CN103182087B - Trimethyl chitosan-graft-polyethylene glycol/nucleic acid brain-targeting micellar and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, and relates to an acetylcholine-receptor-mediated trimethyl chitosan-graft-polyethylene glycol/nucleic acid brain-targeting micellar and a preparation method thereof. According to the invention, trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide is obtained through the steps that: chitosan is sequentially subjected to quaternary ammonium modification such that trimethyl chitosan is obtained; the amino group of trimethyl chitosan is subjected to a reaction with an active group of two-end-activated polyethylene glycol, such that trimethyl chitosan-graft-polyethylene glycol is obtained; and another active group of polyethylene glycol is subjected to a reaction with brain-targeting functional peptide RVG. According to the invention, ion composite micellar is adopted as a carrier, and brain-targeting functional peptide RVG is adopted as a targeting molecule, such that nucleic acid medicine can actively target and position through blood-brain barrier and brain. The ion composite micellar provided by the invention is formed through static composition of positively charged trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide and negatively charged nucleic acid. With the invention, nucleic acid medicine defects such as easy in-vivo degradation, poor stability, and low transfection efficiency are solved.

Description

A kind of trimethyl chitosan-graft-polyethylene glycol/nucleic acid brain targeting micelles and its preparation method

technical field

The invention belongs to the field of medical gene therapy, and relates to a preparation method and technology of a novel high-efficiency non-viral vector, in particular to a trimethyl chitosan-graft-polyethylene glycol-brain targeting function mediated by an acetylcholine receptor Peptide/nucleic acid brain targeting micelles and methods for their preparation.

Background technique

The number of people suffering from neurodegenerative and other brain diseases is increasing year by year in the world, but there are still few effective drugs for treating such diseases. Nucleic acid drugs have become the forefront of medical research due to their high specificity, rapid action, safety, and high efficiency.

However, there are insurmountable problems in the application of nucleic acid drugs: first, there are a large number of nucleases in the body, which can hydrolyze the phosphodiester bond of nucleic acid, making the drug degraded and ineffective before reaching the target; second, nucleic acid drugs The permeability to the cell membrane is poor, and it is difficult to reach the target site through the cell membrane. The current methods to solve the above problems mainly use viral or non-viral vectors to realize the administration of nucleic acid drugs. Among them, using the viral shell as a carrier may cause the host's serum immune response. In contrast, non-viral vectors have many advantages such as low immunogenicity, low cost, and no limit on the volume of gene drugs, and have become an important research direction for nucleic acid drug carriers. The non-viral vectors currently studied mainly include liposomes, nanoparticles and polycations. This type of carrier carries nucleic acid with high-density positively charged cationic lipids or cationic polymer materials and negatively charged nucleic acids to form liposomes, nanoparticles, micelles and other delivery systems with strong controllability and low toxicity by means of electrostatic interaction. .

Due to the existence of the blood-brain barrier (BBB) between the central nervous system and the systemic circulation, many potential drugs (especially nucleic acid drugs) are restricted from entering the brain from the blood to exert their effects. At present, there are mainly four methods for promoting the passage of drugs through the BBB. Among them, artificially opening the dense connection on the BBB will non-selectively allow harmful substances and drugs to enter the brain at the same time; structural modification of drugs may reduce or change the efficacy of drugs and pharmacokinetic parameters in vivo; changing the route of administration mainly includes cervical Arterial infusion and nasal administration, the former is easy to cause damage to the brain, while the latter has low bioavailability and is difficult to meet clinical requirements; the above-mentioned shortcomings do not exist through receptor mediation, and it is currently a hotspot in the study of brain drug delivery systems.

In 2007, Priti Kumar et al. (Kumar P, Wu HQ, McBride JL, et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature, 2007, 448: 39-43) reported a 29-amino acid The small peptide RVG has targeting properties of acetylcholine receptors, which are mainly distributed on brain cells, including brain capillary endothelial cells used as a BBB model, which allows the peptide-modified carrier to efficiently penetrate the BBB and target at the same time. to the capacity of brain cells. Experiments have proved that the small peptide can not only pass through the BBB by itself, but also can carry FITC-labeled siRNA into the brain through the 9-polyarginine connected at the carboxy-terminal. After intravenous injection, siRNA effectively silenced the target gene, and no FITC fluorescence was detected in the spleen and liver, which indicated that the small peptide had strong encephalotropic and brain cell-targeting properties.

Graft copolymers containing PEG side chains and polycation backbones and negatively charged nucleic acids can self-assemble into polyion complex micelles through electrostatic complexation. As a nucleic acid drug brain-targeted delivery system, the micelles have the following advantages: The core/shell structure can not only prolong the residence time of the drug in the blood, but also protect the drug from contact with enzymes; the micelles have small particle size, strong penetrating power, and good stability. Active targeting can be achieved after connecting the brain-targeting functional peptide. direction; can pass through the blood-brain barrier; the preparation process is mild, and the drug activity can be kept to the greatest extent.

In summary, the present invention has prepared nucleic acid polyion composite micelles modified with brain-targeting functional peptide RVG to realize the active targeting of nucleic acid drugs through the blood-brain barrier and the brain, and to solve the problem of easy degradation, instability and Clinical application defects such as low transfection efficiency.

Contents of the invention

The purpose of the present invention is to prepare a brain-targeted micelle of trimethyl chitosan-graft-polyethylene glycol-brain-targeted functional peptide/nucleic acid mediated by acetylcholine receptors, so as to realize the passage of nucleic acid drugs Active targeting of the blood-brain barrier and the brain, and solving its clinical application defects such as easy degradation, instability and low transfection efficiency in vivo.

The present invention uses siRNA (sequence 5′-GUGCCUACCUGGACAAGAAdTdT-3′) designed for Alzheimer’s disease as a model drug of nucleic acid drugs, and the technical scheme adopted is as follows (as shown in Figure 1).

Chitosan (Chitosan) is quaternized to produce trimethyl chitosan (TMC), and polyethylene glycol is added to trimethyl chitosan to produce trimethyl chitosan-graft-polyethylene Diol, then, the brain-targeted functional peptide RVG is connected to the other end of the polyethylene glycol to prepare the acetylcholine receptor-mediated trimethyl chitosan-graft-polyethylene glycol-brain-targeted functional peptide (TMC-g-PEG-RVG), so that it has the function of penetrating the blood-brain barrier and targeting the brain. Finally, the material and the negatively charged nucleic acid are self-assembled into brain-targeting micelles through electrostatic complexation.

The preparation of trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide of the present invention comprises the following steps:

1) Purification of chitosan:

The weight average molecular weight of the chitosan in the invention is 5-500kD, and the deacetylation degree is 80%-95%.

The specific purification steps are as follows:

Chitosan was dissolved in 1% acetic acid aqueous solution, and after suction filtration, the pH value of the filtrate was adjusted to 7.0 with ammonia water to precipitate chitosan, and then freeze-dried after suction filtration.

2) Preparation of trimethyl chitosan:

The present invention selects iodomethane as methylation reagent, and concrete preparation method is as follows:

                                                

Add the purified chitosan (Chitosan) into N-methylpyrrolidone (NMP), and stir and swell at room temperature for 12 hours. Add sodium iodide (NaI), sodium hydroxide (NaOH) solution and methyl iodide (CH ₃ I), and react in a water bath at 60°C for 30-60 minutes to complete the first step of methylation. Add sodium hydroxide solution and methyl iodide to continue the reaction for 30-120 minutes to complete the second step of methylation. Add absolute ethanol to the reaction solution and stir for 1 hour to precipitate trimethyl chitosan. After the precipitate is centrifuged, it is washed 3 times with ethanol and 3 times with ether, and then vacuum-dried. After drying, the product was dissolved in 50 g·L ^-1 aqueous sodium chloride solution to exchange I ions for Cl ions, dialyzed in distilled water for 72 hours, and the dialysate was freeze-dried to obtain trimethyl chitosan (TMC).

By changing the reaction time of the first step of methylation and the second step of methylation, a series of TMCs with different degrees of quaternization can be prepared. The degree of quaternization of the trimethyl chitosan prepared by the invention is 10%-99%, and the molar percentage.

3) Preparation of trimethyl chitosan-graft-polyethylene glycol:

Concrete preparation steps are as follows:

 

Trimethyl chitosan (TMC) was reacted with polyethylene glycol activated at both ends in water, the reaction temperature was 4–50°C, and the reaction time was 3–480 minutes. After the reaction, the unreacted polyethylene glycol is removed by ultrafiltration and centrifugation, and the concentrated solution is diluted with distilled water, then ultrafiltration and centrifugation are repeated three times, and finally the ultrafiltrate is freeze-dried to obtain the product.

When the amount of polyethylene glycol used in the above preparation method was 3 times the quality of TMC, the polyethylene glycol grafting rate of the product obtained was 34%; when it was 1.5 times the quality of TMC, the polyethylene glycol grafting rate of the product obtained was 14%; when being 1 times the mass of TMC, the polyethylene glycol grafting rate of the resulting product is 5%.

The general formula of the polyethylene glycol is R ₁ -(CH ₂ CH ₂ O) _n -R ₂ , where n=20–200, and R ₁ is an active group that can react with an amino group to form a polyethylene glycol amide bond Groups, including succinimide groups, acetic acid groups, acetaldehyde groups, isocyanate groups, isocyansulfuric acid groups, acrylic acid groups, nitrophenol groups, _R2 is an active compound that can react with sulfhydryl groups groups, including maleimide groups, vinylsulfone groups, and thiol groups.

4) Preparation of trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide (TMC-g-PEG-RVG):

In the present invention, the brain-targeting functional peptide RVG is selected as the targeting molecule to endow micelles with the function of actively targeting the brain.

The brain-targeting functional peptide RVG (sequence YTIWMPENPRGTPCDIFTNSRGKRASNG) selected in the present invention is characterized in that it contains 29 amino acids (YTIWMPENPRPGTPCDIFTNSRGKRASNG), has acetylcholine receptor targeting, and has a sulfhydryl group (-SH) in its molecular structure.

The specific preparation steps are as follows:

 

Trimethyl chitosan-graft-polyethylene glycol was reacted with brain-targeting functional peptide RVG in pH7–8 phosphate buffer, the reaction temperature was 15–30°C, and the reaction time was 8–24 hours. After the reaction, the unreacted RVG was removed by ultrafiltration and centrifugation, and the obtained concentrated solution was diluted with distilled water and then ultrafiltered three times. Finally, the ultrafiltrate was freeze-dried to obtain TMC- g -PEG-RVG.

  The above-mentioned trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide can self-assemble with negatively charged nucleic acids into active brain-targeting micelles. The specific preparation steps are as follows:

Trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide (TMC-g-PEG-RVG) was dissolved in water, and then sterilized through a 0.22 μm filter membrane. Dissolve nucleic acids in water. Take a certain amount of the above two solutions and mix at room temperature, vortex for 5–30 s to mix evenly, and let stand at room temperature for 15–60 minutes.

The present invention prepares micelles with a +/-charge ratio of TMC-PEG-RVG and siRNA of 1:4-64:1, a particle size distribution of 30-700nm, and a Zeta potential of +1mV-+15mV.

The trimethyl chitosan-grafted-polyethylene glycol-brain targeting functional peptide/siRNA brain active targeting micelles mediated by acetylcholine receptors prepared by the present invention are neurons with acetylcholine receptors expressed on the cell surface The transfection rate of -2a cells was 83%, which was significantly improved compared with the 0.41% transfection rate of naked siRNA.

Confocal microscope imaging shows that the trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide/siRNA brain active targeting micelles mediated by acetylcholine receptors prepared by the present invention can enter in a large amount The results of Neuro-2a cells expressing acetylcholine receptors on the surface are consistent with the results of flow cytometry.

Description of drawings

Fig. 1 is a technical solution diagram of the present invention.

FIG. 2 is a graph showing the results of infrared spectrum characterization of Example 2.

Fig. 3 is example 2 H-NMR characterizing result figure.

Fig. 4 is example 5H-NMR characterization result figure.

Fig. 5 is example 6 H-NMR characterization result figure.

Fig. 6 is the graph of electrophoresis results of Example 7.

FIG. 7 is a diagram of the confocal microscope results of Example 9. FIG.

Detailed ways

The purification of example 1 chitosan

Add 13.8g of chitosan into 1120mL of 1% acetic acid aqueous solution, stir magnetically for 40 minutes to dissolve it, filter with suction, add ammonia water dropwise to adjust the pH value of the filtrate to 7.0 to precipitate chitosan, and filter with 0.45μm filter membrane, The chitosan product on the filter membrane was washed 3 times with double distilled water, and freeze-dried.

The preparation of example 2 N-trimethyl chitosan

sample 1

Weigh 0.5 g of purified chitosan (molecular weight 50 kD, degree of deacetylation 95%) into a 50 mL three-necked flask, add 20 mL of N-methylpyrrolidone, and stir and swell at room temperature for 12 hours. Add 1.2 g of sodium iodide, 2.8 mL of sodium hydroxide solution with a mass concentration of 0.2 g·mL ^-1 and 3 mL of methyl iodide, and stir in a water bath at 60 °C for 45 minutes to complete the first step of methylation. Add 2.8 mL of sodium hydroxide solution and 1.25 mL of methyl iodide to react for 45 minutes to complete the second step of methylation. The reaction solution was added to 200 mL of absolute ethanol and stirred for 1 hour to precipitate trimethyl chitosan. The precipitate was centrifuged (5000 rpm, 15 minutes) and washed three times with ethanol, three times with ether, and then dried in vacuum. After drying, the product was dissolved in 20 mL of 50 g L ^-1 sodium chloride aqueous solution to exchange I ions for Cl ions, and was purified by dialysis in distilled water in a dialysis bag with a molecular weight cut-off of 12–14 kD for 48 hours, and the dialysate was freeze-dried. In N-trimethyl chitosan (TMC).

Take 5 mg of purified chitosan raw material (Chitosan) and synthesized N-trimethyl chitosan (TMC) respectively, and compress it with KBr for infrared spectrum scanning. The results are shown in Figure 2. It can be seen from Figure 2 that compared with chitosan (Chitosan), the synthesized N-trimethyl chitosan (TMC) has a ν _NH peak near 3200 cm ^-1 and a β _NH peak at 1590 cm ^-1 significantly weakened; and a new δ _CH3 peak appeared at 1475 cm ^-1 , which proved that -NH ₂ of chitosan was quaternized to generate -N ⁺ (CH ₃ ) ₃ .

Take 5 mg of synthesized N-trimethyl chitosan (TMC), and use D ₂ O as solvent for ¹ H-NMR (300 MHz) detection, the results are shown in Figure 3. The peak of chemical shift 3.343 in Figure 3 is -N ⁺ (CH ₃ ) ₃ in TMC, and the degree of quaternization of amino groups in chitosan can be calculated by formula 1 to be 32%.

%DQ=[[(CH ₃ ) ₃ ]/[H]×1/9]×100 (1)

In the formula, %DQ is the degree of quaternization of amino groups in chitosan, [(CH ₃ ) ₃ ] is the peak area integral value of the trimethyl group peak with a chemical shift of 3.343, and [H] is the chemical shift at 5.0- 5.7 Integrated peak area of ¹ H peak in m-chitosan.

sample 2

Weigh 0.5 g of purified chitosan (molecular weight 100kD, degree of deacetylation 80%) into a 50 mL three-necked flask, add 20 mL of N-methylpyrrolidone, and stir and swell at room temperature for 12 hours. Add 1.2 g of sodium iodide, 2.8 mL of sodium hydroxide solution with a mass concentration of 0.2 g·mL ^-1 and 3 mL of methyl iodide, and stir in a water bath at 60 °C for 60 minutes to complete the first step of methylation. Add 2.8 mL of sodium hydroxide solution and 1.25 mL of methyl iodide to react for 120 minutes to complete the second step of methylation. The reaction solution was added to 200 mL of absolute ethanol and stirred for 1 hour to precipitate trimethyl chitosan. The precipitate was centrifuged (5000 rpm, 15 minutes) and washed three times with ethanol, three times with ether, and then dried in vacuum. After drying, the product was dissolved in 20 mL of 50 g·L ^-1 sodium chloride l aqueous solution to exchange I ^-1 for Cl ^-1 , and purified by dialysis with distilled water in a dialysis bag with a molecular weight cut-off of 12-14 kD for 48 hours, and the dialysate Freeze dried. According to NMR, the quaternization rate is 99%.

The preparation of example 3 trimethyl chitosan-graft-polyethylene glycol

sample 1

10 mg of trimethyl chitosan and 15 mg of maleimide-polyethylene glycol-succinimide (MAL-PEG-SCM) of sample 1 in Example 2 were dissolved in 2 mL of distilled water and magnetically stirred at room temperature for 6 hours , ultrafiltration and centrifugation to remove unreacted PEG modifiers, the obtained concentrated solution was diluted with distilled water and then ultrafiltration and centrifugation, repeated 3 times, and finally the ultrafiltrate was freeze-dried.

Get the trimethyl chitosan (TMC) of example 2 sample 1, the synthetic trimethyl chitosan-graft-polyethylene glycol (TMC-PEG-MAL) and maleimide-polyethylene glycol - 5 mg each of succinimide (MAL-PEG-SCM) was characterized by ¹ H-NMR (300 MHz) using D ₂ O as a solvent, and the results are shown in Fig. 4 . In Figure 4, compared with TMC-PEG-MAL, the peak with a chemical shift of 3.713 is the peak of the -CH ₂ CH ₂ - group in polyethylene glycol (see Figure 4 MAL-PEG-SCM), with a chemical shift of 6.881 The peak is the peak corresponding to H in maleimide (MAL) in MAL-PEG-SCM. At the same time, the doublet corresponding to H in succinimide (SCM) with a chemical shift of about 2.9 in MAL-PEG-SCM is in TMC-PEG-MAL disappears, all of the above prove that polyethylene glycol has been grafted successfully. Can calculate the graft rate of polyethylene glycol by formula 2 to be 15.4%.

Grafting rate (%)=[PEG]/([H]×248)×100 （2）

In the formula, [PEG] is the peak area integral value of the -CH ₂ CH ₂ - group peak with a chemical shift of 3.713 in TMC-PEG-MAL, and [H] is ¹ H in the chitosan skeleton with a chemical shift of 5.0-5.7 The peak area integral value of the peak, 248 is the number of protons in each polyethylene glycol chain.

sample 2

10 mg of trimethyl chitosan and 30 mg of maleimide-polyethylene glycol-succinimide (MAL-PEG-SCM) of sample 1 in Example 2 were dissolved in 2 mL of distilled water, and magnetically stirred at room temperature for 6 hours , ultrafiltration and centrifugation to remove unreacted PEG modifiers, the obtained concentrated solution was diluted with distilled water and then ultrafiltration and centrifugation, repeated 3 times, and finally the ultrafiltrate was freeze-dried. According to NMR, the grafting rate of polyethylene glycol was 34%.

Example 4 The preparation of trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide (TMC-PEG-RVG)

Trimethyl chitosan-graft-polyethylene glycol 10 mg and 6 mg RVG of sample 2 in example 3 were dissolved in the phosphate buffer saline of 2 mL pH7, magnetic stirring at room temperature 24 hours, ultrafiltration centrifugation (5000g, 1 Hours) to remove unreacted RVG, the obtained concentrate was diluted with distilled water and then ultrafiltered and centrifuged, repeated 3 times, and finally the ultrafiltrate was freeze-dried to obtain TMC-PEG-RVG.

The product was characterized by ¹ H-NMR (300 MHz) using D ₂ O as a solvent, and the results are shown in Figure 5 . In Figure 5, compared with TMC-PEG-MAL, TMC-PEG-RVG and TMC-PEG-MAL, the two groups of peaks with chemical shifts of 0.6-2.0 and 6.7-7.6 should be the peaks of brain-targeting functional peptide RVG, and the chemical shift is 6.881 The peak (the peak of MAL in TMC-PEG-MAL) disappeared, all of the above proved that the brain-targeting functional peptide RVG has been successfully inserted.

Example 5 Preparation of acetylcholine receptor-mediated trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide/siRNA brain-targeting micelles

TMC-PEG-RVG 1.4mg of Example 4 was dissolved in 161 μl of DEPC-treated water, and then sterilized by a 0.22 μm filter membrane. 16.5 μg siRNA was dissolved in 480 μl DEPC-treated water. Take 80 μl of 5 parts of the above-mentioned siRNA solution, and add 5, 10, 20, 40, and 80 μl of the above-mentioned TMC-PEG-RVG solution respectively to prepare the +/- charge ratio of TMC-PEG-RVG and siRNA as 2:1, 4:1, For 8:1, 16:1 and 32:1 micelles, vortex for 20s to mix, and let stand at room temperature for 30 minutes.

The siRNA-loaded micelles of each +/- charge ratio were subjected to electrophoresis experiments with siRNA as a control, and the results are shown in Figure 2. The first band in Figure 2 is the electrophoresis band of the siRNA control, and the remaining bands are +/- charge ratios of 32:1, 16:1, 8:1, 4:1 and 2 from left to right: 1 Electrophoresis band of micelles loaded with siRNA. It can be seen that when the +/- charge ratio reaches 32:1, the siRNA band on the electrophoresis graph has disappeared, indicating that the siRNA has been fully complexed with TMC-PEG-RVG at this time, and is entrapped and protected by micelles.

Table 1 shows the Zeta potential measurement results of siRNA-loaded micelles with each +/- charge ratio.

Table 1 Size distribution and Zeta potential of micelles loaded with siRNA

Example 6 Acetylcholine receptor-mediated PEGylated N-trimethylchitosan siRNA brain-targeted micelles transfected into Neuro-2a cells by flow cytometry

Neuro-2a model cells were cultured in 12-well plates (10 ⁴ /well) for 24 hours, and the medium was replaced with serum-free medium. Among them, 66 μl of PBS with pH 7.4 was added to each well of 3 wells of the blank group; 66 μl of FAM fluorescently labeled siRNA solution (equivalent to 100 pmol siRNA per well) was added to each well of 3 wells of the control group; 3 wells of the preparation group were added to each well 66 μl of FAM-siRNA-loaded micelles with a +/- charge ratio of 32:1 from Example 7 (equivalent to 100 pmol siRNA per well), cultured for 4 hours, washed twice with PBS of pH 7.4, added to complete culture medium for culture . After 16 hours, the cells were digested and centrifuged, and the cells in each well were redispersed in 300 μl of PBS for flow cytometry detection. The test results showed that the transfection rate of the siRNA control was 0.41%, and the transfection rate of the preparation group was 83.42%. , it can be seen that acetylcholine receptor-mediated PEGylated N-trimethyl chitosan siRNA brain-targeting micelles significantly increased the transfection rate of Neuro-2a cells expressing acetylcholine receptors on the surface.

Example 7 Acetylcholine receptor-mediated PEGylated N-trimethyl chitosan siRNA brain-targeted micelles transfection of Neuro-2a cells by confocal microscopy imaging experiment

Neuro-2a model cells were cultured in 6-well plates (10 ⁴ /well) for 24 hours, and the medium was replaced with serum-free medium. Add 66 μl of FAM-siRNA-loaded micelles with a +/- charge ratio of 32:1 (equivalent to 100 pmol siRNA per well) of Example 7 to each well, incubate for 4 hours, wash 3 times with PBS of pH 7.4, add completely culture medium. After 4 hours, they were fixed with 4% paraformaldehyde at 37°C, stained with DAPI, fixed with 50% glycerol, and sent to a confocal microscope for testing. The results are shown in Figure 3.

Claims (9)

1. a trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide/nucleic acid brain targeting micelle, is characterized in that, described micelle is made of positively charged trimethyl shell Glycan-graft-polyethylene glycol-brain-targeting functional peptide and negatively charged nucleic acid are self-assembled through electrostatic complexation, and the prepared micelles of trimethyl chitosan-graft-polyethylene glycol- The +/- charge ratio of brain-targeting functional peptide and nucleic acid is 1:4-64:1, and the trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide is chitosan in sequence After quaternization modification, trimethyl chitosan is obtained, and the amine group of trimethyl chitosan reacts with an active group of polyethylene glycol activated at both ends to obtain trimethyl chitosan-graft-polyethylene glycol Alcohol, obtained by reacting another active group of polyethylene glycol with the brain-targeting functional peptide RVG, the brain-targeting functional peptide is RVG, contains 29 amino acids, and its sequence is YTIWMPENPRPGTPCDIFTNSRGKRASNG. 2. trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide/nucleic acid brain targeting micelles according to claim 1, is characterized in that, described chitosan weight average molecular weight It is 10-150kD, and the degree of deacetylation is 80%-95%. 3. trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide/nucleic acid brain targeting micelles according to claim 1, is characterized in that, the quaternization rate is 10%-99 %, mole percent. 4. trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide/nucleic acid brain targeting micelles according to claim 1, is characterized in that, the general formula of described polyethylene glycol R ₁ -(CH ₂ CH ₂ O) _n -R ₂ , where n=20–200, R ₁ is an active group that can react with amino groups to form polyethylene glycol amide bonds, selected from succinimide groups group, acetic acid group, acetaldehyde group, isocyanate group, isocyansulfuric acid group, acrylic acid group, nitrophenol group, _R2 is an active group that can react with sulfhydryl, selected from maleyl imine group, vinylsulfone group, thiol group. 5. a preparation method of trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide/nucleic acid brain targeting micelles as claimed in claim 1, is characterized in that, comprises the steps: (1) Purification of chitosan; (2) Preparation of trimethyl chitosan; (3) Preparation of trimethyl chitosan-graft-polyethylene glycol; (4) Preparation of trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide; (5) Trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide can self-assemble with negatively charged nucleic acids to form active brain-targeting micelles. 6. The preparation method according to claim 5, characterized in that the reaction temperature in step (3) is 4-50°C, the reaction time is 3-480 minutes, and the grafting rate of polyethylene glycol is 5%-35 %. 7. according to the preparation method described in claim 5, it is characterized in that, in step (4): trimethyl chitosan-graft-polyethylene glycol is mixed with brain target in the phosphate buffer of pH6-9 React to the functional peptide RVG, the reaction temperature is 15-30°C, and the reaction time is 8-24 hours. 8. The preparation method according to claim 5, characterized in that, in step (5), trimethyl chitosan-graft-polyethylene glycol-brain targeting functional peptide is dissolved in DEPC-treated water , and then pass through a 0.22 μm filter membrane to sterilize, dissolve the nucleic acid in DEPC-treated water, mix the above two solutions at room temperature, and vortex for 5-30 s to mix evenly. 9. The preparation method according to claim 5, characterized in that, the particle size distribution of the prepared micelles is 10-700nm, and the Zeta potential is +1mV-+15mV.