CN116869962A - 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 DSPE-TK-PEG 3400 Synthesis of apoE, synthesis of manganese dioxide nanoenzyme, preparation of manganese dioxide nanoenzyme liposome, M 2 Extracting microglial cell membrane and preparing bionic cell membrane nano carrier. It has excellent brain targeting properties, and promotes microglial cell targeting to M 2 Phenotypic conversion inhibition in AD brainThe immune system is in an overactive state, so that the ROS are efficiently cleared, the oxidative stress is relieved, the AD progress is delayed in an early stage, the neuron functions are protected, and the memory and cognitive ability of AD patients are recovered. 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 (AIzheimer 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. With the increase in average human life, the prevalence of Alzheimer's disease is increasing, and about 5000 tens of thousands of people are worldwide demented in 2022, with an estimated increase in 2050 to 1.52 million. Recently, data published by the China's aging Association indicates that about 1507 tens of thousands of Alzheimer's disease patients exist in the elderly 60 years old and older in China, and the number is the first in the world.
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,
DSPE-TK-PEG 3400 -synthesis of apoE: dissolving active ester of hydroxy polyethylene glycol in solvent, adding propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, reacting, concentrating, precipitating, and drying to obtain HO-PEG 3400 -TK-NH 2 HO-PEG 3400 -TK-NH 2 Dissolving in solvent, adding distearoyl phosphatidylEthanol amine modified active ester and triethylamine, concentrating, precipitating and drying to obtain NHS-PEG 3400 -TK-DSPE, weighing NHS-PEG 3400 Dissolving TK-DSPE in buffer solution, adding apoE mimic 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 in buffer; 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;
M 2 extraction of microglial cell membranes: induction of microglial cells as M 2 Centrifuging and collecting microglial cells, and extracting M by lyophilization or sucrose gradient centrifugation 2 Microglial cell membranes;
preparation of a cell membrane bionic nano-carrier: subjecting said M 2 Mixing microglial cell membrane with AAG/BM-Lip-apoE according to the mass ratio of 1:4-4:1, repeatedly freezing and thawing, passing through polycarbonate membrane by using 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 M 2 The mass ratio of microglial cell membrane to 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 preferable scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the DSPE-TK-PEG 3400 -synthesis of apoE, said HO-PEG 3400 -TK-NH 2 Is prepared by dissolving 1.5g of active ester of hydroxy polyethylene glycol in 10mL of chloroform, adding propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, reacting at room temperature for 24h, concentrating under reduced pressure, precipitating with glacial ethyl ether, filtering, and vacuum drying to obtain HO-PEG 3400 -TK-NH 2 。
As a preferable scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the DSPE-TK-PEG 3400 -in the synthesis of apoE, the NHS-PEG 3400 Preparation of TK-DSPE 1.0gHO-PEG was weighed 3400 -TK-NH 2 Dissolving in 5mL chloroform, adding distearoyl phosphatidyl ethanolamine modified active ester and triethylamine, reacting at room temperature for 24h, concentrating under reduced pressure, pouring into glacial ethyl ether for precipitation, filtering, and vacuum drying to obtain NHS-PEG 3400 -TK-DSPE。
As a preferable scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the DSPE-TK-PEG 3400 Synthesis of apoE comprising weighing 0.1g NHS-PEG 3400 Dissolving TK-DSPE 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 lyophilizing the dialysate to obtain 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 preferable scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the freeze-drying method is to make M 2 Adding phosphate buffer solution into microglial cells, mixing, and concentrating cell suspension at a concentration of 1×10 7 Adding proper amount of protease inhibitor per mL, squeezing via liposome extruder, centrifuging at 4deg.C for 3000 g/10 min, collecting supernatant 10000 g/10 min, centrifuging at 4deg.C, and lyophilizing to obtain M 2 Microglial cell membranes.
As a preferable scheme of the preparation method of the bionic nanoenzyme for treating Alzheimer's disease, the sucrose gradient centrifugation method is to carry out M 2 Microglial cells were resuspended in Tris-magnesium salt buffer at 4℃at a cell suspension concentration of 1X 10 7 Adding protease inhibitor at a ratio of 0.25M/mL, squeezing by liposome extruder, adding 1M sucrose solution, mixing to obtain final concentration of 0.25M,3000g,10min, centrifuging at 4deg.C, collecting supernatant 10000g,10min, centrifuging at 4deg.C, collecting precipitate, washing with 0.25M sucrose solution, centrifuging, removing supernatant, collecting precipitate to obtain M 2 Microglial cell membranes.
The invention has the beneficial effects that: construction of M according to the invention 2 Microglial cell membrane (M) 2 CM) fused MnO 2 Bionic nano-enzyme drug delivery system and ROS (reactive oxygen species) -responsive DSPE-TK-PEG (direct-acting-TK-polyethylene glycol) designed and synthesized 3400 The apoE fragment is jointly involved in assembly and is compositely loaded with heat shock protein inhibitor 17-AAG (AAG/BM-Lip-apoE@M) 2 CM). By M 2 Interaction of key proteins on microglial cell membrane and brain microvascular endothelial cells realizes efficient brain delivery and promotes M overactive under neuroinflammation 1 Conversion of microglial cells to anti-inflammatory M 2 The type effectively improves the inflammatory environment in the brain of the AD patient; mnO (MnO) 2 The nano enzyme (BM) can remove a large amount of intracellular ROS, effectively relieve the oxidative stress state in the brain and trigger DSPE-TK-PEG 3400 apoE cleavage and release of apoE mimetic peptide effects transfer and clearance of Amyloid-beta (Abeta) to microglia, while ROS promote rapid release of 17-AAG, effecting efficient clearance of phosphorylated Tau protein (p-Tau) within neurons. Expecting M to be designed 2 The bionic nano-enzyme drug delivery system fused with microglial cell membranes can track the whole disease course of the disease in a brand-new treatment thought, effectively save the cognitive dysfunction of AD patients, and provide a scheme for reference for clinical treatment of 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, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is DSPE-TK-PEG 3400 -schematic synthesis of apoE.
FIG. 2 is a Bio-MS diagram of the synthesized product.
FIG. 3 is a schematic view of 1 H-NMR chart.
FIG. 4 is DSPE-TK-PEG 3400 ROS-responsive Bio-MS contrast map of apoE.
Fig. 5 is a TEM and XRD pattern of BM.
FIG. 6 is DSPE-TK-mPEG 3400 -apoE proportion and ultrasound time screening results.
FIG. 7 is a BCA assay for AAG/BM-Lip-apoE@M 2 CM surface membrane protein content.
FIG. 8 is AAG/BM-Lip-apoE and AAG/BM-Lip-apoE@M 2 CM particle size and Zeta potential.
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 shows phagocytosis of Abeta by BV2 cells after treatment of each group 1-42 Is a laser confocal map of (2).
FIG. 19 is a WB plot and corresponding semi-quantitative analysis of p-Tau-associated proteins after treatment of each group.
FIG. 20A beta 1-42 SH-SY5Y cell viability under 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 M 2 SDS-PAGE electrophoresis of different CM 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, DAPI 4',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 neuron fiber tangle, OD optical density value, OAokadaic acid, PAGE polyacrylamide gel electrophoresis, PVDF polydifluoroethylene membrane, ROS reactive oxygen species, [ Ru (dpP) 3 ]Cl 2 Tris (4, 7-diphenyl-1, 10-phenanthroline) ruthenium dichloride, sodium SDS dodecyl sulfate, SP senile plaques, TEMED tetramethyl ethylenediamine, thT thioflavin T, tris trimethylol aminomethane, TMB 3,4, 5-trimethoxybenzaldehyde and 17-AAG 17-acrylamide.
Example 1:
DSPE-TK-PEG 3400 -synthesis of apoE:
1.5g of NHS-PEG was weighed out 3400 dissolving-OH (hydroxy polyethylene glycol active ester) in 10mL chloroform, adding 1.0g NH 2 -TK-NH 2 (propane-2, 2-diylbis (sulfadiazine) diethylamine) and triethylamine react for 24 hours at room temperature, and after decompression concentration, a large amount of diethyl ether is poured into the mixture for precipitation, the product is collected by filtration, and the product is dried in vacuum to obtain HO-PEG 3400 -TK-NH 2 . 1.0g HO-PEG was weighed out 3400 -TK-NH 2 Dissolving in 5mL chloroform, adding appropriate amount of DSPE-NHS (distearoyl phosphatidyl ethanolamine modified active ester) and triethylamine, reacting at room temperature for 24 hr, collecting the filtered product, and vacuum drying to obtain HO-PEG 3400 -TK-DSPE. Weighing 0.5-gHO-PEG 3400 Dissolving TK-DSPE in 5mL acetonitrile, adding appropriate amount of DSC and triethylamine, reacting at room temperature overnight, collecting the filtered product according to the method, and vacuum drying to obtain NHS-PEG 3400 -TK-DSPE. Weigh 0.1gNHS-PEG 3400 Dissolving TK-DSPE in 0.1M PBS solution with pH of 8.0, adding 0.25g apoE mimetic peptide, stirring at room temperature for 24 hr, dialyzing in dialysis bag (MW: 3500 Da) for purification for 24 hr, and lyophilizing the dialysate to obtain DSPE-TK-PEG product 3400 apoE. Adopting biological mass spectrum (Bio-MS) and nuclear magnetic hydrogen spectrum 1 H-NMR) for product structure identification.
DSPE-TK-PEG 3400 ROS responsiveness of apoE: weigh 5mg DSPE-TK-PEG 3400 apoE, add2mL of PBS buffer (10 mM, pH 7.4) was uniformly dispersed, and 2mL of H was added thereto 2 O 2 The solution (200. Mu.M) was incubated at 37℃for 3h and dialyzed in a dialysis bag (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 were weighed, dissolved in a proper amount of chloroform, and then 3mL of diethyl ether was added to be mixed uniformly to obtain an oil phase. Weighing 60mg BM nano enzyme and right amount of DSPE-TK-PEG 3400 apoE was dissolved as an aqueous phase by adding 0.6mL of PBS (pH 7.4). 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 5mL 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 preparation process of the AAG/BM-Lip-apoE are not added with 17-AAG and DSPE-TK-PEG 3400 -apoE to obtain BM-Lip; the Lip-apoE can be prepared without adding BM and 17-AAG; the BM-Lip-apoE can be prepared without adding 17-AAG; no addition of 17-AAG, DSPE-TK-PEG 3400 apoE and BM, consisting of DOTAP and cholesterol alone, namely Lip. Fixing other prescriptions and processes respectively, and sequentially inspecting DSPE-TK-PEG 3400 -apoE the proportion of functional fragments to DOTAP is 5% (0.75 mg), 8% (1.2 mg) and 10% (1.5 mg), respectively, and the particle size or potential of AAG/BM-Lip-apoE prepared at ultrasonic times of 3min,6min and 9min, optimize the optimal prescription and process.
M 2 Microglial cell membrane (M) 2 CM) extraction and characterization: mouse microglial cells (BV 2) were cultured and induced with IL-4 (20 ng/mL) for 24h to differentiate into M 2 Microglia cells were collected by centrifugation at 1000r/min for 3 min. The cell membrane is extracted by lyophilization and sucrose gradient centrifugation, respectively. The method (method 1) comprises the following specific steps: adding PBS into the collected cells, and mixing to obtain a cell suspension with a concentration of 1×10 7 And adding a proper amount of protease inhibitor into the mixture per mL, and uniformly mixing the mixture. 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 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 Adding proper amount of protease inhibitor into the mixture per mL, and mixing the mixture evenly. Squeezing for 20 times by liposome extruder, adding 1M sucrose solution, mixing to final concentration of 0.25M, centrifuging (3000 g,10min, 4deg.C), collecting supernatant, centrifuging (10000 g,10min, 4deg.C), collecting cell precipitate, washing with 0.25M sucrose solution, centrifuging, and removing supernatant to obtain precipitate at bottom of tube 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. Thus, sucrose gradient centrifugation was subsequently selected as the final cell membrane M 2 CM extraction method.
Preparation of a cell membrane bionic nano-carrier:
membrane lipid ratio screening: m to be extracted 2 CM and prepared AAG/BM-Lip-apoE are in qualitySequentially mixing the materials according to the weight ratio of 1:4-4:1, repeatedly freezing and thawing (4 ℃/45 ℃) and sequentially passing through polycarbonate membranes of 400nm and 200nm by a liposome extruder, further centrifuging for 10min by 10000g, and purifying to remove M which is not loaded successfully 2 CM to obtain AAG/BM-Lip-apoE@M 2 CM. Lip and M 2 Co-extrusion of CM with liposome extruder to obtain lip@M 2 CM; BM-Lip-apoE and M 2 Co-extruding CM with liposome extruder to obtain BM-Lip-apoE@M 2 CM. BCA kit for determining AAG/BM-Lip-apoE@M 2 CM membrane protein content, according to M 2 Correlation of CM membrane protein content and membrane lipid mass ratio, the membrane lipid ratio with highest cell membrane coating efficiency was determined.
Investigation of ROS scavenging ability in vitro: h 2 O 2 Is eliminated: preparation of 1,5, 10, 20, 50, 100. Mu.M H 2 O 2 Standard solution, 500. Mu.L sample was added to 50. Mu.M sample red and 0.1U/mL HRP, and the mixture was reacted at 37℃for 10 minutes after mixing. Absorbance at 570nm (OD) was measured using UV-vis and OD570 was plotted nm -a concentration standard curve. Respectively taking 3mL BM and AAG/BM-Lip-apoE@M 2 CM addition equivalent 200. Mu. M H 2 O 2 Solution, 3mL PBS solution was added with equal amount of H 2 O 2 Placing the solution as control group in a constant temperature shaking oven at 37deg.C, respectively sucking 500 μl of the reaction solution at 0min,10min,20min,30min,40min,50min,60min, adding duplex red and HRP, mixing, measuring OD value at 570nm, and calculating to obtain residual H in the system 2 O 2 Content, and percent degradation versus time.
Proper BM and AAG/BM-Lip-apoE@M are taken 2 CM and 20mL H 2 O 2 (100 mu M) solution mixing, digital camera photographing recording O 2 Pictures were generated and O in solution at different time points was measured with a portable oximeter 2 Concentration variation and plotting O 2 A time-dependent curve is generated.
Hydroxyl radical (. OH) and superoxide radical anion (O) 2 ·- ) Cleaning experiment: evaluation of superoxide anion (O) Using Electron Spin Resonance (ESR) experiments 2 ·- ) And hydroxyl radical (. Cndot.) OH). O (O) 2 ·- The 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. Adding BM and AAG/BM-Lip-apoE@M into the above prepared solution respectively 2 CM, untreated control, samples test record. With Abeta 1-42 Interaction evaluation of (c): fluorescence co-localization analysis: preparation of Dil-labeled AAG/BM-Lip-apoE@M 2 CM(Dil-AAG/BM-Lip-apoE@M 2 CM). mu.L of Dil-AAG/BM-Lip-apoE@M was taken 2 CM and 100. Mu.L of FITC-Abetse:Sub>A 1-42 (1 mg/mL) was incubated 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
DSPE-TK-PEG 3400 Structural identification of 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, compared with DSPE-TK-PEG 3400 apoE theoretical molecular weight is consistent. FIG. 3A is DSPE-TK-PEG 3400 apoE 1 FIG. 3B is an H-NMR chart of apoE mimetic peptides 1 H-NMR FIG. 3C is NH 2 -TK-NH 2 A kind of electronic device 1 H-NMR chart. Wherein the peak having a chemical shift value of about 3.5ppm in FIG. 3A is PEG 3400 The peak having a chemical shift value of about 4.2ppm is a characteristic peak of hydroxyl hydrogen in DSPE, the multiple peak of about 7.2ppm in fig. 3B is a characteristic peak of benzene ring in Phe in apoE polypeptide, and the characteristic peak of benzene ring appears at the position corresponding to fig. 3A. B, C, d and e in FIG. 3C are characteristic peaks of methylene, methyl and amino hydrogens, respectively, of TK, the synthesis product DSPE-TK-PEG 3400 apoE 1 The H-NMR chart likewise shows peaks at the corresponding b, c and d positions (FIG. 3A). Bio-MS 1 The H-NMR results all indicate DSPE-TK-PEG 3400 apoE was successfully synthesized.
DSPE-TK-PEG 3400 ROS responsiveness of apoE: as can be seen in FIG. 4 Bio-MS, DSPE-TK-PEG 3400 apoE has a molecular weight of about 9492. At the same time as H 2 O 2 The molecular weight of the solution after reaction was reduced by about 8683, and PEG 3400 The molecular weight of the apoE fragment corresponds. Description of the invention in the channel H 2 O 2 After treatment, DSPE-TK-PEG 3400 Fragmentation of TK fragment in apoE structure, disruption of structure into PEG 3400 apoE fragment and 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, respectively, with Mn (IV) in manganese dioxide 2p3 / 2 And Mn (IV) 2p1/2 Correspondingly, 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° with MnO 2 The XRD standard pattern card comparison of (c) is consistent.
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, DSPE-TK-mPEG 3400 The proportion of apoE has a remarkable effect on the Zeta potential, and the Zeta potential value is conversely reduced along with the increase of the proportion, which shows that DSPE-TK-mPEG 3400 The hydrophilic long chains in apoE fragments will form a certain shielding effect on the potential of the liposome surface, thus lowering the absolute value of the Zeta potential to a certain extent. 8% DSPE-TK-mPEG was chosen in view of the subsequent fusion effect with the cell membrane 3400 The addition ratio of apoE is used as the optimal prescription. 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: quantitative determination of AAG/BM-Lip-apoE@M by BCA kit 2 CM membrane protein content to determine the optimal membrane lipid mass ratio. As can be seen from FIG. 7, with increasing membrane lipid ratio, AAG/BM-Lip-apoE@M 2 CM membrane protein content increases with increasing membrane lipid ratio greater than 1:1 AAG/BM-Lip-apoE@M 2 CM membrane protein content gradually tends to be saturated, and more cell membranes are continuously added on the basis, after centrifugation, obvious cell membrane precipitation at the bottom of the tube can be found, 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 preferred as the optimal adding ratio of the cell membranes and the liposome.
AAG/BM-Lip-apoE@M 2 Characterization of 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 AAG/BM-Lip-apoE@M 2 123.37 + -1.11 nm of CM; and due to the neutralization of negative charge of cell membrane, zeta potential is reduced from the original 33.79 +/-0.51 mV to AAG/BM-Lip-apoE@M 2 CM 13.66.+ -. 1.34mV, thus demonstrated successful fusion of cell membrane with liposome, and under TEM observation (FIG. 9), AAG/BM-Lip-apoE and AAG/BM-Lip-apoE@M 2 CM has vesicle-like structure, and the two have small difference in particle size, and are fused with cell membrane to form AAG/BM-Lip-apoE@M 2 The vesicle bilayer membrane structure of CM is more evident. 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, M was extracted 2 CM and BV2 cells are substantially identical in protein expression at the same location and AAG/BM-Lip-apoE@M after membrane fusion 2 CM also retains almost all of the original protein information of BV2 cells, while AAG/BM-Lip-apoE, which does not interact with the cell membrane, does not show any protein bands, thus demonstrating successful modification of the vector by the cell membrane. In fig. 10, a: BV2 cells; b: m is M 2 CM；C：AAG/BM-Lip-apoE@M 2 CM；D：AAG/BM-Lip-apoE。
Western Blotting experiment: as can be seen from the WB results of FIG. 11, there are a large number of specific proteins, integrin. Alpha.4 and Mac-1, enriched in M 2 CM and AAG/BM-Lip-apoE@M 2 Efficient transfer of membrane protein information to AAG/BM-Lip-apoE@M on the surface of CM 2 Brain delivery of CM, as well as active uptake of microglia and neurons, have positive effects. In fig. 11, a: BV2 cells; b: m is M 2 CM；C：AAG/BM-Lip-apoE@M 2 CM。
Investigation of ROS scavenging ability in vitro:
H 2 O 2 is eliminated: FIG. 12 is H 2 O 2 From the graph of percent degradation versus time, BM and AAG/BM-Lip-apoE@M were found 2 CM vs H in the system 2 O 2 The consumption of (2) is faster, and H in the system is generated after 10, 20 and 50min of reaction 2 O 2 The reduction rates were 59.1%,60.6% and 69.9%, respectively, and 1h had reached 71.9%; and H is 2 O 2 Control group at each time point H before and after incubation 2 O 2 The degradation percentage is relatively stable, and the degradation percentage is only about 22.1 percent in 1 h. From this, BM and AAG/BM-Lip-apoE@M 2 CM can effectively remove H in the reaction system 2 O 2 Presumably, the AAG/BM-Lip-apoE@M 2 CAT-like enzyme activity of BM in CM structures and DSPE-TK-PEG 3400 TK bond cleavage on apoE fragment consuming H in System 2 O 2 Related to the following.
OH and 2 ·- is eliminated: use of ESR analysis means for evaluation of BM and AAG/BM-Lip-apoE@M 2 CM vs. OH and O 2 ·- The control group had a strong ESR signal (1:2:2:1) in BM and AAG/BM-Lip-apoE@M 2 After CM treatment, the peak intensity was significantly reduced, indicating BM and AAG/BM-Lip-apoE@M 2 CM can consume effectively. BM and AAG/BM-Lip-apoE@M 2 After CM treatment 2 At the same ESR signal (1:1:1:1) in the control group, the characteristic peak was also significantly reduced, indicating BM and AAG/BM-Lip-apoE@M 2 CM can also effectively remove O 2 ·- . The experimental results show that AAG/BM-Lip-apoE@M 2 CM has a sensitive ROS scavenging capacity and can be effectively applied to relieving oxidative stress microenvironment in the brain of an AD patient.
In fig. 12, a: post treatment H of different Carriers 2 O 2 Percent degradation. B: ESR spectrum of OH scavenging. C: o (O) 2 ·- ESR profile of the purge.
Example 3:
in vitro evaluation of brain targeting:
mouse brain microvascular endothelial cells(bEnd.3) cultivation: the cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% diabody at 37℃in 5% CO 2 Culturing in a cell culture 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 105 cells/well. Changing liquid every other day, measuring transendothelial cell resistance (TEER) until TEER reaches 200Ω/cm 2 I.e. successfully build the BBB model in vitro. The upper chamber medium was aspirated and diluted with 1640 medium C6-BM-Lip-apoE and C6-BM-Lip-apoE@M were added 2 CM (DOTAP concentration: 100. Mu.g/mL) aspirates 200. Mu.L of lower chamber liquid at 5min,10min,15min,30min,60min, respectively, while supplementing an equal volume of blank 1640 medium. 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, the liquid crystal display device comprises a liquid crystal display device,permeability (nmol/s), C 0 (nmol/mL) is the initial concentration of C6 in the upper chamber, A (cm) 2 ) Is the surface area of the Transwell membrane.
ZO-1 immunofluorescence experiment: bEnd.3 cells at 1X 10 4 The density of each hole is inoculated in a confocal small dish with a cover, after 24 hours of culture, the original serum-containing culture medium is discarded, and PBS solution, BM-Lip-apoE and BM-Lip-apoE@M are respectively added 2 CM is incubated for 1h, after tissue fixing liquid is fixed for 10min,rabbit anti-mouse ZO-1 dilution (1:200) was added and incubated overnight at 4 ℃. Washed 3 times with PBS and incubated with anti-Cy 3 labeled goat anti-rabbit IgG (1:300) 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 a cell density of about 1X 10 5 And/or holes. After 24h of incubation, the medium with serum was discarded and LPS (100 ng/mL) was added to induce M 1 Microglial cells. Respectively adding PBS solution, lip-apoE and lip@M 2 CM, BM-Lip and BM-Lip-apoE@M 2 CM, DOTAP concentration of 120. Mu.g/mL and BM concentration of 500. Mu.g/mL) for 1h, and FITC-Abetse:Sub>A was added to each group 1-42 15min of treatment and 3 washes 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, BM-Lip-apoE@M 2 The Papp values of the CM group reached 2.6X10 in 60min 9 cm/s, significantly higher than BM-Lip-apoE and PBS control, BM-Lip-apoE had a Papp value of 1.4X10 at the same time 9 cm/s, differences in the structure of the vector indicate M 2 CM and DSPE-TK-PEG 3400 The apoE fragment can effectively assist BM-Lip-apoE@M 2 CM was distributed from the Transwell upper chamber to the lower chamber across bEnd.3 monolayer cells. This may be the same as M 2 ICAM-1/VCAM-1 binding of the Interginα4 and Mac-1 proteins expressed on CM and bEnd.3 cell surface reversibly opens the BBB tight junction, and apoE mimetic peptides and the LRP1 receptor-mediated active transport pathway on bEnd.3 cell surface are involved in the distribution across the BBB into the brain. In fig. 13, a: PBS; b: BM-Lip-apoE; c: BM-Lip-apoE@M 2 CM; n=3, and represents that the 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 linked related proteins, and its expression is closely related to the integrity of endothelial cell closely linked, so ZO-1 protein on brain microvascular endothelial cell bEnd.3 can be directly labeled by immunofluorescenceReflecting BM-Lip-apoE@M 2 CM transmembrane transport mechanism. 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. And BM-Lip-apoE@M 2 CM experiments showed that bEnd.3 cells had significantly lower red fluorescence intensity and a significant portion of the red fluorescence was absent, demonstrating that BM-Lip-apoE@M 2 CM can reduce the expression of the tight junction related protein ZO-1, disrupting the tight junction between endothelial cells. This is in accordance with M 2 Interginα4 and Mac-1 proteins after CM coating in BM-Lip-apoE@M 2 The presentation of CM surfaces is closely related, which can reversibly open BBB tight junctions with ICAM-1/VCAM-1 binding to the surface of bend.3 cells, increasing BBB permeability, 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 by flow cytometry, CD206 being M 2 Protein specific markers on microglia, CD86 being M 1 Protein specific markers on microglia. And M is as follows 0 、M 1 In comparison with the control group, M in FIG. 15 2 Increased fluorescence intensity of CD206 marker in group of IL-4 induced microglia, whereas M 2 The fluorescence intensity of the panel CD86 marker was reduced, indicating that it had been successfully differentiated into M 2 Microglial cells. Further comparative investigation of M in LPS-induced differentiation of each experimental group 1 The transformation promotion trend on the microglial cell model shows that the Lip-apoE@M 2 The fluorescence intensity of CD206 after CM is taken up by cells is obviously higher than that of M 0 、M 1 Control group was enhanced and was associated with M 2 The control group had comparable intensity, while Lip-apoE@M 2 Fluorescence intensity of CM and M 0 、M 1 The control group was significantly weaker than the control group, indicating M 2 CM coating can promote M 1 Microglial cellsTo M 2 Microglial cell transformation; BM-Lip-apoE and M 1 After co-incubation of microglial cells, the fluorescence intensity of the CD206 marker is higher than that of M 2 Control group and Lip-apoE@M 2 CM experimental group is further enhanced, and the fluorescence intensity of CD86 marker is obviously weakened, which shows that the efficient elimination of ROS by BM nano-enzyme can relieve M 1 The state of microglial overactivation under phenotype, contributing to M 1 Microglial cell direction M 2 Phenotypic conversion, improving neuroinflammation induced by ROS as a pro-inflammatory factor. And M is 1 Microglial cell passes through BM-Lip-apoE@M 2 After CM treatment, the fluorescence intensity of CD206 is further enhanced, which is obviously higher than that of Lip-apoE@M 2 The CM and BM-Lip-apoE groups were more pronounced and the fluorescence intensity of the CD86 marker was further reduced compared to that of the other groups, indicating more M 1 Differentiation of microglial cells into M 2 Phenotyping, anti-inflammatory M 2 The higher proportion of microglial cells effectively relieves the abnormal immune activation state of microglial cells and AD neuroinflammatory environment caused by the microglial cells, and the abnormal immune activation state is matched with M 2 CM and BM synergy is closely related.
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, M is equal to 0 Differentiation of microglial cells into M after LPS induction compared to resting state 1 Type, with slightly enhanced migration capacity, whereas microglial differentiation to M upon IL-4 stimulation 2 The migration ability of the model is obviously enhanced. And M is as follows 1 And M 2 Compared with the control group, each experimental group obviously quickens the migration speed of microglia after being treated respectively, and BM-Lip-apoE@M 2 The migration ability after CM processing is most remarkable. As can be seen from the scratch test of FIG. 17, M after 12h of cell growth 0 The control group still has obvious scratch area, and M is induced 1 And M 2 Cell scratch area was reduced after transformation, and M 2 Phenotypic group cell gap ratio M 1 The smaller phenotype groups indicate faster cell migration crawling. After packet processing, compared with M 1 The phenotype group showed significantly faster cell migration, with BM-Lip-apoE@M 2 Massive cell alignment of CM groupThe centers of the scratches are converged, the original scratches are 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 microglial differentiation into M 2 Phenotypically, can enhance the migration capacity of microglia such that M 2 Microglia deposit more rapidly around pathological proteins and around neuronal debris, facilitating 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, M is equal to 0 Differentiation of microglial cells into M after LPS induction compared to resting state 1 Type, with slightly enhanced migration capacity, whereas microglial differentiation to M upon IL-4 stimulation 2 The migration ability of the model is obviously enhanced. And M is as follows 1 And M 2 Compared with the control group, each experimental group obviously quickens the migration speed of microglia after being treated respectively, and BM-Lip-apoE@M 2 The migration ability after CM processing is most remarkable. As can be seen from the scratch test of FIG. 17, M after 12h of cell growth 0 The control group still has obvious scratch area, and M is induced 1 And M 2 Cell scratch area was reduced after transformation, and M 2 Phenotypic group cell gap ratio M 1 The smaller phenotype groups indicate faster cell migration crawling. After packet processing, compared with M 1 The phenotype group showed significantly faster cell migration, with BM-Lip-apoE@M 2 The CM group has a large number of cells converging 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 microglial differentiation into M 2 Phenotypically, can enhance the migration capacity of microglia such that M 2 Microglia deposit more rapidly around pathological proteins and around neuronal debris, facilitating subsequent phagocytic clearance.
Examination of microglial phagocytic Capacity establishing M 1 Microglial cell model, and microglial cell pair is observed by contrast of laser confocal microscopePhagocytic clearance ability of aβ. In FIG. 18, the green fluorescence is FITC-labeled Abeta 1-42 Red fluorescence is a lysosome labeled by LVso-Tracker Red probe, and blue fluorescence is a cell nucleus labeled by DAPI. As shown in FIG. 18, BV2 cells and FITC-Abetse:Sub>A 1-42 After incubation, aβ 1-42 The green fluorescence of (2) and the red fluorescence of lysosomes are still scattered in a large quantity in cytoplasm, and only a small amount of the fluorescence is overlapped to show weak orange fluorescence, which indicates that the Abeta which can be metabolized and cleared by lysosomes without effective intervention means 1-42 Is limited. BV2 cells after uptake of Lip-apoE, Aβ 1-42 The green fluorescence of (2) and the red fluorescence of lysosomes begin to overlap, and the orange fluorescence after red-green overlapping is presented, which shows that apoE mimic peptide can carry Abeta 1-42 Transport to lysosomal metabolic clearance. lip@M 2 CM, BM-Lip and BM-Lip-apoE@M 2 After CM and BV2 cells are incubated, green fluorescence and red fluorescence are overlapped in a large amount, orange fluorescence is more obvious, and all three preparation groups can promote M 1 Transformation differentiation of microglial cells and induction of transformed M 2 The migration capacity of microglia is enhanced, and Abeta of phagocytosis pathology can be more actively identified 1-42 Deposition, enhancement of Abeta 1-42 Ability to transport to lysosomes and promote more aβ on this basis 1-42 Is cleared by lysosomal metabolism. BM-Lip-apoE@M having both pro-cell transformation and apoE mimetic peptide effects 2 CM recorded on confocal laser microscopy, which resulted in microglial cells exhibiting greater phagocytic capacity and binding to aβ 1-42 Abnormal protein removal effect.
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 AAG/BM-Lip-apoE@M compared to the OA control group and the 17-AAG experimental group 2 The expression of Hsp40, hsp70 and p-Tau in the CM group was significantly reduced.
Neuroprotection: with Abeta 1-42 Stimulation of SH-SY5Y cells establishes a model of neurocytotoxicity. As shown in the experimental results of CCK-8 in FIG. 20, abeta 1-42 ControlCell viability was significantly reduced in the group by AAG/BM-Lip-apoE@M 2 The viability of the CM treated cells was much higher than in the control and other experimental groups. In fig. 20, a: a control group; b: aβ 1-42 ；C：AAG-Lip-apoE@M 2 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: with Abeta 1-42 Stimulation of SH-SYSY cells establishes a model of nerve cytotoxicity. To evaluate BM-Lip-apoE and lip@M 2 CM combined effect, according to Chou-Talalay combined drug index method and application of CompuSy software calculation, BM-Lip-apoE and lip@M are obtained 2 CM combination (1:1 mass ratio) at 0.125IC50, 0.25IC50, 0.5IC50 and 1.0IC50 (Table 1), the Cl values obtained were less than 1, indicating BM-Lip-apoE and lip@M 2 CM combined action (the mass ratio of the two is 1:1) has synergistic action, and the action concentration is minimum in CI value at the respective IC50, and the synergistic neuroprotection is optimal.
Table 1 AD evaluation of the neuroprotective effect of mice on synergistic therapeutic effect (n=3)
| Concentration of action | Cell viability (%) | CI value | Interaction determination |
| 0.125IC 50 | 54.6±2.2 | 0.884±0.100 | ++ |
| 0.25IC 50 | 68.9±1.4 | 0.617±0.050 | ++ |
| 0.5IC 50 | 78.4±0.5 | 0.514±0.040 | ++ |
| 1.0IC 50 | 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 group shows good brain targeting, the brain of the mice from 2h to 8h shows high-intensity fluorescence distribution, and the brain fluorescence gradually increases along with the time extension, and the brain from 12h to 24h still has stronger fluorescence accumulation, which indicates that DiR-BM-lip@M 2 CM and DiR-BM-Lip-apoE@M 2 CM can be distributed faster into the brain and can reside in the brain with a gradual increase and stability over a considerable period of time. And at each observation time point, diR-BM-Lip-apoE@M 2 The brain fluorescence intensity of the CM group mice is higher than that of DiR-BM-lip@M 2 CM group. Comparison of DiR-BM-Lip and DiR-BM-lip@M 2 Differences in CM brain fluorescence distribution indicate M 2 The CM coating has a key effect on enhancing the brain targeting delivery characteristic, can obviously improve the brain entering efficiency and prolong the residence time in the brain, and realizes the effective delivery of AD therapeutic drugs. Contrast DiR-BM-lip@M 2 CM and DiR-BM-Lip-apoE@M 2 CM structural differences, due to further modification of apoE mimetic peptides, exhibit more pronounced brain targeting characteristics. 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 intravenous DiR-BM-Lip, diR-BM-lip@M in mice 2 CM and DiR-AAG/BM-Lip-apoE@M 2 Distribution in the brain at 2h,4h,8h,12h and 24h after CM, diR-BM-Lip-apoE@M at all time points measured 2 The accumulation of CM in brain is higher than that of DiR-BM-Lip and DiR-BM-lip@M 2 The CM reaches 109.5ng/mg in 4 hours, which is obviously higher than DiR-BM-Lip and DiR-BM-lip@M 2 30.68ng/mg and 87.4ng/mg of CM. Three preparation groups were selected for 4h to map the distribution of heart, liver, spleen, lung, kidney and brain 22B, diR-BM-Lip-apoE@M 2 The distribution of CM in the brain is considerable, consistent with the results of live 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 2 CM; n=3, 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, the track of the mouse sailing route was significantly increased in the platform quadrant, wherein AAG/BM-Lip-apoE@M 2 The route of travel for the CM group of mice was close to the sham group, essentially near the platform. As can be seen from the figure, the escape latency of the sham operation group is 7.26+ -4.88 s, the AD model group is 35.58+ -2.55 s, and the sham operation group is subjected to AAG/BM-Lip-apoE, AAG-Lip-apoE@M 2 CM，BM-Lip-apoE@M 2 CM，AAG/BM-Lip@M 2 CM and AAG/BM-Lip-apoE@M 2 The escape latency decreased to 23.96.+ -. 4.89s, 20.32.+ -. 4.32s, 17.74.+ -. 4.21s, 17.85.+ -. 6.58s and 11.17.+ -. 6.37s after two weeks of CM tail intravenous injection, respectively. The residence time of each group of mice in the platform quadrant is 37.03+/-1.45% in the sham operation group, 18.80+/-5.24% in the AD model group, and the mice are subjected to AAG/BM-Lip-apoE and AAG-Lip-apoE@M 2 CM，BM-Lip-apoE@M 2 CM，AAG/BM-Lip@M 2 CM and AAG/BM-Lip-apoE@M 2 CM was increased to 19.97±6.52%,24.87±3.68%,24.87±5.40%,26.33±6.52% and 34.17±2.95%, respectively, after treatment. The number of times of mice passing through the platform is 4.8+/-0.84 in the sham operation group, 1.6+/-0.55 in the AD model group, and the mice pass through AAG/BM-Lip-apoE and AAG-Lip-apoE@M 2 CM，BM-Lip-apoE@M 2 CM，AAG/BM-Lip@M 2 CM and AAG/BM-Lip-apoE@M 2 CM was increased to 2.60± 1.14,3.00 ± 1.00,3.25 ± 1.26,3.00 ±1.00 and 4.4±1.14, respectively, after treatment. From the above results, it can be seen that AAG/BM-Lip-apoE@M 2 CM treatment is most prominent, and can improve learning and memory in mice. The A/P/B is AAG/BM-Lip-apoE; A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; A/L/B@CM is AAG/BM-lip@M 2 CM; A/P/B@CM is AAG/BM-Lip-apoE@M 2 CM; n=12, p < 0.01, represents the statistical difference between the group and the AD model group.
Nesting experiment: as can be seen from the nesting condition of the mice and the analysis result of the corresponding data in FIG. 24, after two weeks of grouping treatment, AAG/BM-Lip-apoE@M 2 The CM preparation group has obvious and obvious difference (p < 0.01) in nesting behavior compared with AD model group, and is basically similar to that of normal mice in false operation group, and the model is proved to be subjected to AAG/BM-Lip-apoE@M 2 AD mice after CM treatment were improved in cognitive behaviours. The A/P/B is AAG/BM-Lip-apoE; A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; A/L/B@CM is AAG/BM-lip@M 2 CM; A/P/B@CM is AAG/BM-Lip-apoE@M 2 CM；n＝12， ** Represents a statistical difference, p < 0.01, compared to the AD model group.
Immunofluorescence: FIG. 25A is a schematic of hippocampal neurons 8-OH from mice of each groupG immunofluorescence analysis results, wherein DAPI blue fluorescence outlines the basic outline of the mouse hippocampus, 8-OHG red fluorescence labeled active oxygen molecules. No red fluorescence was observed in the normal mice group, stronger red fluorescence spots appeared in the hippocampus of the AD model group, and red fluorescence spots were reduced to different degrees after two weeks of tail intravenous injection of each preparation group, wherein AAG/BM-Lip-apoE@M 2 The fluorescent spots of the CM group are basically disappeared, which indicates that the level of the hippocampal 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 on brain sections of mice in each group, DAPI blue fluorescence labeling nuclei, green fluorescence labeling CD206 and CD86 labeling CD86, respectively, wherein the red fluorescence of CD86 is stronger and the green fluorescence of CD206 is weaker in the AD model group, microglia exhibiting over-activated M 1 Phenotypic status, in which the green fluorescence of CD206 in the brain was increased to different extents and the red fluorescence of CD86 was decreased to different extents after treatment with each formulation group, AAG/BM-Lip-apoE@M 2 The CM group red fluorescence is basically disappeared, the green fluorescence intensity is most obvious, and the result shows that the microglial cells in the brain of mice after stem prognosis of each preparation group are mostly anti-inflammatory M 2 Phenotypes, which are beneficial for improving 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@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; 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 brain sections Abeta of mice in each group 1-42 And p-Tau protein immunohistochemical analysis results, the AD model group can see the Abeta amyloid deposition and p-Tau protein aggregate with obvious orange color under a microscope, the deposition is consistent with the typical pathological characteristics of AD mice, and normal mice hardly see abnormal protein deposition. After two weeks of drug administration treatment, AD mice had a.beta.in the brain 1-42 And the expression of phospho-Tau (Ser 396) is obviously reduced, and the deposition or aggregation of abnormal protein under a microscope is basically disappeared, wherein AAG/BM-Lip-apoE@M 2 The most obvious effect after CM administration treatment shows that the medicine can effectively improve Abeta 1-42 Deposition state in brain and correction of p-Tabnormal entanglement of au protein, reducing damage to neurons by the au protein and the au protein, and recovering physiological functions of neurons. The A/P/B is AAG/BM-Lip-apoE; A/P@CM is AAG-Lip-apoE@M 2 CM; P/B@CM is BM-Lip-apoE@M 2 CM; 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 surviving neurons in the hippocampus of mice in the sham group was large and arranged in order, while the number of surviving neurons in the hippocampus of mice in the AD model group was significantly reduced and different degrees of shrinkage deep staining occurred, the pathological changes of cells in the hippocampus of mice were reduced after treatment with each group, the number of surviving neurons was increased and arranged in order, and the number of neurons in the hippocampus of mice was increased and arranged in order 2 The CM group was similar to the sham group, and restored to the normal state of neurons.
In conclusion, the invention successfully constructs M 2 MnO fused with microglial cell membrane 2 Bionic nano-enzyme drug delivery system AAG/BM-Lip-apoE@M 2 CM, which has excellent brain targeting properties, promotes microglial cell targeting to M 2 Phenotype transformation inhibits the state of overactivation of the immune system in AD brain, efficiently clears ROS, relieves oxidative stress, and delays the possibility of AD progression in early stages; AAG/BM-Lip-apoE@M 2 CM can also directly clear abnormal pathological marker proteins at the AD downstream target spot, protect neuron functions and restore the memory and cognitive ability 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,
DSPE-TK-PEG 3400 -synthesis of apoE: dissolving active ester of hydroxy polyethylene glycol in solvent, adding propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, reacting, concentrating, precipitating, and drying to obtain HO-PEG 3400 -TK-NH 2 HO-PEG 3400 -TK-NH 2 Dissolving in solvent, adding distearoyl phosphatidyl ethanolamine modified active ester and triethylamine, reacting, concentrating, precipitating, and drying to obtain NHS-PEG 3400 -TK-DSPE, weighing NHS-PEG 3400 Dissolving TK-DSPE in buffer solution, adding apoE mimic 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 in buffer; 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;
M 2 Extraction of microglial cell membranes: induction of microglial cells as M 2 Centrifuging and collecting microglial cells, and extracting M by lyophilization or sucrose gradient centrifugation 2 Microglial cell membranes;
preparation of a cell membrane bionic nano-carrier: subjecting said M 2 Mixing microglial cell membrane with AAG/BM-Lip-apoE according to the mass ratio of 1:4-4:1, repeatedly freezing and thawing, passing through polycarbonate membrane by using 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 M 2 The mass ratio of microglial cell membrane to 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 nano-enzyme for treating Alzheimer's disease according to claim 1 or 2, wherein 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.
4. The method for preparing the bionic nanoenzyme for treating alzheimer's disease according to claim 1 or 2, characterized in that: the DSPE-TK-PEG 3400 -synthesis of apoE, said HO-PEG 3400 -TK-NH 2 Is prepared by dissolving 1.5g of active ester of hydroxy polyethylene glycol in 10mL of chloroform, adding propane-2, 2-diylbis (sulfadiazine) diethylamine and triethylamine, reacting at room temperature for 24h, concentrating under reduced pressure, precipitating with glacial ethyl ether, filtering, and vacuum drying to obtain HO-PEG 3400 -TK-NH 2 。
5. The method for preparing the bionic nanoenzyme for treating Alzheimer's disease according to claim 4, which is characterized in that: the DSPE-TK-PEG 3400 -in the synthesis of apoE, the NHS-PEG 3400 Preparation of TK-DSPE 1.0gHO-PEG was weighed 3400 -TK-NH 2 Dissolving in 5mL chloroform, adding distearoyl phosphatidyl ethanolamine modified active ester and triethylamine, reacting at room temperature for 24h, concentrating under reduced pressure, pouring into glacial ethyl ether for precipitation, filtering, and vacuum drying to obtain NHS-PEG 3400 -TK-DSPE。
6. The method according to claim 4 for treating alrThe preparation method of the bionic nano-enzyme for treating the Alzheimer disease is characterized by comprising the following steps of: the DSPE-TK-PEG 3400 Synthesis of apoE comprising weighing 0.1g NHS-PEG 3400 Dissolving TK-DSPE 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 lyophilizing the dialysate to obtain 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 to make M 2 Adding phosphate buffer solution into microglial cells, mixing, and concentrating cell suspension at a concentration of 1×10 7 Adding proper amount of protease inhibitor per mL, squeezing via liposome extruder, centrifuging at 4deg.C for 3000 g/10 min, collecting supernatant 10000 g/10 min, centrifuging at 4deg.C, and lyophilizing 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 comprises the steps of 2 Microglial cells were resuspended in Tris-magnesium salt buffer at 4℃at a cell suspension concentration of 1X 10 7 Adding protease inhibitor at a ratio of 0.25M/mL, squeezing by liposome extruder, adding 1M sucrose solution, mixing to final concentration of 0.25M,3000g,10min, centrifuging at 4deg.C, and collectingCentrifuging the supernatant fluid 10000g at 4deg.C for 10min, collecting precipitate, washing with 0.25M sucrose solution, centrifuging, discarding supernatant, collecting precipitate to obtain M 2 Microglial cell membranes.
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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