ROS (reactive oxygen species) response type step-by-step targeting nano-micelle for regulating polarization and iron metabolism imbalance of inflammatory microglial cells of Alzheimer's disease and preparation method of ROS response type step-by-step targeting nano-micelle

Abstract

The invention discloses an ROS (reactive oxygen species) response type step-by-step targeting nano-micelle for regulating polarization and iron metabolism imbalance of inflammatory microglial cells of Alzheimer's disease and a preparation method of the ROS response type step-by-step targeting nano-micelle, and relates to the technical field of biological medicines. The preparation method comprises the following steps: carrying out ultrasonic mixing reaction on aspirin modified glucan, DSPE-PEG2000-TK-PEG2000-KLVFFAED, DSPE-PEG2000-MG1 and icariin, so as to obtain the ROS response type step-by-step targeting nano-micelle. The preparation method comprises the following steps: carrying out ultrasonic mixing reaction on the aspirin modified glucan, the DSPE-PEG2000-TK-PEG2000-KLVFFAED, the DSPE-PEG2000-MG1 The ROS response type step-by-step targeting nano-micelle prepared by the preparation method disclosed by the invention is combined with a multi-stage targeting strategy, so that not only can the blood brain barrier be spanned, but also the precise delivery of drugs, the regulation and control of microglial cell functions and the breaking of vicious circle of iron metabolism disorder and oxidative stress in an AD focus area can be realized, and a new thought and a new solution are provided for AD treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a ROS-responsive step-by-step targeted nanomicelle for regulating polarization of inflammatory microglial cells and iron metabolism imbalance in Alzheimer's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a serious degenerative disease of the central nervous system. With the aging of society, the prevalence of AD has increased year by year, placing a heavy burden on society and families. Due to the complex and unclear etiology of AD, there is currently no effective treatment. Existing single-target drugs cannot prevent the disease from worsening, and have large side effects and limited efficacy. Although β-amyloid protein (Aβ) has been widely studied and clinically tested as a hot target for AD treatment in recent years, the results show that strategies targeting Aβ alone are not sufficient to effectively treat AD. Therefore, innovative therapies are urgently needed to break the current treatment dilemma.

[0003] The latest research reveals that the vicious cycle between abnormal activation of microglia and oxidative stress and inflammatory microenvironment in the brain is a key link in the pathological progression of AD. Microglia, as immune cells in the brain, are regulated by neuronal signals. However, in AD patients, neuronal loss leads to abnormal activation of microglia, producing a large amount of reactive oxygen species (ROS) and inflammatory factors, further aggravating neuroinflammation and toxic protein deposition in the brain. Microglia can exhibit dual phenotypes of M1 and M2, among which M1 microglia are associated with inflammation and neurological damage, while M2 microglia help to clear toxic proteins and reduce inflammation. Therefore, regulating the polarization of microglia, inhibiting M1 and activating M2, is a potential new strategy for AD treatment. In addition, the role of dysregulated iron metabolism in neurodegenerative diseases has received increasing attention. Excessive accumulation of iron not only exacerbates oxidative stress, but also interacts with the activation of microglia to form a vicious cycle. This makes regulating microglial function, improving iron metabolism and controlling oxidative stress an important way to break the progression of AD.

[0004] The molecular formula of icariin (ICA) is: 33 H 40 O 15 , CAS No. 489-32-7, structural formula is as follows:

[0005]

[0006] As an active ingredient extracted from the traditional Chinese medicine Epimedium, icariin has shown the potential to inhibit the polarization of M1 microglia and promote their polarization to M2, which can effectively reduce neuroinflammation. At the same time, icariin can also regulate iron metabolism, reduce iron accumulation in the brain, reduce oxidative stress, and thus delay the pathological progression of AD. However, the poor water solubility of icariin limits its clinical application. Summary of the invention

[0007] The purpose of the present invention is to provide a ROS-responsive step-by-step targeted nanomicelle that regulates polarization and iron metabolism imbalance of inflammatory microglia in Alzheimer's disease and a preparation method thereof to solve the problems existing in the above-mentioned prior art. The ROS-responsive step-by-step targeted nanomicelle can not only cross the blood-brain barrier, but also achieve precise drug delivery in the AD lesion area, regulate microglial function, break the vicious cycle of iron metabolism imbalance and oxidative stress, and thus provide new ideas and solutions for AD treatment.

[0008] Micelles have important advantages as a nanoscale drug delivery system. The micelle structure spontaneously formed by amphiphilic molecules can encapsulate hydrophobic drugs and significantly improve the water solubility and bioavailability of drugs. Amphiphilic polymers are synthesized by modifying dextran with aspirin to form stable micelles, which can effectively load icariin with poor water solubility and enhance its delivery efficiency in vivo. In addition, in order to solve the problem that the blood-brain barrier (BBB) ​​hinders the entry of drugs into the brain; after the drugs enter the brain, it is difficult to concentrate in the lesion site, resulting in a significant reduction in efficacy and bringing safety hazards to normal brain tissue. The present invention combines the unique ROS overexpression characteristics in the AD brain and designs a ROS-responsive step-by-step targeted loading icariin nano-micelle. By introducing ketal thiols (TK) into the nano drug delivery system, it is selectively broken in the high ROS environment in the brain, releasing the drug while accurately targeting M1 microglia. The system uses KLVFFAED peptide as a primary brain-targeting ligand to help micelles cross the BBB; after entering the brain, under the action of ROS, KLVFFAED falls off, exposing the secondary targeting peptide MG1, further enhancing the specific effect of micelles on M1 microglia. This multi-level response mechanism significantly increases the accumulation of icariin in the lesion area, reduces the nonspecific uptake of healthy tissues, and achieves high-efficiency, low-toxicity precision treatment.

[0009] Based on this, the present invention provides the following solutions:

[0010] The present invention provides a method for preparing a ROS-responsive stepwise targeted nano-micelle for regulating polarization of inflammatory microglial cells and imbalance of iron metabolism in Alzheimer's disease, comprising: preparing aspirin-modified dextran, DSPE-PEG 2000 -TK-PEG2000 -KLVFFAED, DSPE-PEG 2000 -MG1 and icariin are subjected to ultrasonic mixing reaction to obtain the ROS-responsive stepwise targeted nanomicelles;

[0011] The DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED is composed of DSPE-PEG 2000 -TK-PEG 2000 -NHS is reacted with a primary targeting peptide under the action of triethylamine; the amino acid sequence of the primary targeting peptide is KLVFFAED;

[0012] The DSPE-PEG 2000 -MG1 is composed of DSPE-PEG 2000 -NHS is reacted with a secondary targeting peptide under the action of triethylamine to obtain the product; the amino acid sequence of the secondary targeting peptide is CHHSSSARC.

[0013] Furthermore, the aspirin-modified dextran is prepared by esterification reaction of aspirin and dextran under the action of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine.

[0014] Furthermore, the mass ratio of the aspirin, the dextran, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and the 4-dimethylaminopyridine is 0.09:0.4:0.096:0.061.

[0015] Furthermore, the DSPE-PEG 2000 -TK-PEG 2000 -NHS, the primary targeting peptide and the triethylamine have a mass volume ratio of 0.1 g:0.11 g:0.3 mL.

[0016] Furthermore, the DSPE-PEG 2000 -NHS, the secondary targeting peptide and the triethylamine have a mass volume ratio of 0.1 g:0.11 g:0.3 mL.

[0017] Furthermore, the aspirin-modified dextran, the DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED, DSPE-PEG 2000 The mass ratio of -MG1 to the icariin is 5:1:1:1.

[0018] Furthermore, the ultrasonic power of the ultrasonic mixing reaction is 500W.

[0019] The present invention also provides a ROS-responsive stepwise targeted nanomicelle prepared according to the above preparation method for regulating polarization of inflammatory microglial cells in Alzheimer's disease and imbalance of iron metabolism.

[0020] The present invention also provides the use of the above-mentioned ROS-responsive step-by-step targeted nano-micelles in the preparation of drugs for treating Alzheimer's disease.

[0021] The present invention also provides a drug for treating Alzheimer's disease, wherein the active ingredient comprises the above-mentioned ROS-responsive step-by-step targeted nano micelles.

[0022] The present invention discloses the following technical effects:

[0023] The present invention innovatively constructs a ROS-responsive step-by-step targeted loaded icariin nanomicelle, which, combined with a multi-level targeting strategy, can not only cross the blood-brain barrier, but also achieve precise drug delivery in the AD lesion area, regulate microglial function, and break the vicious cycle of iron metabolism disorders and oxidative stress, thereby providing new ideas and solutions for AD treatment.

[0024] Compared with the prior art, the ROS-responsive step-by-step targeted nanomicelle system of the present invention shows significant innovation and advantages in the treatment of AD. Most traditional AD treatment methods focus on a single target, such as β-amyloid protein (Aβ), which has certain effects but cannot prevent the progression of the disease and is accompanied by large side effects. The present invention breaks through this limitation through a multi-target, multi-level response mechanism. The nanomicelles use thiol ketal bonds (TK) to achieve a specific response to the high oxidative stress environment of the brain, and through the multi-level targeting strategy of KLVFFAED peptide and MG1 peptide, it can not only effectively cross the blood-brain barrier (BBB), but also accurately target M1 microglia, regulate their polarization state, reduce neuroinflammation, and improve iron metabolism disorders and oxidative stress at the same time, breaking the vicious cycle in the progression of AD.

[0025] In addition, in order to solve the problem of poor water solubility and low in vivo utilization of the therapeutic drug icariin, the present invention effectively encapsulates the poorly water-soluble icariin through the hydrophobic core of the nano-micelles, significantly improving its water solubility and bioavailability. This micelle system not only improves the stability and delivery efficiency of icariin in the body, but also ensures the precise release and accumulation of the drug at the lesion site, reduces the non-specific uptake of healthy tissues, thereby achieving efficient and low-toxic AD treatment, and provides a new technical solution for the application of icariin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 A roadmap for the synthesis of aspirin-modified dextran;

[0028] Figure 2 This is the H NMR spectrum of aspirin-modified dextran;

[0029] Figure 3 DSPE-PEG is the primary targeting molecule 2000 -TK-PEG 2000 -H NMR spectrum of KLVFFAED;

[0030] Figure 4 DSPE-PEG is a secondary targeting molecule 2000 -H NMR spectrum of MG1;

[0031] Figure 5 The infrared images of Dextran, Aspirin and Aspirin-modified Dextran (Asp-Dex);

[0032] Figure 6 Infrared images of aspirin-modified dextran, icariin, MDA@ICA, and KMDA@ICA;

[0033] Figure 7 is the critical micelle concentration diagram of KMDA@ICA;

[0034] Figure 8 This is a comparison of the appearance of ICA aqueous solution and KMDA@ICA solution;

[0035] Fig. 9 It is the statistical diagram of micelle size of each group;

[0036] Fig.10 It is the statistical diagram of micelle potential of each group;

[0037] Fig.11 Transmission electron microscopy images of KMDA@ICA before oxidation (A) and after oxidation (B);

[0038] Fig.12 This is a graph showing the results of measuring the uptake of different micelles by M1 polarized BV.2 cells using fluorescence microscopy;

[0039] Fig.13This is a quantitative analysis of the uptake of different micelles by M1 polarized BV.2 cells measured by fluorescence microscopy;

[0040] Fig.14 The figure shows the results of measuring the uptake of different micelles by M1 polarized BV.2 cells using flow cytometry;

[0041] Fig.15 This is a quantitative analysis of the uptake of different micelles by M1 polarized BV.2 cells measured by flow cytometry;

[0042] Fig.16 In vitro release curves of each group;

[0043] Fig.17 This is a diagram showing the results of in vivo imaging of small animals to observe the long-circulation effects of different preparations in rats;

[0044] Fig.18 This is a result diagram of in vivo imaging of small animals to observe the distribution of different preparations in mice;

[0045] Fig.19 The figure shows the results of the iron content test of BV.2 cells after treatment with different groups;

[0046] Fig. 20 Fluorescence microscopy images of the expression of M1 polarization markers in BV.2 cells after treatment with different groups;

[0047] Fig.21 Fluorescence microscopy images of the expression of M2 polarization markers in BV.2 cells after treatment with different groups;

[0048] Fig. 22 The results of the water maze experiment are used to evaluate the improvement of the cognitive ability of AD mice by each preparation group; A is a statistical analysis of the time in the quadrant where the original platform is located in the water maze experiment after the AD mice are treated with different preparation groups; B is a statistical analysis of the number of crossing the original platform area in the water maze experiment after the AD mice are treated with different preparation groups; C is a statistical analysis of the nesting experiment scores after the AD mice are treated with different preparation groups;

[0049] Fig.23 The statistical graphs show the expression of glutathione (A), superoxide dismutase (B) and malondialdehyde (C), key molecules of oxidative stress in the brain of mice in each group;

[0050] Fig.24 The figures are statistical graphs of the levels of inflammatory factors in the brain tissues of mice in each group; A is a statistical graph of the content of tumor necrosis factor-α in the brains of AD mice after being treated with different preparations; B is a statistical graph of the content of interleukin-6 in the brains of AD mice after being treated with different preparations; C is a statistical graph of the content of interleukin-1β in the brains of AD mice after being treated with different preparations. DETAILED DESCRIPTION

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0054] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0055] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0056] Example 1

[0057] 1. Experimental instruments and materials

[0058] 1. Experimental instruments: DZKW-S-4 electric constant temperature water bath (Shanghai Medical Equipment Company Medical Equipment Factory No. 5); JY92-IIN ultrasonic cell crusher (Ningbo Xinzhi Biotechnology Co., Ltd.); SG3300H ultrasonic cleaner (Shanghai Guante Ultrasonic Instrument Co., Ltd.); RE52CS rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); XS105 one-hundred-thousandth balance (Mettler Toledo); Ti-S fluorescent inverted microscope (Nikon Company, Japan); HBS-1096A microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.); 1100 liquid chromatograph (UV detector, Dalian Yilite); C18 chromatographic column (250mm×4.6mm, 5μm, Shanghai Yuexu). Sephadex G-50 column (Shanghai Hualan Chemical Technology Co., Ltd.); dialysis bag (Beijing Solebold Technology Co., Ltd., molecular weight cutoff 500-12000Da); polycarbonate membrane (Milipore Co., Ltd., USA); 500A nanoparticle size and Zeta potential analyzer (Anton Paar, Austria); JEM-2000EX transmission electron microscope (Japan Electronics Co., Ltd.); SW-CJ-1D single-person clean bench (Shanghai Sujing Industrial Co., Ltd.); gas-jacketed carbon dioxide cell culture incubator (Thermo Flesher Scientific (Asheville) LLC, USA); FACSCalibur flow cytometer (BD Company, USA); multispectral in vivo imaging system (Carestream Health, Inc., USA); RM2235 paraffin slicer (Leica, Germany); BrukerAvance III HD 600MHz; Shimadzu Fourier transform infrared spectrometer IRTracer-100.

[0059] 2. Experimental materials: icariin, dextran, aspirin, anhydrous dimethyl sulfoxide (Shanghai Aladdin Biochemical Technology Co., Ltd.); distearoylphosphatidylethanolamine-polyethylene glycol 2000-NHS (DSPE-PEG 2000 -NHS), distearoylphosphatidylethanolamine-polyethylene glycol 2000-ketone thioacetate-polyethylene glycol 2000-NHS (DSPE-PEG 2000 -TK-PEG 2000-NHS), peptides (Xi'an Ruixi Biotechnology Co., Ltd.); iNOS, iron content determination kits (Abcam, USA); CD206 antibody (Biolegend); TNF-α ELISA kit, IL-1β ELISA kit, IL-6 ELISA kit (Beijing Solebow Technology Co., Ltd.) GSH kit, SOD kit, MDA kit (Nanjing Jiancheng Biotechnology Co., Ltd.); DMSO-d6 (Cambridge Isotope Laboratories, Inc.); the remaining reagents were of analytical grade, and water was purified water.

[0060] 2. Experimental Methods

[0061] 1. Preparation of Nanomicelles

[0062] 1.1 Preparation of membrane materials and targeting ligands

[0063] The membrane material aspirin modified dextran (Asp-Dex) is prepared by esterification reaction of raw materials aspirin and dextran (MW2000) under the action of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·Cl) and 4-dimethylaminopyridine (DMAP). The main process is as follows:

[0064] A 100 mL three-necked bottle was fixed on an iron stand on a magnetic stirrer. 0.09 g of aspirin, 0.061 g of 4-dimethylaminopyridine, 0.096 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, a magnetic stirrer and 10 mL of anhydrous dimethyl sulfoxide (DMSO) were added to the three-necked bottle in sequence. Nitrogen was repeatedly filled three times to deoxygenate, and stirred at 500 rpm for 0.5 hours (0.5-1 hour can achieve the same effect) at room temperature under nitrogen protection to activate the carboxyl group. 0.4 g of dextran was dissolved in 10 mL of anhydrous DMSO and ultrasonically treated to fully dissolve it. The dissolved dextran solution was slowly added dropwise to the above reaction system and continued to stir at room temperature for 48 hours. After the reaction was completed, 10 times the volume of cold anhydrous ethanol was added and allowed to stand overnight to precipitate the product. The next day, the precipitate was collected and transferred to a dialysis bag (molecular weight cutoff 500Da), and dialyzed with distilled water for 48 hours, with the water replaced every 12 hours. The dialyzed product was freeze-dried to finally obtain aspirin-modified dextran (Asp-Dex).

[0065] Primary targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED by DSPE-PEG 2000 -TK-PEG 2000-NHS is reacted with KLVFFAED peptide under the action of triethylamine. The main process is as follows:

[0066] Weigh 0.1 g DSPE-PEG 2000 -TK-PEG 2000 -NHS was dissolved in 3 mL N,N-dimethylformamide (DMF), and 0.11 g of the primary targeting peptide (amino acid sequence: KLVFFAED) and 0.3 mL of triethylamine were added to dissolve completely, and the mixture was reacted at room temperature for 12 h. The reaction solution was transferred to a dialysis bag (molecular weight cutoff: 2000 Da), and dialyzed in pure water for 24 h. The dialysate was collected and freeze-dried to obtain the primary targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED.

[0067] Secondary targeting molecule DSPE-PEG 2000 -MG1 is made of raw material DSPE-PEG 2000 -NHS and the secondary targeting peptide, MG1 peptide (amino acid sequence: CHHSSSARC), were synthesized by reaction under the action of triethylamine. The main process is as follows:

[0068] 0.1 g DSPE-PEG 2000 -NHS was dissolved in 3 mL N,N-dimethylformamide (DMF), 0.11 g MG1 polypeptide and 0.3 mL triethylamine were added to dissolve completely, and the mixture was reacted at room temperature for 12 h; the reaction solution was transferred to a dialysis bag (molecular weight cutoff 2000 Da), dialyzed in pure water for 24 h, and the dialysate was collected and freeze-dried to obtain the secondary targeting molecule DSPE-PEG 2000 -MG1.

[0069] 1.2 Preparation of ROS-responsive stepwise targeted nanomicelles

[0070] Accurately weigh 10 mg Asp-Dex and 2 mg DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED and 2mg DSPE-PEG 2000 -MG1 in a 15 mL centrifuge tube, add 5 mL PBS as solvent to dissolve the above materials, then add 4mLThe ethyl acetate solution containing icariin (ICA) (containing 2 mg of ICA) was placed in an ultrasonic cell disruptor and sonicated for 10 min (power: 500 W, single ultrasonic time 10 s, stop interval 5 s). The solution was transferred to a round-bottom flask and rotary evaporated under reduced pressure in a 50°C water bath to remove the ethyl acetate. Finally, the micelle solution was squeezed through a microporous filter membrane with a pore size of 0.22 μm twice to make the micelle particle size distribution more uniform. The filtrate was collected to obtain ROS-responsive stepwise targeted nanomicelles (KMAD@ICA).

[0071] During the preparation of nanomicelles, ICA and DSPE-PEG were not added. 2000 -TK-PEG 2000 -KLVFF and DSPE-PEG 2000 -MG1 to obtain blank micelles (Asp-Dex); no DSPE-PEG was added 2000 -TK-PEG 2000 -KLVFF to obtain MG1 modified micelles (MAD@ICA); without adding DSPE-PEG 2000 -MG1 to obtain ROS-sensitive micelles (KAD@ICA); without adding DSPE-PEG 2000 -TK-PEG 2000 -KLVFF and DSPE-PEG 2000 -MG1 to obtain ICA micelles (Asp-Dex@ICA); DSPE-PEG 4000 -KLVFF instead of DSPE-PEG 2000 -TK-PEG 2000 -KLVFF obtains ROS-insensitive micelles.

[0072] 1.3 Preparation of fluorescent probe micelles

[0073] Coumarin (Cou) micelles and DiR micelles were prepared by the same method as above, wherein ICA was replaced by coumarin or DiR, respectively.

[0074] 2. Identification of the properties of nanomicelles

[0075] 2.1 Material structure characterization

[0076] 20 mg of nanomicelle sample was dissolved in 0.5 mL of DMSO-d6 and transferred to a nuclear magnetic resonance tube for hydrogen nuclear magnetic resonance spectrum testing.

[0077] Take 2 mg of nano-micelle sample and mix it with KBr in a ratio of 1:100, grind it and press it into a tablet; put the pressed KBr wafer into a bracket, and then put it into an infrared detector for detection.

[0078] 2.2 Determination of critical micelle concentration

[0079] The critical micelle concentration (CMC) was determined using pyrene as a fluorescent probe. The specific process was as follows: First, the micelles were diluted to different concentrations (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 mg / mL), and then a final concentration of 6×10 -7 mol / L of pyrene, the mixture was incubated at room temperature in the dark for 12 h to allow pyrene to embed into the hydrophobic core of the micelles; the fluorescence spectrum was recorded using an excitation wavelength of 340 nm and an emission wavelength range of 300-400 nm. Calculation of I1(I 373 ) / I3(I 384 ) and plot the ratio versus micelle concentration. The CMC is determined by the intersection of the two linear regions in the graph.

[0080] 2.3 Particle size and Zeta potential determination

[0081] Take 200 μL of micelles and dilute to 2 mL with distilled water. Use a 500A nanoparticle size and Zeta potential analyzer to measure the particle size and Zeta potential of different micelles by dynamic light scattering. Each preparation is measured 3 times, and each cycle is set to measure 10 times.

[0082] 2.4 Transmission electron microscopy observation of micelle morphology

[0083] Observation of KMAD@ICA morphology by transmission electron microscopy. Take an appropriate amount of micelles and dilute them to a suitable concentration. Drop a drop on the copper grid for electron microscopy, stain it with 2% phosphotungstic acid, and observe its morphology by transmission electron microscopy after drying. The acceleration voltage of the transmission electron microscope is set to 120 kV.

[0084] 2.5KMAD@ICA microenvironment response evaluation

[0085] 100 μM hydrogen peroxide was added to the KMAD@ICA solution and incubated for 1 h. Dynamic laser scattering was used to analyze the change in KMAD@ICA particle size, and transmission electron microscopy was used to observe the morphology of KMAD@ICA to evaluate its intelligent response behavior.

[0086] 2.6 In vitro release evaluation of nanomicelles

[0087] The in vitro release behavior of nanomicelles was investigated by dialysis bag method. The release medium was PBS solution containing 5% Tween 80 and PBS solution containing 5% Tween 80 and 100μM hydrogen peroxide. 1mL of ICA standard solution, Asp-Dex@ICA and KMAD@ICA were respectively placed in dialysis bags with a molecular weight cutoff of 8-14kDa, immersed in 20mL of release medium, and the release system was placed in a shaker at 37°C and 100rpm. 0.5mL of release medium was taken out at the set time points (2, 6, 12, 24 and 48h), and 0.5mL of fresh release medium was added in time. Each preparation was repeated 3 times. The amount of ICA in each sample was determined by HPLC, and the drug release amount of various preparations at each time point was calculated.

[0088] In vitro release rate (%) = amount of drug released / total drug amount × 100%.

[0089] 3. Evaluation of the targeting properties of ROS-responsive step-by-step targeted micelles in vitro

[0090] 3.1 Cell culture

[0091] Mouse microglial cells (BV.2) were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) in a 37°C, 5% CO2-95% air cell culture incubator and passaged when the confluence reached 90%.

[0092] 3.2 Evaluation of micelle-responsive targeting of M1 polarized microglia

[0093] BV2 cells were cultured at a density of 1 × 10 5 The cells were inoculated in a culture dish. After LPS (1 μg / mL) was used to induce the cells for 24 h, the cells were washed with PBS, and the culture medium containing KMAD@Cou and non-ROS-responsive Cou micelles was added to different culture dishes. After 2 h, the culture medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, PDAI stained the cell nucleus for 10 min, and the distribution of fluorescence in the cells was observed using a fluorescence microscope.

[0094] Flow cytometry was used to detect the evaluation of micelle responsiveness targeting M1 polarized microglia. The specific method is the same as above. After culturing for 2 hours, the culture medium was discarded and washed with PBS to remove excess micelles that were not taken up. The cells were digested with trypsin, centrifuged and washed, and then pre-cooled PBS was added to resuspend the cells. The fluorescence intensity of the probe bound to the cells was quantitatively analyzed by flow cytometry.

[0095] 4. Evaluation of the long-term circulation effect and targeting of different micelles in vivo

[0096] 4.1 Investigation of long-cycle effects

[0097] SD rats were randomly divided into the following 6 groups (n=3):

[0098] (1) Normal saline (Control); (2) Free DiR (Free DiR); (3) DiR micelles (Asp-Dex@DiR); (4) MG1 modified DiR micelles (MAD@DiR); (5) ROS responsive stepwise targeted DiR micelles (KMAD@DiR). Since ICA has no fluorescence, ICA was replaced by the in vivo fluorescent dye DiR.

[0099] Each group of rats was given the above preparations once through tail vein injection. The DiR dose in each administration group was ensured to be equal, all at 3 μg / rat. 200 μL of whole blood was collected at different time points (1, 3, 6, 12, 18, 24, 36, 48, 72, 96 h) into a 2 mL centrifuge tube containing an anticoagulant, mixed, and transferred to a 96-well plate. The fluorescence signal intensity of each preparation was observed using a small animal in vivo imaging analyzer to evaluate its long-circulation effect in rats.

[0100] 4.2 In vivo targeting evaluation

[0101] 4.2.1 In vivo imaging

[0102] In vivo imaging experiments were used to investigate the in vivo targeting of different agents. The drug was replaced with the fluorescent reagent DiR. The mice were divided into the following groups:

[0103] (1) Normal saline (Control); (2) Free DiR (Free DiR); (3) DiR micelles (Asp-Dex@DiR); (4) MG1 modified DiR micelles (MAD@DiR); (5) ROS responsive stepwise targeted DiR micelles (KMAD@DiR). The DiR dose in each drug administration group was the same, 2 μg / mouse.

[0104] The mice were anesthetized with isoflurane, and the fluorescence signals in each group of mice were monitored using a living imaging system. Fluorescence imaging was performed on the mice at different time points (1, 3, 6, 12, 18, 24, 36, 48, and 72 h) to observe the fluorescence signal intensity in vivo.

[0105] 5. Evaluation of the pharmacodynamics of micelles against AD

[0106] 5.1 Iron content detection.

[0107] Iron content was measured using an iron content assay kit according to the manufacturer's instructions. BV.2 cells were cultured at 1 × 10 6 The cells were inoculated at a density of 100 μmol / well in a six-well plate and cultured overnight.-1 ) and treated with ammonium ferric citrate FAC (600 μmol L -1 After all treatments were completed, the cells were washed twice with cold PBS, lysed with lysis buffer, and further treated with 30 μL of iron detection reagent for 30 min. The absorbance was measured with an enzyme reader at a wavelength of 550 nm.

[0108] 5.2 Evaluation of the effect of micelles on regulating microglial M1 / M2 polarization

[0109] Cell immunofluorescence staining was used to evaluate the polarization of microglia. BV2 cells were seeded in cell culture dishes at a density of 1×10 6 cells. FAC (200 μmol L -1 ) induced the cells for 24 hours, LPS (1 μg / mL) induced the cells for 6 hours, washed the cells with PBS, and added the culture medium containing PBS, Asp-Dex@ICA, KAD@ICA or KMAD@ICA to different cell plates, with cells without LPS induction as blank controls. After 36 hours, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Then the cells were exposed to 0.3% TritonX-100 for 10 minutes, blocked with staining blocking buffer at room temperature for 1 hour, and primary antibody CD206 (1:50 volume dilution) and anti-iNOS (1:200 volume dilution) were added to the cells of different groups and incubated with the cell samples at 4°C overnight, washed three times with PBS, and FITC-labeled secondary antibody (1:250 volume dilution) was added and incubated in the iNOS wells for 1 hour at room temperature. Cy5-labeled secondary antibody (1:250 volume dilution) was added and incubated in the CD206 wells for 1 hour at room temperature. Finally, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), washed with PBS, and observed and photographed under a fluorescence microscope.

[0110] 5.3 Animal grouping and drug administration

[0111] Six-month-old APP / PS1 double transgenic mice were randomly divided into four groups: AD group, free ICA group, Asp-Dex@ICA group and KMAD@ICA group; C57BL / 6 mice of the same age were taken as wild blank control group (Wild). The Wild group and AD group were given equal volumes of normal saline. The Free ICA group, Asp-Dex@ICA group and KMAD@ICA group were injected into the tail vein once every two days at a dose of 5 mg / kg of ICA. The above mice were given behavioral observations for two consecutive months. After the observation, the mice were euthanized for pharmacodynamic analysis.

[0112] 5.4 Morris water maze test

[0113] The Morris water maze experiment was used to investigate the spatial learning and memory ability of mice after drug treatment. The specific process is as follows: First, a positioning navigation experiment was conducted. The first 4 days were the training phase, and the formal experiment was conducted on the 5th day. The experiment recorded the swimming distance of the mice. The mice were placed at the center of the four quadrants in the pool, and the escape latency was set to 60 seconds. If the mouse found the platform, it was allowed to stay on the platform for 15 seconds; if the platform was not found, the mouse was guided to climb onto the platform and stay for 15 seconds. On the 6th day, the platform was removed, and the mouse was placed in the quadrant opposite to the original platform. The number of times the mouse crossed the original platform within 60 seconds and the time it stayed in the quadrant were measured, and the entire movement trajectory was recorded to evaluate the learning and memory ability of the mouse.

[0114] 5.5 Nesting experiment

[0115] After the water maze experiment, a nesting experiment was conducted to evaluate the changes in cognitive ability, motor ability, and coordination of each group of mice after drug treatment. The specific experimental process is as follows: the mice were placed in a clean cage, and a 5 square centimeter nesting cotton pad was provided to the mice in a corner of the cage. The nesting conditions of the mice were recorded at different time points, and finally the nests were scored according to the method reported in the literature, from 1 to 5 (very poor / no nesting-best nesting).

[0116] 5.6 Preparation of brain tissue samples

[0117] 1 hour after the last administration, mice in each group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), fixed on the operating table, exposed the heart, inserted the syringe from the left ventricle into the aorta, opened the right atrial appendage, and perfused the pre-cooled saline through the heart. When the liver turned white and the liquid flowing out of the atrial appendage was clear, the perfusion was stopped. The mouse skull was cut open and the whole brain was quickly taken out. The brain tissue was divided into two along the midline, and the left half of the brain was placed in a cryopreservation tube, frozen and stored for standby use. Cold PBS solution was added to the left half of the brain tissue of each group of mice, stirred at high speed under ice bath until homogenized, centrifuged at 10000rpm for 10 minutes at 4°C using a high-speed refrigerated centrifuge, and the supernatant was aspirated into an EP tube for biochemical kit detection.

[0118] 5.7 Detection of oxidative stress level in the brain

[0119] Biochemical kits were used to detect the content of malondialdehyde (MDA) and glutathione (GSH), which represent the level of oxidative stress in the brain of mice in each treatment group, and the activity of superoxide dismutase (SOD). The experimental operation steps were strictly carried out in accordance with the instructions of the kit. The experimental steps are as follows: cold PBS solution was added to the brain tissue of each group of mice, stirred at high speed under ice bath until homogenized, centrifuged at 10000rpm at 4℃ for 10 minutes using a high-speed refrigerated centrifuge, and the supernatant was aspirated into an EP tube, and the content of malondialdehyde (MDA) and glutathione (GSH) in the brain tissue and the activity of superoxide dismutase (SOD) were detected strictly in accordance with the manufacturer's instructions.

[0120] 5.8 Detection of neuroinflammation levels in the brain

[0121] ELISA kits were used to detect the levels of inflammatory factors TNF-α, IL-1β and IL-6 in the brain of mice in each treatment group. The experimental operation steps were strictly carried out in accordance with the instructions of the kit. The experimental steps are as follows:

[0122] First, restore the kit to room temperature; then add the display agent A, B and stop solution to the blank well for zero adjustment; add 50μL of diluted standard to the standard well, add 50μL of standard / sample diluent and 50μL of biotin antigen working solution to the zero well; add 50μL of sample and 50μL of biotin antigen working solution to the sample well; shake gently, cover with sealing film, and incubate at 37℃ for 30min; dilute the washing solution for later use; discard the liquid in the well, fill each well with washing solution, wash for 30s and discard, wash 5 times; then add 50μL avidin-HRP, shake gently, cover with sealing film, incubate at 37℃ for 30min; peel off the sealing film, discard the liquid in the wells, fill each well with washing solution and wash for 30s and then discard, wash 5 times, and pat dry; add 50μL of color developer A and 50μL of color developer B to the wells in turn, shake gently, develop at 37℃ in the dark for 10min, then add 50μL of stop solution to each well to stop; finally, adjust the blank well to zero, and measure the absorbance value of each well in sequence at a wavelength of 450nm; calculate the levels of IL-1β, IL-6 and TNF-α in each sample.

[0123] 6. Statistical Analysis

[0124] GraphpadPrism 9.0 software was used for statistical analysis. All experimental data were expressed as Mean ± SD. One-way analysis of variance was used for comparison among multiple groups, and LSD-t test was used for further pairwise comparison. P < 0.05 indicated that the difference was statistically significant.

[0125] 3. Results

[0126] 1. Structural characterization of Asp-Dex

[0127] The synthesis formula of Asp-Dex is as follows Figure 1 As shown, the NMR spectrum of Asp-Dex is as follows Figure 2 As shown in the spectrum, the characteristic peaks of Asp in Asp-Dex (chemical shift of benzene ring 7-8ppm, methyl 2-3ppm) and the characteristic peaks of Dex (3-5ppm) are clearly visible. The infrared image of Asp-Dex is shown in Figure 5 As shown, compared with Dex, Asp-Dex has a range of 1735-1750cm -1 The peak intensity and displacement of the ester group at the displacement point changed. The above results prove that Asp-Dex was successfully synthesized.

[0128] 2. Structural Characterization of Targeting Molecules

[0129] Secondary targeting molecule DSPE-PEG 2000 -MG1 H NMR spectrum is as follows Figure 3 As shown, DSPE-PEG is clearly visible in the spectrum 2000 -The characteristic peak of the peptide in MG1 (chemical shift 7-8.5ppm) proves the secondary targeting molecule DSPE-PEG 2000 -MG1 was successfully synthesized. The primary targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED H NMR spectrum is as follows Figure 4 As shown in the spectrum, the characteristic peaks of KLVFFAED polypeptide (chemical shift 7-7.5 ppm) and the characteristic peaks of ketalthiool (TK) bond (chemical shift 2.5-3 ppm, 1.2-1.7 ppm) are clearly visible, proving that the primary targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED was successfully synthesized.

[0130] 3. Structural Characterization of Micelles

[0131] The infrared spectrum of the prepared micelle KMAD@ICA is shown in Figure 6 As shown in the figure, it can be seen that the characteristic peak of ICA disappears compared with KMAD@ICA (shift 1250-1750cm -1 , 2750-3000cm -1 ) showed that ICA was successfully encapsulated in the micelles. Figure 7 The CMC of the micelles was shown to be 0.00796 mg / mL, which is significantly lower than the effective concentration of micelles in the blood (about 0.5 mg / mL), thus preventing the micelles from being degraded due to dilution when entering the body. Figure 8The figure shows the appearance comparison of ICA free aqueous solution and KMAD@ICA. It can be clearly seen from the figure that many insoluble particles are suspended in the free aqueous solution, while the micelle solution is clear and transparent, indicating that preparing ICA into micelles significantly increases the solubility of the drug. Fig. 9 The sizes of different formulation groups are shown, among which the particle size of Asp-Dex is 62.90±0.12nm, the particle size of Asp-Dex@ICA is 63.26±0.16nm, the particle size of MAD@ICA is 77.13±0.71nm, the particle size of KMAD@ICA is 120.72±2.71nm, and the particle size of KMAD@ICA in the presence of hydrogen peroxide is 116.10±1.35nm; from the results, it can be seen that after ICA is encapsulated in micelles, the size of the micelles increases slightly, and the modification of the targeting molecule increases the size of the micelles. In the presence of hydrogen peroxide, the TK bond in the primary targeting molecule in KMAD@ICA responsively breaks, the outermost KLVFFAED molecule falls off, and the micelles show size shrinkage. Fig.10 The Zeta potential of different preparation groups is shown, among which the potential of Asp-Dex is -1.43±0.12 mV, the potential of Asp-Dex@ICA is -1.87±0.058 mV, the potential of MAD@ICA is -3.97±0.32 mV, the potential of KMAD@ICA is -5.27±0.32 mV, and the potential of KMAD@ICA in the presence of hydrogen peroxide is -3.7±0.87 mV.

[0132] 4. Evaluation of ROS Responsiveness of Micelles

[0133] Fig.11 A in the middle is a transmission electron microscope photo of micelles, which shows that the micelles are spherical and have a particle size of about 100 nm; Fig.11 Figure B is a transmission electron microscopy photo of micelles after hydrogen peroxide oxidation. It can be seen from the figure that some micelles are in a cleaved state, proving that the prepared KMAD@ICA has ROS responsiveness. In addition, fluorescence microscopy was used to observe the uptake of coumarin-loaded KMAD micelles and non-ROS responsive micelles without TK bonds in M1 polarized microglia. The results showed that Fig.12 and Fig.13 As shown, the fluorescence signal of the non-ROS-responsive micelles was significantly weaker than that of the KMAD group, indicating that the KMAD micelles could responsively cleave the outermost primary targeting molecules in the high-level oxidative environment of M1 polarized microglia, exposing the secondary M1 microglia targeting peptide MG1 and increasing uptake. Fig.14 and Fig.15Flow cytometry was used to detect the uptake of coumarin-loaded KMAD micelles and micelles without TK bonds in M1 polarized microglia. The experimental results showed that the fluorescence signal of the KMAD group was significantly stronger than that of the micelles without ROS-sensitive bonds, which further supported the effect of KMAD micelles in step-by-step targeting of ROS response.

[0134] Fig.16 The in vitro release results of micelles were shown. At 48h, the in vitro release rate of ICA free drug was (92.33±3.97)%, the in vitro release rate of Asp-Dex@ICA was (70.59±0.77)%, the in vitro release rate of KMAD@ICA was (70.16±0.44)%, and the in vitro release rate of KMAD@ICA+H2O2 was (90.07±2.89)%. The experimental results showed that KMAD@ICA was co-incubated with release media containing / without H2O2. Within 48 hours, the release rate of ICA in the release medium containing H2O2 was significantly increased compared with the release medium without H2O2. This indicates that ICA loaded in KMAD@ICA can be responsively released in an oxidative environment. This property can support the selective release of drugs from micelles in pathological environments.

[0135] 5. Evaluation of micelle long-circulation effect

[0136] DiR was used instead of ICA as a fluorescent probe to observe the circulation behavior of each preparation in rat blood after intravenous administration. Blood samples were collected at predetermined time points to observe the fluorescence intensity in the blood. Fig.17 As shown in the figure, the circulation time of each preparation in the blood is in the following order: KMAD@DiR>MAD@DiR>Asp-Dex@DiR>Free DiR. Compared with other micellar preparations, the retention rate of Free DiR in the blood is extremely low, and its fluorescence signal is almost invisible. The above results show that KMAD@DiR has a good long circulation effect in vivo.

[0137] 6. Evaluation of the targeting properties of micelles in vivo

[0138] The in vivo fluorescence imaging system was used to observe the distribution of fluorescence signals in mice at different time points. Fig.18 . In the Free DiR group, almost no fluorescence signal was observed in the brain during the period of 1-72h. However, the KMAD@DiR group had the strongest fluorescence signal in the brain at each time point. The order of DiR fluorescence intensity at different time points is as follows: KMAD@DiR>MAD@DiR>Asp-Dex@DiR>Free DiR. The above results show that KMAD@DiR has better brain targeting and long circulation effect.

[0139] 7. Evaluation of the efficacy of micelles against AD

[0140] 7.1 Determination of the ability of micelles to regulate intracellular iron content

[0141] Since excess intracellular iron ions can stimulate the Fenton reaction, which in turn triggers oxidative stress and drives microglial M1 polarization, regulating the intracellular iron ion content is beneficial to regulating microglial polarization. Fig.19 As shown in Figure 2, the results of iron content determination showed that FAC stimulation significantly increased the iron level in BV.2 cells. ICA, Asp-Dex@ICA and KMAD@ICA treatments were able to reduce this abnormally elevated iron level, and KMAD@ICA had the best effect compared to the other treatment groups.

[0142] 7.2 Evaluation of the ability of micelles to regulate microglial M1 / M2 polarization

[0143] Iron metabolism disorder and oxidative stress can drive the polarization of microglia M1. After intervention with different preparations, the expression of iNOS, a marker of microglia M1 polarization, was shown in Figure 2 Fig. 20 As shown, the fluorescence signal showed a trend of gradual weakening. Asp-Dex@ICA, KAD@ICA, and KMAD@ICA all downregulated the expression of iNOS. Compared with the Asp-Dex@ICA group without targeted modification, the targeted modification group had a stronger ability to downregulate iNOS; and compared with the KAD@ICA group without MG1 modification, the KMAD@ICA group had a stronger ability to downregulate iNOS.

[0144] In addition, the expression of microglial M2 polarization marker CD206 was Fig.21 As shown, the fluorescence signal showed a trend of gradual enhancement. Asp-Dex@ICA, KAD@ICA, and KMAD@ICA all upregulated the expression of CD206. Compared with the Asp-Dex@ICA group without targeted modification, the targeted modification group had a stronger ability to upregulate CD206; and compared with the KAD@ICA group without MG1 modification, the KMAD@ICA group had a stronger ability to upregulate CD206.

[0145] 7.3 Evaluation of the ability of micelles to improve the behavior of AD mice

[0146] First, a water maze experiment was used to evaluate the improvement of cognitive ability of AD mice by each preparation group. Fig. 22A and B show that compared with the wild group mice, the number of times and time that the AD group mice crossed the platform were reduced, while KMAD@ICA administration increased the number and time that the AD mice crossed the platform, and there was a significant difference compared with the AD group (P<0.05). The above experimental results show that KMAD@ICA can significantly improve the cognitive function of AD mice, and its efficacy is better than that of ICA free drug and Asp-Dex@ICA group.

[0147] Fig. 22 Figure C shows the results of the nest-building experiment. The mice in the Wild group were able to build a complete, three-dimensional, and comfortable nest, while the mice in the AD group only messed up the cotton pad and were unable to chew and carry the cotton pad to build a normal nest. After treatment with KMAD@ICA, the mice's ability to build nests improved; compared with the AD group, KMAD@ICA significantly improved the nest-building ability of AD mice (P<0.05).

[0148] 7.4 Micelles improve oxidative stress in the brain of AD mice

[0149] The biochemical kit was used to detect the expression of key oxidative stress molecules SOD, GSH, and MDA in the brain of each group of mice after treatment. The experimental results are as follows: Fig.23 As shown in the figure, the levels of antioxidant molecules SOD and GSH in the brain of AD mice decreased significantly, and the content of oxidation product MDA increased dramatically, indicating that the brain of AD mice was in a state of oxidative stress; after drug treatment, the level of oxidative stress in the mouse brain decreased; compared with the AD group, KMAD@ICA can significantly increase the activity of antioxidant molecules SOD and the content of GSH in the brain of AD mice, and the difference is statistically significant; among all drug groups, the KMAD@ICA group has the strongest effect on reducing the MDA content in the brain of AD mice.

[0150] 7.5 Micelles improve neuroinflammation in the brain of AD mice

[0151] The levels of inflammatory factors in the brain tissue of each group of mice were detected using a kit. The results are as follows: Fig.24 As shown, compared with the Wild group mice, the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the brain of AD mice were significantly increased; compared with the AD group, the ICA free drug group significantly reduced the levels of inflammatory factors in the brain of AD mice. Encapsulating ICA in micelles enhanced the therapeutic effect of ICA, and the improvement effect of KMAD@ICA was significantly higher than that of the Asp-Dex@ICA group.

[0152] In summary, through the above implementation examples, it can be seen that KMAD@ICA can drive the polarization of microglia M2, inhibit iron overload, and can achieve step-by-step targeting through ROS response to achieve precise drug delivery to M1 microglia in the brain after crossing the BBB, thereby exerting a multi-pathway and multi-target comprehensive anti-AD effect.

[0153] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing ROS-responsive stepwise targeted nanomicelles for regulating polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease, characterized in that: Including aspirin-modified dextran, DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED, DSPE-PEG 2000 -MG1 and icariin are subjected to ultrasonic mixing reaction to obtain the ROS-responsive stepwise targeted nanomicelles; The DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED is composed of DSPE-PEG 2000 -TK-PEG 2000 -NHS is reacted with a primary targeting peptide under the action of triethylamine; the amino acid sequence of the primary targeting peptide is KLVFFAED; The DSPE-PEG 2000 -MG1 is composed of DSPE-PEG 2000 -NHS is reacted with a secondary targeting peptide under the action of triethylamine to obtain the product; the amino acid sequence of the secondary targeting peptide is CHHSSSARC.

2. The preparation method according to claim 1, characterized in that: The aspirin-modified dextran is prepared by esterification reaction of aspirin and dextran under the action of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the aspirin, the dextran, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and the 4-dimethylaminopyridine is 0.09:0.4:0.096:0.

061.

4. The preparation method according to claim 1, characterized in that: The DSPE-PEG 2000 -TK-PEG 2000 -NHS, the primary targeting peptide and the triethylamine have a mass volume ratio of 0.1 g:0.11 g:0.3 mL.

5. The preparation method according to claim 1, characterized in that: The DSPE-PEG 2000 -NHS, the secondary targeting peptide and the triethylamine have a mass volume ratio of 0.1 g:0.11 g:0.3 mL.

6. The preparation method according to claim 1, characterized in that: The aspirin-modified dextran, the DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED, DSPE-PEG 2000 The mass ratio of -MG1 to the icariin is 5:1:1:

1.

7. The preparation method according to claim 1, characterized in that: The ultrasonic power of the ultrasonic mixing reaction is 500W.

8. A ROS-responsive stepwise targeted nanomicelle for regulating polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease, prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the ROS-responsive stepwise targeted nanomicelles as claimed in claim 8 in the preparation of a drug for treating Alzheimer's disease.

10. A drug for treating Alzheimer's disease, characterized in that: The active ingredient comprises the ROS-responsive stepwise targeted nanomicelles as described in claim 8.

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