CN118340741A - Preparation method and application of bionic nano-enzyme for treating Alzheimer's disease - Google Patents
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Abstract
The invention discloses a preparation method and application of bionic nanoenzyme for treating Alzheimer's disease, comprising synthesis of DSPE-TK-PEG 3400 -apoE, synthesis of manganese dioxide nanoenzyme, preparation of manganese dioxide nanoenzyme liposome, extraction of M 2 type microglial cell membrane and preparation of a cell membrane bionic nanocarrier. The compound has excellent brain targeting property, promotes microglial cells to transform into M 2 phenotype to inhibit the excessive activation state of an immune system in AD brain, efficiently eliminates ROS, relieves oxidative stress, delays AD progression in an early stage, protects neuron functions, and restores memory and cognitive ability of AD patients. Is expected to provide a brand new and efficient multi-target treatment strategy for clinical prevention and treatment of AD.
Description
Technical Field
The invention belongs to the technical field of drug research and development, and particularly relates to a preparation method and application of bionic nanoenzyme for treating Alzheimer's disease.
Background
Alzheimer's disease (Alzheimer disease, AD) is a central neurodegenerative disease which is a common occurrence in the elderly and is characterized by cognitive decline and behavioral disorders, and places a heavy burden on the home and society. The prevalence of Alzheimer's disease is increasing with the increase in average human life.
Alzheimer's Disease (AD) is a central nervous system degenerative disease with hidden onset, and symptoms such as AD clinical cognitive dysfunction are closely related to the change of micro-environments in brain and amyloid deposition and neurofibrillary tangles caused by the change.
Therefore, the method is used for intervention by a plurality of therapeutic means aiming at multiple targets of AD disease progression, and has important significance for AD prevention or cure.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments.
As one of the aspects of the invention, the invention provides a preparation method of bionic nanoenzyme for treating Alzheimer's disease, which is characterized by comprising the following steps: comprising the steps of (a) a step of,
Synthesis of DSPE-TK-PEG 3400 -apoE: dissolving hydroxyl polyethylene glycol active ester in a solvent, adding propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, reacting, concentrating, precipitating, drying to obtain HO-PEG 3400-TK-NH2, dissolving HO-PEG 3400-TK-NH2 in the solvent, adding distearoyl phosphatidylethanolamine modified active ester and triethylamine, reacting, concentrating, precipitating, drying to obtain NHS-PEG 3400 -TK-DSPE, weighing NHS-PEG 3400 -TK-DSPE, dissolving in a buffer solution, adding apoE simulated peptide, reacting, purifying, and drying to obtain DSPE-TK-PEG 3400 -apoE;
Synthesis of manganese dioxide nano enzyme: preparing a potassium permanganate aqueous solution and a bovine serum albumin aqueous solution, dropwise adding the potassium permanganate aqueous solution into the bovine serum albumin aqueous solution, stirring for reaction, dialyzing for purification, and drying to obtain manganese dioxide nano-enzyme;
Preparing manganese dioxide nano enzyme liposome: preparing an oil phase: weighing (2, 3-dioleoyl-propyl) -trimethylamine, cholesterol and 17-acrylamide, dissolving in a solvent, and adding diethyl ether; preparing an aqueous phase: weighing manganese dioxide nano-enzyme and DSPE-TK-PEG 3400 -apoE and dissolving in a buffer solution; dropwise adding the water phase into the oil phase, mixing in a water bath, distilling under reduced pressure, removing the organic solvent, adding water to form aqueous suspension, performing ultrasonic treatment for 6min, and filtering by a microporous filter membrane to obtain AAG/BM-Lip-apoE;
extraction of M 2 type microglial cell membranes: inducing microglial cells into M 2 microglial cells, centrifugally collecting, and extracting M 2 microglial cell membranes by adopting a lyophilization method or a sucrose gradient centrifugation method;
Preparation of a cell membrane bionic nano-carrier: mixing the M 2 microglial cell membrane with AAG/BM-Lip-apoE according to the mass ratio of 1:4-4:1, repeatedly freezing and thawing, passing through a polycarbonate membrane by a liposome extruder, centrifuging, and purifying to obtain AAG/BM-Lip-apoE@M 2 CM.
As a preferable scheme of the preparation method of the bionic nano-enzyme for treating Alzheimer's disease, in the preparation of the cell membrane bionic nano-carrier, the mass ratio of the M 2 type microglial cell membrane to the AAG/BM-Lip-apoE is 1:1; the repeated freeze thawing treatment is carried out by sequentially carrying out repeated freeze thawing treatment at 4 ℃ and 45 ℃.
As a preferable scheme of the preparation method of the bionic nano-enzyme for treating Alzheimer's disease, in the preparation of the cell membrane bionic nano-carrier, a liposome extruder is used for passing through a polycarbonate membrane, and a liposome extruder is used for sequentially passing through a polycarbonate membrane of 400nm and a polycarbonate membrane of 200 nm.
As a preferred scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, in the synthesis of DSPE-TK-PEG 3400 -apoE, the preparation of HO-PEG 3400-TK-NH2 is to weigh 1.5g of active ester of hydroxyl polyethylene glycol and dissolve in 10mL of chloroform, add propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, react for 24 hours at room temperature, decompress and concentrate, then pour into glacial ethyl ether for precipitation, filter and dry in vacuum to obtain HO-PEG 3400-TK-NH2.
As a preferred scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, in the synthesis of DSPE-TK-PEG 3400 -apoE, the preparation of NHS-PEG 3400 -TK-DSPE is to weigh 1.0g of HO-PEG 3400-TK-NH2 and dissolve in 5mL of chloroform, add distearoyl phosphatidylethanolamine modified active ester and triethylamine, react for 24 hours at room temperature, decompress and concentrate, then pour into glacial ethyl ether for precipitation, filter and dry in vacuum to obtain NHS-PEG 3400 -TK-DSPE.
As a preferred scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the synthesis of DSPE-TK-PEG 3400 -apoE comprises the steps of weighing 0.1g of NHS-PEG 3400 -TK-DSPE, dissolving in 0.1M phosphate buffer solution with pH of 8.0, adding apoE mimic peptide, stirring at room temperature for reaction for 24 hours, dialyzing and purifying in a 3500Da dialysis bag for 24 hours, and freeze-drying the dialysate to obtain a product DSPE-TK-PEG 3400 -apoE.
As a preferred scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the synthesis of the manganese dioxide nanoenzyme is to weigh 31.6mg of potassium permanganate, add 3mL of water to dissolve to obtain potassium permanganate aqueous solution, weigh 250mg of bovine serum albumin, add 7mL of water to dissolve to obtain bovine serum albumin aqueous solution, add the potassium permanganate aqueous solution into the bovine serum albumin aqueous solution drop by drop, stir and react for 1.5h at 37 ℃, dialyse and purify for 24h in 8000-14000Da dialysis bags, and freeze-dry dialysate to obtain the manganese dioxide nanoenzyme.
As a preferred scheme of the preparation method of the bionic nano-enzyme for treating Alzheimer's disease, the freeze-drying method comprises the steps of adding M 2 microglial cells into phosphate buffer solution, uniformly mixing, adding a proper amount of protease inhibitor, extruding the mixture back and forth through a liposome extruder, centrifuging the mixture at 3000g,10min and 4 ℃, collecting 10000g of supernatant, centrifuging the supernatant at 10min and 4 ℃, and finally freeze-drying the supernatant to obtain M 2 microglial cell membranes.
As a preferred scheme of the preparation method of the bionic nano-enzyme for treating Alzheimer's disease, the sucrose gradient centrifugation method is to re-suspend M 2 microglial cells with Tris-magnesium salt buffer solution at 4 ℃, the concentration of cell suspension is 1 multiplied by 10 7/mL, add a proper amount of protease inhibitor, squeeze back and forth through a liposome extruder, add 1M sucrose solution to mix until the final concentration is 0.25M,3000g,10min, centrifuge at 4 ℃, collect the supernatant 10000g,10min, further centrifuge at 4 ℃, take the precipitate, wash the precipitate with 0.25M sucrose solution and centrifuge, discard the supernatant, and take the precipitate to obtain M 2 microglial cell membrane.
The invention has the beneficial effects that: the invention constructs a MnO 2 bionic nanoenzyme drug delivery system fused with M 2 microglial cell membranes (M 2 CM), and the MnO 2 bionic nanoenzyme drug delivery system and a designed and synthesized ROS responsive DSPE-TK-PEG 3400 -apoE fragment are jointly involved in assembly and are compositely loaded with a heat shock protein inhibitor 17-AAG (AAG/BM-Lip-apoE@M 2 CM). The interaction of key proteins on the M 2 type microglial cell membrane and brain microvascular endothelial cells is utilized to realize high-efficiency brain delivery, and the M 1 type microglial cells overactivated under neuroinflammation are promoted to be converted into anti-inflammatory M 2 type, so that the inflammatory environment in the brain of an AD patient is effectively improved; mnO 2 nano enzyme (BM) can remove a large amount of intracellular ROS, trigger DSPE-TK-PEG 3400 -apoE to break while effectively relieving oxidative stress state in brain, release apoE mimic peptide to realize transfer and removal of beta Amyloid-beta protein (Abeta) to microglial cells, ROS promote 17-AAG to be released rapidly at the same time, and realize effective removal of phosphorylated Tau protein (p-Tau) in neurons. The bionic nano-enzyme drug delivery system fused with the M 2 microglial cell membrane, which is expected to be designed, can effectively save the cognitive dysfunction of AD patients by tracking the whole disease course with a brand-new treatment thought, and provides a scheme for reference for the clinical treatment of the brain neurodegenerative diseases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, in which:
FIG. 1 is a schematic representation of the synthesis of DSPE-TK-PEG 3400 -apoE.
FIG. 2 is a Bio-MS diagram of the synthesized product.
FIG. 3 is a 1 H-NMR chart.
FIG. 4 is a comparison of ROS-responsive Bio-MS of DSPE-TK-PEG 3400 -apoE.
Fig. 5 is a TEM and XRD pattern of BM.
FIG. 6 shows the proportion of DSPE-TK-mPEG 3400 -apoE and the result of ultrasonic time screening.
FIG. 7 shows the determination of the protein content of the AAG/BM-Lip-apoE@M 2 CM surface film by the BCA method.
FIG. 8 shows the particle size and Zeta potential of AAG/BM-Lip-apoE and AAG/BM-Lip-apoE@M 2 CM.
Fig. 9 is a TEM image of different carriers.
FIG. 10 is a SDS-PAGE electrophoresis after membrane lipid fusion.
FIG. 11 is a WB pattern after membrane lipid fusion.
FIG. 12 is an in vitro ROS scavenging capacity.
Fig. 13 is an apparent permeability coefficient.
FIG. 14 is a photograph of ZO-1 immunofluorescent staining and ZO-1 fluorescent intensity values.
FIG. 15 shows the fluorescence intensity measurements for CD206 and CD86 from flow cytometric analysis.
FIG. 16 is a graph of the Transwell migration profile and relative cell mobility after each set of treatments.
FIG. 17 is a graph of cell scratch and cell mobility after each group treatment.
Fig. 18 is a laser confocal plot of BV2 cells phagocytosis of aβ 1-42 after each group treatment.
FIG. 19 is a WB plot and corresponding semi-quantitative analysis of p-Tau-associated proteins after treatment of each group.
FIG. 20 shows SH-SY5Y cell viability under Abeta 1-42 stimulation.
FIG. 21 is a photograph of a mouse living body at various time points.
Fig. 22 shows the distribution of groups in the brain and 4h in vivo tissue distribution at different time points.
FIG. 23 shows the results of the water maze test.
Figure 24 is a mouse nesting experiment.
FIG. 25 is an immunofluorescence experiment.
FIG. 26 is a diagram of the immunohistochemistry of the brain regions Aβ1-42 and p-Tau of mice after treatment.
FIG. 27 is a diagram of Nissl staining of brain regions of mice after treatment.
FIG. 28 is a SDS-PAGE electrophoresis of M 2 CM from different extraction methods.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
The invention relates to an abbreviation and a Chinese name thereof: AD Alzheimer's disease, abeta amyloid, APP amyloid precursor protein, BBB blood brain barrier, BCA biquinolinecarboxylic 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 neuronal fiber entanglement, OD optical density value, OAokadaic acid, PAGE polyacrylamide gel electrophoresis, PVDF polydifluoroethylene membrane, ROS active oxygen, [ Ru (dPp) 3]Cl2 tris (4, 7-diphenyl-1, 10-phenanthroline) dichlorophenanthropomorphic ruthenium, SDS sodium dodecyl sulfate, senile plaque, TEMED tetramethyl ethylenediamine, thT thio T, tris-trimethyl aminomethane, 3,4, 17-AAG-trimethoxy-17.
Example 1:
synthesis of DSPE-TK-PEG 3400 -apoE:
1.5g of NHS-PEG 3400 -OH (hydroxy polyethylene glycol active ester, purchased from Sigma company) is weighed and dissolved in 10mL of chloroform, 1.0g of NH 2-TK-NH2 (propane-2, 2-diylbis (sulfadiyl) diethylamine) and triethylamine are added for reaction at room temperature for 24 hours, and after decompression concentration, a large amount of diethyl ether is poured for precipitation, the product is collected by filtration, and vacuum drying is carried out, thus obtaining HO-PEG 3400-TK-NH2. 1.0g of HO-PEG 3400-TK-NH2 is weighed and dissolved in 5mL of chloroform, a proper amount of DSPE-NHS (distearoyl phosphatidyl ethanolamine modified active ester, purchased from Corden Pharma company) and triethylamine are added, after reaction for 24 hours at room temperature, the filtered product is collected according to the method, and the HO-PEG 3400 -TK-DSPE is obtained after vacuum drying. Weighing 0.5g of HO-PEG 3400 -TK-DSPE, dissolving in 5mL of acetonitrile, adding a proper amount of DSC and triethylamine, reacting overnight at room temperature, collecting a filtered product according to the method, and vacuum drying to obtain NHS-PEG 3400 -TK-DSPE. Weighing 0.1g of NHS-PEG 3400 -TK-DSPE, dissolving in 0.1M PBS solution with pH of 8.0, adding 0.25g of apoE mimetic peptide (sequence RLASHLRKLRKRLLREEQAQQIRLQAEAFQARLKSWFEPLVEDM) or apoE protein, stirring at room temperature for reaction for 24h, dialyzing in a dialysis bag (MW: 3500 Da) for purification for 24h, and freeze-drying the dialysate to obtain the product DSPE-TK-PEG 3400 -apoE. Product structure identification was performed using biological mass spectrometry (Bio-MS) and nuclear magnetic resonance (1 H-NMR).
ROS responsiveness of DSPE-TK-PEG 3400 -apoE: 5mg of DSPE-TK-PEG 3400 -apoE was weighed, added to 2mL of PBS buffer (10 mM, pH 7.4) to disperse uniformly, then added to 2mL of H 2O2 solution (200. Mu.M) to incubate at 37℃for 3H, and dialyzed in dialysis bags (MW: 3500 Da) for 24H. The dialyzed sample was freeze-dried and the products were identified using Bio-MS.
Synthesis and characterization of manganese dioxide nanoenzyme: 31.6mg of potassium permanganate was weighed out, and 3mL of distilled water was added for dissolution. Another 250mg of Bovine Serum Albumin (BSA) was weighed and dissolved in 7mL of deionized water. Dropwise adding the potassium permanganate solution into the BSA solution, stirring at 37 ℃ for reaction for 1.5h, dialyzing and purifying for 24h in a dialysis bag (MW: 8000-14000 Da), and freeze-drying the dialyzate to obtain manganese dioxide (BM) nano-enzyme, and preserving at 4 ℃ for later use. And respectively adopting a Transmission Electron Microscope (TEM), a UV-2600i ultraviolet visible spectrum (UV-vis), a Fourier transform infrared spectrum (FT-IR), an X-ray photoelectron spectroscopy (XPS) and an X-ray diffraction (XRD) to carry out structural identification on the BM nano enzyme.
Preparation and prescription process optimization of manganese dioxide nano enzyme liposome: manganese dioxide nano-enzyme liposome (AAG/BM-Lip-apoE) is prepared by adopting a reverse evaporation method. 15mg of DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine), 3.5mg of cholesterol and 20mg of 17-AAG are weighed, dissolved in a proper amount of chloroform, and then 3m L diethyl ether is added to be uniformly mixed as an oil phase. Another 60mg BM nanoenzyme and an appropriate amount of DSPE-TK-PEG 3400 -apoE were weighed and dissolved in 0.6mL PBS (pH 7.4) as an aqueous phase. Dropwise adding the water phase into the oil phase (the volume ratio of the oil phase to the water phase is 6:1), performing water bath ultrasonic treatment for 10min, and performing reduced pressure distillation on a rotary evaporator to remove the organic solvent. After the gel appeared, the depressurization was stopped and 5m L of deionized water was added to form an aqueous suspension. Transferring to an ultrasonic cell pulverizer for 6min at 100w, and sequentially filtering with 0.45 μm and 0.22 μm microporous filter membrane to obtain AAG/BM-Lip-apoE. The prescription and the preparation process of the AAG/BM-Lip-apoE can prepare BM-Lip without adding 17-AAG and DSPE-TK-PEG 3400 -apoE; the Lip-apoE can be prepared without adding BM and 17-AAG; the BM-Lip-apoE can be prepared without adding 17-AAG; 17-AAG, DSPE-TK-PEG 3400 -apoE and BM are not added, and the Lip is formed by DOTAP and cholesterol only. Fixing other prescriptions and processes respectively, sequentially examining the particle size or the potential of the AAG/BM-Lip-apoE prepared when the proportion of DSPE-TK-PEG 3400 -apoE functional fragments to DOTAP is 5% (0.75 mg), 8% (1.2 mg) and 10% (1.5 mg) respectively and the ultrasonic time is 3min,6min and 9min, and optimizing the optimal prescriptions and processes.
Extraction and characterization of microglial cell membranes of type M 2 (M 2 CM): mouse microglia (BV 2) were cultured and induced with IL-4 (20 ng/mL) for 24h to differentiate into M 2 -type microglia, 1000r/min, cells were collected by centrifugation for 3 min. The cell membrane is extracted by lyophilization and sucrose gradient centrifugation, respectively. The method (method 1) comprises the following specific steps: the collected cells are added into PBS and mixed uniformly, so that the concentration of the cell suspension is 1X 10 7/mL, and then a proper amount of protease inhibitor is added and mixed uniformly. Squeezing for about 20 times by liposome extruder, centrifuging (3000 g,10min, 4deg.C), collecting supernatant, centrifuging (10000 g,10min, 4deg.C), and lyophilizing to obtain M 2 -type microglial cell membrane (M 2 CM). The sucrose gradient centrifugation (method 2) comprises the following specific steps: the collected cells were resuspended in Tris-magnesium salt buffer at 4℃to give a cell suspension concentration of 1X 10 7 cells/mL, and then mixed with an appropriate amount of protease inhibitor. Squeezing for 20 times by liposome extruder, adding 1M sucrose solution, mixing to obtain final concentration of 0.25M, centrifuging (3000 g,10min, 4deg.C), collecting supernatant, centrifuging (10000 g,10min, 4deg.C), collecting cell precipitate, washing the obtained cell precipitate with 0.25M sucrose solution, centrifuging, and removing supernatant to obtain precipitate at bottom of tube which is M 2 CM. The cell membrane extracted by the above two methods is preserved at-20deg.C. As can be seen from SDS-PAGE electrophoresis (FIG. 28), the cell membrane proteins extracted by lyophilization are concentrated at about 25kDa molecular weight, and the whole protein information is less retained, while the cell membrane extracted by sucrose gradient centrifugation has protein expression at different molecular weight positions, and has abundant protein information. Therefore, sucrose gradient centrifugation was subsequently selected as the extraction method for the final cell membrane M 2 CM.
Preparation of a cell membrane bionic nano-carrier:
membrane lipid ratio screening: sequentially mixing the extracted M 2 CM and the prepared AAG/BM-Lip-apoE according to the mass ratio of 1:4-4:1, repeatedly freezing and thawing (4 ℃/45 ℃) and processing, sequentially passing through a polycarbonate film of 400nm and 200nm by a liposome extruder, further centrifuging for 10min by 10000g, and purifying to remove unsuccessfully loaded M 2 CM to obtain AAG/BM-Lip-apoE@M 2 CM. Co-extruding the Lip and M 2 CM by a liposome extruder to obtain lip@M 2 CM; and (3) co-extruding the BM-Lip-apoE and the M 2 CM by a liposome extruder to obtain the BM-Lip-apoE@M 2 CM. The BCA kit is used for measuring the content of AAG/BM-Lip-apoE@M 2 CM membrane protein, and determining the membrane lipid ratio with the highest membrane coating efficiency according to the correlation between the content of M 2 CM membrane protein and the membrane lipid mass ratio.
Investigation of ROS scavenging ability in vitro: clearance of H 2O2: 1,5, 10, 20, 50, 100. Mu.M H 2O2 standard solution was prepared, 500. Mu.L of each sample was taken and 50. Mu.M Amplex red and 0.1U/mL HRP were added, and after mixing, the mixture was reacted at 37℃for 10min. Absorbance at 570nm (OD) was measured using UV-vis and OD570 nm -concentration standard curve was plotted. 3mL of BM and AAG/BM-Lip-apoE@M 2 CM are respectively taken, 200 mu M H 2O2 equivalent solution is added, 3mL of PBS solution is added, H 2O2 equivalent solution is taken as a control group, the control group is placed in a constant temperature shaking box at 37 ℃, 500 mu L of reaction solution is respectively absorbed at 0min,10min,20min,30min,40min,50min and 60min, ampliex red and HRP are added, OD value at 570nm is measured after uniform mixing, residual H 2O2 content in the system is calculated, and degradation percentage-time curve is drawn.
Appropriate amounts of BM and AAG/BM-Lip-apoE@M 2 CM were mixed with 20mL of H 2O2 (100. Mu.M) solution, the pictures were taken with a digital camera to record O 2, the change in concentration of O 2 in the solution at different time points was measured with a portable oximeter, and a time-dependent plot of O 2 was generated.
Hydroxyl radical (·oh) and superoxide anion (O 2 ·-) scavenging experiments: the scavenging of superoxide radical anions (o 2 ·-) and hydroxyl radicals (. OH) was evaluated by Electron Spin Resonance (ESR) experiments. The O 2 ·- clean up experiment steps were as follows: a proper amount of 10mM xanthine solution and 1U/mL xanthine oxidase solution are taken and mixed evenly, 20 mu L of DMPO and 180 mu L of buffer solution are added, and the reaction is inoculated for 5min. BM and AAG/BM-Lip-apoE@M 2 CM were added to the solutions prepared as described above, respectively, and the untreated control group was sampled and tested. Interaction evaluation with aβ 1-42: fluorescence co-localization analysis: preparation of Dil-labeled AAG/BM-Lip-apoE@M 2CM(Dil-AAG/BM-Lip-apoE@M2 CM). mu.L of Dil-AAG/BM-Lip-apoE@M 2 CM was incubated with 100. Mu.L of FITC-Abetse:Sub>A 1-42 (1 mg/mL) at 37℃for 2h. Samples were added drop wise to slides and observed under an overhead fluorescence microscope after sealing.
Example 2:
Example 1 experimental results:
structural identification of DSPE-TK-PEG 3400 -apoE: FIG. 2 is a Bio-MS plot of the synthesized product, from which the m/z values can be deduced that the molecular weight of the product is about 9492, consistent with the theoretical molecular weight of DSPE-TK-PEG 3400 -apoE. FIG. 3A is a 1 H-NMR diagram of DSPE-TK-PEG 3400 -apoE, FIG. 3B is a 1 H-NMR diagram of apoE mimetic peptide, and FIG. 3C is a 1 H-NMR diagram of NH 2-TK-NH2. Wherein, the peak with chemical shift value of about 3.5ppm in FIG. 3A is the characteristic peak of PEG 3400, the peak with chemical shift value of about 4.2ppm is the characteristic peak of hydroxyl hydrogen in DSPE, the multiple peak with chemical shift value of about 7.2ppm in FIG. 3B is the characteristic peak of benzene ring in Phe in apoE simulated peptide, and the corresponding position in FIG. 3A also shows the characteristic peak of benzene ring. B, C, d and e in FIG. 3C are characteristic peaks of methylene, methyl and amino hydrogens, respectively, of TK, and the 1 H-NMR plot of the synthesized product DSPE-TK-PEG 3400 -apoE likewise shows peaks at the corresponding b, C and d positions (FIG. 3A). Both Bio-MS and 1 H-NMR results indicated successful synthesis of DSPE-TK-PEG 3400 -apoE.
ROS responsiveness of DSPE-TK-PEG 3400 -apoE: as seen in FIG. 4 for Bio-MS, DSPE-TK-PEG 3400 -apoE has a molecular weight of about 9492. The molecular weight after reaction with H 2O2 solution was reduced by about 8683, consistent with the molecular weight of the PEG 3400 -apoE fragment. It was demonstrated that the TK fragment in the DSPE-TK-PEG 3400 -apoE structure was cleaved after H 2O2 treatment, and the structure was broken down into a PEG 3400 -apoE fragment and a DSPE fragment. Thus, DSPE-TK-PEG 3400 -apoE is ROS responsive.
Characterization of manganese dioxide nanoenzyme: successful preparation of BM was identified using TEM, XPS and XRD analysis. When observed under TEM (FIG. 5), BM exhibits relatively uniform spherical particles, which are uniformly dispersed and have a particle size of about 10 nm. XPS analysis results show that BM has characteristic absorption at 641.8eV and 653.5eV, which respectively correspond to Mn (IV) 2p3/2 and Mn (IV) 2p1/2 in manganese dioxide, and show that the valence state of Mn element in BM is +4. In the XRD pattern, BM showed a characteristic diffraction absorption peak at 2θ=21.06°, consistent with the XRD standard pattern card contrast for MnO 2.
Screening a prescription process of the manganese dioxide nano enzyme liposome: the prescription and process screening results of AAG/BM-Lip-apoE are shown in FIG. 6. As can be seen from fig. 6, the proportion of DSPE-TK-mPEG 3400 -apoE has a significant effect on the Zeta potential, and as the proportion increases, the Zeta potential value decreases inversely, which indicates that the hydrophilic long chain in the DSPE-TK-mPEG 3400 -apoE fragment can 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. The addition ratio of 8% DSPE-TK-mPEG 3400 -apoE was chosen as the optimal prescription amount, considering the subsequent fusion effect with the cell membrane. As can be seen from fig. 6, the ultrasonic time has a certain effect on the particle size of the liposome, and helps the liposome to further increase the dispersion degree within a certain range, and the particle size tends to decrease, but the relatively long ultrasonic time increases the probability of collision between particles, so that the particle size of the liposome increases. Therefore, the ultrasonic time is preferably 6 minutes.
Membrane lipid ratio screening: the AAG/BM-Lip-apoE@M 2 CM membrane protein content was quantitatively determined by BCA kit to determine the optimal membrane lipid mass ratio. As shown in FIG. 7, as the membrane lipid ratio increases, the AAG/BM-Lip-apoE@M 2 CM membrane protein content increases, and when the membrane lipid ratio is greater than 1:1, the AAG/BM-Lip-apoE@M 2 CM membrane protein content gradually tends to saturate, and more cell membranes are continuously added on the basis, obvious cell membrane precipitation at the bottom of the tube can be found after centrifugation, and when the membrane lipid ratio is 1:1, the optimal fusion effect of the cell membranes can be realized, and accordingly, 1:1 is preferable as the optimal addition ratio of the cell membranes and the liposome.
Characterization of AAG/BM-Lip-apoE@M 2 CM: the average particle size and Zeta potential results are shown in FIG. 8. After fusion with cell membrane, the average particle size increased from 108.23 + -1.92 nm to 123.37 + -1.11 nm of AAG/BM-Lip-apoE@M 2 CM; and due to the neutralization effect of negative charges of the cell membrane, the Zeta potential is reduced from the original 33.79 +/-0.51 mV to 13.66+/-1.34 mV of AAG/BM-Lip-apoE@M 2 CM, so that the cell membrane and the liposome can be proved to be successfully fused, and under TEM observation (figure 9), the AAG/BM-Lip-apoE and the AAG/BM-Lip-apoE@M 2 CM both show vesicle-shaped structures, the particle sizes of the AAG/BM-Lip-apoE@M 2 CM are not greatly different, and the vesicle bilayer membrane structure of the AAG/BM-Lip-apoE@M 2 CM is more obvious after the AAG/BM-apoE@M 2 CM is fused with the cell membrane. In fig. 9, a: AAG/BM-Lip-apoE; b: AAG/BM-Lip-apoE@M 2 CM.
SDS-PAGE electrophoresis experiment: as can be seen from the SDS-PAGE electrophoresis of FIG. 10, the extracted M 2 CM was substantially identical to BV2 cells in protein expression at the same position, and the AAG/BM-Lip-apoE@M 2 CM also almost retained all the original protein information of BV2 cells after membrane fusion, while AAG/BM-Lip-apoE which did not interact with the cell membrane did not show any protein bands, thus proving successful modification of the carrier by the cell membrane. In fig. 10, a: BV2 cells; b: m 2CM;C：AAG/BM-Lip-apoE@M2 CM; d: AAG/BM-Lip-apoE.
Western Blotting experiment: from the WB results of fig. 11, it is seen that there are a large number of specific proteins Integrin α4 and Mac-1 enriched on the surface of M 2 CM and AAG/BM-Lip-apoe@m 2 CM, and that efficient transfer of membrane protein information has a positive effect on brain delivery of AAG/BM-Lip-apoe@m 2 CM, as well as active uptake of microglia and neurons. In fig. 11, a: BV2 cells; b: m 2CM;C：AAG/BM-Lip-apoE@M2 CM.
Investigation of ROS scavenging ability in vitro:
clearance of H 2O2: FIG. 12 is a graph showing the percent degradation versus time of H 2O2, showing that BM and AAG/BM-Lip-apoE@M 2 CM consume H 2O2 in the system faster according to the curve results, and the H 2O2 reduction rates in the system are 59.1%,60.6% and 69.9% respectively after 10, 20 and 50min of reaction, and the 1H has reached 71.9%; the degradation percentage of H 2O2 in the H 2O2 control group at each time point before and after incubation is stable, and the degradation percentage is only about 22.1% in 1H. From this, it is understood that both B M and AAG/BM-Lip-apoE@M 2 CM can effectively scavenge H 2O2 in the reaction system, presumably related to the CAT-like activity of BM in the AAG/BM-Lip-apoE@M 2 CM structure, as well as H 2O2 in the TK bond cleavage consumption system on the DSPE-TK-PEG 3400 -apoE fragment.
Cleaning of OH and O 2 ·-: ESR analysis means were used to evaluate the clearance of BM and AAG/BM-Lip-apoE@M 2 CM to OH and O 2 ·-, and the control group had a strong ESR signal (1:2:2:1), and after treatment with BM and AAG/BM-Lip-apoE@M 2 CM, the peak intensity was significantly reduced, indicating that BM and AAG/BM-Lip-apoE@M 2 CM could effectively consume OH. After BM and AAG/BM-Lip-apoE@M 2 CM treatment, 2 ·- had significantly reduced characteristic peaks at the same ESR signal (1:1:1) in the control group, indicating that BM and AAG/BM-Lip-apoE@M 2 CM also effectively cleared O 2 ·-. The experimental results show that AAG/BM-Lip-apoE@M 2 CM has sensitive ROS scavenging capability and can be effectively applied to relieving oxidative stress microenvironment in the brain of an AD patient.
In fig. 12, a: percent degradation of H 2O2 after treatment with different carriers. B: ESR profile of OH scavenging. C: ESR profile of O 2 ·- clearance.
Example 3:
in vitro evaluation of brain targeting:
mouse brain microvascular endothelial cells (bend.3) culture: cells were cultured in 1640 medium containing 10% fetal bovine serum, 1% diabody, at 37 ℃,5% co 2 in a cell incubator. When the number of cells in the bottle is about 80-90%, the medium in the bottle is discarded, and after 3 times of washing with PBS, 2mL of pancreatin is added for digestion. The digestion was then terminated by adding complete medium. Cells were collected by centrifugation at 1200r/min for 3 min. The cell sediment is added with a proper amount of culture medium to be blown uniformly and then transferred into a cell culture bottle for continuous culture.
Transmembrane transport studies: the bEnd.3 cells in the logarithmic growth phase were added to the Transwell plate upper chamber to a density of about 1X 10 5 cells/well. Changing liquid every other day, measuring the trans-endothelial cell resistance (TEER), and successfully establishing an in vitro BBB model when the TEER reaches 200 omega/cm 2. The upper chamber medium was aspirated, and C6-BM-Lip-apoE and C6-BM-Lip-apoE@M 2 CM (DOTAP concentration: 100. Mu.g/mL) diluted with 1640 medium were added, and 200. Mu.L of lower chamber liquid was aspirated at 5min,10min,15min,30min,60min, respectively, while an equal volume of blank 1640 medium was replenished. The fluorescence intensity at each time point C6 was measured, and a PBS solution of C6 was used as a control group. The apparent permeability coefficients (apparent permeability coefficient, papp) were calculated for each of the above measured C6 fluorescence intensities as follows to evaluate and compare the capacity and differences in transmembrane transport. TEER was assayed at various stages of the experiment (0 h, 0.5h and 24 h) to assess the integrity of the BBB.
Wherein,For permeability (nmol/s), C 0 (nmol/mL) is the initial concentration of C6 in the upper chamber, and A (cm 2) is the surface area of the Transwell membrane.
ZO-1 immunofluorescence experiment: bEnd.3 cells were inoculated into capped confocal dishes at a density of 1X10 4 cells/well, incubated for 24h, the original serum-containing medium was discarded, PBS solution, BM-Lip-apoE and BM-Lip-apoE@M 2 CM were added separately to incubate for 1h, the tissue fixative was fixed for 10min, and then rabbit anti-mouse ZO-1 diluent (1:200) was added and incubated overnight at 4 ℃. PBS was washed 3 times and then anti-Cy 3-labeled goat anti-rabbit IgG (1:300) was added and incubated for 50min at room temperature. After washing 3 times with PBS and staining nuclei with DAPI for 5min, they were observed under a laser confocal microscope.
Examination of microglial phagocytic Capacity: BV2 cells in logarithmic growth phase were plated in 24-well plates to give cell densities of about 1X 10 5 cells/well. After 24h of incubation, the medium containing serum was discarded and LPS (100 ng/mL) was added to induce M 1 -type microglia. PBS solution, lip-apoE, lip@M 2 CM, BM-Lip and BM-Lip-apoE@M 2 CM were added respectively, DOTAP concentrations were 120. Mu.g/mL and BM concentrations were 500. Mu.g/mL, and each group was incubated for 1h, treated with FITC-Abetse:Sub>A 1-42 for 15min, and washed 3 times with PBS. Lysosomes were stained with Lyso-Tracker probe for 30min and washed 3 times with PBS. After 10min fixation with tissue fixative, nuclei were stained with DAPI for 5min and observed under a laser confocal fluorescence microscope.
Calculation of apparent permeability coefficient: as shown in FIG. 13, the Papp value of the BM-Lip-apoE@M 2 CM group reached 2.6X10 9 CM/s in 60min, which was significantly higher than that of the BM-Lip-apoE and PBS control groups, and the BM-Lip-apoE had Papp value of 1.4X10 9 CM/s in the same time, the differences in vector structure indicated that the M 2 CM and DSPE-TK-PEG 3400 -apoE fragments could effectively assist in the distribution of BM-Lip-apoE@M 2 CM from the Transwell upper chamber across bEnd.3 monolayer cells to the lower chamber. This may be related to the ICAM-1/VCAM-1 binding of Intergin α4 and Mac-1 proteins expressed on M 2 CM and bEnd.3 cell surface reversibly opening the BBB tight junction, and the distribution of apoE mimetic peptides and the LRP1 receptor-mediated active transport pathways across the BBB into the brain. In fig. 13, a: PBS; b: BM-Lip-apoE; c: BM-Lip-apoE@M 2CM;n＝3,＊ represents that this group has a statistical difference from the PBS group, p < 0.05.
Investigation of ZO-1 immunofluorescence: ZO-1 belongs to one of the closely connected related proteins, and the expression of the ZO-1 is closely related to the integrity of the closely connected endothelial cells, so that the ZO-1 protein on the brain microvascular endothelial cells bEnd.3 can directly reflect the transmembrane transport mechanism of BM-Lip-apoE@M 2 CM through immunofluorescence. As shown in fig. 14, blue fluorescence was DPAI-labeled nuclei, and red fluorescence-labeled ZO-1 was encircled outside the contours of the cell membrane to express the integrity of the tight junctions. After the PBS control group and the BM-Lip-apoE experimental group are contacted with bEnd.3 cells, the cell edges all show complete red fluorescence profiles, and the fluorescence intensity is higher, which indicates that the integrity of the original tight connection of the cells is not changed after the BM-Lip-apoE is contacted with the cells, and is consistent with the PBS control group. The BM-Lip-apoE@M 2 CM experimental group obviously reduces the red fluorescence intensity of bEnd.3 cells, and a considerable part of red fluorescence is absent, which proves that the BM-Lip-apoE@M 2 CM can reduce the expression of a tight junction related protein ZO-1 and disturb the tight junction between endothelial cells. This is closely related to the presentation of Intergin a 4 and Mac-1 proteins on the BM-Lip-apoe@m 2 CM surface after M 2 CM coating, which can reversibly open the BBB tight junction in conjunction with ICAM-1/VCAM-1 binding to the surface of the bend.3 cells, increasing the permeability of the BBB, enabling efficient delivery to the brain.
Microglial transformation evaluation:
Immunofluorescence and flow result analysis: FIG. 15 shows the fluorescence intensity measurements of CD206 and CD86 from flow cytometric analysis, CD206 being a protein-specific marker on type M 2 microglia and CD86 being a protein-specific marker on type M 1 microglia. the increase in fluorescence intensity of the CD206 marker in the IL-4 induced microglia of the M 2 group and the decrease in fluorescence intensity of the CD86 marker of the M 2 group in fig. 15 compared to the M 0、M1 control group indicate that they have been successfully differentiated into M 2 type microglia. Further comparing and examining the transformation promotion trend of each experimental group on an M 1 type microglial cell model induced to differentiate by LPS, the result shows that the fluorescence intensity of CD206 is obviously enhanced compared with that of an M 0、M1 control group after Lip-apoE@M 2 CM is ingested by cells, The fluorescence intensity of Lip-apoE@M 2 CM is obviously reduced compared with that of the M 0、M1 control group, which shows that the M 2 CM coating can promote the conversion of M 1 microglia cells to M 2 microglia cells; After the BM-Lip-apoE and the M 1 microglial cells are incubated together, the fluorescence intensity of the CD206 mark is further enhanced compared with that of an M 2 control group and an Lip-apoE@M 2 CM experimental group, the fluorescence intensity of the CD86 mark is obviously reduced, The high-efficiency clearance of BM nanoenzyme to ROS can relieve the excessive activation state of microglial cells under M 1 phenotype, promote the conversion of M 1 microglial cells to M 2 phenotype, and improve the neuroinflammation caused by ROS as a pro-inflammatory factor. The fluorescence intensity of the CD206 is further enhanced after the M 1 type microglial cells are treated by BM-Lip-apoE@M 2 CM, which is obviously more obvious than that of Lip-apoE@M 2 CM and BM-Lip-apoE groups, And the fluorescence intensity of the CD86 marker is further reduced compared with the prior art, which indicates that more M 1 microglial cells are differentiated into M 2 phenotype, so that the proportion of anti-inflammatory M 2 microglial cells is higher, thereby effectively relieving the abnormal immune activation state of microglial cells and the AD neuroinflammatory environment caused by the abnormal immune activation state, This is closely related to the synergy of M 2 CM and BM.
Investigation of microglial migration behaviour: and (3) establishing a Transwell model of BV2 cells, and examining the influence of microglial cell transformation on migration behaviors. as can be seen from fig. 16, the migration ability of microglial cells differentiated into M 1 type after LPS induction was slightly enhanced, while that of microglial cells differentiated into M 2 type after IL-4 stimulation was significantly enhanced, compared with M 0 resting state. Compared with the control groups M 1 and M 2, the migration speed of microglial cells is obviously accelerated after each experimental group is treated respectively, and the migration capacity of BM-Lip-apoE@M 2 CM is most obvious after the treatment. As can be seen from the scratch test of FIG. 17, after 12h of cell growth, the M 0 control group still has a more obvious scratch area, the cell scratch area is reduced after M 1 and M 2 transformation is induced, and the cell gap of the M 2 phenotype group is smaller than that of the M 1 phenotype group, indicating that the migration and crawling speed of the cells is faster. After grouping treatment, compared with the M 1 phenotype group, the cell migration is obviously accelerated, wherein a large number of cells in the BM-Lip-apoE@M 2 CM group are converged towards the center of the scratch, the original scratch is not obvious, and the migration and growth behaviors after treatment of each experimental group are consistent with the Transwell experimental results. In combination with the results of microglial transformation, it is speculated that each formulation group promotes the differentiation of microglial cells into the M 2 phenotype, which enhances the migration capacity of microglial cells, allows the M 2 microglial cells to more rapidly deposit on pathological proteins and aggregate around neuronal debris, and is beneficial to exerting subsequent phagocytic clearance.
Investigation of microglial migration behaviour: and (3) establishing a Transwell model of BV2 cells, and examining the influence of microglial cell transformation on migration behaviors. as can be seen from fig. 16, the migration ability of microglial cells differentiated into M 1 type after LPS induction was slightly enhanced, while that of microglial cells differentiated into M 2 type after IL-4 stimulation was significantly enhanced, compared with M 0 resting state. Compared with the control groups M 1 and M 2, the migration speed of microglial cells is obviously accelerated after each experimental group is treated respectively, and the migration capacity of BM-Lip-apoE@M 2 CM is most obvious after the treatment. As can be seen from the scratch test of FIG. 17, after 12h of cell growth, the M 0 control group still has a more obvious scratch area, the cell scratch area is reduced after M 1 and M 2 transformation is induced, and the cell gap of the M 2 phenotype group is smaller than that of the M 1 phenotype group, indicating that the migration and crawling speed of the cells is faster. After grouping treatment, compared with the M 1 phenotype group, the cell migration is obviously accelerated, wherein a large number of cells in the BM-Lip-apoE@M 2 CM group are converged towards the center of the scratch, the original scratch is not obvious, and the migration and growth behaviors after treatment of each experimental group are consistent with the Transwell experimental results. In combination with the results of microglial transformation, it is speculated that each formulation group promotes the differentiation of microglial cells into the M 2 phenotype, which enhances the migration capacity of microglial cells, allows the M 2 microglial cells to more rapidly deposit on pathological proteins and aggregate around neuronal debris, and is beneficial to exerting subsequent phagocytic clearance.
Examination of microglial phagocytic Capacity: and (3) establishing an M 1 microglial cell model, and comparing and observing the phagocytic clearance capacity of the microglial cells to Abeta by a laser confocal microscope. In FIG. 18, the green fluorescence is FITC-labeled Abeta 1-42, the red fluorescence is Lyso-TRACKERRED probe-labeled lysosomes, and the blue fluorescence is DAPI-labeled nuclei. As shown in fig. 18, after incubation of BV2 cells with FITC-aβ 1-42, green fluorescence of aβ 1-42 and red fluorescence of lysosomes remained largely scattered in the cytoplasm, and only se:Sub>A small amount of coincidence was seen to exhibit weak orange fluorescence, indicating that the amount of aβ 1-42 that could be cleared by lysosome metabolism without effective intervention was limited. After BV2 cells ingest Lip-apoE, the green fluorescence of Abeta 1-42 and the red fluorescence of lysosomes start to overlap, and the orange fluorescence after red-green superposition is presented, which indicates that apoE mimic peptide can carry Abeta 1-42 to transport to lysosome for metabolic clearance. After the Lip@M 2 CM, the BM-Lip and the BM-Lip-apoE@M 2 CM are incubated with BV2 cells, green fluorescence and red fluorescence are overlapped in a large amount, orange fluorescence is more obvious, and all three preparation groups can promote the transformation and differentiation of M 1 microglial cells, The M 2 type microglial cell migration capacity after induced transformation is enhanced, and the deposition of the phagocytic Abeta 1-42 can be more actively identified, the capacity of Abeta 1-42 in transferring to lysosomes is enhanced, and on the basis, more Abeta 1-42 is promoted to be metabolically cleared by the lysosomes. And BM-Lip-apoE@M 2 CM with the functions of cell transformation promotion and apoE mimic peptide is recorded by a laser confocal microscope, so that microglial cells show stronger phagocytic capacity and an effect of eliminating Abeta 1-42 abnormal protein.
SH-SY5Y intracellular p-Tau clearance under OA induction: western blotting experiment: a p-Tau cell model is established by using OA induced SH-SY5Y cells, and the expression of Hsp40, hsp70 and p-Tau after each group of treatment of the p-Tau cell model is analyzed by adopting a WB method. FIG. 19 shows that the expression of Hsp40, hsp70 and p-Tau was significantly reduced in the AAG/BM-Lip-apoE@M 2 CM group compared to the OA control group and the 17-AAG experimental group.
Neuroprotection: stimulation of SH-SY5Y cells with Abeta 1-42 establishes a model of neurocytotoxicity. As shown in the CCK-8 experimental results of FIG. 20, the survival rate of cells in the Abeta 1-42 control group is obviously reduced, and the survival rate of cells after AAG/BM-Lip-apoE@M 2 CM treatment is far higher than that of the control group and other experimental groups. In fig. 20, a: a control group; b: aβ 1-42;C：AAG-Lip-apoE@M2 CM; d: AAG/BM-Lip-apoE; e: AAG/BM-Lip-apoE@M2CM; n=3, p < 0.01 represents a statistical difference between this group and the aβ1-42 group.
Evaluation of the synergistic effect of neuroprotection: stimulation of SH-SY5Y cells with Abeta 1-42 establishes a model of neurocytotoxicity. In order to evaluate the combined action effect of BM-Lip-apoE and lip@M 2 CM, according to the Chuu-Talalay combined drug index method and CompuSy software calculation, the combined action index Cl values (Table 1) of BM-Lip-apoE and lip@M 2 CM (the mass ratio of the two is 1:1) at 0.125IC50, 0.25IC50, 0.5IC50 and 1.0IC50 are obtained, the obtained Cl values are all smaller than 1, the combined action of BM-Lip-apoE and lip@M 2 CM (the mass ratio of the two is 1:1) has a synergistic action, the Cl value of the action concentration is minimum at the respective IC50, and the synergistic neuroprotection effect is optimal.
Table 1 evaluation of neuroprotective effect of AD mice on synergistic therapeutic effect (n=3)
| Concentration of action | Cell viability (%) | Cl value | Interaction determination |
| 0.125IC50 | 54.6±2.2 | 0.884±0.100 | ++ |
| 0.25IC50 | 68.9±1.4 | 0.617±0.050 | ++ |
| 0.5IC50 | 78.4±0.5 | 0.514±0.040 | ++ |
| 1.0IC50 | 86.3±0.4 | 0.379±0.050 | +++ |
Note that: ++ + synergistic effect the effect is stronger; ++ moderate synergy
Example 4:
evaluation of brain targeting: as shown in fig. 21, at each observation time point, the brains of the DiR-BM-Lip group mice always have no obvious fluorescence distribution, and weak fluorescence appears only at 4h and 8h, which indicates that the speed and the degree of the DiR-BM-Lip delivery to the brains are extremely limited and the brain targeting property is poor. In contrast, diR-BM-lip@M 2 CM and DiR-BM-Lip-apoE@M 2 CM groups exhibit good brain targeting, high-intensity fluorescence distribution is exhibited in the brains of mice from 2h to 8h, and the fluorescence of the brains gradually increases with time, and strong fluorescence accumulation is still exhibited in the brains from 12h to 24h, indicating that DiR-BM-lip@M 2 CM and DiR-BM-Lip-apoE@M 2 CM can be distributed into the brains more rapidly, and can reside in the brain in a gradual and steady manner over a relatively long period of time. And at each observation time point, the brain fluorescence intensity of the mice in the DiR-BM-Lip-apoE@M 2 CM group is higher than that of the mice in the DiR-BM-lip@M 2 CM group. The difference of the brain fluorescence distribution of DiR-BM-Lip and DiR-BM-lip@M 2 CM is compared, which shows that the coating of M 2 CM has a critical effect on enhancing the brain targeting delivery characteristic, can remarkably improve the brain entering efficiency and prolong the residence time in the brain, and realizes the effective delivery of AD therapeutic drugs. the difference of DiR-BM-lip@M 2 CM and DiR-BM-Lip-apoE@M 2 CM structure is compared, and the apoE mimic peptide is further modified, so that more obvious brain targeting characteristics are presented. In FIG. 21, diR-L/B is DiR-BM-Lip; diR-L/B@CM is DiR-BM-lip@M 2 CM; DiR-P/B@CM is DiR-BM-Lip-apoE@M 2 CM.
FIG. 22A shows the distribution of DiR-BM-Lip, diR-BM-lip@M 2 CM and DiR-AAG/BM-Lip-apoE@M 2 CM in the brain after intravenous injection of the mice, at 2h,4h,8h,12h and 24h, the accumulation of DiR-BM-Lip-apoE@M 2 CM in the brain was higher than that of DiR-BM-Lip and DiR-BM-lip@M 2 CM, at 4h, at 109.5ng/mg, significantly higher than that of DiR-BM-Lip and DiR-BM-lip@M 2 CM at 30.68ng/mg and 87.4ng/mg. The distribution of the three preparation groups in the heart, liver, spleen, lung, kidney and brain is plotted in the figure 22B, and the distribution of DiR-BM-Lip-apoE@M 2 CM in the brain is quite considerable, which is consistent with the results of in vivo imaging of small animals. In FIG. 22, diR-L/B is DiR-BM-Lip; diR-L/B@CM is DiR-BM-lip@M 2 CM; diR-P/B@CM is DiR-BM-Lip-apoE@M 2CM;n＝3,＊＊＊ representing a statistical difference, P < 0.001.
Water maze experiment: fig. 23 is a map of the navigation route (a) of the positioning of the mice in the water maze circular table of each group, the escape latency (B) of each group, the residence time (C) of each group in the platform quadrant, and the number of times each group passed the platform (D). The sham routes are substantially clustered near the platform and pass through the platform at multiple locations; the AD model group route is disordered, the AD model group walks around the pool, and the position of the platform can not be found basically in the test period; after each formulation group administration treatment, the track of the route of the mice was significantly increased in the platform quadrant, with the route of the AAG/BM-Lip-apoE@M 2 CM group mice approaching the sham group, essentially near the platform. as can be seen from the figure, the escape latency of the sham operation group was 7.26.+ -. 4.88s, the AD model group was 35.58.+ -. 2.55s, and the escape latency was reduced to 23.96.+ -. 4.89s after two weeks of intravenous injection through AAG/BM-Lip-apoE, AAG-Lip-apoE@M 2CM,BM-Lip-apoE@M2CM,AAG/BM-Lip@M2 CM and AAG/BM-Lip-apoE@M 2 CM tail vein, 20.32.+ -. 4.32s, 17.74.+ -. 4.21s, 17.85.+ -. 6.58s and 11.17.+ -. 6.37s. The residence time of each group of mice in the platform quadrant is 37.03+/-1.45%, the sham operation group is 18.80+/-5.24%, the AD model group is respectively increased to 19.97+/-6.52% after being treated by AAG/BM-Lip-apoE, AAG-Lip-apoE@M 2CM,BM-Lip-apoE@M2CM,AAG/BM-Lip@M2 CM and AAG/BM-Lip-apoE@M 2 CM, 24.87.+ -. 3.68%, 24.87.+ -. 5.40%, 26.33.+ -. 6.52% and 34.17.+ -. 2.95%. The number of times that each group of mice passed through the platform was 4.8.+ -. 0.84 in the sham surgery group and 1.6.+ -. 0.55 in the AD model group, and the mice were increased to 2.60.+ -. 1.14,3.00.+ -. 1.00 after treatment with AAG/BM-Lip-apoE, AAG-Lip-apoE@M 2CM,BM-Lip-apoE@M2CM,AAG/BM-Lip@M2 CM and AAG/BM-Lip-apoE@M 2 CM, respectively, 3.25.+ -. 1.26,3.00.+ -. 1.00 and 4.4.+ -. 1.14. From the above results, it is clear that AAG/BM-Lip-apoE@M 2 CM treatment is most prominent and can improve learning and memory ability of mice. The A/P/B is AAG/BM-Lip-apoE; the A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; The A/L/B@CM is AAG/BM-lip@M 2 CM; A/P/B@CM is AAG/BM-Lip-apoE@M 2CM;n＝12,＊＊, which represents that the group has a statistical difference from the AD model group, P < 0.01.
Nesting experiment: as can be seen from the nesting situation of the mice and the corresponding data analysis results in FIG. 24, after two weeks of grouping treatment, the nesting behaviors of the mice in the AAG/BM-Lip-apoE@M 2 CM preparation group are obviously and remarkably different (p < 0.01) compared with those of the mice in the AD model group, and are basically similar to those of the normal mice in the sham operation group, so that the cognitive behaviours of the AD mice treated by the AAG/BM-Lip-apoE@M 2 CM preparation group are improved. The A/P/B is AAG/BM-Lip-apoE; the A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; the A/L/B@CM is AAG/BM-lip@M 2 CM; A/P/B@CM is AAG/BM-Lip-apoE@M 2CM;n＝12,＊＊, which represents a statistical difference, P < 0.01, compared to the AD model group.
Immunofluorescence: FIG. 25A is a graph showing the results of 8-OHG immunofluorescence analysis of hippocampal neurons from each group of mice, wherein DAPI blue fluorescence outlines the basic outline of hippocampal regions of mice, and 8-OHG red fluorescence labels active oxygen molecules. No red fluorescence is observed in the normal mice, stronger red fluorescence spots appear in the hippocampus of the AD model group, the red fluorescence spots are reduced to different degrees after two weeks of tail vein injection of each preparation group, wherein the fluorescence spots of the AAG/BM-Lip-apoE@M 2 CM group are basically disappeared, which indicates that the level of the hippocampus 8-OHG is greatly reduced after treatment, and the aggregation state of active oxygen in the brain is effectively improved. FIG. 25B shows the results of immunofluorescence analysis of CD206 and CD86 in brain sections of mice in each group, DAPI blue fluorescence marks nuclei, green fluorescence and red fluorescence marks CD206 and CD86, respectively, wherein the red fluorescence of CD86 in brain sections of mice in the AD model group is strong, the green fluorescence of CD206 is weak, microglial cells exhibit an excessively activated M 1 phenotype, the green fluorescence of CD206 in the brain is enhanced to different extents after treatment with each preparation group, the red fluorescence of CD86 shows a different extent of attenuation, the red fluorescence of AAG/BM-Lip-apoE@M 2 CM group is substantially lost, the green fluorescence intensity is most remarkable, and the results show that microglial cells in brains of mice in each preparation group are the anti-inflammatory M 2 phenotype, which is beneficial for improving the inflammatory environment in brains of AD patients. In the figure, A/P/B is AAG/BM-Lip-apoE; the A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; the A/L/B@CM is AAG/BM-lip@M 2 CM; A/P/B@CM is AAG/BM-Lip-apoE@M 2 CM.
Immunohistochemistry: FIG. 26 shows the results of immunohistochemical analysis of Abeta 1-42 and p-Tau protein in brain sections of mice from each group, the apparent orange Abeta amyloid deposition and p-Tau protein aggregation were seen in the AD model group under the microscope, consistent with the typical pathological characteristics of AD mice, while almost no abnormal protein deposition was seen in normal mice. After two weeks of administration treatment, the expression of Abeta 1-42 and phospho-Tau (Ser 396) in the brain of the AD mice is obviously reduced, and the deposition or aggregation of abnormal proteins under a microscope is basically disappeared, wherein the effect is most obvious after the AAG/BM-Lip-apoE@M 2 CM is administered, which shows that the effect is most obvious, can effectively improve the deposition state of Abeta 1-42 in the brain, correct the abnormal entanglement of p-Tau proteins, reduce the damage of the Abeta 1-42 and the phospho-Tau protein to neurons and restore the physiological functions of the neurons. The A/P/B is AAG/BM-Lip-apoE; the A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; the A/L/B@CM is AAG/BM-lip@M 2 CM; A/P/B@CM is AAG/BM-Lip-apoE@M 2 CM.
Nib staining: nissl staining can indirectly reflect changes in neuronal morphology and survival. According to the results shown in fig. 27, the number of the surviving neurons in the hippocampus of the mice in the sham operation group is more and the neurons are arranged orderly, while the number of the surviving neurons in the hippocampus of the mice in the AD model group is obviously reduced and different degrees of shrinkage deep staining occur, the pathological changes of the cells in the hippocampus of the mice are reduced after the mice are treated by each group of administration, the number of the surviving neurons is increased and the neurons are arranged orderly, and the AAG/BM-Lip-apoE@M 2 CM group is similar to the sham operation group and is recovered to the normal state of the neurons.
In conclusion, the MnO 2 bionic nanoenzyme drug delivery system AAG/BM-Lip-apoE@M 2 CM with M 2 type microglial cell membrane fusion is successfully constructed, has excellent brain targeting characteristics, promotes microglial cells to transform to M 2 phenotype to inhibit the state of excessive activation of an AD brain immune system, effectively eliminates ROS to relieve oxidative stress, and delays the possibility of AD progress in an early stage; AAG/BM-Lip-apoE@M 2 CM can also directly clear abnormal pathological marker proteins at an AD downstream target, protect neuronal functions and restore memory and cognitive abilities of AD patients. The multifunctional bionic nano drug delivery system designed for the multiple causes of the AD is expected to provide a brand new and efficient multi-target treatment strategy for clinical prevention and treatment of the AD.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. A preparation method of bionic nano-enzyme for treating Alzheimer's disease, which is characterized by comprising the following steps: comprising the steps of (a) a step of,
Synthesis of DSPE-TK-PEG 3400 -apoE: dissolving hydroxyl polyethylene glycol active ester in a solvent, adding propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, reacting, concentrating, precipitating, drying to obtain HO-PEG 3400-TK-NH2, dissolving HO-PEG 3400-TK-NH2 in the solvent, adding distearoyl phosphatidylethanolamine modified active ester and triethylamine, reacting, concentrating, precipitating, drying to obtain NHS-PEG 3400 -TK-DSPE, weighing NHS-PEG 3400 -TK-DSPE, dissolving in a buffer solution, adding apoE simulated peptide, reacting, purifying, and drying to obtain DSPE-TK-PEG 3400 -apoE; the apoE mimetic peptide is capable of carrying beta amyloid for transport to lysosome for metabolic clearance and has brain targeting characteristics;
Synthesis of manganese dioxide nano enzyme: preparing a potassium permanganate aqueous solution and a bovine serum albumin aqueous solution, dropwise adding the potassium permanganate aqueous solution into the bovine serum albumin aqueous solution, stirring for reaction, dialyzing for purification, and drying to obtain manganese dioxide nano-enzyme;
Preparing manganese dioxide nano enzyme liposome: preparing an oil phase: weighing (2, 3-dioleoyl-propyl) -trimethylamine, cholesterol and 17-acrylamide, dissolving in a solvent, and adding diethyl ether; preparing an aqueous phase: weighing manganese dioxide nano-enzyme and DSPE-TK-PEG 3400 -apoE and dissolving in a buffer solution; dropwise adding the water phase into the oil phase, mixing in a water bath, distilling under reduced pressure, removing the organic solvent, adding water to form aqueous suspension, performing ultrasonic treatment for 6min, and filtering by a microporous filter membrane to obtain AAG/BM-Lip-apoE;
extraction of M 2 type microglial cell membranes: inducing microglial cells into M 2 microglial cells, centrifugally collecting, and extracting M 2 microglial cell membranes by adopting a lyophilization method or a sucrose gradient centrifugation method;
Preparation of a cell membrane bionic nano-carrier: mixing the M 2 microglial cell membrane with AAG/BM-Lip-apoE according to the mass ratio of 1:4-4:1, repeatedly freezing and thawing, passing through a polycarbonate membrane by a liposome extruder, centrifuging, and purifying to obtain AAG/BM-Lip-apoE@M 2 CM.
2. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1, wherein the method comprises the following steps: in the preparation of the cell membrane bionic nano-carrier, the mass ratio of the M 2 type microglial cell membrane to the AAG/BM-Lip-apoE is 1:1; the repeated freeze thawing treatment is carried out by sequentially carrying out repeated freeze thawing treatment at 4 ℃ and 45 ℃.
3. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1 or 2, characterized in that: in the preparation of the cell membrane bionic nano-carrier, a liposome extruder is used for passing through a polycarbonate membrane, and the liposome extruder is used for sequentially passing through a polycarbonate membrane of 400nm and a polycarbonate membrane of 200 nm.
4. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1 or 2, characterized in that: in the synthesis of DSPE-TK-PEG 3400 -apoE, the preparation of HO-PEG 3400-TK-NH2 is to weigh 1.5g of active ester of hydroxyl polyethylene glycol and dissolve in 10mL of chloroform, add propane-2, 2-diylbis (sulfadiyl) diethylamine and triethylamine, react for 24 hours at room temperature, decompress and concentrate, then pour into glacial ethyl ether for precipitation, filter and dry in vacuum to obtain HO-PEG 3400-TK-NH2.
5. The method for preparing the bionic nanoenzyme for treating Alzheimer's disease according to claim 4, which is characterized in that: in the synthesis of DSPE-TK-PEG 3400 -apoE, the preparation of NHS-PEG 3400 -TK-DSPE is to weigh 1.0gHO-PEG 3400-TK-NH2, dissolve in 5mL chloroform, add distearoyl phosphatidyl ethanolamine modified active ester and triethylamine, react for 24 hours at room temperature, decompress and concentrate, then pour into glacial ethyl ether for precipitation, filter, and dry in vacuum to obtain NHS-PEG 3400 -TK-DSPE.
6. The method for preparing the bionic nanoenzyme for treating Alzheimer's disease according to claim 4, which is characterized in that: the synthesis of DSPE-TK-PEG 3400 -apoE comprises weighing 0.1g of NHS-PEG 3400 -TK-DSPE, dissolving in 0.1M phosphate buffer solution with pH of 8.0, adding apoE mimic peptide, stirring at room temperature for reaction for 24h, dialyzing in 3500Da dialysis bag for purification for 24h, and freeze-drying the dialysate to obtain the product DSPE-TK-PEG 3400 -apoE.
7. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1 or 2, characterized in that: the synthesis of the manganese dioxide nano enzyme comprises the steps of weighing 31.6mg of potassium permanganate, adding 3mL of water for dissolution to obtain a potassium permanganate aqueous solution, weighing 250mg of bovine serum albumin, adding 7mL of water for dissolution to obtain a bovine serum albumin aqueous solution, dropwise adding the potassium permanganate aqueous solution into the bovine serum albumin aqueous solution, stirring at 37 ℃ for reaction for 1.5h, dialyzing and purifying for 24h in a 8000-14000Da dialysis bag, and freeze-drying the dialyzate to obtain the manganese dioxide nano enzyme.
8. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1 or 2, characterized in that: the freeze-drying method is that M 2 microglial cells are added into phosphate buffer solution and mixed evenly, the concentration of cell suspension is 1 multiplied by 10 7/mL, then a proper amount of protease inhibitor is added, the mixture is extruded back and forth through a liposome extruder, 3000g is centrifuged at 10min and 4 ℃, 10000g of supernatant is collected, 10min is further centrifuged at 4 ℃, and finally the supernatant is taken and freeze-dried to obtain M 2 microglial cell membranes.
9. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1 or 2, characterized in that: the sucrose gradient centrifugation method is characterized in that M 2 microglial cells are resuspended by using a Tris-magnesium salt buffer solution with the temperature of 4 ℃, the concentration of cell suspension is 1 multiplied by 10 7/mL, a proper amount of protease inhibitor is added, the mixture is squeezed back and forth by a liposome extruder, then 1M sucrose solution is added for mixing to obtain the final concentration of 0.25M,3000g,10min and 4 ℃ for centrifugation, then 10000g of supernatant fluid is collected, 10min and 4 ℃ for further centrifugation, sediment is taken, then the sediment is washed by using 0.25M sucrose solution for centrifugation, and the supernatant fluid is discarded, so that the M 2 microglial cell membrane is obtained.
10. The use of the bionic nanoenzyme prepared by the preparation method of claim 1 in preparing a medicament for treating Alzheimer's disease.
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