection of selenium-complexed SOD / CAT cascade catalytic nanomaterials reducing HEI-OC1 cell apoptosis;

[0032] Fig.11 This is a graph showing the results of using CCK-8 assay to detect the ability of different concentrations of selenium-complexed SOD / CAT cascade catalytic nanomaterials to inhibit the killing of HEI-OC1 cells by ferroptosis inducer (Erastin);

[0033] Fig.12 This is the result of tail vein injection of selenium-complexed SOD / CAT cascade catalytic nanomaterials to treat noise (114dB, 2-20kHz)-induced noise-induced hearing loss (NIHL) mouse model. DETAILED DESCRIPTION

[0034] In order to better explain the present invention, the present invention is described in detail with reference to the embodiments of the present invention, and the main contents of the present invention are further illustrated in conjunction with specific examples, but the content of the present invention is not limited to the following examples. If no specific technology or conditions are indicated in the examples, the technology or conditions described in the literature in this area or the product instructions are used. If the manufacturer of the reagents or instruments is not indicated, they are all conventional products that can be obtained commercially.

[0035] Although the pathophysiology of noise-induced hearing loss (NIHL) remains incompletely understood, there is considerable evidence that reactive oxygen species (ROS) play a central role as a major etiology. Due to the high metabolic demands of the cochlea, sensory hair cells, stria vascularis, and spiral ganglion neurons, in particular, are susceptible to ROS-induced damage, including superoxide anions (O 2 · - ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (·OH) etc.

[0036] Antioxidant enzymes include superoxide dismutase (SOD) and catalase (CAT), which are essential for neutralizing free radicals to prevent cell damage. However, the redox balance in cells is disrupted, and the two enzymes play a limited role, requiring catalytic power to be provided from the outside. Cascade catalytic nanoplatforms are a good choice, and the two enzymes can be loaded into the nanoplatform to enhance the cascade catalytic activity between SOD and CAT, converting superoxide anions into oxygen. The present invention endows the material with the ability to respond to ROS as a cascade catalytic nanoplatform through the use of a diselenide crosslinker (3,3-diselenodipyric acid di(N-succinimidyl) ester). And the cascade catalytic nanoplatform has the advantages of easy manufacture, versatility and modifiability, and shows great prospects in biomedicine. Their application in the treatment of NIHL has not yet been explored.

[0037] The present invention is further described in detail below through examples and related performance tests.

[0038] Preparation Example 1 Preparation of Diselenide Crosslinking Agent

[0039] Sodium borohydride (2.8 g) and selenium (2.8 g) were placed in an ice-cold three-necked flask equipped with a condenser, an inlet adapter, and a drop funnel under a nitrogen atmosphere. Water (150 mL) was added under stirring. After about 15 min, the ice bath was removed and the second portion of elemental selenium (2.8 g) was added to the colorless mixture. Then stirred for 3 h. 2 Se 2 The brown-red aqueous solution was cooled to room temperature and stirred in 50 mL of H 2 3-Bromopropanol (10 g) was added to the mixture. The obtained yellow solution was stirred for another 12 h, the aqueous layer was extracted with ethyl acetate, and the aqueous layer was back-extracted with sodium chloride, dried, and the solvent was concentrated in vacuo to obtain a diselenide crosslinker precursor powder.

[0040] The precursor powder (1 g) was dissolved in 15 mL of anhydrous tetrahydrofuran in a two-necked flask under a nitrogen atmosphere, and then succinimide (1.1 g) was added and stirred continuously. After 30 minutes, a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.65 g) in 15 mL of anhydrous tetrahydrofuran was added dropwise to the mixture at 0°C and stirred overnight at room temperature. Then, impurities were filtered off and tetrahydrofuran was removed using a rotary evaporator to obtain the diselenide crosslinker 3,3-diselenodipropionic acid di(N-succinimide) ester.

[0041] Example 1 Preparation of Selenium Composite SOD / CAT Cascade Catalytic Nanomaterials

[0042] The synthesis of selenium-composite SOD / CAT cascade catalytic nanomaterials is mainly divided into two steps.

[0043] Initially, 1 mg of catalase (CAT), 0.5 mg of superoxide dismutase (SOD) and 7 mg of human serum albumin (HSA) were dissolved in 1 mL of ultrapure water, mixed evenly, and then 4 mL of glutaraldehyde solution (25 wt %) and another 8 mL of ultrapure water were added, and stirred at 1150 rpm for 12 hours at room temperature.

[0044] In the second stage, 120 mg of diselenide crosslinker (3,3-diselenodipronate (N-succinimidyl) ester) was added to the stirred mixed solution, and the mixed solution was stirred at 1150 rpm for 12 hours at room temperature. The material was collected and dialyzed with PBS for 24 hours to obtain selenium-complexed SOD / CAT cascade catalytic nanomaterials, which were stored at 4°C for further use.

[0045] <Performance Test>

[0046] 1. Transmission electron microscopy test

[0047] The transmission electron microscopy (TEM) of the selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1 is as follows: Figure 1 As shown, it can be seen from the TEM image that the selenium composite SOD / CAT cascade catalytic nanomaterial obtained by the present invention is a uniform spherical structure.

[0048] 2. Hydrodynamic size test

[0049] The selenium composite SOD / CAT cascade catalytic nanomaterial prepared in Example 1 is dispersed in ultrapure water as follows Figure 2 As shown, it can be seen that dynamic light scattering (DLS) measurements indicate that the hydrodynamic size of the niobium carbide nanozyme is approximately 80 nm.

[0050] 3. In vitro CAT enzyme activity assay

[0051] The catalase (CAT)-like activity of the selenium-composite SOD / CAT cascade catalytic nanomaterials was evaluated using a portable dissolved oxygen meter that can detect the O generated by the decomposition of hydrogen peroxide. 2 Briefly, 1 mL of selenium-composite SOD / CAT cascade catalytic nanomaterials (120 μg / mL) was added to 9 mL of H 2 O 2 Observe and record the different concentrations of H 2 O 2 The corresponding oxygen is generated.

[0052] The results are as follows Figure 3 As shown, Figure 3 It shows that in the selenium-containing composite SOD / CAT cascade catalytic nanomaterials and H 2 O 2 In aqueous solution, as H 2 O 2 The increase in concentration, H 2 O 2 Decomposed to produce O 2 , and at higher H 2 O 2 At concentrations, O 2 The generation of CAT increased significantly, proving that the CAT enzyme activity of the selenium-composite SOD / CAT cascade catalyzed nanomaterials could remove H 2 O 2 .

[0053] 4. In vitro SOD enzyme activity assay

[0054] The total superoxide dismutase colorimetric test kit (NBT) was used to determine the SOD enzyme activity of the selenium composite SOD / CAT cascade catalytic nanomaterial. The principle of the assay is that Nitor-TB, as a colorless indicator, can react with the superoxide anion (O 2 · - ) reaction produces a water-soluble formazan dye with a characteristic absorption peak at 560nm. Since SOD enzyme can catalyze the superoxide anion (O 2 · - ) undergoes a disproportionation reaction, so this reaction step can be inhibited by SOD. Therefore, the activity of SOD is negatively correlated with the amount of formazan dye produced. The colorimetric analysis of the Nitor-TB product can be used to calculate O 2 · - Clearance rate.

[0055] The results are as follows Figure 4 As shown in Figure 2, with the increase of the concentration of selenium composite SOD / CAT cascade catalytic nanomaterials, O 2 ·- The clearance rate increased, confirming that the SOD enzyme activity of the selenium-composite SOD / CAT cascade catalyzed nanomaterials could clear O 2 · - .

[0056] 5. Determination of the ability to scavenge hydroxyl radicals (·OH) in vitro

[0057] The hydroxyl radical scavenging capacity colorimetric test kit was used for determination. The determination principle is H 2 O 2 / Fe 2+ After the Fenton reaction generates ·OH, 1,10-phenanthroline can effectively capture the generated hydroxyl radicals and the remaining Fe 2+ It reacts with it to generate a colored substance with a characteristic absorption peak at 510nm. After adding a substance with scavenging ability, the colored substance decreases, so the ability to scavenge ·OH can be judged based on the numerical change of the absorbance value at 510nm.

[0058] The results are as follows Figure 5 As shown, with the increase of the concentration of selenium composite SOD / CAT cascade catalytic nanomaterials, the ·OH scavenging rate increased, indicating that selenium composite SOD / CAT cascade catalytic nanomaterials can scavenge hydroxyl radicals in a concentration-dependent manner.

[0059] 6. In vitro scavenging ability of ABTS free radicals (ABTS + ) determination

[0060] The total antioxidant capacity (T-AOC) colorimetric assay kit (ABTS enzyme catalysis method) was used. The assay principle is that ABTS is oxidized to green ABTS under the action of appropriate oxidants. + , in the presence of antioxidants ABTS· + The production of will be suppressed, according to ABTS· + The change in the characteristic absorption peak at 734 nm can be used to measure and calculate the total antioxidant capacity of the sample.

[0061] The results are as follows Figure 6 As shown, with the increase of the concentration of selenium-complexed SOD / CAT cascade catalytic nanomaterials, the total antioxidant capacity increased, indicating that it was able to scavenge ABTS free radicals.

[0062] 7. Biocompatibility test of different concentrations of selenium-composite SOD / CAT cascade catalytic nanomaterials

[0063] The cytotoxicity of different concentrations of selenium-composite SOD / CAT cascade catalytic nanomaterials was evaluated by CCK-8 method. HEI-OC1 cells were seeded in 96-well plates and cultured overnight. The culture medium was removed, and fresh DMEM culture medium containing different concentrations of selenium-composite SOD / CAT cascade catalytic nanomaterials (0, 15, 30, 60 and 120 μg / mL) was added to the 96-well plate and incubated with Otology Animal Institute-Corti 1 (HEI-OC1) cells for another 24 h. After reaching the set time point, the cells were incubated with CCK-8 solution for 30 min, and then the absorbance at 450 nm was detected by microplate reader to evaluate cell viability.

[0064] The results are as follows Figure 7 As shown, after incubating HEI-OC1 cells with different concentrations of selenium-composite SOD / CAT cascade catalytic nanomaterials for 24 hours, the cell activity was always maintained at a high level, indicating that the toxicity of the selenium-composite SOD / CAT cascade catalytic nanomaterial itself to cells is negligible, and the selenium-composite SOD / CAT cascade catalytic nanomaterial shows good biocompatibility within the experimental concentration range.

[0065] 8. Protective effect of different concentrations of selenium-composite SOD / CAT cascade catalytic nanomaterials on cells

[0066] In order to examine the different modes of H 2 O 2 Cell viability was measured by CCK-8. 2 O 2 In order to develop a cell model of oxidative stress induced by H 2 O 2 Next, the cells were incubated with different concentrations of selenium-complexed SOD / CAT cascade catalytic nanomaterials (0, 30, 40, 60, and 120 μg / mL) for 3 h, and then replaced with 600 μM H 2 O 2 The cells were incubated with fresh DMEM at 33 °C for 24 h to evaluate the effect of the selenium-complexed SOD / CAT cascade catalytic nanomaterials on H 2 O 2 Activity of induced oxidative stress. After washing twice, 100 μL of dissolved CCK-8 working solution was added to each well and incubated at 33 °C for 1 hour. The cells were then incubated with CCK-8 solution for 30 min using a multifunctional enzyme labeling instrument, and the absorbance at 450 nm was measured using a microplate reader to assess cell viability.

[0067] The results are as follows Figure 8As shown in Figure 2, with the addition of selenium-composite SOD / CAT cascade catalytic nanomaterials, the activity of HEI-OC1 cells gradually increased, weakening the H 2 O 2 (600μM) killing ability on HEI-OC1 cells.

[0068] 9. Detection of the effect of selenium-composite SOD / CAT cascade catalytic nanomaterials on ROS levels in HEI-OC1 cells

[0069] Flow cytometry was used to detect the effect of selenium composite SOD / CAT cascade catalytic nanomaterials on the level of ROS in HEI-OC1 cells. The experimental method was as follows: HEI-OC1 cells were seeded in a 6-well plate and incubated for 24 hours and divided into three groups. The control group was normal growth cells; 2 O 2 The group was treated with 2 mM H 2 O 2 The cells were incubated with fresh DMEM for 12 h. The treatment group was pretreated with selenium-composite SOD / CAT cascade catalytic nanomaterials (120 μg / mL) for 3 h, and then 2 mM H 2 O 2 The cells were then resuspended in 0.25% trypsin-EDTA, centrifuged and resuspended in 1 mL phosphate buffer containing 10 μM DCFH, incubated at room temperature in the dark for 30 min, and then analyzed by flow cytometry.

[0070] The results are as follows Fig. 9 As shown, with H 2 O 2 Compared with the control group, the addition of selenium-complexed SOD / CAT cascade catalytic nanomaterials greatly reduced the level of ROS in HEI-OC1 cells.

[0071] 10. Detection of the effect of selenium-composite SOD / CAT cascade catalytic nanomaterials on HEI-OC1 cell apoptosis

[0072] Flow cytometry was used to detect the effect of selenium composite SOD / CAT cascade catalytic nanomaterials (SCGS) on HEI-OC1 cell apoptosis. The experimental method was as follows: HEI-OC1 cells were seeded in a 6-well plate and incubated for 24 hours and divided into three groups. The control group was normal growing cells; 2 O 2 The group was treated with 6 mM H 2 O 2 The cells were incubated with fresh DMEM for 6 h. The treatment group was pretreated with selenium-composite SOD / CAT cascade catalytic nanomaterials (120 μg / mL) for 3 h, and then 6 mM H 2 O 2The cells were then resuspended in 0.25% trypsin-EDTA, centrifuged and resuspended in 1 mL of phosphate buffered saline containing PI and Annexin-V, incubated at room temperature in the dark for 30 min, and then analyzed by flow cytometry.

[0073] The results are as follows Fig.10 As shown, with H 2 O 2 Compared with the control group, the addition of selenium-complexed SOD / CAT cascade catalytic nanomaterials greatly inhibited the apoptosis of HEI-OC1 cells.

[0074] 11. Detection of the effect of selenium-composite SOD / CAT cascade catalytic nanomaterials on ferroptosis of HEI-OC1 cells

[0075] Flow cytometry was used to detect the effect of selenium-composite SOD / CAT cascade catalytic nanomaterials on apoptosis of HEI-OC1 cells. The experimental method was as follows: HEI-OC1 cells were seeded in 6-well plates and incubated for 24 hours, and treated with selenium-composite SOD / CAT cascade catalytic nanomaterials (0, 20, 30, 40, 60, 90 and 120 μg / mL) for 3 hours at 33°C, and then replaced with fresh DMEM containing 15 μM Erastin for 24 hours at 33°C to evaluate the activity of selenium-composite SOD / CAT cascade catalytic nanomaterials in inhibiting erastin-induced ferroptosis. After washing twice, 100 μL of dissolved CCK-8 working solution was added to each well and incubated at 33°C for 1 hour. The absorbance was then measured at 450 nm using a multifunctional enzyme labeling instrument. The cells were incubated with CCK-8 solution for 30 minutes, and then the absorbance at 450 nm was detected using an enzyme reader to evaluate cell viability.

[0076] The results are as follows Fig.11 As shown, compared with the Erastin group, the addition of selenium-complexed SOD / CAT cascade catalytic nanomaterials greatly inhibited the ferroptosis of HEI-OC1 cells.

[0077] 12. Selenium-complexed SOD / CAT cascade catalytic nanomaterials for the treatment of noise-induced hearing loss (NIHL) mice induced by noise (114dB, 2-20kHz)

[0078] Fifteen 4-week-old male C57BL / 6 mice were randomly divided into three groups, with 5 mice in each group, including a normal control group, a noise treatment group (114dB, 2-20kHz) induced permanent hearing loss group, and a noise treatment group treated with selenium-complexed SOD / CAT cascade catalytic nanomaterials. The three groups of mice were treated in different ways as follows:

[0079] Normal control group: raised under normal environment.

[0080] Noise injury group (NE): Mice with NIHL were induced by exposure to 114 dB, 2-20 kHz noise for 2 hours.

[0081] Drug treatment group (NE+SCGS): One day and one hour before exposure to 114 dB, 2-20 kHz noise for 2 hours, 125 μl of normal saline containing 120 μg / ml selenium-complex SOD / CAT cascade catalytic nanomaterials was injected into the tail vein, and the above treatment was performed once a day for three days after the noise exposure.

[0082] The hearing of mice in different groups was detected by auditory brainstem response (ABR).

[0083] The results are as follows Fig.12 As shown, compared with the noise damage group, the selenium-composite SOD / CAT cascade catalytic nanomaterial significantly alleviated the hearing loss of mice and lowered the ABR threshold. 14 days after noise exposure, the drug-treated group restored the hearing level to a normal hearing level. Therefore, the selenium-composite SOD / CAT cascade catalytic nanomaterial can effectively treat noise-induced hearing loss.

[0084] The above results show that the selenium composite SOD / CAT cascade catalytic nanomaterial of the present invention has the ability to remove H 2 O 2 , hydroxyl radical (·OH), superoxide anion (O 2 ·- ), ABTS free radicals and other free radicals, and can cross the blood-labyrinth barrier to reach the inner ear through systemic administration to exert a good noise-induced hearing loss (NIHL) therapeutic effect. It is suitable for systemic administration. Selenium-complex SOD / CAT cascade catalytic nanomaterials are a convenient, safe and ideal noise-induced hearing loss treatment drug. The present invention provides a new approach for the treatment of NIHL.

Claims

1. A selenium composite SOD / CAT cascade catalytic nanomaterial, characterized in that: The invention is prepared by self-assembly and cross-linking of catalase, superoxide dismutase, human serum albumin, glutaraldehyde and diselenide cross-linking agent; the diselenide cross-linking agent is 3,3-diselenodiprotionic acid di(N-succinimide) ester.

2. The selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 1, characterized in that: The selenium composite SOD / CAT cascade catalytic nano material is a spherical particle structure.

3. The selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 2, characterized in that: The particle size of the selenium composite SOD / CAT cascade catalytic nano material is 50-200nm.

4. The method for preparing the selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 1, characterized in that: The steps include: S1, dissolving catalase, superoxide dismutase and human serum albumin in ultrapure water, adding glutaraldehyde solution to form a mixed solution, and stirring the mixed solution at room temperature; S2. Add 3,3-diselenodipronate (N-succinimide) to the mixed solution and continue stirring, collect the material, and obtain the selenium composite SOD / CAT cascade catalytic nanomaterial after dialysis.

5. The method for preparing the selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 4, characterized in that: In step S1, the mass ratio of catalase, superoxide dismutase and human serum albumin added is (0.5-2):(0.5-2):10, the concentration of the glutaraldehyde solution is 20wt%-25wt%, the stirring time is 12-24 hours, and the stirring speed is 1000-1200rpm.

6. The method for preparing the selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 5, characterized in that: In the step S1, the mass ratio of glutaraldehyde to human serum albumin is (2-3):10, and the concentration of glutaraldehyde in the mixed solution is 0.05-0.1 mg / ml.

7. The method for preparing the selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 4, characterized in that: The mass ratio of 3,3-diselenedipropionic acid bis(N-succinimide) added in step S2 to human serum albumin added in step S1 is (10-20):1, the concentration of 3,3-diselenedipropionic acid bis(N-succinimide) in the mixed solution is 8-12 mg / ml, the stirring time in step S2 is 12-24 hours, and the stirring speed is 1000-1200 rpm.

8. Use of the selenium composite SOD / CAT cascade catalytic nanomaterial according to any one of claims 1 to 3 or the selenium composite SOD / CAT cascade catalytic nanomaterial prepared according to the preparation method according to any one of claims 5 to 7 in the preparation of a drug for treating noise-induced hearing loss.

9. The use of the selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 8 in the preparation of a drug for treating noise-induced hearing loss, characterized in that: The noise-induced hearing loss includes an increase in the hearing threshold.

10. Use of the selenium composite SOD / CAT cascade catalytic nanomaterial according to claim 8 in the preparation of a drug for treating noise-induced hearing loss, characterized in that: The medicine for treating noise-induced hearing loss is a systemic administration preparation.

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