CN110652594B - Multi-target-point therapeutic micelle for regulating and controlling Alzheimer disease microenvironment and preparation method thereof - Google Patents
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Abstract
The invention belongs to the field of pharmaceutical preparations, and relates to a multi-target therapeutic micelle for regulating and controlling an Alzheimer disease microenvironment and a preparation method thereof. The preparation is prepared into a nano drug delivery system by insoluble drugs, synthesized polypeptide modified polyethylene glycol polyamino acid polymer materials, injection solvents and the like, the preparation adopts a polypeptide modified polyethylene glycol polyamino acid polymer material targeting RAGE, and the insoluble drugs are encapsulated in a self-assembly mode in an aqueous phase solution, so that the preparation method is simple, and the particle size distribution of nanoparticles is uniform; by targeting RAGE receptors, micelles can cross the aberrant blood brain barrier and accumulate at AD foci; by bonding ROS-sensitive phenylboronate functional groups as hydrophobic blocks, the drug delivery system can realize the controllable release of drugs in an AD microenvironment, and simultaneously remove focus ROS to effectively normalize and regulate the microenvironment so as to exert AD curative effect.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a multi-target therapeutic micelle for regulating and controlling an Alzheimer disease microenvironment and a preparation method thereof.
Background
Alzheimer's Disease (AD) is a common neurodegenerative disease, and is characterized by central nerve cell loss and advanced brain atrophy as the main pathological features, which are clinically manifested as overall dementia symptoms such as dysfunction in memory and cognition. However, after decades of pathological mechanism studies, the pathogenesis of AD is still not clear. Clinical practice shows that currently, the cholinergic drugs used in clinic can only compensate the neurotransmitter lost due to neuron apoptosis, and cannot prevent the disease from worsening; in recent years, antibody drugs developed according to the "amyloid hypothesis" have failed clinical research, and although amyloid deposits in brain are effectively cleared, dementia symptoms of patients cannot be reversed, so that AD patients fall into a situation of no medicine and no medicine, and therefore, AD treatment strategies different from the "sheep death and reinforcement" type are in urgent need of development.
It is well known in the art that during the development of complex diseases, the formation of microenvironment of a specific focus is often accompanied. In the case of AD, the interaction between multiple cells, such as diseased nerve cells, damaged vascular endothelial cells, and abnormally activated microglia, collectively lead to a chronic pro-inflammatory oxidative stress microenvironment. The cells are regulated by various abnormal biochemical signals such as Reactive Oxygen Species (ROS), abeta and the like in a focus microenvironment to gradually generate phenotypic and functional changes, so that the progress of the disease course of AD is promoted, and the responsiveness of nerve cells to the current therapeutic drugs is weakened; by utilizing the process of bidirectional communication between the pathological cells and the microenvironment, the microenvironment regulation and control can simultaneously influence various cells to realize multi-target treatment, and the method is a potential development direction of AD drugs. In recent years, nano-drug delivery systems with disease microenvironment regulation function are frequently reported. The drug delivery system can achieve a synergistic effect with the drug by normalizing and regulating various biochemical molecules in the microenvironment while delivering the therapeutic drug to the target cells, so as to enhance the overall therapeutic effect of the drug delivery system, however, the current AD nano-drug delivery strategy mainly focuses on the delivery of a single target drug to nerve cells, and lacks the regulation aiming at the AD microenvironment; therefore, there is a need to develop a nano-drug delivery system that can target AD foci and improve their microenvironment.
Studies have shown that receptor for advanced glycation end products (RAGE) plays an important role in the development of the microenvironment for AD disease; along with the activation of microglia, the expression quantity of RAGE receptors is up-regulated in nerve cells, microglia and vascular endothelial cells in a plurality of AD models, and the RAGE receptors are applied to the diagnosis of AD as a target point of imaging at present; RAGE receptors on brain capillary endothelial cells have the physiological role of transporting peripheral A β into the brain, and overexpression of RAGE receptors under the stimulation of inflammatory factors significantly increases intracerebral transport of A β. It has been reported that the short peptide KLVFFAED (Ab peptide for short) mimics the site of action of A.beta.with RAGE and competes for inhibition of binding of A.beta.to brain capillary endothelial cells.
Based on the basis and the current situation of the prior art, the inventor of the application aims to provide a multi-target therapeutic micelle for regulating and controlling an Alzheimer disease microenvironment and a preparation method thereof.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a multi-target therapeutic micelle for regulating and controlling an Alzheimer disease microenvironment and a preparation method thereof.
In the invention, a polypeptide-modified polyethylene glycol polyamino acid polymer material targeting RAGE is used for encapsulating an insoluble model drug to prepare a nano micelle preparation, and through targeting RAGE receptors, micelles cross an abnormal blood brain barrier and accumulate in AD focuses; the drug delivery system realizes the controllable release of the drug in an AD microenvironment by a mode of bonding ROS-sensitive phenylboronate functional groups as hydrophobic blocks, and simultaneously clears away ROS on focus to carry out effective normalization regulation on the microenvironment so as to exert AD curative effect.
Specifically, the multi-target treatment micelle for regulating and controlling the microenvironment of the Alzheimer's disease is prepared from a loaded drug, a synthesized polypeptide-modified polyethylene glycol polylysine phenylboronate derivative and an injection solvent, wherein the polypeptide-modified polymer material is 5-20 mg/mL, the drug concentration is 0.2-1 mg/mL, and the mass ratio of the drug to the polypeptide-modified polymer material is 1: 5-1: 20.
More specifically, the present invention is directed to a method for producing,
the polymer micelle nano preparation loaded with the insoluble drug comprises a polymer material, a polypeptide and an injection solvent, and the nano preparation is loaded with the insoluble drug; the particle size of the nanoparticles is 40-100 nm;
in the present invention, the loading substance of the micelle is a poorly soluble drug such as curcumin or a poorly soluble fluorescent probe;
according to the invention, the polymer material is polyethylene glycol polylysine derivative, and a phenylboronic acid ester functional group is bonded on a side chain amino group of lysine;
in the invention, the polypeptide is a ligand molecule which can be specifically combined with a RAGE receptor on the surface of a blood brain barrier, namely Ab peptide, and the sequence is KLVFFAED;
in the invention, the injection solvent is water for injection or normal saline.
Further, the invention provides a method for synthesizing an Ab peptide modified polymer material, which comprises the following steps:
(1) Weighing N 6 Dissolving carbobenzoxy-L-lysine and triphosgene in tetrahydrofuran, reacting at 50-60 ℃ for 5-10 hours under the protection of nitrogen, precipitating by using anhydrous n-hexane, and performing suction filtration and drying to obtain a lysine monomer; in the reaction liquid, triphosgene is N 6 The mole number of the carbobenzoxy-L-lysine is 0.3 to 0.5 time, and the volume of the normal hexane is 5 to 10 times of that of the tetrahydrofuran;
(2) Weighing the lysine monomer obtained in the step (1) and polyethylene glycol, dissolving in anhydrous N' N-dimethylformamide, reacting for 24-72 hours at 55-65 ℃ under the protection of nitrogen, precipitating by using anhydrous ether, and performing suction filtration and drying to obtain a white solid. Dissolving the white solid in a mixed solution of trifluoroacetic acid and hydrobromic acid (containing 30 percent of acetic acid), reacting for 3 to 8 hours at room temperature, dialyzing by using pure water and freeze-drying; the lysine monomer is 35 to 45 times of the mole number of the polyethylene glycol; the trifluoroacetic acid is 10 to 30 times of the volume of hydrobromic acid (containing 30 percent of acetic acid).
(3) Weighing bifunctional polyethylene glycol, reacting according to the step (2), dialyzing by using pure water and freeze-drying;
(4) Weighing the functionalized polyethylene glycol polylysine polymer obtained in the step (3), ab peptide, sodium ascorbate, cuprous iodide and N, N-diisopropylethylamine, dissolving in N' N-dimethylformamide, stirring and reacting at 25-50 ℃ for 0.5-24 hours under the protection of nitrogen, dialyzing by using pure water and freeze-drying. The Ab peptide is 1-20 times of the mole number of the polyethylene glycol in the step (3), the sodium ascorbate is 5-50 times of the mole number of the polyethylene glycol, the cuprous iodide is 1-10 times of the mole number of the polyethylene glycol, and the N, N-diisopropylethylamine is 1-10 times of the mole number of the polyethylene glycol;
(5) Weighing p-hydroxymethylphenylboronic acid, pinacol and anhydrous sodium sulfate, mixing the p-hydroxymethylphenylboronic acid, pinacol and anhydrous sodium sulfate in tetrahydrofuran, stirring at room temperature for 12-24 hours, filtering to remove precipitates, removing a solvent under reduced pressure, redissolving with ethyl acetate, washing with pure water and saturated salt water, drying an organic phase with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain a phenylboronic acid ester activation precursor; in the reaction solution, pinacol is 1 to 5 times of the mole number of the p-hydroxymethylphenylboronic acid, and anhydrous sodium sulfate is 10 to 30 times of the mole number of the p-hydroxymethylphenylboronic acid;
(6) Weighing the phenylboronic acid ester activated precursor obtained in the step (5), polyethylene glycol polylysine polymer, 4-dimethylaminopyridine and triethylamine, mixing in N' N-dimethylformamide, reacting at room temperature for 24-48 hours under the protection of nitrogen, dialyzing by using pure water, and freeze-drying to obtain the phenylboronic acid ester bonded polyethylene glycol polylysine derivative. The phenylboronic acid ester activating precursor is 40-60 times of the mole number of the polyethylene glycol polylysine polymer, and the 4-dimethylaminopyridine is 40-60 times of the mole number of the polyethylene glycol polylysine polymer; the triethylamine is 40-60 times of the mole number of the polyethylene glycol polylysine polymer.
Further, the invention provides a preparation method of the micro-environment regulation type polymer micelle loaded with the insoluble drug, which comprises the following steps:
(1) Preparing an N' N-dimethylformamide solution of the loaded medicine with the volume mass concentration of 10-20 mg/mL;
(2) Weighing polyethylene glycol polylysine derivatives without Ab peptide modification and with Ab peptide modification, dissolving the polyethylene glycol polylysine derivatives in N 'N-dimethylformamide, adding the N' N-dimethylformamide solution of the medicine in the step (1) into the solution, filling the mixed solution into a dialysis bag, and dialyzing by using 10mM phosphate buffer solution with the pH value of 7.0-7.5 to obtain Ab peptide modified polymer micelles loaded with the medicine; the concentration of the polyethylene glycol polylysine derivative solution is 5-20 mg/mL, the mass of the medicine is 0.1-0.3 time of that of the polymer material, and the polyethylene glycol polylysine derivative without Ab peptide modification is 1.5-9 times of that of the polyethylene glycol polylysine derivative with Ab peptide modification; the molecular interception of the dialysis bag is 2000-4000.
The invention has the advantages and effects that:
1. the polymeric micelle prepared by a dialysis method is used as a drug carrier, the polymeric material can be self-assembled by the hydrophilic and hydrophobic properties of the material, the preparation process is simple and feasible, and the prepared nanoparticles have uniform particle size distribution and the particle size of 40-100 nm.
2. The polymer micelle nanoparticles prepared by using Ab peptide modified polymer material containing phenylboronate functional groups keep stable blood circulation after being injected into an organism to realize long circulation, and then loaded therapeutic drugs are effectively delivered to AD focus parts through RAGE targeting action of Ab peptide; the phenylboronate structure can promote the degradation of micelle and the release of the drug in a proinflammatory microenvironment with a high oxidation level, increase the accumulation of the drug in brain tissues and improve the AD curative effect of the drug
Drawings
FIG. 1 is a scheme showing the synthesis method of Ab peptide modified polyethylene glycol polylysine phenylboronate derivatives.
Fig. 2 is a Nuclear Magnetic Resonance (NMR) spectrum of Ab peptide modified polyethylene glycol polylysine phenylboronate derivatives.
Fig. 3 is a particle size distribution diagram and a photomicrograph of curcumin-loaded Ab peptide-modified polymeric micelle nanoparticles, wherein a is the particle size distribution diagram of the polymeric micelle, and B is the photomicrograph of the polymeric micelle.
FIG. 4 is a graph showing in vitro degradation behavior of Ab peptide-modified polyethylene glycol polylysine phenylboronate derivatives.
Fig. 5 is a fluorescent image of brain of mice injected with different nanoparticles at tail vein for 4h, wherein A is a live body picture and B is an isolated tissue picture.
Fig. 6 shows the therapeutic effect of AD after injection of free curcumin solution and different nanoparticles into tail vein of mice, wherein a is water maze path diagram and B is brain amyloid deposition diagram.
Detailed Description
The present invention will be described in detail with reference to examples.
Example 1
The Ab peptide modified polymer micelle nanoparticle preparation for loading insoluble AD therapeutic drug curcumin is reported to have multiple action mechanisms of anti-inflammation, antioxidation and the like, is suitable for multi-target treatment of AD, and the polymer synthesis method is shown in figure 1 and comprises the following steps:
1) Synthesis of Ab peptide-modified polyethylene glycol polylysine phenylboronate derivatives
(1) Weighing N 6 1g of carbobenzoxy-L-lysine and 0.3g of triphosgene in 10mL of tetrahydrofuran under the protection of nitrogenReacting at 50 ℃ for 5 hours, precipitating by using 50mL of anhydrous n-hexane, and performing suction filtration and drying to obtain a lysine monomer;
(2) Weighing 210mg of lysine monomer obtained in the step (1) and 100mg of polyethylene glycol, dissolving in 3mL of anhydrous N' N-dimethylformamide, reacting at 55 ℃ for 24 hours under the protection of nitrogen, precipitating by using 30mL of anhydrous ether, and performing suction filtration and drying to obtain a white solid; dissolving 100mg of white solid in a mixture of 1mL of trifluoroacetic acid and 0.1mL of hydrobromic acid (containing 30% acetic acid), reacting at room temperature for 3 hours, dialyzing with pure water, and lyophilizing;
(3) Weighing 100mg of difunctional polyethylene glycol, reacting according to the step (2), dialyzing by using pure water and freeze-drying;
(4) Weighing 20mg of the functionalized polyethylene glycol polylysine polymer obtained in the step (3), 4mg of Ab peptide, 12mg of sodium ascorbate, 1.5mg of cuprous iodide and 1 mu L of N, N-diisopropylethylamine, dissolving the mixture in 5mL of N' N-dimethylformamide, stirring the mixture for reaction at 25 ℃ in the dark under the protection of nitrogen for 8 hours, dialyzing the mixture by using pure water, and freeze-drying the mixture;
(5) Weighing 1g of p-hydroxymethylphenylboronic acid, 1.2g of pinacol and 9g of anhydrous sodium sulfate, mixing the materials in tetrahydrofuran, stirring the mixture at room temperature for 12 hours, filtering out a precipitate, removing the solvent under reduced pressure, redissolving the precipitate with 30mL of ethyl acetate, washing the redissolved solution with 10mL of pure water for 3 times, washing the redissolved solution with 10mL of saturated saline solution for 1 time, drying an organic phase with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain a phenylboronic acid ester activated precursor;
(6) Weighing 400mg of the phenylboronate activated precursor obtained in the step (5), 300mg of polyethylene glycol polylysine polymer, 190mg of 4-dimethylaminopyridine and 150 mu L of triethylamine, mixing in N' N-dimethylformamide, reacting at room temperature for 48 hours under the protection of nitrogen, dialyzing by using pure water, and freeze-drying to obtain a phenylboronate bonded polyethylene glycol polylysine derivative;
2) Preparation of curcumin-loaded Ab peptide-modified polymer micelle nanoparticle preparation
(1) Preparing an N' N-dimethylformamide solution of curcumin with the volume mass concentration of 10 mg/mL;
(2) Weighing 9mg of polyethylene glycol polylysine derivative without Ab peptide modification and 1mg with Ab peptide modification, dissolving in 2mL of N '-dimethylformamide, adding 100 μ L of N' -dimethylformamide solution of curcumin in the step (1) into the solution, filling the mixed solution into a dialysis bag with molecular cut-off of 2000, and dialyzing with 10mM phosphate buffer solution with pH value of 7.0 to obtain Ab peptide modified polymer micelle loaded with curcumin;
the Ab peptide modified polyethylene glycol polylysine phenylboronate derivative is successfully synthesized by the method (shown in figure 2), the prepared micelle is spherical particles, the particle size distribution is uniform, the average particle size is about 60nm (shown in figure 3), the synthesized polymer material is investigated by an in-vitro degradation experiment of the material, and the result shows that after hydrogen peroxide is added into a medium in a pulse mode, the material has a similar pulse type gradual degradation behavior, and the material is proved to have the characteristic of oxidation-sensitive degradation (shown in figure 4).
Example 2 Ab peptide-modified Polymer micelle nanoparticle formulation loaded with near-Infrared Probe
1) Synthesis of Ab peptide-modified polyethylene glycol polylysine phenylboronate derivatives
(1) Weighing N 6 Dissolving 1g of carbobenzoxy-L-lysine and 0.45g of triphosgene in 10mL of tetrahydrofuran, reacting for 8 hours at 55 ℃ under the protection of nitrogen, precipitating by using 80mL of anhydrous n-hexane, and performing suction filtration and drying to obtain a lysine monomer;
(2) Weighing 240mg of lysine monomer obtained in the step (1), 100mg of polyethylene glycol, dissolving the lysine monomer and the polyethylene glycol in 3mL of anhydrous N' N-dimethylformamide, reacting at 60 ℃ for 48 hours under the protection of nitrogen, precipitating by using 30mL of anhydrous ether, carrying out suction filtration and drying to obtain a white solid, dissolving 100mg of the white solid in a mixed solution of 1mL of trifluoroacetic acid and 0.05mL of hydrobromic acid (containing 30% acetic acid), reacting at room temperature for 6 hours, dialyzing by using pure water, and freeze-drying;
(3) Weighing 100mg of difunctional polyethylene glycol, reacting according to the step (2), dialyzing by using pure water and freeze-drying;
(4) Weighing the functionalized polyethylene glycol polylysine polymer obtained in the step (3) at the weight of 20mg, ab peptide at the weight of 8mg, sodium ascorbate at the weight of 24mg, cuprous iodide at the weight of 3mg, and N, N-diisopropylethylamine at the weight of 2 mu L, dissolving the obtained product in N-dimethylformamide at the weight of 5mLN', stirring the obtained product in the absence of light at the temperature of 37 ℃ under the protection of nitrogen for reaction for 12 hours, dialyzing the obtained product by using pure water, and freeze-drying the obtained product;
(5) Weighing 1g of p-hydroxymethylphenylboronic acid, 2.5g of pinacol and 18g of anhydrous sodium sulfate, mixing the materials in tetrahydrofuran, stirring the mixture at room temperature for 18 hours, filtering out a precipitate, removing a solvent under reduced pressure, redissolving the precipitate with 30mL of ethyl acetate, washing the redissolved solution with 10mL of pure water for 3 times, washing the redissolved solution with 10mL of saturated saline solution for 1 time, drying an organic phase with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain a phenylboronic acid ester activated precursor;
(6) Weighing 500mg of the phenylboronate activated precursor obtained in the step (5), 300mg of polyethylene glycol polylysine polymer, 240mg of 4-dimethylaminopyridine and 200 mu L of triethylamine, mixing in N' N-dimethylformamide, reacting at room temperature for 36 hours under the protection of nitrogen, dialyzing by using pure water, and freeze-drying to obtain a phenylboronate bonded polyethylene glycol polylysine derivative;
2) Preparation of Ab peptide-modified polymer micelle nanoparticle preparation loaded with near-infrared probe
(1) Weighing 8mg of polyethylene glycol polylysine derivative without Ab peptide modification and 2mg with Ab peptide modification, dissolving in 1mL of N-dimethylformamide, adding N' -dimethylformamide solution containing 1mg of BODIPY near-infrared probe into the solution, filling the mixed solution into a dialysis bag with molecular cut-off of 3500, and dialyzing with 10mM phosphate buffer solution with pH value of 7.4 to obtain Ab peptide modified polymer micelle loaded with BODIPY;
by intravenous injection of different polymeric micellar nanoparticles into the mouse tail, the results showed significant accumulation of Ab peptide-modified polymeric micellar nanoparticles in brain tissue (as shown in fig. 5).
Example 3 curcumin loaded Ab peptide modified polymeric micelle nanoparticle formulation
1) Synthesis of Ab peptide-modified polyethylene glycol polylysine phenylboronate derivatives
(1) Weighing N 6 Dissolving 1g of carbobenzoxy-L-lysine and 0.5g of triphosgene in 10mL of tetrahydrofuran, reacting at 55 ℃ for 10 hours under the protection of nitrogen, precipitating by using 100mL of anhydrous n-hexane, and performing suction filtration and drying to obtain a lysine monomer;
(2) Weighing 270mg of lysine monomer obtained in the step (1) and 100mg of polyethylene glycol, dissolving in 3mL of anhydrous N' N-dimethylformamide, reacting at 65 ℃ for 72 hours under the protection of nitrogen, precipitating by using 30mL of anhydrous ether, and filtering and drying to obtain a white solid. 100mg of a white solid was dissolved in a mixture of 1mL of trifluoroacetic acid and 0.03mL of hydrobromic acid (containing 30% acetic acid), reacted at room temperature for 8 hours, dialyzed against pure water and lyophilized;
(3) Weighing 100mg of difunctional polyethylene glycol, reacting according to the step (2), dialyzing by using pure water and freeze-drying;
(4) Weighing the functionalized polyethylene glycol polylysine polymer obtained in the step (3) at the weight of 20mg, ab peptide at the weight of 20mg, sodium ascorbate at the weight of 60mg, cuprous iodide at the weight of 8mg, and N, N-diisopropylethylamine at the weight of 5 muL, dissolving the obtained product in N-dimethylformamide at the weight of 5mLN, stirring the obtained solution at the temperature of 50 ℃ in the dark under the protection of nitrogen, reacting the obtained product for 24 hours, dialyzing the obtained product by using pure water, and freeze-drying the obtained product;
(5) Weighing 1g of p-hydroxymethylphenylboronic acid, 5g of pinacol and 30g of anhydrous sodium sulfate, mixing the materials in tetrahydrofuran, stirring the mixture at room temperature for 24 hours, filtering out a precipitate, removing a solvent under reduced pressure, redissolving the precipitate with 30mL of ethyl acetate, washing the redissolved solution with 10mL of pure water for 3 times, washing the redissolved solution with 10mL of saturated saline solution for 1 time, drying an organic phase with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain a phenylboronic acid ester activation precursor;
(6) Weighing 600mg of the phenylboronate activated precursor obtained in the step (5), 300mg of polyethylene glycol polylysine polymer, 300mg of 4-dimethylaminopyridine and 250 mu L of triethylamine, mixing in N' N-dimethylformamide, reacting at room temperature for 48 hours under the protection of nitrogen, dialyzing by using pure water, and freeze-drying to obtain a phenylboronate bonded polyethylene glycol polylysine derivative;
2) Preparation of curcumin-loaded Ab peptide-modified polymer micelle nanoparticle preparation
(1) Preparing an N' N-dimethylformamide solution of curcumin with the volume mass concentration of 20 mg/mL;
(2) Weighing 6mg of polyethylene glycol polylysine derivative without Ab peptide modification and 4mg with Ab peptide modification, dissolving in 0.5mLN 'N-dimethylformamide, adding 150 μ L of N' N-dimethylformamide solution of curcumin obtained in the step (1) into the solution, filling the mixed solution into a dialysis bag with molecular cut-off of 4000, and dialyzing with 10mM phosphate buffer solution with pH value of 7.5 to obtain Ab peptide modified polymer micelle loaded with curcumin;
the results show that the Ab peptide modified polymer micelle nanoparticles can significantly improve the cognitive function of mice by injecting a free curcumin solution and different polymer micelle nanoparticles into the tail veins of the mice, and the water maze path shows that the mice in a treatment group can memorize the position of a platform and swim in the quadrant of the platform for a long time; brain sections showed a significant reduction in amyloid deposition in the brain of the treated mice (as shown in figure 6).
Claims (3)
1. A multi-target treatment micelle for regulating and controlling an Alzheimer's disease microenvironment is characterized in that a nano drug delivery system is prepared by a loaded insoluble drug with an Alzheimer's disease brain regulating and controlling function, a synthesized polyethylene glycol polylysine bonded phenyl borate derivative modified by a polypeptide with an Alzheimer's disease brain microenvironment targeting function and an injection solvent; wherein the polypeptide modified polymer material is 5-20mg/mL, the medicine concentration is 0.2-1mg/mL, and the mass ratio of the medicine to the polypeptide modified polymer material is 1: 5~1: 20;
the polymer material is a polyethylene glycol polylysine bonded phenylboronate derivative, and phenylboronate is connected to a lysine side chain through a covalent bond;
the polypeptide is a specific ligand-Ab peptide of a RAGE receptor, and the polypeptide sequence is KLVFFAED;
the entrapped drug is slightly soluble drug curcumin with the function of Alzheimer brain regulation.
2. The multi-target therapeutic micelle for regulating the microenvironment of alzheimer's disease according to claim 1, wherein said injection vehicle is water for injection or physiological saline.
3. The method of preparing a multi-target therapeutic micelle that modulates the microenvironment of alzheimer's disease of claim 1, comprising the steps of:
1) Synthesis of Ab peptide-modified polyethylene glycol polylysine phenylboronate derivatives
(1) Weighing 1g of N6-benzyloxycarbonyl-L-lysine and 0.3-0.5 g of triphosgene, dissolving in 10mL of tetrahydrofuran, reacting at 50-60 ℃ for 5-10 hours under the protection of nitrogen, precipitating by using 50-100 mL of anhydrous N-hexane, and carrying out suction filtration and drying to obtain a lysine monomer;
(2) Weighing 210-270 mg of lysine monomer obtained in the step (1) and 100mg of polyethylene glycol, dissolving in 3mL of anhydrous N' N-dimethylformamide, reacting at 55-65 ℃ for 24-72 hours under the protection of nitrogen, precipitating by using 30mL of anhydrous ether, carrying out suction filtration and drying to obtain a white solid, dissolving 100mg of the white solid in 1mL of trifluoroacetic acid and 0.03-0.1 mL of hydrobromic acid mixed solution containing 30% acetic acid, reacting at room temperature for 3-8 hours, dialyzing by using pure water, and freeze-drying;
(3) Weighing 100mg of azide/amino bifunctional polyethylene glycol, reacting with a lysine monomer according to the step (2), dialyzing by using pure water and freeze-drying to obtain an azide functionalized polyethylene glycol polylysine polymer;
(4) Weighing the functionalized polyethylene glycol polylysine polymer 20mg, ab peptide 4-20 mg, sodium ascorbate 12-60 mg, cuprous iodide 1.5-8mg and N, N-diisopropylethylamine 1-5 mu L obtained in the step (3), dissolving in 5mL of N' -N-dimethylformamide, stirring and reacting at 25-50 ℃ in the dark for 8-24 hours under the protection of nitrogen, dialyzing by using pure water, and freeze-drying;
(5) Weighing 1g of p-hydroxymethylphenylboronic acid, 1.2-5 g of pinacol and 9-30 g of anhydrous sodium sulfate, mixing the materials in tetrahydrofuran, stirring at room temperature for 12-24 hours, filtering out a precipitate, removing a solvent under reduced pressure, redissolving with 30mL of ethyl acetate, washing with 10mL of pure water for 3 times, washing with 10mL of saturated saline solution for 1 time, drying an organic phase with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain a phenylboronic acid ester activation precursor;
(6) Taking 400-600 mg of phenylboronate activated precursor obtained in the step (5), 300mg of polyethylene glycol polylysine polymer or Ab modified polyethylene glycol polylysine polymer, 190-300 mg of 4-dimethylaminopyridine and 150-250 mu L of triethylamine, mixing in N' N-dimethylformamide, reacting at room temperature for 24-48 hours under the protection of nitrogen, dialyzing with pure water and freeze-drying to obtain phenylboronate bonded polyethylene glycol polylysine derivative;
2) Preparation of Ab peptide modified polymer micelle nanoparticle preparation loaded with insoluble drug
(1) Preparing an N' N-dimethylformamide solution of the loaded medicine with the volume mass concentration of 10-20 mg/mL;
(2) 6-9 mg of Ab peptide-free modified and 1-4 mg of Ab peptide-modified polyethylene glycol polylysine phenylboronate derivatives are weighed and dissolved in 0.5-2mL of N '-dimethylformamide, 50-150 μ L of the N' -dimethylformamide solution of the carried drug in the step (1) is added into the solution, the mixed solution is put into a dialysis bag with molecular cut-off of 2000-4000, and is dialyzed by using 10mM phosphate buffer solution with the pH value of 7.0-7.5, so that the Ab peptide-modified polymer micelle loaded with the insoluble drug is prepared.
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