A micelle for alleviating neurotoxicity and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine development, and specifically relates to a micelle for alleviating neurotoxicity, and its preparation method and application. The preparation method of the micelle of the present invention comprises the following steps: S1. preparing N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselendipropionate); S2. preparing L ‑ Cystine ‑ (Polyethylene glycol ester) 2-(diselenedipropionate) 2; S3. Preparing micelles that alleviate neurotoxicity; further, to further improve the alleviation of neurotoxicity, antioxidant active factors such as schisandrin B can also be loaded. The micelles of the present invention can alleviate neurotoxicity caused by oxidative stress, and the micelles formed after loading schisandrin B are more effective, thereby being used for the efficient prevention and treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine development, and specifically relates to a micelle for alleviating neurotoxicity, and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are a general term for oxygen-containing free radicals and peroxides that are prone to forming free radicals and are related to oxygen metabolism in organisms. They are a type of one-electron reduction product of oxygen in the body, generated when electrons leak out of the respiratory chain before being transferred to terminal oxidases, consuming approximately 2% of oxygen.

[0003] Reactive oxygen species are generated in a variety of ways, primarily through redox reactions within organisms, particularly within organelles such as mitochondria and chloroplasts. Understanding how these reactions occur, which involve multiple organelles and metabolic pathways, can help us better understand their role in living organisms and their relationship to disease.

[0004] An imbalance in the body's redox balance leads to excessive production of reactive oxygen species. Brain neurons are poorly able to scavenge free radicals. Their high unsaturated fatty acid content, high oxygen consumption, and relatively weak antioxidant defense mechanisms make them highly susceptible to oxidative damage. Single antioxidant active ingredients often suffer from poor water solubility, targeting, and gastrointestinal stability. The blood-brain barrier, in particular, hinders the effectiveness of antioxidants. Oxidative damage caused by excess oxygen free radicals is directly involved in the pathogenesis of various neurodegenerative diseases, including Alzheimer's.

[0005] Therefore, it is urgent to develop substances that can alleviate neurotoxicity by resisting oxidative stress, so as to effectively prevent and treat neurodegenerative diseases. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a micelle that alleviates neurotoxicity, which can alleviate the neurotoxicity caused by oxidative stress and thus can be used for the efficient prevention and treatment of neurodegenerative diseases.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a micelle for alleviating neurotoxicity, wherein the micelle has a structure as shown in formula (I):

[0009]

[0010] Specifically, n in the structural formula (I) is 40 to 180.

[0011] Preferably, n is 45 in the structural formula.

[0012] In a second aspect, the present invention provides a method for preparing micelles for alleviating neurotoxicity, the preparation method comprising the following steps:

[0013] S1. Preparation of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2;

[0014] Dissolve N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2 and 3,3'-diselenedipropionic anhydride in dichloromethane, add N,N'-dicyclohexylcarbodiimide and react at room temperature. After removing the insoluble matter, precipitate in ether and dry in vacuum to obtain a crude product.

[0015] The crude product was completely dissolved in deionized water and transferred to a dialysis bag. Deionized water was used as the dialysis medium for dialysis to remove unreacted polyethylene glycol. The dialysate was collected and freeze-dried to obtain N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2;

[0016] S2. Preparation of L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2;

[0017] N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester)-2-(diselenodiprotate) 2 was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid, hydrolyzed at room temperature, precipitated in ether and dried in vacuum to obtain L-cystine-(polyethylene glycol ester)-2-(diselenodiprotate) 2;

[0018] S3. Preparation of micelles to alleviate neurotoxicity;

[0019] L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 is completely dissolved in deionized water and then transferred to a dialysis bag. Deionized water is used as a dialysis medium for dialysis. The dialysate is collected and freeze-dried to obtain micelles that alleviate neurotoxicity.

[0020] Preferably, the preparation method further comprises the following steps:

[0021] S4. Encapsulated antioxidant activity factors;

[0022] The antioxidant active factors and L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 are dissolved in tetrahydrofuran, and the resulting solution is transferred to a dialysis bag. Dialysis is performed using deionized water as a dialysis medium, and the dialysate is collected and freeze-dried to obtain micelles that encapsulate the antioxidant active factors and alleviate neurotoxicity.

[0023] Preferably, the method further comprises the step of preparing N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2, comprising:

[0024] The N,N'-bis(tert-butyloxycarbonyl)-L-cystine represented by formula (II) is subjected to esterification reaction with polyethylene glycol 2000, 4000 or 8000 under the action of N,N'-dicyclohexylcarbodiimide to obtain the product.

[0025]

[0026] More preferably, the method further comprises the step of preparing 3,3'-diselenodiprolic anhydride, comprising:

[0027] 3,3'-diselenodiproic acid represented by formula (III) is refluxed with acetyl chloride to obtain the product.

[0028]

[0029] Preferably, the mass ratio of the antioxidant active factor to the L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 is 1:2-8.

[0030] Preferably, the antioxidant activity factor is selected from one or more of schisandrin B, curcumin and resveratrol.

[0031] Preferably,

[0032] The mass ratio of the antioxidant active factor to the L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 is 1:3,

[0033] The antioxidant activity factor is schisandrin B.

[0034] Preferably,

[0035] The reaction time at room temperature in step S1 is 12 to 48 hours.

[0036] The vacuum drying time in step S1 is 12 hours.

[0037] The dialysis time in step S1 is 72h.

[0038] The time of hydrolysis at room temperature in step S2 is 2 to 6 hours.

[0039] The dialysis time in step S3 is 72 h.

[0040] Preferably, in step S1, the molar ratio of the N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2 to the 3,3-diselenodiprolic anhydride is 68:163.

[0041] Preferably, in step S2, the molar concentration of the N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 and the mixed solvent of dichloromethane and trifluoroacetic acid is 5.7 mmol / L.

[0042] More preferably, the volume ratio of dichloromethane to trifluoroacetic acid in the mixed solvent of dichloromethane and trifluoroacetic acid is 2:1.

[0043] Preferably, the molecular weight cut-off of the dialysis bag is 3500 Da.

[0044] In a third aspect, the present invention provides the use of the micelles described in the present invention in the preparation of drugs for alleviating neurotoxicity caused by oxidative stress.

[0045] In a fourth aspect, an oral medication is provided, comprising a safe and effective amount of the micelles according to the present invention.

[0046] In the present invention, a safe and effective amount refers to an amount of a compound or composition that is sufficient to significantly induce a positive beneficial effect, preferably a positive beneficial effect of alleviating neurotoxicity caused by oxidative stress, including the beneficial effects disclosed herein in an independent or combined form, but the amount is low enough to avoid serious side effects, that is, to provide a reasonable benefit-risk ratio within the scope of reasonable judgment of the technician.

[0047] In a fifth aspect, the invention provides the use of the micelles described in the present invention in the preparation of oral medications.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention prepares a micelle containing multiple disulfide bonds and diselenide bonds and having synergistic anti-oxidative stress properties by deprotecting a substance obtained by sequentially esterifying N,N'-bis(tert-butyloxycarbonyl)-L-cystine with polyethylene glycol and 3,3'-diselenodiprotonic anhydride to obtain a carrier L-cystine-(polyethylene glycol ester)2-(diselenodiprotonic ester)2, and then assembling an antioxidant active factor with the carrier L-cystine-(polyethylene glycol ester)2-(diselenodiprotonic ester)2 to form a micelle loaded with the antioxidant active factor.

[0050] Because the occurrence and development of aging and neurodegenerative diseases are closely related to oxidative stress, wild-type (N2) Caenorhabditis elegans and its Alzheimer's mutant (CL4176) were selected as model organisms to evaluate the biosafety and antioxidant stress-reducing effects of neurotoxicity-mitigating micelles and micelles encapsulating antioxidant active factors. The study demonstrated that these micelles, while exhibiting excellent biosafety, not only reduced the accumulation of lipofuscin in N2 C. elegans due to normal aging but also extended lifespan and alleviated neurotoxicity by increasing antioxidant enzyme activity and reducing the lipid peroxide malondialdehyde content. The study further confirmed that after the Alzheimer's mutant (CL4176) was treated with neurotoxicity-alleviating micelles and neurotoxicity-alleviating micelles encapsulating antioxidant active factors, its antioxidant enzyme activities were generally improved and MDA content was reduced, while the increase rate of SOD and GSH-Px activities exceeded that of N2 type Caenorhabditis elegans, further confirming the broad prospects of neurotoxicity-alleviating micelles and neurotoxicity-alleviating micelles encapsulating antioxidant active factors for the efficient prevention and treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 These are the hydrogen nuclear magnetic resonance spectra of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2(A) and L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2(B).

[0052] Figure 2 Infrared spectra of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2(A) and L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2(B).

[0053] Figure 3 The critical micelle concentration curves are shown for the micelles (A) prepared in Example 1 and the micelles@schisandrin B (B) prepared in Example 2.

[0054] Figure 4 The hydrodynamic diameter (D) of the micelles @ Schisandrae Chinensis B prepared in Example 2 before (A) and after (B) H2O2 treatment for 48 h h )Change curve chart.

[0055] Figure 5 Transmission electron micrographs of the micelles@Schisandrin B prepared in Example 2 before (A) and after (B) oxidation by H2O2 for 48 h.

[0056] Figure 6The release curves of the micelles@Schisandrin B prepared in Example 2 in normal physiological environment (A) and in the presence of H2O2 (B).

[0057] Figure 7 Survival curves of N2 type Caenorhabditis elegans (A) and CL4176 type Caenorhabditis elegans (B) that were untreated and exposed to the micelles prepared in Example 1, schisandrin B, and micelles@schisandrin B prepared in Example 2.

[0058] Figure 8 Fluorescence images of N2 C. elegans without treatment (A) and after exposure to micelles prepared in Example 1 (B), schisandrin B (C), and micelles@schisandrin B prepared in Example 2 (D), as well as a graph of lipofuscin fluorescence intensity in the nematodes (E).

[0059] Figure 9 Fluorescence images of N2 type Caenorhabditis elegans without treatment (A) and exposed to micelles prepared in Example 1 (B), schisandrin B (C), and micelles@schisandrin B prepared in Example 2 (D), as well as a graph of ROS fluorescence intensity in the nematodes (E).

[0060] Figure 10 Fluorescence images of CL4176 type Caenorhabditis elegans untreated (A) and exposed to micelles prepared in Example 1 (B), schisandrin B (C), and micelles@schisandrin B prepared in Example 2 (D), as well as a graph of ROS fluorescence intensity in the nematodes (E).

[0061] Figure 11 CAT activity (A), SOD activity (B), GSH-Px activity (C), and MDA content (D) of N2 C. elegans exposed to the micelles prepared in Example 1, schisandrin B, and micelles@schisandrin B prepared in Example 2; N2 C. elegans not exposed to the samples served as the control group.

[0062] Figure 12 CAT activity (A), SOD activity (B), GSH-Px activity (C), and MDA content (D) of CL4176 C. elegans exposed to the micelles prepared in Example 1, schisandrin B, and micelles@schisandrin B prepared in Example 2; CL4176 C. elegans not exposed to the samples served as the control group. DETAILED DESCRIPTION

[0063] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention.

[0064] Example 1

[0065] This embodiment provides a method for preparing micelles that alleviate neurotoxicity, the preparation method comprising the following steps:

[0066] S1. Preparation of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2;

[0067] N,N'-bis(tert-butoxycarbonyl)-L-cystine-(polyethylene glycol ester) 2 (0.68 mM) and 3,3'-diselenodiprotonic anhydride (1.63 mM) were dissolved in 50 mL of anhydrous dichloromethane, and N,N'-dicyclohexylcarbodiimide was added to react at room temperature for 48 h. After removing the insoluble matter, the mixture was precipitated in ether and the precipitate was collected. The precipitate was dried under vacuum at room temperature for 12 h to obtain a crude product. The crude product was completely dissolved in deionized water and transferred to a dialysis bag (MWCO: 3500 Da). The dialysis was carried out for 72 h using deionized water as the dialysis medium to remove unreacted polyethylene glycol. The dialysate was collected and freeze-dried to obtain N,N'-bis(tert-butoxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprotonic anhydride) 2.

[0068] S2. Preparation of L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2;

[0069] N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2 (0.12 mM) was dissolved in 21 mL of a mixed solvent of dichloromethane and trifluoroacetic acid (the volume ratio of dichloromethane to trifluoroacetic acid was 2:1), hydrolyzed at room temperature for 4 h, and then rotary evaporated. The concentrate was precipitated in excess ether and dried in vacuum to obtain L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2.

[0070] S3. Preparation of micelles to alleviate neurotoxicity;

[0071] L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 is completely dissolved in deionized water and then transferred to a dialysis bag. Deionized water is used as a dialysis medium for dialysis. The dialysate is collected and freeze-dried to obtain micelles that alleviate neurotoxicity (referred to as micelles for short).

[0072] like Figure 1 As shown in (A), the characteristic chemical shift of the methylene group in polyethylene glycol appears at 3.65 ppm. After esterification with 3,3'-diselenodiprotation to form N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprotation) 2, -Se CH2 CH 2 -、-SeCH2- CH2-The chemical shifts of the characteristic protons appeared at 2.85 and 3.10 ppm, indicating the successful preparation of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2. Figure 1 As shown in (B), at 1.43ppm (CH3)3 The chemical signal of C-proton disappeared, indicating that L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 was successfully prepared.

[0073] The chemical structures of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselendipropionate) 2 and L-cystine-(polyethylene glycol ester) 2-(diselendipropionate) 2 were further characterized by infrared spectroscopy. Figure 2 As shown in (A), the -COC-, -CH stretching vibration and terminal -OH characteristic peaks of polyethylene glycol in N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselendipropionate) 2 appear at 1113, 2884 and 3436 cm -1 In addition, 527, 805, 1700cm -1 The characteristic peaks attributed to -C-Se, -Se-Se-, and -C=O further prove the successful preparation of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprotate) 2. After deprotection, the bis(tert-butyloxycarbonyl) deprotection forms a primary amino group, therefore, Figure 2 As can be seen in (B), the asymmetric stretching vibration of -NH2 appears at 3566cm -1 , another 1650cm -1 The stretching vibration of primary amine -NH appears, 1061cm -1 CN stretching vibration appeared at , confirming that L-cystine-(polyethylene glycol ester) 2-(diselenodiprotational ester) 2 had been successfully prepared.

[0074] The preparation steps of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2 include:

[0075] The N,N'-bis(tert-butyloxycarbonyl)-L-cystine represented by formula (II) is subjected to esterification reaction with polyethylene glycol 2000, 4000 or 8000 under the action of N,N'-dicyclohexylcarbodiimide to obtain the product.

[0076] The preparation steps of 3,3'-diselenedipropionic anhydride include:

[0077] 3,3'-diselenodiproic acid represented by formula (III) is refluxed with acetyl chloride to obtain the product.

[0078]

[0079] Example 2

[0080] This embodiment provides a method for preparing micelles that encapsulate antioxidant active factors and alleviate neurotoxicity, the preparation method comprising the following steps:

[0081] The antioxidant active factor schisandrin B (10 mg) and L-cystine-(polyethylene glycol ester) 2-(diselenodipronate) 2 (30 mg) prepared in Example 1 were dissolved in tetrahydrofuran, and the resulting solution was transferred to a dialysis bag and dialyzed with deionized water as the dialysis medium for 72 hours. After the end, the unencapsulated schisandrin B was filtered out, the filtrate was collected, and freeze-dried to obtain a powder that is the neurotoxicity-alleviating micelle that encapsulates the antioxidant active factor (referred to as micelles@schisandrin B).

[0082] Example 3

[0083] This example provides drug loading and encapsulation efficiency measurements.

[0084] Prepare 0.05-10 mg L -1 The ultraviolet absorbance of the Schisandrin B-ethanol standard solution at 216 nm was measured, and the standard curve of Schisandrin B was obtained after linear fitting, as shown in the first formula:

[0085] First formula:

[0086] C (mg L -1 )=(A-0.1301) / 0.2102(R 2 =0.9936)

[0087] Wherein, C represents the concentration of Schisandrin B-ethanol standard solution, and A represents the ultraviolet absorbance of the standard solution at 216 nm.

[0088] The neurotoxicity-alleviating micelles prepared in Example 2 were dissolved in ethanol, and their UV absorbance at 216 nm was measured. The absorbance was substituted into the first formula to calculate the mass of schisandrin B embedded in the micelles. The drug loading and encapsulation efficiency were calculated using the second and third formulas.

[0089] Second formula:

[0090] Drug loading (%) = (mass of embedded Schisandrin B / mass of micelles prepared in Example 2) × 100%;

[0091] The third formula:

[0092] Encapsulation efficiency (%) = (mass of encapsulated Schisandrin B / mass of initially added Schisandrin B) × 100%;

[0093] Based on this calculation, the drug loading capacity of schisandrin B is 31.2% and the encapsulation efficiency is 83.4%.

[0094] Example 4

[0095] This example verifies the micellization behavior.

[0096] To confirm the formation of micelles, the critical micelle concentrations of the micelles prepared in Example 1 and the micelles prepared in Example 2 were measured by fluorescence spectrometer. Pyrene was used as a fluorescent probe and an acetone solution of pyrene (5×10 -4 M). Take a certain amount of this solution and add it to the sample tubes respectively. After evaporation for 2 h in the dark, add 4 mL of 5×10 -5 ~5×10 -1 mg mL -1 The micelles prepared in Example 1 or the micelles prepared in Example 2 @ Schisandrae Chinensis B were stirred overnight. The excitation wavelength was 350 nm, and the emission spectrum from 350 to 550 nm was measured with a slit width of 2 nm. The critical micelle concentration (CMC) was calculated as the ratio of the fluorescence intensity at 383 and 372 nm (I 383 / I 372 ) as the ordinate and the logarithm of the micelle concentration (Log C) as the abscissa, which corresponds to the concentration at the intersection of the tangent lines of the curve.

[0097] like Figure 3 As shown in (A), the CMC value of the micelles prepared in Example 1 is 44.6 mg L -1 This indicates that the micelles with 3,3'-diselenedipropionic anhydride as the hydrophobic core and polyethylene glycol and cystine as the hydrophilic shell have good stability. Figure 3 As shown in (B), the hydrophobic Schisandrin B can generate hydrophobic interactions and hydrogen bonds with 3,3'-diselenedipropionic anhydride, which reduces the CMC value of the micelles prepared in Example 2 to 19.3 mg L -1 , indicating that the encapsulation of hydrophobic schisandrin B can enhance the stability of micelles.

[0098] Example 5

[0099] In this example, particle size analysis was performed.

[0100] The oxidative stress microenvironment was set up to study the hydrodynamic diameter (D h The hydrodynamic diameter (D) of the micelles @ Schisandrin B prepared in Example 2 before (A) and after (B) H2O2 oxidation for 48 h was measured by laser particle size Zeta potential instrument. h ) and polydispersity index (PDI).

[0101] like Figure 4As shown in (A), the micelles prepared in Example 2 @ Schisandrae Chinensis B h The particle size of the micelles was 135 nm, and the polydispersity index (PDI) was only 0.266, indicating that the micelle size distribution was relatively uniform. The micelle size also increased significantly after 48 h of H2O2 oxidation. This is because the micelles were slowly oxidized to form selenious acid, which caused the new assembly to swell. The PDI eventually increased to 0.508. Figure 4 As shown in (B), oxidation changes the particle size distribution curve of the micelles from a single peak to a double peak, further confirming that exogenous oxidative stimulation can change the micelles@schisandrin B prepared in Example 2 and lead to the release of schisandrin B.

[0102] Example 6

[0103] In this embodiment, microscopic morphology observation was performed.

[0104] 100 μL of the micelles@schisandrin B solution prepared in Example 2 was dripped onto the carbon support film, and the microscopic morphology of the micelles@schisandrin B prepared in Example 2 was observed using a transmission electron microscope (accelerating voltage 80 keV). The test was conducted at 25°C.

[0105] To further confirm the effect of oxidative stimulation on the micromorphology of micelles, transmission electron microscopy was used to observe the micromorphology of micelles after different treatments. Figure 5 As shown in (A), the micelles@Schisandrin B prepared in Example 2 have a clear core-shell structure without any treatment, and the average particle size is 235.18±6.83nm; after 48h of H2O2 oxidation, the original core-shell structure of the micelles is no longer visible. While a new sheet-like assembly is formed, a small amount of Schisandrin B nanoparticles appear in the field of view, as shown in FIG. Figure 5 As shown in (B), the above-mentioned changes in particle size and morphology of the micelles indicate that they are highly sensitive to oxidative stimulation. Such structural changes lay the foundation for the subsequent sustained release of schisandrin B at the site of oxidative stress.

[0106] Example 7

[0107] This example provides a demonstration of the release of antioxidant active ingredients.

[0108] The micelles@Schisandra chinensis B powder prepared in Example 2 were dissolved in pH 7.4 PBS and pH 7.4 PBS containing H2O2 and then encapsulated in a dialysis bag with a molecular weight cut-off of 3500 (the concentration of the micelle solution was 1 mg mL -1 ), immersed in a beaker filled with 200 mL of the corresponding solvent, and regularly taking 4 mL of dialysate and replenishing the same amount of dialysate. After completion, the ultraviolet absorbance of the dialysate taken out at 216 nm was measured, and the cumulative release of schisandrin B was calculated according to the fourth formula:

[0109] The fourth formula:

[0110] Cumulative release amount (%) = (M t / M o )×100%;

[0111] Among them, M t 、M o represent the release amount at time t and the total mass of schisandrin B embedded in the micelles, respectively.

[0112] like Figure 6 As shown in (A), the final release amount of Schisandrin B from the micelles@Schisandrin B prepared in Example 2 under normal physiological conditions is only 2.65±0.10%, which is negligible, indicating that the micelles can avoid the side effects of the active factor on normal tissues; Figure 6 As shown in (B), the micelles@Schisandrin B prepared in Example 2 were exposed to oxidative stress in the presence of H₂O₂. The diselenide bonds in the micelles were oxidized to selenious acid, causing the micelles to rupture. Schisandrin B then exhibited a sustained release, with a cumulative release of 66.21±1.80% at the end of the release period. These results demonstrate that the micelles@Schisandrin B prepared in Example 2 respond to oxidative stress, enabling the slow release of the encapsulated antioxidant active ingredient.

[0113] Example 8

[0114] This example provides the effects of micelles on the survival rate of Caenorhabditis elegans.

[0115] L4 stage N2 and CL4176 type Caenorhabditis elegans were transferred to 24-well plates containing micelles (0.688 mg / mL) prepared in Example 1, schisandrin B (0.312 mg / mL) and micelles@schisandrin B (1.0 mg / mL) prepared in Example 2, and 1 mL of PBS buffer and 10 μL of 5-fluorouracil (1.25 mg / mL) were added to the well plates. Caenorhabditis elegans without sample were used as the control group. This was recorded as day 0, and the plates were transferred every two days. During this process, the number of deaths was observed and recorded until all nematodes died, and a nematode survival curve was drawn. The experiment was repeated three times and calculated according to the fifth formula.

[0116] The fifth formula:

[0117] Survival rate (%) = (number of surviving nematodes / total number of nematodes) × 100%;

[0118] Depend on Figure 7As shown in (A), the average lifespan, median lifespan, and maximum lifespan of N2 C. elegans exposed to micelles, schisandrin B, and micelles@schisandrin B were all extended compared to the control group. Taking the maximum lifespan as an example, the maximum lifespans of the three groups were extended by 9.09%, 9.09%, and 27.27% compared to the control group (as shown in Table 1). Figure 7 As shown in Figure (B), the lifespan of CL4176 C. elegans exposed to micelles, schisandrin B, and micelles@schisandrin B was also significantly extended. Compared with the control group, the maximum lifespan extension rates of the three groups of nematodes were the most significant, reaching 11.11%, 11.11%, and 33.33%, respectively (as shown in Table 2). These results confirm the biosafety of the two micelles prepared in Examples 1 and 2, and also preliminarily demonstrate the beneficial effect of micelles in extending nematode lifespan. Moreover, the maximum lifespan extension rates of N2 C. elegans and CL4176 C. elegans exposed to micelles@schisandrin B were particularly significant, indicating that micelles@schisandrin B has the most significant effect in extending nematode lifespan.

[0119] Table 1 Lifespan of N2-type Caenorhabditis elegans

[0120]

[0121] Table 2 Lifespan of CL4176 Caenorhabditis elegans

[0122]

[0123] Example 9

[0124] This example provides the determination of lipofuscin content.

[0125] L4-stage N2 C. elegans were cultured on NGM plates containing micelles, schisandrin B, or micelles + schisandrin B for 10 days. After anesthesia with 10 mmol / L tetramisole hydrochloride, the cells were fixed. Twenty randomly selected N2 C. elegans from each group were imaged under a fluorescence microscope using excitation and emission wavelengths of 380 nm and 430 nm, respectively. The fluorescence intensity of lipofuscin in the nematodes was analyzed using ImageJ.

[0126] The fluorescent molecules in the cell age pigment granules lipofuscin are generally considered to be the end product of lipid peroxidation. They increase with age and are therefore also called aging pigments. Lipofuscin can be deposited in various tissues and organs. If deposited in the brain, it can cause neuronal damage and is one of the pathogenesis of Alzheimer's disease. The fluorescence intensity of nematodes is proportional to the content of lipofuscin in their body. Figure 8As shown in (A), (B), (C), (D), and (E), the fluorescence intensity of N2-type Caenorhabditis elegans after co-incubation with micelles, schisandrin B, and micelles@schisandrin B is in the following order: control group > micelles > schisandrin B > micelles@schisandrin B. In other words, both micelles and micelles@schisandrin B can eliminate the accumulation of lipofuscin caused by aging, indicating that micelles and micelles@schisandrin B can delay the pace of aging, and micelles@schisandrin B can delay the pace of aging more significantly.

[0127] Example 10

[0128] This example provides a qualitative determination of ROS levels.

[0129] C. elegans strains N2 and CL4176 were washed three times with M9 buffer. 2',7'-dichlorofluorescein diacetate (DCFA-DH) was then diluted to 50 μM, and 1 mL of this solution was added to each well of a 24-well plate. The experimental nematodes were then transferred to a 24-well plate, sealed, and protected from light. After incubation in the dark at 20°C for 1 hour, the nematodes were washed three more times with M9 buffer to remove any residual fluorescent dye. The washed nematodes were transferred to a centrifuge tube and anesthetized with 1 mL of a pre-prepared 60 μM levamisole solution. The sample containing the nematodes was then placed dropwise onto a glass slide and imaged under a confocal laser scanning fluorescence microscope. Fluorescence images were acquired at an excitation wavelength of 485 nm. Twenty nematodes were randomly selected from each group for imaging, and relative fluorescence intensity was analyzed using ImageJ software.

[0130] ROS has a strong oxidative capacity and can bind to most macromolecules in the body, such as proteins, lipids, and DNA, thereby causing cell damage. The accumulation of cell damage is the main cause of aging. Figure 9 As shown in (A), (B), (C), (D), and (E), the total reactive oxygen species (ROS) level in N2-type Caenorhabditis elegans was qualitatively determined using fluorescence imaging technology. The order of fluorescence intensity was control group > micelles > schisandrin B > micelles@schisandrin B. Since the fluorescence intensity in the nematode body is proportional to the ROS concentration, it was demonstrated that both micelles and micelles@schisandrin B can effectively eliminate ROS in the nematode body, with micelles@schisandrin B having the best effect. Figure 10 As shown in (A), (B), (C), (D), and (E), micelles and micelles@schizandrin B also showed excellent ROS scavenging effects on CL4176 type Caenorhabditis elegans, indicating that micelles and micelles@schizandrin B have great potential to alleviate brain damage caused by reactive oxygen species by resisting oxidative stress; among them, micelles@schizandrin B had the best effect.

[0131] Example 11

[0132] This example provides the determination of antioxidant enzyme activity and malondialdehyde (MDA) content.

[0133] Synchronized L1-stage N2 and CL4176 C. elegans were exposed to the same conditions starting 10 days later. The experiment was terminated after 10 days. The treated nematodes were collected, washed three times with M9 buffer to ensure cleanliness, and then transferred to sterile centrifuge tubes. 2 mL of PBS buffer was added to each tube, and two sterilized steel balls were added as a grinding medium. The tubes were vortexed for 30 seconds, repeated three times with a 20-second interval, to break the worms and evenly grind them. The suspension was homogenized, all performed under cold conditions. The tubes were then centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected and transferred to a fresh centrifuge tube for total protein quantification. OD values ​​were measured to calculate superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content. Each assay was performed in triplicate. The above data were considered significant when P < 0.05 (*), highly significant when P < 0.01 (**), and extremely significant when P < 0.001 (***).

[0134] SOD can convert O2 - Decomposed into H2O2 and O2, CAT can further decompose H2O2 into H2O and O2; GSH-Px can catalyze the conversion of reduced glutathione into oxidized glutathione, converting toxic peroxides into non-toxic hydroxyl compounds. This process can promote the decomposition of H2O2 and protect the structure and function of cell membranes from interference and damage by peroxides. Figure 11 As shown in (A), (B), (C), and (D), as shown in Table 3, the antioxidant enzyme activity of N2 C. elegans treated with micelles, schisandrin B, and micelles@schisandrin B was significantly increased compared with the control group, and the content of malondialdehyde (MDA), the end product of lipid peroxidation, was significantly reduced. In particular, the GSH-Px activity increased by 70.64%. This means that micelles and micelles@schisandrin B can delay aging by increasing antioxidant enzyme activity and inhibiting lipid peroxide content; among them, micelles@schisandrin B had the most significant effect in delaying aging.

[0135] Depend on Figure 12As shown in (A), (B), (C), and (D), as shown in Table 4, after the CL4176 type of Caenorhabditis elegans was treated with micelles, schisandrin B, and micelles@schisandrin B, the increase rates of SOD activity and GSH-Px activity exceeded those of the N2 type of Caenorhabditis elegans, while the antioxidant enzyme activities were generally improved and the MDA content was reduced. This further confirmed the broad prospects of micelles and micelles@schisandrin B for the efficient prevention and treatment of neurodegenerative diseases; among them, micelles@schisandrin B had a more obvious effect in the prevention and treatment of neurodegenerative diseases.

[0136] Table 3 Changes in antioxidant enzyme activities in N2-type Caenorhabditis elegans

[0137]

[0138] Table 4 Changes in antioxidant enzyme activities in CL4176 nematodes

[0139]

[0140] Similarly, in order to further illustrate the beneficial effects of the present invention, the following examples are provided:

[0141] Example 2.1 is provided. Compared with Example 2, the difference of Example 2.1 is that the antioxidant active factor schisandrin B of the present invention is replaced by curcumin;

[0142] Example 2.2 is provided. Compared with Example 2, the difference of Example 2.2 is that the antioxidant active factor schisandrin B of the present invention is replaced by resveratrol;

[0143] Micelles encapsulating antioxidant activity factors and alleviating neurotoxicity were prepared using the preparation method of the present invention and tested according to the testing methods of Examples 4, 5, 6, 7, 8, 9, 10, and 11. The neurotoxicity-alleviating effects of the micelles encapsulating antioxidant activity factors prepared in Examples 2.1 and 2.2 were observed. The results were similar to those of the micelles@schisandrin B prepared in Example 2.

[0144] Similarly, in order to further illustrate the beneficial effects of the present invention, the following comparative examples are provided:

[0145] Comparative Example 2.1 is provided. Compared with Example 2, the difference of Comparative Example 2.1 is that the mass ratio of the antioxidant active factor of the present invention to the L-cystine-(polyethylene glycol ester) 2-(diselendipropionate) 2 is adjusted to 1:1;

[0146] Comparative Example 2.2 is provided. Compared with Example 2, the difference of Comparative Example 2.2 is that the mass ratio of the antioxidant active factor of the present invention to the L-cystine-(polyethylene glycol ester) 2-(diselendipropionate) 2 is adjusted to 1:9;

[0147] Combined with the preparation method of the present invention, micelles encapsulating antioxidant active factors for alleviating neurotoxicity were prepared and tested according to the test methods of Examples 8, 9, 10, and 11. The effects of the micelles encapsulating antioxidant active factors for alleviating neurotoxicity prepared in Comparative Examples 2.1 and 2.2 were observed. The results were the same as those in the control group. Figure 7-12 The experimental group of schisandra chinensis B was similar.

[0148] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0149] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A micelle for alleviating neurotoxicity, characterized in that: The micelle has a structure as shown in formula (I): In the structural formula (I), n is 40, 45 or 180.

2. The method for preparing the neurotoxicity-relieving micelles according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Preparation of N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2; Dissolve N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2 and 3,3'-diselenedipropionic anhydride in dichloromethane, add N,N'-dicyclohexylcarbodiimide and react at room temperature. After removing the insoluble matter, precipitate in ether and dry in vacuum to obtain a crude product. The crude product was completely dissolved in deionized water and transferred to a dialysis bag. Deionized water was used as the dialysis medium for dialysis to remove unreacted polyethylene glycol. The dialysate was collected and freeze-dried to obtain N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2; S2. Preparation of L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2; N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester)-2-(diselenodiprotate) 2 was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid, hydrolyzed at room temperature, precipitated in ether and dried in vacuum to obtain L-cystine-(polyethylene glycol ester)-2-(diselenodiprotate) 2; S3. Preparation of micelles to alleviate neurotoxicity; L-cystine-(polyethylene glycol ester)-2-(diselenedipropionate) 2 was completely dissolved in deionized water and then transferred to a dialysis bag. Deionized water was used as the dialysis medium for dialysis. The dialysate was collected and freeze-dried to obtain micelles that alleviate neurotoxicity. The preparation method further comprises the following steps: S4. Encapsulated antioxidant activity factors; The antioxidant active factor and L-cystine-(polyethylene glycol ester)-2-(diselendipropionate) 2 were dissolved in tetrahydrofuran, and the resulting solution was transferred to a dialysis bag. The dialysis was performed using deionized water as a dialysis medium. The dialysate was collected and freeze-dried to obtain micelles encapsulating the antioxidant active factor and alleviating neurotoxicity. The preparation method of the N,N'-bis(tert-butyloxycarbonyl)-L-cystine-(polyethylene glycol ester) 2 comprises the following steps: The N,N'-bis(tert-butyloxycarbonyl)-L-cystine represented by formula (II) is subjected to an esterification reaction with polyethylene glycol 2000, 4000 or 8000 in the presence of N,N'-dicyclohexylcarbodiimide to obtain the product; The mass ratio of the antioxidant active factor to the L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 is 1:2-8; The antioxidant activity factor is selected from one or more of schisandrin B, curcumin and resveratrol.

3. The preparation method according to claim 2, characterized in that The mass ratio of the antioxidant active factor to the L-cystine-(polyethylene glycol ester) 2-(diselenodiprolate) 2 is 1:3, The antioxidant activity factor is schisandrin B.

4. The preparation method according to claim 2, characterized in that The reaction time at room temperature in step S1 is 12 to 48 hours. The vacuum drying time in step S1 is 12 hours. The dialysis time in step S1 is 72h. The time for hydrolysis at room temperature in step S2 is 2 to 6 hours.

5. Use of the micelle according to claim 1 in preparing a drug for alleviating Alzheimer's disease caused by oxidative stress.

6. The use according to claim 5, characterized in that The drug is an oral drug.

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