A preparation method and application of bionic nanozyme for treating Alzheimer's disease

Abstract

The invention discloses a preparation method and application of a bionic nanozyme for treating Alzheimer's disease, comprising DSPE-TK-PEG 3400 The synthesis of apoE, the synthesis of manganese dioxide nanozyme, the preparation of manganese dioxide nanozyme liposomes, the extraction of M2 microglial cell membrane, and the preparation of cell membrane bionic nanocarriers. It has excellent brain targeting properties, promotes the transformation of microglia to M2 phenotype, inhibits the over-activation of the immune system in the AD brain, efficiently removes ROS and relieves oxidative stress, delays the progression of AD in the early stages, protects neuronal function, and restores memory and cognitive ability in AD patients. It is expected to provide a new and efficient multi-target treatment strategy for the clinical prevention and treatment of AD.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research and development, and specifically relates to a preparation method and application of a bionic nanozyme for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a central nervous system degenerative disease that occurs frequently in the elderly and is characterized by cognitive decline and behavioral disorders.

[0003] Alzheimer's disease (AD) is a degenerative disease of the central nervous system with an insidious onset. Clinical symptoms of AD, such as cognitive impairment, are closely related to changes in the brain microenvironment and the resulting amyloid protein deposition and neurofibrillary tangles.

[0004] Therefore, using multiple therapeutic interventions to target multiple targets in the progression of AD is of great significance for the prevention or cure of AD. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a method for preparing a bionic nanozyme for treating Alzheimer's disease, characterized in that: comprising:

[0007] DSPE-TK-PEG 3400 -apoE synthesis: dissolve the active ester of hydroxy polyethylene glycol in a solvent, add propane-2, 2-diylbis(sulfonamide)diethylamine and triethylamine, react, concentrate, precipitate, and dry to obtain HO-PEG 3400 -TK-NH2, HO-PEG 3400 -TK-NH2 is dissolved in a solvent, and distearoylphosphatidylethanolamine modified active ester and triethylamine are added. After the reaction, the mixture is concentrated, precipitated, and dried to obtain NHS-PEG 3400 -TK-DSPE, weigh NHS-PEG 3400 -TK-DSPE was dissolved in buffer, apoE mimetic peptide was added, and after reaction, purification and drying, DSPE-TK-PEG was obtained. 3400 -apoE;

[0008] Synthesis of manganese dioxide nanozyme: prepare potassium permanganate aqueous solution and bovine serum albumin aqueous solution, add the potassium permanganate aqueous solution dropwise into the bovine serum albumin aqueous solution, stir to react, dialyze to purify, and dry to obtain manganese dioxide nanozyme;

[0009] Preparation of manganese dioxide nanozyme liposomes: Prepare the oil phase: weigh (2,3-dioleoyl-propyl)-trimethylamine, cholesterol, 17-propenylamine, dissolve in a solvent, and add ether; prepare the water phase: weigh manganese dioxide nanozyme and DSPE-TK-PEG 3400 -apoE is dissolved in a buffer solution; the aqueous phase is added dropwise to the oil phase, mixed in a water bath, distilled under reduced pressure, the organic solvent is removed, water is added to form an aqueous suspension, ultrasonically treated for 6 minutes, and filtered through a microporous filter membrane to obtain AAG / BM-Lip-apoE;

[0010] Extraction of M2 microglial cell membrane: Induce microglia into M2 microglia, collect by centrifugation, and extract M2 microglial cell membrane by freeze-drying or sucrose gradient centrifugation;

[0011] Preparation of cell membrane biomimetic nanocarriers: The M2 microglial cell membrane and AAG / BM-Lip-apoE were mixed at a mass ratio of 1:4 to 4:1, subjected to repeated freeze-thaw treatments, passed through a polycarbonate membrane using a liposome extruder, centrifuged, and purified to obtain AAG / BM-Lip-apoE@M2CM.

[0012] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease described in the present invention, in the preparation of the cell membrane bionic nanocarrier, the mass ratio of the M2 microglial cell membrane to AAG / BM-Lip-apoE is 1:1; the repeated freeze-thaw treatment is a repeated freeze-thaw treatment at 4°C and 45°C.

[0013] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease described in the present invention, in the preparation of the cell membrane bionic nanocarrier, the liposome extruder is used to pass through a polycarbonate membrane, which is a 400nm and a 200nm polycarbonate membrane in sequence.

[0014] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease of the present invention, the DSPE-TK-PEG 3400 -apoE synthesis, the HO-PEG 3400 -TK-NH2 was prepared by weighing 1.5 g of hydroxy polyethylene glycol active ester and dissolving it in 10 mL of chloroform, adding propane-2, 2-diylbis(sulfonamide)diethylamine and triethylamine, reacting at room temperature for 24 h, concentrating under reduced pressure, pouring into ice ether for precipitation, filtering, and vacuum drying to obtain HO-PEG 3400 -TK-NH2.

[0015] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease of the present invention, the DSPE-TK-PEG3400 -apoE synthesis, the NHS-PEG 3400 -TK-DSPE was prepared by weighing 1.0 g HO-PEG 3400 -TK-NH2 was dissolved in 5 mL of chloroform, and distearoylphosphatidylethanolamine modified active ester and triethylamine were added. After reacting at room temperature for 24 h, the mixture was concentrated under reduced pressure and poured into ice ether for precipitation, filtered, and vacuum dried to obtain NHS-PEG 3400 -TK-DSPE.

[0016] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease of the present invention, the DSPE-TK-PEG 3400 -apoE synthesis, including weighing 0.1g NHS-PEG 3400 -TK-DSPE was dissolved in 0.1M pH 8.0 phosphate buffer, apoE mimetic peptide was added, stirred at room temperature for 24 hours, dialyzed and purified in a 3500Da dialysis bag for 24 hours, and the dialyzate was freeze-dried to obtain the product DSPE-TK-PEG 3400 -apoE.

[0017] As a preferred embodiment of the preparation method of the bionic nanozyme for treating Alzheimer's disease described in the present invention, the synthesis of the manganese dioxide nanozyme is as follows: 31.6 mg of potassium permanganate is added to 3 mL of water to dissolve to obtain a potassium permanganate aqueous solution, 250 mg of bovine serum albumin is added to 7 mL of water to dissolve to obtain a bovine serum albumin aqueous solution, the potassium permanganate aqueous solution is added dropwise to the bovine serum albumin aqueous solution, the reaction is stirred at 37° C. for 1.5 hours, the solution is dialyzed and purified in a 8000-14000Da dialysis bag for 24 hours, and the dialysate is freeze-dried to obtain the manganese dioxide nanozyme.

[0018] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease of the present invention, the freeze-drying method is to add M2 microglia to phosphate buffer and mix them evenly, and the concentration of the cell suspension is 1×10 7 / mL, then add an appropriate amount of protease inhibitors, squeeze back and forth through a liposome extruder, centrifuge at 3000g, 10min, 4℃, collect the supernatant, further centrifuge at 10000g, 10min, 4℃, and finally lyophilize the supernatant to obtain M2 microglial cell membrane.

[0019] As a preferred embodiment of the method for preparing the bionic nanozyme for treating Alzheimer's disease of the present invention, the sucrose gradient centrifugation method is to resuspend the M2 microglia in 4°C Tris-magnesium salt buffer, and the concentration of the cell suspension is 1×10 7 / mL, add an appropriate amount of protease inhibitors, squeeze back and forth through a liposome extruder, add 1M sucrose solution and mix to make the final concentration of 0.25M, 3000g, 10min, 4℃ centrifuge, collect the supernatant, 10000g, 10min, 4℃ further centrifuge, take the precipitate, wash the precipitate with 0.25M sucrose solution and centrifuge, discard the supernatant, and take the precipitate to obtain M2 microglial cell membrane.

[0020] Beneficial effects of the present invention: The present invention constructs a MnO2 bionic nanoenzyme drug delivery system fused with M2 microglial cell membrane (M2CM), and designs and synthesizes ROS-responsive DSPE-TK-PEG 3400 -apoE fragments are assembled together and loaded with heat shock protein inhibitor 17-AAG (AAG / BM-Lip-apoE@M2CM). The key proteins on the membrane of M2 microglia interact with brain microvascular endothelial cells to achieve efficient brain delivery, and promote the conversion of over-activated M1 microglia under neuroinflammation into anti-inflammatory M2 microglia, effectively improving the inflammatory environment in the brain of AD patients; MnO2 nanozymes (BM) remove a large amount of intracellular ROS, effectively alleviating the oxidative stress state in the brain, while triggering DSPE-TK-PEG 3400 -apoE breaks, releasing apoE mimetic peptides to transfer amyloid-β protein (Aβ) to microglia for removal, while ROS promotes the rapid release of 17-AAG to effectively remove phosphorylated Tau protein (p-Tau) in neurons. It is expected that the proposed bionic nanozyme drug delivery system with M2 microglial membrane fusion can effectively save the cognitive dysfunction of AD patients with a new treatment idea, track the entire course of the disease, and provide a reference for the clinical treatment of brain neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0022] Figure 1 DSPE-TK-PEG 3400 -Schematic diagram of the synthesis of apoE.

[0023] Figure 2 Bio-MS diagram of the synthetic product.

[0024] Figure 3 for 1 H-NMR spectrum.

[0025] Figure 4 DSPE-TK-PEG 3400-Bio-MS comparison of ROS responsiveness of apoE.

[0026] Figure 5 TEM image and XRD pattern of BM.

[0027] Figure 6 DSPE-TK-mPEG 3400 -ApoE percentage and ultrasound time screening results.

[0028] Figure 7 The BCA method was used to determine the surface membrane protein content of AAG / BM-Lip-apoE@M2CM.

[0029] Figure 8 Particle size and Zeta potential of AAG / BM-Lip-apoE and AAG / BM-Lip-apoE@M2CM.

[0030] Fig. 9 TEM images of different carriers.

[0031] Fig.10 This is the SDS-PAGE electrophoresis diagram after membrane lipid fusion.

[0032] Fig.11 This is the WB image after membrane lipid fusion.

[0033] Fig.12 is the ROS scavenging ability in vitro.

[0034] Fig.13 is the apparent permeability coefficient.

[0035] Fig.14 The following are the images of ZO-1 immunofluorescence staining and the fluorescence intensity values ​​of ZO-1.

[0036] Fig.15 The fluorescence intensity of CD206 and CD86 was measured by flow cytometry.

[0037] Fig.16 The Transwell migration diagrams and relative migration rates of cells after treatment in each group.

[0038] Fig.17 Shown are the cell scratch images and cell migration rates after treatment in each group.

[0039] Fig.18 The phagocytosis of Aβ by BV2 cells after treatment in each group 1-42 Laser confocal image.

[0040] Fig.19 Western blot images of p-Tau-related proteins and corresponding semi-quantitative analysis after treatment in each group.

[0041] Fig. 20 Aβ 1-42 Survival rate of SH-SY5Y cells under stimulation.

[0042] Fig.21 These are in vivo imaging images of mice at different time points.

[0043] Fig. 22 The distribution of each group in the brain at different time points and the tissue distribution in the body at 4 hours.

[0044] Fig.23 Results of the water maze experiment.

[0045] Fig.24 Nesting experiments for mice.

[0046] Fig.25 For immunofluorescence experiments.

[0047] Fig.26 Immunohistochemistry of Aβ1-42 and p-Tau in the brain of mice after treatment.

[0048] Fig. 27 This is the Nissl staining image of the mouse brain area after treatment.

[0049] Fig.28 The SDS-PAGE electrophoresis diagram of M2CM extracted by different methods. DETAILED DESCRIPTION

[0050] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0051] The abbreviations and Chinese names involved in the present invention are: AD Alzheimer's disease, Aβ amyloid protein, APP amyloid precursor protein, BBB blood-brain barrier, BCA bicinchoninic acid, BSA bovine serum albumin, CM cell membrane, CNS central nervous system, DMSO dimethyl sulfoxide, DAPI4′, 6′-diamidino-2-phenylindole, ESR electron spin resonance, FITC fluorescein isothiocyanate, FRET fluorescence resonance energy transfer, H&E hematoxylin-eosin, Hsp4040 heat shock protein, Hsp7070 heat shock protein, Hsp9090 heat shock protein, HRP horseradish peroxidase, IHC immunohistochemistry, MPS reticuloendothelial system, NFT neurofibrillary tangles, OD optical density value, OA okadaic acid, PAGE polyacrylamide gel electrophoresis, PVDF polyvinyl difluoride membrane, ROS reactive oxygen species, [Ru(dpp)3]Cl2 tris(4,7-biphenyl-1,10-phenanthroline) ruthenium dichloride, SDS sodium dodecyl sulfate, SP senile plaques, TEMED tetramethylethylenediamine, ThT thioflavin T, Tris trishydroxymethylaminomethane, TMB 3,4,5-trimethoxybenzaldehyde, 17-AAG 17-propenylamine.

[0052] Embodiment 1:

[0053] DSPE-TK-PEG 3400 -Synthesis of apoE:

[0054] Weigh 1.5 g NHS-PEG 3400 -OH (hydroxy polyethylene glycol active ester, purchased from Sigma) was dissolved in 10 mL of chloroform, 1.0 g of NH2-TK-NH2 (propane-2, 2-diylbis(sulfonamide)diethylamine) and triethylamine were added, and the mixture was reacted at room temperature for 24 h. After concentration under reduced pressure, the mixture was poured into a large amount of icy ether for precipitation, the product was collected by filtration, and vacuum dried to obtain HO-PEG 3400 -TK-NH2. Weigh 1.0g HO-PEG 3400 -TK-NH2 was dissolved in 5 mL of chloroform, and appropriate amounts of DSPE-NHS (distearoylphosphatidylethanolamine modified active ester, purchased from Corden Pharma) and triethylamine were added. After reacting at room temperature for 24 h, the filtered product was collected according to the above method and vacuum dried to obtain HO-PEG 3400 -TK-DSPE. Weigh 0.5 g HO-PEG 3400 -TK-DSPE was dissolved in 5 mL of acetonitrile, and appropriate amounts of DSC and triethylamine were added. The reaction was allowed to proceed overnight at room temperature. The filtered product was collected according to the above method and vacuum dried to obtain NHS-PEG. 3400 -TK-DSPE. Weigh 0.1 g NHS-PEG 3400-TK-DSPE was dissolved in 0.1M PBS solution (pH 8.0), and 0.25g apoE mimetic peptide (sequence RLASHLRKLRKRLLREEQAQQIRLQAEAFQARLKSWFEPLVEDM) or apoE protein was added. After stirring at room temperature for 24 hours, it was dialyzed and purified in a dialysis bag (MW: 3500Da) for 24 hours. The dialyzate was freeze-dried to obtain the product DSPE-TK-PEG 3400 -apoE. Bio-MS and H-NMR spectrometry ( 1 H-NMR) was used to identify the product structure.

[0055] DSPE-TK-PEG 3400 - ROS responsiveness of apoE: Weigh 5 mg DSPE-TK-PEG 3400 -apoE, add 2mL PBS buffer (10mM, pH 7.4) to disperse evenly, then add 2mL H2O2 solution (200μM) and incubate at 37℃ for 3h, dialyze in a dialysis bag (MW: 3500Da) for 24h. The dialyzed sample was freeze-dried and the product was identified by Bio-MS.

[0056] Synthesis and characterization of manganese dioxide nanozyme: Weigh 31.6 mg of potassium permanganate and add 3 mL of distilled water to dissolve. Weigh 250 mg of bovine serum albumin (BSA) and add 7 mL of deionized water to dissolve. Add the above potassium permanganate solution dropwise to the BSA solution, stir and react at 37°C for 1.5 hours, dialyze and purify in a dialysis bag (MW: 8000-14000Da) for 24 hours, and freeze-dry the dialysate to obtain manganese dioxide (BM) nanozyme, which is stored at 4°C for later use. Transmission electron microscopy (TEM), UV-2600i ultraviolet visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) were used to identify the structure of BM nanozyme.

[0057] Preparation and formulation process optimization of manganese dioxide nanozyme liposomes: Manganese dioxide nanozyme liposomes (AAG / BM-Lip-apoE) were prepared by reverse evaporation method. Weigh 15 mg DOTAP ((2,3-dioleoyl-propyl)-trimethylamine), 3.5 mg cholesterol and 20 mg 17-AAG, dissolve them in an appropriate amount of chloroform, and then add 3 mL of ether to mix as the oil phase. Weigh 60 mg BM nanozyme and an appropriate amount of DSPE-TK-PEG 3400-apoE, add 0.6mL of PBS (pH 7.4) to dissolve as the aqueous phase. Add the aqueous phase dropwise into the oil phase (the volume ratio of the oil and water phase is 6:1), and after water bath ultrasonication for 10 minutes, perform vacuum distillation on a rotary evaporator to remove the organic solvent. When a gel-like substance appears, stop the decompression operation, add 5mL of deionized water to form an aqueous suspension. Transfer to an ultrasonic cell crusher and ultrasonicate at 100w for 6 minutes, then filter with 0.45μm and 0.22μm microporous filter membranes in turn to obtain AAG / BM-Lip-apoE. The above prescription and preparation process of AAG / BM-Lip-apoE do not add 17-AAG and DSPE-TK-PEG 3400 -apoE can be used to obtain BM-Lip; Lip-apoE can be obtained without adding BM and 17-AAG; BM-Lip-apoE can be obtained without adding 17-AAG; without adding 17-AAG, DSPE-TK-PEG 3400 -apoE and BM, and Lip is composed only of DOTAP and cholesterol. 3400 -The particle size or potential of AAG / BM-Lip-apoE prepared when the ratio of apoE functional fragment to DOTAP was 5% (0.75 mg), 8% (1.2 mg) and 10% (1.5 mg), and the ultrasonic time was 3 min, 6 min and 9 min, and the optimal prescription and process were optimized.

[0058] Extraction and characterization of M2 microglial cell membrane (M2CM): Mouse microglial cells (BV2) were cultured and induced to differentiate into M2 microglial cells with IL-4 (20 ng / mL) for 24 h. The cells were collected by centrifugation at 1000 r / min for 3 min. The cell membrane was extracted by freeze-drying and sucrose gradient centrifugation, respectively. The specific steps of freeze-drying (method 1) were as follows: the collected cells were added to PBS and mixed to a concentration of 1×10 7 / mL, then add an appropriate amount of protease inhibitors and mix well. Squeeze back and forth through a liposome extruder for about 20 times, centrifuge (3000g, 10min, 4℃), collect the supernatant and further centrifuge (10000g, 10min, 4℃), and finally freeze-dry the supernatant to obtain M2 microglial cell membrane (M2CM). The specific steps of sucrose gradient centrifugation (method 2) are as follows: resuspend the collected cells in 4℃ Tris-magnesium salt buffer to a cell suspension concentration of 1×10 7 / mL, add an appropriate amount of protease inhibitors and mix well. Squeeze back and forth through a liposome extruder for about 20 times, then add 1M sucrose solution and mix to make the final concentration 0.25M, centrifuge (3000g, 10min, 4℃), take the supernatant and further centrifuge (10000g, 10min, 4℃) to get the cell precipitate, then wash the obtained cell precipitate with 0.25M sucrose solution and centrifuge, discard the supernatant, and the precipitate at the bottom of the tube is M2CM. The cell membranes extracted by the above two methods are stored at -20℃. From the SDS-PAGE electrophoresis diagram ( Fig.28 ) It can be seen that the cell membrane proteins extracted by freeze-drying are concentrated around 25kDa molecular weight, and the overall protein information is less, while the cell membrane extracted by sucrose gradient centrifugation has protein expression at different molecular weight positions and has rich protein information. Therefore, sucrose gradient centrifugation was subsequently selected as the final cell membrane M2CM extraction method.

[0059] Preparation of cell membrane biomimetic nanocarriers:

[0060] Screening of membrane lipid ratio: The extracted M2CM and the prepared AAG / BM-Lip-apoE were mixed in a mass ratio of 1:4 to 4:1, and after repeated freeze-thaw cycles (4℃ / 45℃), they were passed through 400nm and 200nm polycarbonate membranes in turn using a liposome extruder, and further centrifuged at 10000g for 10min to purify and remove the unsuccessfully loaded M2CM to obtain AAG / BM-Lip-apoE@M2CM. Lip and M2CM were co-extruded using a liposome extruder to obtain Lip@M2CM; BM-Lip-apoE and M2CM were co-extruded using a liposome extruder to obtain BM-Lip-apoE@M2CM. The BCA kit was used to determine the content of AAG / BM-Lip-apoE@M2CM membrane protein, and the membrane lipid ratio with the highest cell membrane coating efficiency was determined based on the correlation between the M2CM membrane protein content and the membrane lipid mass ratio.

[0061] In vitro ROS scavenging ability investigation: H2O2 scavenging: Prepare 1, 5, 10, 20, 50, 100 μM H2O2 standard solutions, take 500 μL of each sample and add 50 μM Amplex red and 0.1 U / mL HRP, mix well and react at 37°C for 10 min. Use UV-vis to measure the absorbance value (OD value) at 570 nm, and plot OD570 nm-Concentration standard curve. Take 3 mL of BM and AAG / BM-Lip-apoE@M2CM and add an equal amount of 200 μM H2O2 solution, and 3 mL of PBS solution and add an equal amount of H2O2 solution as the control group. Place them in a 37°C constant temperature oscillating box and absorb 500 μM at 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. μ L reaction solution, add Amplex red and HRP, mix well and measure the OD value at 570nm, calculate the remaining H2O2 content in the system, and draw a degradation percentage-time curve.

[0062] An appropriate amount of BM and AAG / BM-Lip-apoE@M2CM was mixed with 20 mL of H2O2 (100 μM) solution. A digital camera was used to take pictures to record the O2 production images. A portable dissolved oxygen meter was used to measure the changes in O2 concentration in the solution at different time points, and the relationship curve between O2 production and time was plotted.

[0063] Hydroxyl radicals (·OH) and superoxide anions (O2 ·- ) Scavenging experiment: Electron spin resonance (ESR) experiment was used to evaluate the superoxide anion (O2 ·- ) and hydroxyl radicals (·OH). O2 ·- The steps of the clearance experiment are as follows: Take an appropriate amount of 10mM xanthine solution and 1U / mL xanthine oxidase solution, mix them, add 20μLDMPO and 180μL buffer, and incubate for 5min. Add BM and AAG / BM-Lip-apoE@M2CM to the above solutions, and the control group without treatment is used for sampling and testing. 1-42 Interaction evaluation: Fluorescence colocalization analysis: Prepare AAG / BM-Lip-apoE@M2CM labeled with Dil (Dil-AAG / BM-Lip-apoE@M2CM). Take 400 μL of Dil-AAG / BM-Lip-apoE@M2CM and 100 μL of FITC-Aβ 1-42 (1 mg / mL) and incubate at 37°C for 2 h. The sample was dropped onto a glass slide, sealed and observed under an upright fluorescence microscope.

[0064] Embodiment 2:

[0065] Example 1 Experimental results:

[0066] DSPE-TK-PEG 3400 -Structural identification of apoE: Figure 2 The Bio-MS image of the synthetic product. From the m / z value in the image, it can be inferred that the molecular weight of the product is about 9492, which is similar to DSPE-TK-PEG3400 -apoE theoretical molecular weight is consistent. Figure 3 A is DSPE-TK-PEG 3400 -apoE 1 H-NMR spectrum, Figure 3 B is apoE mimetic peptide 1 H-NMR spectrum, Figure 3 C is NH2-TK-NH2 1 H-NMR spectrum. Figure 3 The peak with a chemical shift value of about 3.5 ppm in A is PEG 3400 The characteristic peak of the chemical shift value of about 4.2ppm is the characteristic peak of the hydroxyl hydrogen in DSPE. Figure 3 The multiple peaks around 7.2 ppm in B are characteristic peaks of the benzene ring in Phe in the apoE mimetic peptide. Figure 3 The characteristic peak of the benzene ring also appears at the position corresponding to A. Figure 3 b, c, d and e in C are the characteristic peaks of methylene, methyl hydrogen and amino hydrogen of TK, respectively. The synthetic product DSPE-TK-PEG 3400 -apoE 1 The H-NMR graph also has peaks at the corresponding b, c, and d positions ( Figure 3 A) Bio-MS and 1 H-NMR results show that DSPE-TK-PEG 3400 -apoE was successfully synthesized.

[0067] DSPE-TK-PEG 3400 - ROS responsiveness of apoE: Figure 4 Bio-MS showed that DSPE-TK-PEG 3400 -apoE molecular weight is about 9492. After reacting with H2O2 solution, the molecular weight is reduced to about 8683, which is similar to PEG 3400 -apoE fragments. This indicates that after H2O2 treatment, DSPE-TK-PEG 3400 -The TK fragment in the apoE structure breaks and the structure disintegrates into PEG 3400 -apoE fragment and DSPE fragment. Therefore, DSPE-TK-PEG 3400 -apoE is ROS-responsive.

[0068] Characterization of manganese dioxide nanozyme: TEM, XPS and XRD analysis were used to identify the successful preparation of BM. Figure 5), BM presents relatively uniform spherical particles, which are evenly dispersed and have a particle size of about 10nm. XPS analysis results show that BM has characteristic absorption at 641.8eV and 653.5eV, which are respectively related to Mn(1V) in manganese dioxide. 2p3 / 2 and Mn(1V) 2p1 / 2 Correspondingly, it shows that the valence state of Mn element in BM is +4. In the XRD spectrum, BM shows a characteristic diffraction absorption peak at 2θ=21.06°, which is consistent with the XRD standard chart of MnO2.

[0069] Formulation and process screening of manganese dioxide nanozyme liposomes: The formulation and process screening results of AAG / BM-Lip-apoE are shown in Figure 6 .Depend on Figure 6 It can be seen that DSPE-TK-mPEG 3400 The proportion of -apoE has a significant effect on the Zeta potential. As its proportion increases, the Zeta potential value decreases, indicating that DSPE-TK-mPEG 3400 The hydrophilic long chain in the apoE fragment will form a certain shielding effect on the potential of the liposome surface, thereby reducing the absolute value of the Zeta potential to a certain extent. Considering the subsequent fusion effect with the cell membrane, 8% DSPE-TK-mPEG was selected 3400 -apoE addition ratio as the optimized prescription amount. Figure 6 It can be seen that the ultrasonic time has a certain effect on the particle size of liposomes. It helps the liposomes to further increase the degree of dispersion within a certain range, showing a trend of decreasing particle size. However, a relatively long ultrasonic time will increase the probability of collision between particles, causing the liposome particle size to increase. Therefore, the preferred ultrasonic time is 6 minutes.

[0070] Screening of membrane lipid ratio: The AAG / BM-Lip-apoE@M2CM membrane protein content was quantitatively determined by BCA kit to determine the optimal membrane lipid mass ratio. Figure 7 It can be seen that with the increase of membrane lipid ratio, the membrane protein content of AAG / BM-Lip-apoE@M2CM increases accordingly. When the membrane lipid ratio is greater than 1:1, the membrane protein content of AAG / BM-Lip-apoE@M2CM gradually tends to saturation, and on this basis, more cell membranes continue to be added. After centrifugation, obvious cell membrane precipitation can be found at the bottom of the tube. When the membrane lipid ratio is 1:1, the optimal fusion effect of the cell membrane can be achieved. Therefore, 1:1 is preferred as the optimal input ratio of cell membrane and liposome.

[0071] Characterization of AAG / BM-Lip-apoE@M2CM: Average particle size and Zeta potential results are shown in Figure 8After fusion with the cell membrane, the average particle size increased from 108.23±1.92nm to 123.37±1.11nm for AAG / BM-Lip-apoE@M2CM; and due to the neutralization of the negative charge of the cell membrane, the Zeta potential decreased from the original 33.79±0.51mV to 13.66±1.34mV for AAG / BM-Lip-apoE@M2CM, which proved that the cell membrane and liposomes were successfully fused, and the TEM observation ( Fig. 9 ), both AAG / BM-Lip-apoE and AAG / BM-Lip-apoE@M2CM showed vesicle-like structures, with little difference in particle size. After fusion with the cell membrane, the vesicle bilayer membrane structure of AAG / BM-Lip-apoE@M2CM was more obvious. Fig. 9 Middle, A: AAG / BM-Lip-apoE; B: AAG / BM-Lip-apoE@M2CM.

[0072] SDS-PAGE electrophoresis experiment: Fig.10 The SDS-PAGE electrophoresis diagram shows that the protein expression of the extracted M2CM and BV2 cells at the same position is basically the same, and after membrane fusion, AAG / BM-Lip-apoE@M2CM also retains almost all the original protein information of BV2 cells, while AAG / BM-Lip-apoE that does not interact with the cell membrane does not show any protein bands. The results prove the successful modification of the carrier by the cell membrane. Fig.10 In the figure, A: BV2 cells; B: M2CM; C: AAG / BM-Lip-apoE@M2CM; D: AAG / BM-Lip-apoE.

[0073] Western Blotting Experiment: Fig.11 The WB results showed that a large number of specific proteins Integrin α4 and Mac-1 were enriched on the surface of M2CM and AAG / BM-Lip-apoE@M2CM. The effective transfer of membrane protein information had a positive effect on the brain delivery of AAG / BM-Lip-apoE@M2CM and the active uptake by microglia and neurons. Fig.11 Middle, A: BV2 cells; B: M2CM; C: AAG / BM-Lip-apoE@M2CM.

[0074] Investigation of ROS scavenging ability in vitro:

[0075] H2O2 removal: Fig.12The figure is a graph showing the relationship between the percentage of H2O2 degradation and time. According to the curve results, BM and AAG / BM-Lip-apoE@M2CM consume H2O2 in the system faster. After 10, 20 and 50 minutes of reaction, the reduction rates of H2O2 in the system are 59.1%, 60.6% and 69.9%, respectively, and 71.9% in 1 hour. The percentage of H2O2 degradation in the H2O2 control group is relatively stable at various time points before and after incubation, and is only about 22.1% in 1 hour. It can be seen that both BM and AAG / BM-Lip-apoE@M2CM can effectively remove H2O2 from the reaction system, which is speculated to be related to the CATase activity of BM in the AAG / BM-Lip-apoE@M2CM structure and the DSPE-TK-PEG 3400 -The breaking of the TK bond on the apoE fragment is related to the consumption of H2O2 in the system.

[0076] OH and O2 ·- ESR analysis was used to evaluate the clearance of OH and O2 by BM and AAG / BM-Lip-apoE@M2CM. ·- The control group had a strong ESR signal (1:2:2:1). After being treated with BM and AAG / BM-Lip-apoE@M2CM, the peak intensity was significantly reduced, indicating that BM and AAG / BM-Lip-apoE@M2CM can effectively consume ·OH. After being treated with BM and AAG / BM-Lip-apoE@M2CM, the characteristic peak of 2·- at the same ESR signal of the control group (1:1:1:1) was also significantly weakened, indicating that BM and AAG / BM-Lip-apoE@M2CM can also effectively scavenge O2 ·- The above experimental results all show that AAG / BM-Lip-apoE@M2CM has sensitive ROS scavenging ability and can be effectively used to alleviate the oxidative stress microenvironment in the brain of AD patients.

[0077] Fig.12 A: H2O2 degradation percentage after treatment with different carriers. B: ESR spectrum of ·OH removal. C: O2 ·- Cleared ESR spectrum.

[0078] Embodiment 3:

[0079] In vitro evaluation of brain targeting:

[0080] Culture of mouse brain microvascular endothelial cells (bEnd.3): The cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% double antibody at 37°C and 5% CO2 in a cell culture incubator. When the number of cells in the bottle is about 80-90%, the medium in the bottle is discarded, washed with PBS three times, and 2mL of trypsin is added for digestion. Then complete medium is added to terminate digestion. Centrifuge at 1200r / min for 3min to collect cells. Add appropriate amount of culture medium to the cell pellet and blow it evenly, then transfer it to a cell culture bottle for further culture.

[0081] Transmembrane transport study: bEnd.3 cells in the logarithmic growth phase were added to the transwell plate chamber to a density of about 1×10 5 The medium was changed every other day, and the transendothelial electrical resistance (TEER) was measured until the TEER reached 200Ω / cm 2 , that is, the in vitro BBB model was successfully established. The culture medium in the upper chamber was discarded, and C6-BM-Lip-apoE and C6-BM-Lip-apoE@M2CM (DOTAP concentration: 100μg / mL) diluted in 1640 culture medium were added. 200μL of the lower chamber liquid was drawn at 5min, 10min, 15min, 30min, and 60min, and an equal volume of blank 1640 culture medium was added at the same time. The fluorescence intensity of C6 was measured at each time point, and the PBS solution of C6 was used as the control group. The above-measured C6 fluorescence intensity was used to calculate the apparent permeability coefficient (Papp) according to the following formula to evaluate and compare the ability and difference of transmembrane transport. TEER was measured at different stages of the experiment (0h, 0.5h and 24h) to evaluate the integrity of the BBB.

[0082]

[0083] in, is the permeability (nmol / s), C0 (nmol / mL) is the initial concentration of C6 in the upper chamber, A (cm 2 ) is the surface area of ​​the Transwell membrane.

[0084] ZO-1 immunofluorescence assay: bEnd.3 cells were cultured at 1×10 4The cells were inoculated at a density of 100 / well in a confocal dish with a lid. After culturing for 24 hours, the original culture medium containing serum was discarded, and PBS solution, BM-Lip-apoE and BM-Lip-apoE@M2CM were added for incubation for 1 hour. After fixation with tissue fixative for 10 minutes, rabbit anti-mouse ZO-1 diluent (1:200) was added and incubated at 4°C overnight. The cells were washed 3 times with PBS, and then anti-Cy3 labeled goat anti-rabbit IgG (1:300) was added and incubated at room temperature for 50 minutes. The cells were washed 3 times with PBS, and the cell nuclei were stained with DAPI for 5 minutes before observation under a laser confocal microscope.

[0085] Investigation of microglial phagocytic ability: BV2 cells in the logarithmic growth phase were plated in a 24-well plate at a cell density of approximately 1×10 5 After 24 h of culture, the original serum-containing medium was discarded and LPS (100 ng / mL) was added to induce M1 microglia. PBS solution, Lip-apoE, Lip@M2CM, BM-Lip and BM-Lip-apoE@M2CM were added respectively (in the above components, the DOTAP concentration was 120 μg / mL and the BM concentration was 500 μg / mL) and incubated for 1 h. FITC-Aβ was then added to each group. 1-42 The cells were treated for 15 min and washed with PBS for 3 times. Lysosomes were stained with Lyso-Tracker probe for 30 min and washed with PBS for 3 times. After fixation with tissue fixative for 10 min, the nuclei were stained with DAPI for 5 min and observed under a laser confocal fluorescence microscope.

[0086] Calculation of apparent permeability coefficient: Fig.13 As shown in Figure 2, the Papp value of the BM-Lip-apoE@M2CM group reached 2.6×10 within 60 min. 9 cm / s, which was significantly higher than that of BM-Lip-apoE and PBS control groups. The Papp value of BM-Lip-apoE was 1.4×10 9 cm / s, the difference in carrier structure shows that M2CM and DSPE-TK-PEG 3400 -apoE fragments can effectively assist BM-Lip-apoE@M2CM to spread from the upper chamber of Transwell across the bEnd.3 monolayer to the lower chamber. This may be related to the binding of Intergin α4 and Mac-1 proteins expressed on M2CM and ICAM-1 / VCAM-1 on the surface of bEnd.3 cells to reversibly open the BBB tight junctions, and the active transport pathway mediated by apoE mimetic peptides and LRP1 receptors on the surface of bEnd.3 cells to cross the BBB and distribute into the brain. Fig.13A: PBS; B: BM-Lip-apoE; C: BM-Lip-apoE@M2CM; n=3, * indicates that this group has statistical difference with PBS group, p<0.05.

[0087] ZO-1 immunofluorescence investigation: ZO-1 is one of the tight junction-related proteins, and its expression is closely related to the integrity of endothelial cell tight junctions. Therefore, immunofluorescence labeling of ZO-1 protein on brain microvascular endothelial cells bEnd.3 can directly reflect the transmembrane transport mechanism of BM-Lip-apoE@M2CM. Fig.14 As shown, the blue fluorescence is the cell nucleus labeled by DPAI, and the red fluorescence-labeled ZO-1 surrounds the cell membrane outline to express the integrity of the tight junction. After the PBS control group and the BM-Lip-apoE experimental group came into contact with bEnd.3 cells, the cell edges showed a complete red fluorescence outline, and the fluorescence intensity was high, indicating that BM-Lip-apoE did not change the integrity of the original tight junctions of the cells after contact with the cells, which was consistent with the PBS control group. The BM-Lip-apoE@M2CM experimental group significantly reduced the red fluorescence intensity of bEnd.3 cells, and a considerable part of the red fluorescence was missing, indicating that BM-Lip-apoE@M2CM can reduce the expression of the tight junction-related protein ZO-1 and disrupt the tight junctions between endothelial cells. This is closely related to the presentation of Intergin α4 and Mac-1 proteins on the surface of BM-Lip-apoE@M2CM after M2CM coating, which can bind to ICAM-1 / VCAM-1 on the surface of bEnd.3 cells to reversibly open the BBB tight junctions, increase the permeability of the BBB, and achieve effective delivery to the brain.

[0088] Evaluation of microglial transformation:

[0089] Immunofluorescence and flow cytometry analysis: Fig.15 The fluorescence intensity of CD206 and CD86 was measured by flow cytometry. CD206 is a protein-specific marker on M2 microglia, and CD86 is a protein-specific marker on M1 microglia. Compared with the M0 and M1 control groups, Fig.15The fluorescence intensity of CD206 marker in IL-4-induced microglia in the M2 group increased, while the fluorescence intensity of CD86 marker in the M2 group decreased, indicating that they had successfully differentiated into M2 microglia. The transformation-promoting trends of each experimental group in the M1 microglia model induced by LPS were further compared and investigated. The results showed that after Lip-apoE@M2CM was taken up by cells, the fluorescence intensity of CD206 was significantly enhanced compared with that of the M0 and M1 control groups, and was equivalent to that of the M2 control group, while the fluorescence intensity of Lip-apoE@M2CM was significantly weakened compared with that of the M0 and M1 control groups, indicating that M2CM coating can promote the transformation of M1 microglia to M2 microglia; after co-incubation of BM-Lip-apoE and M1 microglia, the fluorescence intensity of CD206 marker was further enhanced compared with that of the M2 control group and the Lip-apoE@M2CM experimental group, and the fluorescence intensity of CD86 marker was significantly weakened, indicating that the efficient clearance of ROS by BM nanozymes can alleviate the over-activated state of microglia under the M1 phenotype, promote the transformation of M1 microglia to the M2 phenotype, and improve neuroinflammation caused by ROS as a proinflammatory factor. After M1 microglia were treated with BM-Lip-apoE@M2CM, the fluorescence intensity of CD206 was further enhanced, which was significantly more significant than that of Lip-apoE@M2CM and BM-Lip-apoE groups. In contrast, the fluorescence intensity of CD86 marker was further reduced, indicating that more M1 microglia differentiated into M2 phenotype, making the proportion of anti-inflammatory M2 microglia higher, thereby effectively alleviating the abnormal immune activation state of microglia and the AD neuroinflammatory environment caused by it, which is closely related to the synergistic effect of M2CM and BM.

[0090] Investigation of microglial migration behavior: A Transwell model of BV2 cells was established to investigate the effect of microglial transformation on its migration behavior. Fig.16 It can be seen that compared with the M0 resting state, microglia differentiated into M1 type after LPS induction, and their migration ability was slightly enhanced, while microglia differentiated into M2 type after IL-4 stimulation, and their migration ability was significantly enhanced. Compared with the M1 and M2 control groups, the migration speed of microglia was significantly accelerated after each experimental group was treated, and the migration ability was most significant after BM-Lip-apoE@M2CM treatment. Fig.17The scratch test showed that after 12 hours of cell growth, the M0 control group still had a relatively obvious scratch area. After inducing M1 and M2 transformation, the cell scratch area decreased, and the cell gap in the M2 phenotype group was narrower than that in the M1 phenotype group, indicating that the cell migration and crawling speed was faster. After group treatment, compared with the M1 phenotype group, the cell migration was significantly accelerated. Among them, a large number of cells in the BM-Lip-apoE@M2CM group converged to the center of the scratch, and the original scratch was no longer obvious. The migration and growth behaviors of each experimental group after treatment were consistent with the results of the Transwell experiment. Combined with the results of microglial transformation, it is speculated that each preparation group promotes microglia to differentiate into the M2 phenotype, which can enhance the migration ability of microglia, so that M2 microglia converge faster around pathological protein deposition and neuronal debris, which is conducive to the subsequent phagocytic clearance.

[0091] Investigation of microglial migration behavior: A Transwell model of BV2 cells was established to investigate the effect of microglial transformation on its migration behavior. Fig.16 It can be seen that compared with the M0 resting state, microglia differentiated into M1 type after LPS induction, and their migration ability was slightly enhanced, while microglia differentiated into M2 type after IL-4 stimulation, and their migration ability was significantly enhanced. Compared with the M1 and M2 control groups, the migration speed of microglia was significantly accelerated after each experimental group was treated, and the migration ability was most significant after BM-Lip-apoE@M2CM treatment. Fig.17 The scratch test showed that after 12 hours of cell growth, the M0 control group still had a relatively obvious scratch area. After inducing M1 and M2 transformation, the cell scratch area decreased, and the cell gap in the M2 phenotype group was narrower than that in the M1 phenotype group, indicating that the cell migration and crawling speed was faster. After group treatment, compared with the M1 phenotype group, the cell migration was significantly accelerated. Among them, a large number of cells in the BM-Lip-apoE@M2CM group converged to the center of the scratch, and the original scratch was no longer obvious. The migration and growth behaviors of each experimental group after treatment were consistent with the results of the Transwell experiment. Combined with the results of microglial transformation, it is speculated that each preparation group promotes microglia to differentiate into the M2 phenotype, which can enhance the migration ability of microglia, so that M2 microglia converge faster around pathological protein deposition and neuronal debris, which is conducive to the subsequent phagocytic clearance.

[0092] Investigation of the phagocytic ability of microglia: An M1 microglia model was established, and the phagocytic clearance ability of microglia on Aβ was observed using laser confocal microscopy. Fig.18 The green fluorescence is FITC-labeled Aβ 1-42 , red fluorescence is lysosomes labeled with Lyso-Tracker Red probe, and blue fluorescence is cell nuclei labeled with DAPI. Fig.18 As shown, BV2 cells were co-stained with FITC-Aβ1-42 After incubation, Aβ 1-42 The green fluorescence of the lysosome and the red fluorescence of the lysosome are still scattered in the cytoplasm, with only a small amount of overlap showing a weak orange fluorescence, indicating that Aβ can be metabolized and cleared by lysosomes without effective intervention. 1-42 The amount of Aβ is limited. After BV2 cells take up Lip-apoE, Aβ 1-42 The green fluorescence of the lysosome and the red fluorescence of the lysosome began to overlap, showing orange fluorescence after the superposition of red and green, indicating that the apoE mimetic peptide can carry Aβ 1-42 After incubation of Lip@M2CM, BM-Lip and BM-Lip-apoE@M2CM with BV2 cells, green fluorescence and red fluorescence overlapped a lot, and orange fluorescence was more obvious. All three preparation groups were able to promote the transformation and differentiation of M1 microglia, and the migration ability of induced transformed M2 microglia was enhanced, which could more actively identify and phagocytose pathological Aβ 1-42 Deposition, enhancement of Aβ 1-42 The ability to transport to lysosomes and promote the production of more Aβ 1-42 The BM-Lip-apoE@M2CM, which promotes cell transformation and acts as an apoE mimetic peptide, enables microglia to show stronger phagocytic ability and Aβ resistance under the laser confocal microscope. 1-42 Abnormal protein clearance effect.

[0093] Clearance of p-Tau in SH-SY5Y cells induced by OA: Western blotting experiment: OA was used to induce SH-SY5Y cells to establish a p-Tau cell model, and the WB method was used to analyze the expression of Hsp40, Hsp70 and p-Tau in the p-Tau cell model after treatment in each group. Fig.19 It can be seen that compared with the OA control group and the 17-AAG experimental group, the expressions of Hsp40, Hsp70 and p-Tau in the AAG / BM-Lip-apoE@M2CM group were significantly decreased.

[0094] Neuroprotection: Using Aβ 1-42 Stimulate SH-SY5Y cells to establish a neuronal cytotoxicity model. Fig. 20 The results of CCK-8 assay showed that Aβ 1-42 The cell survival rate in the control group was significantly reduced, while the survival rate of cells treated with AAG / BM-Lip-apoE@M2CM was much higher than that in the control group and other experimental groups. Fig. 20 Middle, A: control group; B: Aβ 1-42; C: AAG-Lip-apoE@M2CM; D: AAG / BM-Lip-apoE; E: AAG / BM-Lip-apoE@M2CM; n=3, ** represents that this group has statistical difference with Aβ1-42 group, p<0.01.

[0095] Evaluation of synergistic effects of neuroprotection: using Aβ 1-42 SH-SY5Y cells were stimulated to establish a neurotoxic model. To evaluate the combined effect of BM-Lip-apoE and Lip@M2CM, the combined effect index Cl values ​​of BM-Lip-apoE and Lip@M2CM (the mass ratio of the two was 1:1) at 0.125IC50, 0.25IC50, 0.5IC50 and 1.0IC50 were obtained according to the Chou-Talalay combined drug index method and calculated using CompuSy software (Table 1). The obtained Cl values ​​were all less than 1, indicating that the combined effect of BM-Lip-apoE and Lip@M2CM (the mass ratio of the two was 1:1) had a synergistic effect, and the Cl value was the smallest at the respective IC50 concentrations, and the synergistic neuroprotective effect was the best.

[0096] Table 1 Evaluation of synergistic therapeutic effects of neuroprotection in AD mice (n=3)

[0097] Concentration Cell survival rate (%) Cl value Interaction Judgment <![CDATA[0.125IC 50 ]]> 54.6±2.2 0.884±0.100 ++ <![CDATA[0.25IC 50 ]]> 68.9±1.4 0.617±0.050 ++ <![CDATA[0.5IC 50 ]]> 78.4±0.5 0.514±0.040 ++ <![CDATA[1.0IC 50 ]]> 86.3±0.4 0.379±0.050 +++

[0098] Note: +++ Strong synergy; ++ Moderate synergy

[0099] Embodiment 4:

[0100] Evaluation of brain targeting: Fig.21As shown in the figure, at each observation time point, there was no obvious fluorescence distribution in the brain of mice in the DiR-BM-Lip group, and only weak fluorescence appeared at 4h and 8h, indicating that the speed and degree of DiR-BM-Lip delivery to the brain were extremely limited, and the brain targeting characteristics were poor. In contrast, the DiR-BM-Lip@M2CM and DiR-BM-Lip-apoE@M2CM groups showed good brain targeting, with high-intensity fluorescence distribution in the mouse brain from 2h to 8h, and the brain fluorescence gradually increased with time. There was still strong fluorescence accumulation in the brain from 12h to 24h, indicating that DiR-BM-Lip@M2CM and DiR-BM-Lip-apoE@M2CM can be distributed to the brain quickly, and can gradually increase and stably reside in the brain for a considerable period of time. And at each observation time point, the fluorescence intensity of the brain of mice in the DiR-BM-Lip-apoE@M2CM group was higher than that of the DiR-BM-Lip@M2CM group. Comparing the differences in brain fluorescence distribution between DiR-BM-Lip and DiR-BM-Lip@M2CM, it is shown that M2CM coating plays a key role in enhancing the characteristics of brain targeted delivery, which can significantly improve the efficiency of brain entry and prolong the residence time in the brain, thus achieving effective delivery of AD therapeutic drugs. Comparing the structural differences between DiR-BM-Lip@M2CM and DiR-BM-Lip-apoE@M2CM, due to the further modification of apoE mimetic peptide, it presents more obvious brain targeting characteristics. Fig.21 In the analysis, DiR-L / B is DiR-BM-Lip; DiR-L / B@CM is DiR-BM-Lip@M2CM; DiR-P / B@CM is DiR-BM-Lip-apoE@M2CM.

[0101] Fig. 22 A shows the distribution of DiR-BM-Lip, DiR-BM-Lip@M2CM and DiR-AAG / BM-Lip-apoE@M2CM in the brain of mice at 2h, 4h, 8h, 12h and 24h after intravenous injection. At all measured time points, the accumulation of DiR-BM-Lip-apoE@M2CM in the brain was higher than that of DiR-BM-Lip and DiR-BM-Lip@M2CM, reaching 109.5ng / mg at 4h, significantly higher than 30.68ng / mg and 87.4ng / mg of DiR-BM-Lip and DiR-BM-Lip@M2CM. The distribution of the three preparation groups in the heart, liver, spleen, lung, kidney and brain at 4h was plotted Fig. 22 B, The distribution of DiR-BM-Lip-apoE@M2CM in the brain is considerable, consistent with the results of small animal in vivo imaging. Fig. 22In the table, DiR-L / B is DiR-BM-Lip; DiR-L / B@CM is DiR-BM-Lip@M2CM; DiR-P / B@CM is DiR-BM-Lip-apoE@M2CM; n=3, *** represents statistical difference, p<0.001.

[0102] Water maze experiment: Fig.23The navigation route map of each group of mice in the water maze truncated table (A), the escape latency of each group of mice (B), the time each group of mice stayed in the platform quadrant (C), and the number of times each group of mice passed the platform (D). The route of the sham operation group was basically concentrated near the platform, and crossed the platform many times; the route of the AD model group was disorganized, wandering around the pool, and basically could not find the platform during the test period; after the drug treatment of each preparation group, the trajectory of the mouse navigation route increased significantly in the platform quadrant, among which the navigation route of the AAG / BM-Lip-apoE@M2CM group mice was close to the sham operation group, basically near the platform. As can be seen from the figure, the escape latency in the sham operation group was 7.26±4.88s, and that in the AD model group was 35.58±2.55s. Two weeks after tail vein injection of AAG / BM-Lip-apoE, AAG-Lip-apoE@M2CM, BM-Lip-apoE@M2CM, AAG / BM-Lip@M2CM and AAG / BM-Lip-apoE@M2CM, the escape latency was reduced to 23.96±4.89s, 20.32±4.32s, 17.74±4.21s, 17.85±6.58s and 11.17±6.37s, respectively. The time that mice in each group stayed in the platform quadrant was 37.03±1.45% in the sham operation group and 18.80±5.24% in the AD model group. After treatment with AAG / BM-Lip-apoE, AAG-Lip-apoE@M2CM, BM-Lip-apoE@M2CM, AAG / BM-Lip@M2CM and AAG / BM-Lip-apoE@M2CM, the time increased to 19.97±6.52%, 24.87±3.68%, 24.87±5.40%, 26.33±6.52% and 34.17±2.95%, respectively. The number of times mice in each group passed through the platform was 4.8±0.84 in the sham operation group and 1.6±0.55 in the AD model group. After treatment with AAG / BM-Lip-apoE, AAG-Lip-apoE@M2CM, BM-Lip-apoE@M2CM, AAG / BM-Lip@M2CM and AAG / BM-Lip-apoE@M2CM, the numbers increased to 2.60±1.14, 3.00±1.00, 3.25±1.26, 3.00±1.00 and 4.4±1.14, respectively. From the above results, it can be seen that AAG / BM-Lip-apoE@M2CM treatment was the most prominent and could improve the learning and memory ability of mice.A / P / B is AAG / BM-Lip-apoE; A / P@CM is AAG-Lip-apoE@M2CM; P / B@CM is BM-Lip-apoE@M2CM; A / L / B@CM is AAG / BM-Lip@M2CM; A / P / B@CM is AAG / BM-Lip-apoE@M2CM; n=12, ** represents that this group has statistical differences from the AD model group, p<0.01.

[0103] Nesting experiment: Fig.24 The nesting behavior of mice and the corresponding data analysis results show that after two weeks of group treatment, the nesting behavior of mice in the AAG / BM-Lip-apoE@M2CM preparation group was significantly different from that in the AD model group (p<0.01), which was basically close to the normal mice in the sham operation group, indicating that the AD mice treated with AAG / BM-Lip-apoE@M2CM had improved cognitive behavior. A / P / B is AAG / BM-Lip-apoE; A / P@CM is AAG-Lip-apoE@M2CM; P / B@CM is BM-Lip-apoE@M2CM; A / L / B@CM is AAG / BM-Lip@M2CM; A / P / B@CM is AAG / BM-Lip-apoE@M2CM; n=12, ** indicates statistical difference compared with the AD model group, p<0.01.

[0104] Immunofluorescence: Fig.25 A is the result of 8-OHG immunofluorescence analysis of hippocampal neurons in each group of mice, where DAPI blue fluorescence outlines the basic outline of the mouse hippocampus, and 8-OHG red fluorescence marks reactive oxygen molecules. No red fluorescence was observed in the normal mouse group, and strong red fluorescent spots appeared in the hippocampus of the AD model group. After two weeks of tail vein injection in each preparation group, the red fluorescent spots decreased to varying degrees, and the fluorescent spots in the AAG / BM-Lip-apoE@M2CM group basically disappeared, indicating that the level of 8-OHG in the hippocampus was greatly reduced after treatment, and the aggregation state of reactive oxygen in the brain was effectively improved. Fig.25B is the immunofluorescence analysis results of CD206 and CD86 in brain slices of mice in each group. DAPI blue fluorescence marks the cell nucleus, green fluorescence and red fluorescence mark CD206 and CD86 respectively. The red fluorescence of CD86 in brain slices of mice in the AD model group was stronger, while the green fluorescence of CD206 was weaker, and the microglia showed an over-activated M1 phenotype. After treatment with each preparation group, the green fluorescence of CD206 in the brain was enhanced to varying degrees, while the red fluorescence of CD86 was weakened to varying degrees. The red fluorescence in the AAG / BM-Lip-apoE@M2CM group basically disappeared, and the green fluorescence intensity was the most significant. The results showed that after intervention in each preparation group, the microglia in the mouse brain were mostly anti-inflammatory M2 phenotypes, which is beneficial to the improvement of the inflammatory environment in the brain of AD patients. In the figure, A / P / B is AAG / BM-Lip-apoE; A / P@CM is AAG-Lip-apoE@M2CM; P / B@CM is BM-Lip-apoE@M2CM; A / L / B@CM is AAG / BM-Lip@M2CM; A / P / B@CM is AAG / BM-Lip-apoE@M2CM.

[0105] Immunohistochemistry: Fig.26 Aβ in brain slices of mice in each group 1-42 The results of immunohistochemical analysis of p-Tau protein showed that the AD model group had obvious orange Aβ amyloid deposition and p-Tau protein aggregates under the microscope, which was consistent with the typical pathological characteristics of AD mice, while the normal group mice had almost no abnormal protein deposition. After two weeks of drug treatment, Aβ in the brain of AD mice 1-42 The expression of phospho-Tau (Ser396) and phospho-Tau (Ser396) was significantly reduced, and the deposition or aggregation of abnormal proteins under the microscope basically disappeared. Among them, the effect of AAG / BM-Lip-apoE@M2CM treatment was the most obvious, indicating that it can effectively improve Aβ 1-42 The deposition state in the brain and the abnormal entanglement of p-Tau protein are corrected, the damage to neurons by both is reduced, and the physiological function of neurons is restored. A / P / B is AAG / BM-Lip-apoE; A / P@CM is AAG-Lip-apoE@M2CM; P / B@CM is BM-Lip-apoE@M2CM; A / L / B@CM is AAG / BM-Lip@M2CM; A / P / B@CM is AAG / BM-Lip-apoE@M2CM.

[0106] Nissl staining: Nissl staining can indirectly reflect the morphological changes and survival of neurons. Fig. 27The results showed that the number of surviving neurons in the hippocampus of mice in the sham operation group was larger and they were arranged more neatly, while the number of surviving neurons in the hippocampus of mice in the AD model group was significantly reduced and showed different degrees of pyknosis and dark staining. After drug treatment in each group, the cell pathological changes in the hippocampus of mice were alleviated, the number of surviving neurons increased and they were arranged neatly. The AAG / BM-Lip-apoE@M2CM group was similar to the sham operation group and the neurons returned to the normal state.

[0107] In summary, the present invention successfully constructs the M2 microglial cell membrane-fused MnO2 bionic nanoenzyme drug delivery system AAG / BM-Lip-apoE@M2CM, which has excellent brain-targeting properties, promotes microglia to transform into M2 phenotypes, inhibits the over-activation of the immune system in the AD brain, efficiently removes ROS and alleviates oxidative stress, and has the possibility of delaying the progression of AD in the early stages; AAG / BM-Lip-apoE@M2CM can directly remove abnormal pathological marker proteins at the downstream targets of AD, protect neuronal function, and restore memory and cognitive ability of AD patients. This multifunctional bionic nano drug delivery system designed for multiple causes of AD is expected to provide a new and efficient multi-target treatment strategy for the clinical prevention and treatment of AD.

[0108] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a bionic nanozyme for treating Alzheimer's disease, characterized in that: include, DSPE-TK-PEG 3400 -apoE synthesis: dissolve the active ester of hydroxy polyethylene glycol in a solvent, add propane-2, 2-diylbis(sulfonamide)diethylamine and triethylamine, react, concentrate, precipitate, and dry to obtain HO-PEG 3400 -TK-NH2, HO-PEG 3400 -TK-NH2 is dissolved in a solvent, and distearoylphosphatidylethanolamine modified active ester and triethylamine are added. After the reaction, the mixture is concentrated, precipitated, and dried to obtain NHS-PEG 3400 -TK-DSPE, weigh NHS-PEG 3400 -TK-DSPE was dissolved in buffer, apoE mimetic peptide was added, and after reaction, purification and drying, DSPE-TK-PEG was obtained. 3400 -apoE; the apoE mimetic peptide can carry Aβ 1-42 Amyloid transport to lysosomes for metabolic clearance and has brain targeting characteristics; Synthesis of manganese dioxide nanozyme: prepare potassium permanganate aqueous solution and bovine serum albumin aqueous solution, add the potassium permanganate aqueous solution dropwise into the bovine serum albumin aqueous solution, stir to react, dialyze to purify, and dry to obtain manganese dioxide nanozyme; Preparation of manganese dioxide nanozyme liposomes: Prepare the oil phase: weigh (2,3-dioleoyl-propyl)-trimethylamine, cholesterol, and 17-AAG, dissolve them in a solvent, and add ether; prepare the water phase: weigh manganese dioxide nanozyme and DSPE-TK-PEG 3400 -apoE is dissolved in a buffer solution; the aqueous phase is added dropwise to the oil phase, mixed in a water bath, distilled under reduced pressure, the organic solvent is removed, water is added to form an aqueous suspension, ultrasonically treated for 6 minutes, and filtered through a microporous filter membrane to obtain AAG / BM-Lip-apoE; Extraction of M2 microglial cell membrane: Induce microglia into M2 microglia, collect by centrifugation, and extract M2 microglial cell membrane by freeze-drying or sucrose gradient centrifugation; Preparation of cell membrane biomimetic nanocarriers: The M2 microglial cell membrane and AAG / BM-Lip-apoE were mixed in a mass ratio of 1:1, subjected to repeated freeze-thaw treatments at 4°C and 45°C, passed through a polycarbonate membrane using a liposome extruder, centrifuged, and purified to obtain AAG / BM-Lip-apoE@M2CM.

2. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 1, characterized in that: In the preparation of the cell membrane bionic nanocarrier, the liposome extruder is used to pass through a polycarbonate membrane, and the liposome extruder is used to pass through a 400 nm and a 200 nm polycarbonate membrane in sequence.

3. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 1 or 2, characterized in that: The DSPE-TK-PEG 3400 -apoE synthesis, the HO-PEG 3400 -TK-NH2 was prepared by weighing 1.5 g of hydroxy polyethylene glycol active ester and dissolving it in 10 mL of chloroform, adding propane-2, 2-diylbis(sulfonamide)diethylamine and triethylamine, reacting at room temperature for 24 h, concentrating under reduced pressure, pouring into ice ether for precipitation, filtering, and vacuum drying to obtain HO-PEG 3400 -TK-NH2.

4. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 3, characterized in that: The DSPE-TK-PEG 3400 -apoE synthesis, the NHS-PEG 3400 -TK-DSPE was prepared by weighing 1.0 g HO-PEG 3400 -TK-NH2 was dissolved in 5 mL of chloroform, and distearoylphosphatidylethanolamine modified active ester and triethylamine were added. After reacting at room temperature for 24 h, it was concentrated under reduced pressure and poured into ice ether for precipitation, filtered, and vacuum dried to obtain NHS-PEG 3400 -TK-DSPE.

5. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 3, characterized in that: The DSPE-TK-PEG 3400 -apoE synthesis, including weighing 0.1 g NHS-PEG 3400 -TK-DSPE was dissolved in 0.1 M pH 8.0 phosphate buffer, and apoE mimetic peptide was added. After stirring at room temperature for 24 h, it was dialyzed and purified in a 3500 Da dialysis bag for 24 h. The dialysate was freeze-dried to obtain the product DSPE-TK-PEG 3400 -apoE.

6. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 1 or 2, characterized in that: The synthesis of the manganese dioxide nanozyme is as follows: 31.6 mg of potassium permanganate is weighed and added to 3 mL of water to dissolve to obtain a potassium permanganate aqueous solution, 250 mg of bovine serum albumin is weighed and added to 7 mL of water to dissolve to obtain a bovine serum albumin aqueous solution, the potassium permanganate aqueous solution is added dropwise to the bovine serum albumin aqueous solution, the reaction is stirred at 37° C. for 1.5 h, the solution is dialyzed and purified in a 8000-14000 Da dialysis bag for 24 h, and the dialyzate is freeze-dried to obtain the manganese dioxide nanozyme.

7. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 1 or 2, characterized in that: The freeze-drying method is to add M2 microglia to phosphate buffer and mix them evenly. The concentration of the cell suspension is 1 × 10 7 / mL, then add an appropriate amount of protease inhibitors, squeeze back and forth through a liposome extruder, centrifuge at 3000g, 10 min, 4°C, collect the supernatant, further centrifuge at 10000g, 10 min, 4°C, and finally lyophilize the supernatant to obtain M2 microglial cell membrane.

8. The method for preparing the bionic nanozyme for treating Alzheimer's disease according to claim 1 or 2, characterized in that: The sucrose gradient centrifugation method is to resuspend M2 microglia in 4°C Tris-magnesium salt buffer, and the cell suspension concentration is 1 × 10 7 / mL, add an appropriate amount of protease inhibitors, squeeze back and forth through a liposome extruder, add 1 M sucrose solution and mix to make the final concentration of 0.25 M, 3000 g, 10 min, 4 ° C after centrifugation, collect the supernatant 10000 g, 10 min, 4 ° C further centrifugation, take the precipitate, then wash the precipitate with 0.25 M sucrose solution and centrifuge, discard the supernatant, take the precipitate to obtain M2 microglial cell membrane.

9. Use of the bionic nanozyme prepared by the preparation method of claim 1 in preparing a drug for treating Alzheimer's disease.

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