A ROS-responsive stepwise targeted nanomicelle for regulating the polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease and its preparation method

Abstract

The present invention discloses a ROS-responsive stepwise targeted nanomicelle for regulating the polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease and a preparation method thereof, relating to the technical field of biomedicine. The preparation method includes ultrasonic mixing reaction of dextran modified with aspirin, DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED, DSPE-PEG 2000 -MG1 and icariin to obtain the ROS-responsive stepwise targeted nanomicelle. The ROS-responsive stepwise targeted nanomicelle prepared by the present invention, 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 the function of microglia, and break the vicious cycle of iron metabolism disorder and oxidative stress, thereby providing new ideas and solutions for the treatment of AD.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a ROS-responsive stepwise targeted nanomicelle for regulating the polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a serious neurodegenerative disease of the central nervous system. With the aggravation of social aging, the prevalence of AD has been increasing year by year, imposing a heavy burden on society and families. Due to the complex and unclear etiology of AD, there is currently no effective treatment method. Existing single-target drugs cannot prevent the progression of the disease, and have large side effects and limited efficacy. Although a large number of studies and clinical trials have been conducted on β-amyloid (Aβ) as a popular target for AD treatment in recent years, the results show that the strategy of targeting only Aβ is 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 the abnormal activation of microglia and the 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 the abnormal activation of microglia, generating 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 types. Among them, M1-type microglia are related to inflammation and nerve damage, while M2-type microglia contribute to the clearance of toxic proteins and the reduction of inflammation. Therefore, regulating the polarization of microglia, inhibiting M1 type and activating M2 type, is a potential new strategy for AD treatment. In addition, the role of iron metabolism disorders in neurodegenerative diseases has been increasingly concerned. The excessive accumulation of iron not only exacerbates oxidative stress, but also interacts with the activation of microglia, forming 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 C 33 H 40 O 15 , with the CAS number 489-32-7, and the structural formula is as follows:

[0005]

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

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

[0008] Micelles, as a nanoscale drug delivery system, have important advantages. The micelle structure spontaneously formed by amphiphilic molecules can encapsulate hydrophobic drugs, significantly improving the water solubility and bioavailability of drugs. By synthesizing an amphiphilic polymer through the modification of dextran with aspirin to form stable micelles, it can effectively load icariin with poor water solubility and enhance its delivery efficiency in vivo. In addition, to solve the problems that the blood-brain barrier (BBB) hinders the entry of drugs into the brain; it is difficult for drugs to concentrate in the lesion area after entering the brain, resulting in a significant reduction in the curative effect and posing a safety hazard to normal brain tissue. The present invention combines the unique feature of overexpression of ROS in the AD brain and designs a ROS-responsive stepwise targeted icariin-loaded nanomicelle. By introducing a ketothiol (TK) bond into the nanodrug delivery system, it selectively breaks under the high-ROS environment in the brain, releasing the drug while precisely targeting M1 microglia. This system uses the KLVFFAED peptide as the first-level brain-targeting ligand to help the micelles cross the BBB; after entering the brain, under the action of ROS, KLVFFAED falls off, exposing the second-level targeting peptide MG1, further enhancing the specific effect of the micelles on M1 microglia. This multi-level response mechanism significantly improves the accumulation of icariin in the lesion area, reduces the non-specific uptake of healthy tissues, and achieves efficient and low-toxic precise treatment.

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

[0010] The present invention provides a preparation method of a ROS-responsive stepwise targeted nanomicelle for regulating the polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease, including aspirin-modified dextran, DSPE-PEG 2000 -TK-PEG2000 -KLVFFAED, DSPE-PEG 2000 The step of obtaining the ROS-responsive stepwise targeting nanomicelles by subjecting -MG1 and icariin to ultrasonic mixing reaction;

[0011] The DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED is prepared by reacting DSPE-PEG 2000 -TK-PEG 2000 -NHS with the first-stage targeting peptide under the action of triethylamine; the amino acid sequence of the first-stage targeting peptide is KLVFFAED;

[0012] The DSPE-PEG 2000 -MG1 is prepared by reacting DSPE-PEG 2000 -NHS with the second-stage targeting peptide under the action of triethylamine; the amino acid sequence of the second-stage targeting peptide is CHHSSSARC.

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

[0014] Further, 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] Further, the mass-to-volume ratio of the DSPE-PEG 2000 -TK-PEG 2000 -NHS, the first-stage targeting peptide, and the triethylamine is 0.1 g:0.11 g:0.3 mL.

[0016] Further, the mass-to-volume ratio of the DSPE-PEG 2000 -NHS, the second-stage targeting peptide, and the triethylamine is 0.1 g:0.11 g:0.3 mL.

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

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

[0019] The present invention also provides a ROS-responsive stepwise targeting nanomicelle for regulating the polarization of inflammatory microglia and iron metabolism imbalance in Alzheimer's disease, which is prepared by the above-mentioned preparation method.

[0020] The present invention also provides the application of the above-mentioned ROS-responsive stepwise targeting nanomicelle in the preparation of a drug for treating Alzheimer's disease.

[0021] The present invention also provides a drug for treating Alzheimer's disease, and the active ingredient comprises the above-mentioned ROS-responsive stepwise targeting nanomicelle.

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

[0023] The present invention innovatively constructs a ROS-responsive stepwise targeting icariin-loaded nanomicelle. Combining with a multi-stage targeting strategy, it can not only cross the blood-brain barrier, but also achieve precise drug delivery in the AD lesion area, regulate the function of microglia, and break the vicious cycle of iron metabolism disorder and oxidative stress, thus providing new ideas and solutions for AD treatment.

[0024] Compared with the prior art, the ROS-responsive stepwise targeting nanomicelle system of the present invention shows significant innovation and advantages in AD treatment. Most traditional AD treatment methods focus on single targets, such as β-amyloid (Aβ). Although it has certain effects, it 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 and multi-stage response mechanism. The nanomicelle uses a ketothioacetal bond (TK) to achieve specific response to the high oxidative stress environment in the brain, and through a multi-stage targeting strategy of KLVFFAED peptide and MG1 peptide, it can not only effectively cross the blood-brain barrier (BBB), but also precisely target M1-type microglia, regulate its polarization state, reduce neuroinflammation, and at the same time improve iron metabolism disorder and oxidative stress, breaking the vicious cycle in the progression of AD.

[0025] In addition, aiming at the problems of poor water solubility and low in vivo utilization rate of the therapeutic drug icariin, the present invention effectively encapsulates icariin with poor water solubility through the hydrophobic core of the nanomicelle, significantly improving its water solubility and bioavailability. This micelle system not only enhances the stability and delivery efficiency of icariin in vivo, but also ensures the precise release and accumulation of the drug at the lesion site, reduces the non-specific uptake of healthy tissues, and thus realizes efficient and low-toxic AD treatment, providing a brand-new technical solution for the application of icariin. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0027] Figure 1 It is the synthetic method roadmap of aspirin-modified dextran;

[0028] Figure 2 It is the 1H NMR spectrum of aspirin-modified dextran;

[0029] Figure 3 It is the first-level targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED's 1H NMR spectrum;

[0030] Figure 4 It is the second-level targeting molecule DSPE-PEG 2000 -MG1's 1H NMR spectrum;

[0031] Figure 5 It is the infrared spectra of dextran, aspirin, and aspirin-modified dextran;

[0032] Figure 6 It is the infrared spectra of aspirin-modified dextran, icariin, MDA@ICA, and KMDA@ICA;

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

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

[0035] Figure 9 It is the statistical graph of the particle size of each group of micelles;

[0036] Figure 10 It is the statistical graph of the zeta potential of each group of micelles;

[0037] Figure 11 It is the transmission electron microscope image of KMDA@ICA before oxidation (A) and the transmission electron microscope image of KMDA@ICA after oxidation (B);

[0038] Figure 12 It is the result graph of the fluorescence microscope to measure the uptake of different micelles by M1 polarized BV.2 cells;

[0039] Figure 13Quantitative analysis chart for the uptake of different micelles by M1 polarized BV.2 cells measured by fluorescence microscopy;

[0040] Figure 14 Result chart for the uptake of different micelles by M1 polarized BV.2 cells measured by flow cytometry;

[0041] Figure 15 Quantitative analysis chart for the uptake of different micelles by M1 polarized BV.2 cells measured by flow cytometry;

[0042] Figure 16 In vitro release curve chart for each group;

[0043] Figure 17 Result chart for the detection of the long - circulating effect of different preparations in rats by small - animal in - vivo imaging;

[0044] Figure 18 Result chart for the distribution of different preparations in mice by small - animal in - vivo imaging;

[0045] Figure 19 Result chart for the detection of iron content in BV.2 cells after treatment with different groups;

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

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

[0048] Figure 22 Result chart for evaluating the improvement of cognitive ability of AD mice in each preparation group using the water maze experiment; among them, A is the statistical analysis chart of the time in the quadrant where the original platform is located in the water maze experiment after treating AD mice with different preparation groups; B is the statistical analysis chart of the number of times passing through the original platform area in the water maze experiment after treating AD mice with different preparation groups; C is the statistical analysis chart of the nest - building experiment score after treating AD mice with different preparation groups.

[0049] Figure 23 Statistical chart for the expression of key molecules of oxidative stress in the brains of mice in each group: glutathione (A), superoxide dismutase (B), and malondialdehyde (C);

[0050] Figure 24 Statistical chart for the levels of inflammatory factors in the brain tissues of mice in each group; among them, A is the statistical chart of the content of tumor necrosis factor - α in the brains of AD mice treated with different preparation groups; B is the statistical chart of the content of interleukin - 6 in the brains of AD mice treated with different preparation groups; C is the statistical chart of the content of interleukin - 1β in the brains of AD mice treated with different preparation groups. Detailed implementation methods

[0051] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0054] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are merely exemplary.

[0055] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0056] Example 1

[0057] I. Experimental Instruments and Materials

[0058] 1. Experimental instruments: DZKW-S-4 electrothermal constant temperature water bath (Medical Device Factory No. 5, Shanghai Medical Instrument Corporation); JY92-IIN ultrasonic cell disruptor (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 fluorescence inverted microscope (Nikon Corporation, Japan); HBS-1096A microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.); 1100 liquid chromatograph (UV detector, Dalian Elite); C18 chromatographic column (250 mm×4.6 mm, 5 μm, Shanghai Yuexu); Sephadex G-50 dextran gel column (Shanghai Hualan Chemical Technology Co., Ltd.); dialysis bag (Beijing Solarbio Science & Technology Co., Ltd., cut-off molecular weight 500 - 12000 Da); polycarbonate membrane (Millipore Corporation, USA); 500 An nano particle size and Zeta potential analyzer (Anton Paar GmbH, Austria); JEM-2000EX transmission electron microscope (JEOL Ltd., Japan); SW-CJ-1D single-person purification workbench (Shanghai Sujing Industrial Co., Ltd.); air-jacketed carbon dioxide cell incubator (Thermo Flesher Scientific (Asheville) LLC, USA); FACSCalibur flow cytometer (BD Biosciences, USA); multispectral in vivo imaging system (Carestream Health, Inc., USA); RM2235 paraffin slicer (Leica Microsystems GmbH, Germany); Bruker Avance III HD 600 MHz; Shimadzu Fourier transform infrared spectrometer IRTracer-100.

[0059] 2. Experimental materials: icariin, dextran, aspirin, anhydrous dimethyl sulfoxide (Shanghai Aladdin Biochemical Technology Co., Ltd.); distearoyl phosphatidylethanolamine-polyethylene glycol 2000-NHS (DSPE-PEG 2000 -NHS), distearoyl phosphatidylethanolamine-polyethylene glycol 2000-ketothiol-polyethylene glycol 2000-NHS (DSPE-PEG 2000 -TK-PEG 2000 -NHS), polypeptide (Xi'an Ruixi Biotechnology Co., Ltd.); iNOS, iron content determination kit (Abcam, USA); CD206 antibody (Biolegend); TNF-α ELISA kit, IL-1β ELISA kit, IL-6 ELISA kit (Beijing Solarbio Science & Technology Co., Ltd.); GSH kit, SOD kit, MDA kit (Nanjing Jiancheng Bioengineering Institute); DMSO-d 6(Cambridge Isotope Laboratories, Inc.); the remaining reagents are all of analytical grade, and the water is purified water.

[0060] II. Experimental methods

[0061] 1. Preparation of nanomicelles

[0062] 1.1 Preparation of film materials and targeting ligands

[0063] The film material aspirin-modified dextran (Asp-Dex) is prepared by the esterification reaction of the 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] Fix a 100 mL three-necked flask on the iron stand of a magnetic stirrer. Add 0.09 g of aspirin, 0.061 g of 4-dimethylaminopyridine, 0.096 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, a magnetic stir bar, and 10 mL of anhydrous dimethyl sulfoxide (DMSO) to the three-necked flask in sequence. Flush with nitrogen three times repeatedly to remove oxygen, and under nitrogen protection, stir at a speed of 500 rpm at room temperature for 0.5 hour (the same effect can be achieved in 0.5 - 1 hour) to activate the carboxyl group. Dissolve 0.4 g of dextran in 10 mL of anhydrous DMSO and ultrasonically treat it to fully dissolve. Slowly drip the dissolved dextran solution into the above reaction system and continue to stir at room temperature for 48 hours. After the reaction is completed, add 10 times the volume of cold anhydrous ethanol and let it stand overnight to precipitate the product. The next day, collect the precipitate and transfer it to a dialysis bag (cut-off molecular weight 500 Da), dialyze with distilled water for 48 hours, and change the water every 12 hours. Freeze-dry the dialyzed product to finally obtain aspirin-modified dextran (Asp-Dex).

[0065] The first-level targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED is prepared from DSPE-PEG 2000 -TK-PEG 2000 -NHS and KLVFFAED peptide react under the action of triethylamine. The main process is as follows:

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

[0067] The second-stage targeting molecule DSPE-PEG 2000 -MG1 was synthesized from the raw material DSPE-PEG 2000 -NHS and the second-stage targeting peptide - MG1 peptide (amino acid sequence: CHHSSSARC) under the action of triethylamine. The main process is as follows:

[0068] Dissolve 0.1 g of DSPE-PEG 2000 -NHS in 3 mL of N,N-dimethylformamide (DMF), add 0.11 g of MG1 polypeptide and 0.3 mL of triethylamine and dissolve completely, and react at room temperature for 12 h; transfer the reaction solution to a dialysis bag (cut-off molecular weight 2000 Da) and dialyze in pure water for 24 h, and collect the dialysate and freeze-dry to obtain the second-stage targeting molecule DSPE-PEG 2000 -MG1.

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

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

[0071] During the preparation of the nanomicelles, without adding ICA, DSPE-PEG 2000 -TK-PEG 2000-KLVFF and DSPE-PEG 2000 -MG1 to obtain blank micelles (Asp-Dex); without adding DSPE-PEG 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); during the preparation process, DSPE-PEG 4000 -KLVFF is used instead of DSPE-PEG 2000 -TK-PEG 2000 -KLVFF to obtain non-ROS-responsive micelles (ROS-insensitive).

[0072] 1.3 Preparation of fluorescent probe micelles

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

[0074] 2. Characterization of the properties of nanomicelles

[0075] 2.1 Material structure characterization

[0076] Dissolve 20 mg of the nanomicelle sample in 0.5 mL DMSO-d 6 , transfer it to a nuclear magnetic resonance tube for nuclear magnetic resonance hydrogen spectrum testing.

[0077] Take 2 mg of the nanomicelle sample and grind it with KBr at a ratio of 1:100 and then press it into a tablet; place the prepared KBr wafer on a holder and then put it on an infrared detector for detection.

[0078] 2.2 Determination of the critical micelle concentration

[0079] The critical micelle concentration (CMC) is determined using pyrene as a fluorescent probe. The specific process is as follows: First, dilute the micelles 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 add a final concentration of 6×10 -7Pyrene at a concentration of 1 (I 373 ) / I 3 (I 384 ) ratio was calculated and the relationship between the ratio and the micelle concentration was plotted. The CMC was determined from the intersection of the two linear regions in the graph.

[0080] 2.3 Determination of particle size and Zeta potential

[0081] Take 200 μL of micelles and dilute to 2 mL with distilled water. The particle size and Zeta potential of different micelles were determined by dynamic light scattering using a 500 Plus type nanoparticle size and Zeta potential analyzer. Each preparation was measured 3 times, with 10 measurements set for each cycle.

[0082] 2.4 Observation of micelle morphology by transmission electron microscopy

[0083] The morphology of KMAD@ICA was observed by transmission electron microscopy. An appropriate amount of micelles was diluted to a suitable concentration, and a drop was placed on a special copper grid for electron microscopy, stained with 2% phosphotungstic acid, and air-dried before observing its morphology by transmission electron microscopy. The accelerating voltage of the transmission electron microscope was set at 120 kV.

[0084] 2.5 Evaluation of the microenvironment response of KMAD@ICA

[0085] 100 μM hydrogen peroxide was added to the KMAD@ICA solution and incubated for 1 h. The change in the particle size of KMAD@ICA was analyzed using a dynamic laser scattering instrument, and the morphology of KMAD@ICA was observed using a transmission electron microscope to evaluate its intelligent response behavior.

[0086] 2.6 In vitro release evaluation of the nanomicelles

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

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

[0089] 3. Evaluation of in vitro targeting of ROS-responsive stepwise targeting micelles

[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), and placed in a cell culture incubator at 37 °C, 5% CO 2 -95% air, and passaged when the confluence reached 90%.

[0092] 3.2 Evaluation of the responsiveness of micelles to target M1-polarized microglial cells

[0093] BV2 cells were seeded in culture dishes at a density of 1×10 5 cells per well. After inducing the cells with LPS (1 μg / mL) for 24 h, the cells were washed with PBS. The culture media containing KMAD@Cou and non-ROS-responsive Cou micelles were added to different culture dishes respectively. After 2 h, the culture media were discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and the nuclei were stained with PDAI for 10 min. The distribution of fluorescence in the cells was observed under a fluorescence microscope.

[0094] Flow cytometry was used to detect the evaluation of the responsiveness of micelles to target M1-polarized microglial cells. The specific method was the same as above. After continued culture for 2 h, the culture media were discarded, and PBS was used to wash away the excess unabsorbed micelles. The cells were digested with trypsin, centrifuged and washed, and then resuspended in pre-cooled PBS. Flow cytometry was used to quantitatively analyze the fluorescence intensity of the probe bound to the cells.

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

[0096] 4.1 Investigation of the long-circulating effect

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

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

[0099] Each group of rats was administered the above-mentioned preparation via tail vein injection once. Ensure that the DiR doses in each dosing group are equal, all being 3 μg / rat. At different time points (1, 3, 6, 12, 18, 24, 36, 48, 72, 96 h), 200 μL of whole blood was collected into a 2 mL centrifuge tube containing anticoagulant. After mixing, it was transferred to a 96-well plate, and a small animal in vivo imaging analyzer was used to observe the fluorescence signal intensity of each preparation to evaluate its long-circulation effect in rats.

[0100] 4.2 In vivo targeting evaluation

[0101] 4.2.1 In vivo imaging

[0102] The in vivo targeting of different preparations was investigated by in vivo imaging experiments. The drug was replaced with the fluorescent reagent DiR. Mice were divided into the following groups:

[0103] (1) Normal saline (Control); (2) Free DiR; (3) DiR micelles (Asp-Dex@DiR); (4) MG1-modified DiR micelles (MAD@DiR); (5) ROS-responsive stepwise targeting DiR micelles (KMAD@DiR). Ensure that the DiR doses in each dosing group are the same, all being 2 μg / mouse.

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

[0105] 5. Evaluation of the anti-AD pharmacodynamics of micelles

[0106] 5.1 Detection of iron content.

[0107] According to the manufacturer's instructions, an iron content assay kit was used to detect the iron content. BV.2 cells were seeded in six-well plates at a density of 1×10 6 cells / well and cultured overnight. The cells were treated with different preparations (ICA concentration was 25 μmol / L -1 ) and co-incubated with ferric ammonium citrate FAC (600 μmol / L -1 ) for 36 h. 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 using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 550 nm.

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

[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 per well. After the cells were induced with FAC (200 μmol / L -1 ) for 24 h, they were induced with LPS (1 μg / mL) for 6 h. The cells were washed with PBS, and the culture media containing PBS, Asp-Dex@ICA, KAD@ICA or KMAD@ICA were added to different cell plates respectively, and the cells without LPS induction were used as the blank control. After 36 h, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. Then the cells were exposed to 0.3% Triton X-100 for 10 min and blocked with staining blocking buffer at room temperature for 1 h. Primary antibodies CD206 (diluted at a volume ratio of 1:50) and anti-iNOS (diluted at a volume ratio of 1:200) were added to the cells in different groups respectively and incubated with the cell samples overnight at 4 °C. After washing three times with PBS, FITC-labeled secondary antibody (diluted at a volume ratio of 1:250) was added and incubated in the iNOS wells at room temperature for 1 h. Cy5-labeled secondary antibody (diluted at a volume ratio of 1:250) was added and incubated in the CD206 wells at room temperature for 1 h. Finally, staining was performed with 4′,6-diamidino-2-phenylindole (DAPI), washed with PBS, and observed and photographed under a fluorescence microscope.

[0110] 5.3 Animal grouping and 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; another group of age-matched C57BL / 6 mice was used as the wild blank control group (Wild). The Wild group and the AD group were given an equal volume of normal saline respectively. The Free ICA group, the Asp-Dex@ICA group and the KMAD@ICA group were injected intravenously into the tail once every two days at a dose of 5 mg / kg of ICA. After the above mice were continuously administered for two months, behavioral tests were performed. After the tests were completed, the mice were euthanized for pharmacodynamic analysis.

[0112] 5.4 Morris water maze experiment

[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, the place navigation experiment was carried out. The first 4 days were the training stage, and the formal experiment was carried out on the 5th day. The swimming distance of the mice was recorded during the experiment. The mice were placed at the center point of the four quadrants in the pool, and the escape latency was set at 60 seconds. If the mouse found the platform, it was allowed to stay on the platform for 15 seconds; if it did not find the platform, 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 and the time it stayed in the quadrant within 60 s were measured, and the entire movement trajectory was recorded to evaluate the learning and memory ability of the mouse.

[0114] 5.5 Nest building experiment

[0115] After the water maze experiment was completed, the nest building experiment was carried out to evaluate the changes in cognitive ability, motor ability, and coordination of mice in each group after drug treatment. The specific experimental process is as follows: The mice were placed in a clean mouse cage, and a 5 cm² nesting cotton pad was provided to the mice in a corner of the cage. The nesting situation of the mice was photographed at different time points, and finally the nest was scored according to the method reported in the literature, from 1 - 5 (very poor / no nesting - best nesting).

[0116] 5.6 Preparation of brain tissue samples

[0117] 1 h after the last administration, the mice in each group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). The mice were fixed on the operating table, the heart was exposed, the syringe was inserted into the aorta from the left ventricle, the right atrium was opened, and pre-cooled normal saline was perfused through the heart. When the liver turned white and the liquid flowing out of the atrium was clear, the perfusion was stopped. The skull of the mouse was cut open, and the whole brain was quickly removed. The brain tissue was divided into two halves along the midline, and the left half brain was placed in a cryotube, frozen and stored for later use. Cold PBS solution was added to the left brain tissue of each group of mice, and it was stirred at high speed in an ice bath until it became homogenized. It was centrifuged at 10000 rpm 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 intracerebral oxidative stress level

[0119] The contents of malondialdehyde (MDA) and glutathione (GSH), and the activity of superoxide dismutase (SOD), which are representative molecules of the oxidative stress level in the brains of mice in each administration group after treatment, were detected using a biochemical kit. The experimental operation steps were carried out strictly according to the instructions of the kit. The experimental steps were as follows: Cold PBS solution was added to the brain tissues of each group of mice, and high-speed stirring was performed in an ice bath until homogenized. Then, centrifugation was carried out at 10,000 rpm for 10 minutes at 4 °C using a high-speed refrigerated centrifuge. The supernatant was aspirated into an EP tube, and the contents of malondialdehyde (MDA) and glutathione (GSH), and the activity of superoxide dismutase (SOD) in the brain tissues were detected strictly according to the manufacturer's instructions.

[0120] 5.8 Detection of the level of neuroinflammation in the brain

[0121] The levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the brains of mice in each administration group after treatment were detected using an ELISA kit. The experimental operation steps were carried out strictly according to the instructions of the kit. The experimental steps were as follows:

[0122] First, the kit was restored to room temperature; then, developer A, B, and stop solution were added to the blank wells to zero the instrument; 50 μL of the diluted standard product was added to the standard wells, 50 μL of the standard product / sample diluent and 50 μL of the biotinylated antigen working solution were added to the zero well; 50 μL of the sample and 50 μL of the biotinylated antigen working solution were added to the sample wells; gently shake to mix evenly, cover with a sealing film, and incubate at 37 °C for 30 min; dilute the washing solution for later use; discard the liquid in the wells, fill each well with the washing solution, wash for 30 s and then discard, wash 5 times; then, 50 μL of avidin-HRP was added to the standard wells and sample wells, gently shake to mix evenly, cover with a sealing film, and incubate at 37 °C for 30 min; remove the sealing film, discard the liquid in the wells, fill each well with the washing solution, wash for 30 s and then discard, wash 5 times, and pat dry; 50 μL of chromogenic agent A and 50 μL of chromogenic agent B were added to the wells in sequence, gently shake to mix evenly, develop color at 37 °C in the dark for 10 min, and then add 50 μL of stop solution to each well to terminate the reaction; finally, zero the instrument with the blank well, and measure the absorbance values of each well in sequence at a wavelength of 450 nm; calculate the levels of IL-1β, IL-6, and TNF-α in each sample.

[0123] 6. Statistical analysis

[0124] Graphpad Prism 9.0 software was used for statistical analysis. The experimental data were all expressed as Mean±SD. One-way ANOVA was used for comparison among multiple groups, and further pairwise comparison was performed using the LSD-t test. P<0.05 indicated that the difference was statistically significant.

[0125] III. Results

[0126] 1. Structural characterization of Asp-Dex

[0127] The synthesis equation of Asp-Dex is as Figure 1 shown, and the nuclear magnetic resonance spectrum of Asp-Dex is as Figure 2 shown. In the spectrum, the characteristic peaks of Asp in Asp-Dex (chemical shift of benzene ring 7 - 8 ppm, methyl 2 - 3 ppm) and the characteristic peaks of Dex (3 - 5 ppm) can be clearly seen. The infrared spectrum of Asp-Dex is as Figure 5 shown. Compared with Dex, the peak intensity and shift of the ester group at 1735 - 1750 cm -1 shift of Asp-Dex have changed. The above results prove the successful synthesis of Asp-Dex.

[0128] 2. Structural characterization of the targeting molecule

[0129] The nuclear magnetic resonance hydrogen spectrum of the secondary targeting molecule DSPE-PEG 2000 -MG1 is as Figure 3 shown. In the spectrum, the characteristic peaks of the polypeptide in DSPE-PEG 2000 -MG1 (chemical shift 7 - 8.5 ppm) can be clearly seen, proving the successful synthesis of the secondary targeting molecule DSPE-PEG 2000 -MG1. The nuclear magnetic resonance hydrogen spectrum of the primary targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED is as Figure 4 shown. In the spectrum, the characteristic peaks of the KLVFFAED polypeptide (chemical shift 7 - 7.5 ppm) and the characteristic peaks of the ketothiol (TK) bond (chemical shift 2.5 - 3 ppm, 1.2 - 1.7 ppm) can be clearly seen, proving the successful synthesis of the primary targeting molecule DSPE-PEG 2000 -TK-PEG 2000 -KLVFFAED.

[0130] 3. Structural characterization of the micelles

[0131] The infrared spectrum of the prepared micelles KMAD@ICA is as Figure 6 shown. From the figure, it can be seen that the characteristic peaks of ICA disappear compared with KMAD@ICA (shift 1250 - 1750 cm -1 , 2750 - 3000 cm -1 ), indicating that ICA has been successfully encapsulated in the micelles. Figure 7 It shows that the CMC of the micelles is 0.00796 mg / mL, which is significantly lower than the effective concentration of the micelles in the blood (about 0.5 mg / mL), and can avoid the degradation of the micelles due to dilution when entering the body. Figure 8The appearance comparison diagram of the free ICA drug solution and KMAD@ICA is shown. It can be clearly seen from the figure that there are many insoluble particles suspended in the free drug solution, while the micelle solution is clear and transparent, indicating that preparing ICA into micelles significantly increases the solubility of the drug. Figure 9 The sizes of different formulation groups are shown. Among them, the particle size of Asp-Dex is 62.90 ± 0.12 nm, the particle size of Asp-Dex@ICA is 63.26 ± 0.16 nm, the particle size of MAD@ICA is 77.13 ± 0.71 nm, the particle size of KMAD@ICA is 120.72 ± 2.71 nm, and the particle size of KMAD@ICA in the presence of hydrogen peroxide is 116.10 ± 1.35 nm; It can be seen from the results 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 first-level targeting molecule in KMAD@ICA responds and breaks, and the outermost KLVFFAED molecules fall off, and the micelles show a size contraction. Figure 10 The Zeta potentials of different formulation groups are shown. Among them, 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. ROS responsiveness evaluation of micelles

[0133] Figure 11 In A, it is the transmission electron microscope photo of the micelles. It can be seen that the micelles are spherical and the particle size is about 100 nm; Figure 11 In B, it is the transmission electron microscope photo of the micelles after hydrogen peroxide oxidation. It can be seen from the figure that some micelles show a lysed state, proving that the prepared KMAD@ICA has ROS responsiveness. In addition, the uptake of KMAD micelles loaded with coumarin and non-ROS-responsive micelles without TK bonds in M1 polarized microglia was observed using a fluorescence microscope. The results are shown as Figure 12 and Figure 13 shown. The fluorescence signal of the non-ROS-responsive micelles is significantly weaker than that of the KMAD group, indicating that the KMAD micelles can respond and lyse the outermost first-level targeting molecules in the high-level oxidative environment of M1 polarized microglia, exposing the second-level M1 microglia targeting peptide MG1 and increasing uptake. Figure 14 and Figure 15To detect the uptake of coumarin-loaded KMAD micelles and micelles without TK bonds in M1 polarized microglia by flow cytometry, 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 ROS-responsive stepwise targeting effect of KMAD micelles.

[0134] Figure 16 The in vitro release results of the micelles were shown. At 48 h, the in vitro release rate of free ICA 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+H 2 O 2 was (90.07 ± 2.89)%. The experimental results showed that when KMAD@ICA was co-incubated with release media with / without H 2 O 2 within 48 hours, compared with the release media without H 2 O 2 , the release rate of ICA in the release media containing H 2 O 2 increased significantly. This indicated that the ICA loaded in KMAD@ICA could be released in a responsive manner in an oxidative environment. This property could support the selective release of drugs by the micelles in the pathological environment.

[0135] 5. Evaluation of the long-circulation effect of micelles

[0136] Using DiR instead of ICA as a fluorescent probe, the circulation behavior of each preparation in the blood of rats after intravenous administration was observed. Blood samples were collected at the predetermined time points to observe the fluorescence intensity in the blood. The results were as Figure 17 shown. The order of the circulation time of each preparation in the blood from long to short was: KMAD@DiR > MAD@DiR > Asp-Dex@DiR > Free DiR. Compared with other micelle preparations, the retention rate of Free DiR in the blood was extremely low, and its fluorescence signal was almost invisible. The above results indicated that KMAD@DiR had a good long-circulation effect in vivo.

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

[0138] The distribution of fluorescence signals in mice at different time points was observed using an in vivo fluorescence imaging system. The results are shown in Figure 18。During the 1 - 72 h period, almost no fluorescence signal was observed in the brains of the Free DiR group mice. However, the KMAD@DiR group mice had the strongest fluorescence signal in the brain at each time point. The order of DiR fluorescence intensity at different time points was as follows: KMAD@DiR > MAD@DiR > Asp - Dex@DiR > Free DiR. The above results indicate that KMAD@DiR has better brain targeting and long - circulation effects.

[0139] 7. Evaluation of the anti - AD pharmacodynamic effects of micelles

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

[0141] Since excessive iron ions in cells can trigger the Fenton reaction, which in turn induces oxidative stress and drives the M1 polarization of microglia. Therefore, regulating the intracellular iron ion content is beneficial for controlling microglia polarization. As Figure 19 shown, the results of iron content determination indicated that FAC stimulation significantly increased the iron level in BV.2 cells. Treatments with ICA, Asp - Dex@ICA, and KMAD@ICA could all reduce this abnormally elevated iron level, and KMAD@ICA had the best effect compared with other drug - administered groups.

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

[0143] Iron metabolism disorders and oxidative stress can drive the M1 polarization of microglia. After intervention with different formulation groups, the expression of the M1 polarization marker iNOS in microglia was as Figure 20 shown, and the fluorescence signal showed a gradually decreasing trend. Asp - Dex@ICA, KAD@ICA, and KMAD@ICA all down - regulated the expression of iNOS. Compared with the non - targeted modified Asp - Dex@ICA group, the targeted modified groups had a stronger ability to down - regulate iNOS; and compared with the KAD@ICA group without MG1 modification, the KMAD@ICA group had a stronger ability to down - regulate iNOS.

[0144] In addition, the expression of the M2 polarization marker CD206 in microglia was as Figure 21 shown, and the fluorescence signal showed a gradually increasing trend. Asp - Dex@ICA, KAD@ICA, and KMAD@ICA all up - regulated the expression of CD206. Compared with the non - targeted modified Asp - Dex@ICA group, the targeted modified groups had a stronger ability to up - regulate CD206; and compared with the KAD@ICA group without MG1 modification, the KMAD@ICA group had a stronger ability to up - regulate CD206.

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

[0146] First, the Morris water maze experiment was used to evaluate the improvement of cognitive ability of AD mice in each preparation group. From Figure 22 As shown in A and B, compared with the Wild group, the number of times and time for AD group mice to cross the platform were both reduced. After treatment with KMAD@ICA, the number of times and time for AD mice to cross the platform increased, and there were significant differences compared with the AD group (P<0.05). The above experimental results indicate that KMAD@ICA can significantly improve the cognitive function of AD mice, and its therapeutic effect is better than that of free ICA and Asp-Dex@ICA groups.

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

[0148] 7.4 Improvement effect of micelles on oxidative stress in the brains of AD mice

[0149] The expression levels of key molecules of oxidative stress in the brains of mice in each group after treatment, namely SOD, GSH, and MDA, were detected using biochemical kits. The experimental results are as Figure 23 shown. The levels of antioxidant molecules SOD and GSH in the brains of AD mice decreased significantly, and the content of oxidative product MDA increased sharply, indicating an oxidative stress state in the brains of AD mice; after treatment, the level of oxidative stress in the brains of mice decreased; compared with the AD group, KMAD@ICA can significantly increase the activity of antioxidant molecule SOD and the content of GSH in the brains of AD mice, and the difference was statistically significant; among the drug administration groups, the KMAD@ICA group had the strongest effect in reducing the content of MDA in the brains of AD mice.

[0150] 7.5 Improvement effect of micelles on neuroinflammation in the brains of AD mice

[0151] The levels of inflammatory factors in the brain tissues of mice in each group were detected using kits. The results are as Figure 24 shown. Compared with the Wild group, the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the brains of AD group mice increased significantly; compared with the AD group, the free ICA group significantly reduced the levels of inflammatory factors in the brains 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, as can be seen from the above embodiments, KMAD@ICA can drive microglial M2 polarization, inhibit iron overload, and can achieve precise drug delivery to M1 microglia in the brain across the BBB through ROS-responsive stepwise targeting, playing a role in comprehensive anti-AD through multiple pathways and multiple targets.

[0153] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall 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; the amino acid sequence of the secondary targeting peptide is CHHSSSARC; 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.

2. The preparation method according to claim 1, 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.

3. 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.

4. 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.

5. 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.

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

7. 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 6.

8. Use of the ROS-responsive stepwise targeted nanomicelles as claimed in claim 7 in the preparation of a drug for treating Alzheimer's disease.

9. A drug for treating Alzheimer's disease, characterized in that: The active ingredient includes the ROS-responsive stepwise targeted nanomicelles described in claim 7.

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