CN102160894A - Benzamide analog mediated brain-targeting delivery system - Google Patents

Abstract

The invention belongs to the field of pharmaceutical preparations, and relates to a brain-targeted drug delivery system, which includes a mediator molecule, a carrier and a drug. The mediator molecule is a benzamide analog, and the carrier is a polycation macromolecule. Valence combination, drug loading by entrapment or adsorption. The present invention uses tracer technology to carry out targeted characterization of in vivo and isolated brains, suggesting that the drug delivery system can cross the blood-brain barrier, deliver gene drugs and diagnostic drugs into the brain, and can be used for the diagnosis and treatment of brain diseases. The present invention can avoid the potential risk of invasive drug administration and the complicated drug administration process, has the advantages of large dosage, simple drug administration, etc., and the molecular weight of the brain-targeting mediating molecule is small, cheap and easy to obtain, and convenient for the industry. change.

Description

Brain Targeted Drug Delivery System Mediated by Benzamide Analogs

technical field

The invention belongs to the field of pharmaceutical preparations, and relates to a brain-targeted drug delivery system, in particular to a brain-targeted drug delivery system mediated by benzamide analogs. The drug delivery system can cross the blood-brain barrier, deliver gene drugs and diagnostic drugs to the brain, and can diagnose and treat brain diseases.

Background technique

There are a variety of receptors in the human brain, such as dopamine receptors, serotonin receptors, acetylcholine receptors, opioid receptors, etc. These receptors play an extremely important role in the physiological activities of the human body. Brain diseases such as brain tumors, central nervous system infections, chronic pain, drug-induced recessiveness, epilepsy, periodic migraine, neurodegenerative diseases, schizophrenia, etc. have a huge impact on human health. However, most active drugs cannot pass through the blood-brain barrier (BBB), which makes the prevention, diagnosis and treatment of many brain diseases difficult.

The blood-brain barrier is a regulatory interface between the blood and the neurons of the brain and spinal cord, and regulates the exchange of substances between the central nervous system (CNS) and peripheral blood. The BBB has a three-layer structure: the inner layer is brain capillary endothelial cells (BMEC) and the tight junctions between them, the middle layer is basement membrane and pericytes, and the outer layer is astrocytes and extracellular matrix, among which BMEC and its tight junctions Connectivity is the main factor that makes up the BBB barrier. Due to the existence of BBB and cerebrospinal fluid barrier (BCSFB), almost all macromolecular drugs and 98% of drugs for treating and diagnosing brain diseases cannot enter the brain and central nervous system. In addition to the fat solubility of the drug itself, the relative molecular weight and the ability to form hydrogen bonds, etc. are the reasons why the drug is difficult to pass through the BBB, the BBB also has an outward flux mechanism, that is, some drugs are transported from the brain through the P-glycopeptide on the BBB. In addition, the "enzymatic blood-brain barrier" existing on the BBB, that is, highly active neuropeptide-degrading enzymes, such as aminopeptidase, endopeptidase, angiotensin-converting enzyme (ACE), etc., combined with capillaries, It also affects the treatment of brain diseases due to the unstable metabolism of drugs coupled with peptides. BBB not only effectively protects brain tissue, but also creates an insurmountable barrier for drugs to treat brain diseases.

At present, the methods for overcoming the BBB to increase drug delivery in the brain are divided into two categories: invasive and non-invasive according to the way of administration. Among them, the invasive administration methods include hyperosmotic shock, carotid artery injection of vasoactive substances and direct intracerebral injection. Although this method is effective, it may cause brain infection, BBB damage, and surgical injury, and the administration method is complicated, and the patient's compliance is poor, so it is not suitable for routine treatment and diagnosis. In contrast, non-invasive drug delivery methods have broader prospects for clinical application. Non-invasive drug delivery methods mainly include: drug structure modification, chemical delivery system, carrier-mediated transport, endocytosis transport and nasal drug delivery system. The first three methods all require a certain chemical modification of the drug, which has high requirements on the chemical structure and physical and chemical properties of the drug, and has great limitations.

Endocytosis transport is a transport mechanism in which the cell membrane invagination forms a quilt, the endocytosis drug or the drug-loading system enters the cell, and then the drug or the drug-loading system is expelled from the cell by exocytosis. The process of engulfing, liquid and solute or macromolecular complexes is divided into receptor-mediated endocytosis and adsorption-mediated endocytosis. Receptor-mediated endocytosis into the brain is to use a large number of receptors on the BBB endothelial cells to obtain the corresponding specific antibodies through cloning, and use them as drug carriers to realize the drug delivery in the brain. For example, by means of genetic engineering, a monoclonal antibody to the insulin receptor on the BBB endothelial cell membrane is cloned, and this is used as a drug carrier to deliver neurodiagnostic drugs and nerve center therapeutic drugs that cannot pass through the BBB to the brain. Success; using the newly discovered transferrin receptor monoclonal antibody (OX26) on the BBB, the nerve growth factor (NGF) was linked to the mouse OX26, and the brain transport of nerve growth factor was realized, and it has been confirmed OX26-NGF complex has a good therapeutic effect on Harding's chorea rats. The disadvantage of this method is that it has strong immunogenicity and animal species selectivity, and when it is applied to the human body, humanized antibodies need to be prepared by genetic engineering technology, and the preparation technology and equipment require high requirements and the process is complicated. Adsorption-mediated endocytosis is the use of cationically modified proteins (such as cationic albumin) that are electrostatically adsorbed on the BBB (including sialic acid moieties on the luminal side and heparan sulfate on the basement membrane side). This electrostatic attraction can stimulate adsorption-mediated endocytosis and transport of proteins into the brain. It has also been reported that the ability of NGF to cross the BBB of rats can be increased after covalently binding NGF to a polyamine (such as putrescine).

Benzamide compounds are dopamine _D2 receptor selective antagonists with high specificity and strong affinity, and their extrapyramidal side effects are very small. According to reports, the structure-activity relationship between this type of drug and the dopamine _D2 receptor at the molecular level is: the N atom of the pyrrole ring is one of the sites where this type of compound interacts with the dopamine _D2 receptor. Larger, more conducive to the interaction between the compound and the COO ^- group on the receptor amino acid residue, _and the stronger the affinity with the dopamine _D2 receptor; At another site, the benzene ring binds to the hydrophobic part of the acceptor molecule. The binding is affected by the charge on the plane of the benzene ring and the space structure of the benzene ring. The groups keep the coplanar spatial structure, the stronger the affinity between the compound and the dopamine _D2 receptor. Therefore, the work done by researchers at home and abroad is mainly aimed at structural modification and transformation of the group on the N atom of the pyrrole ring and the side chain of the benzene ring of this type of compound, and has synthesized and studied a series of derivatives successively.

Polyethyleneimine (PEI) is a positively charged polymer material rich in N atoms. It has similar properties to polylysine (PLL), dendrimer PAMAM, and cationic liposomes. It can be combined with DNA, RNA Nanoparticles are formed by electrostatic interaction and are widely used in gene transfection and gene therapy. PEI, PAMAM, etc. can non-specifically bind to the negatively charged capillary epithelial cells on the cell membrane, and are widely used in the construction of targeted drug delivery systems. Currently, there are transferrin-PEG-PAMAM, lactoferrin-PEG-PAMAM brain Related reports on targeted drug delivery systems; folic acid, thyroid hormone, and fibroblast growth factor have also been used to modify PEI to realize the construction of targeted therapy systems for tumors, liver and other parts. At present, there are no reports on the construction of PEI brain-targeting vectors modified by benzamide analogs at home and abroad.

Contents of the invention

The purpose of the present invention is to provide a new brain-targeted drug delivery system, in particular to a brain-targeted drug delivery system mediated by benzamide analogs. The present invention constructs a benzamide analog-polyethyleneimine drug delivery system capable of penetrating the blood-brain barrier and realizing intracerebral drug delivery. The drug delivery system can deliver gene drugs and diagnostic drugs into the brain through intravenous administration, and play therapeutic and diagnostic functions.

The brain-targeted drug delivery system of the present invention includes a mediator molecule, a carrier and a drug, the mediator molecule is a benzamide analog, the carrier is a polycation macromolecule, and the two are covalently combined to form a drug-loading system. Drug loading by entrapment or adsorption can significantly increase the amount of drug entering the brain through the blood-brain barrier.

The drug delivery system is made of nanoparticles or micelles, and the particle diameter is 10-500nm.

In the present invention, the polycationic macromolecule carrier is prepared from the following polymer materials:

Polyethyleneimine (PEI) and polyethylene glycol-polyethyleneimine copolymer (PEG-PEI), wherein: PEI is linear or branched, preferably branched, with a molecular weight of 600-100000Da, preferably 10000-30000Da; The molecular weight of PEG is 1000-20000Da, preferably 2000-5000Da; the polyethylene glycol part of the above PFG-PEI can be monomethoxypolyethylene glycol, or one end is methoxy group, and the other end is other active groups Polyethylene glycol derivatives, PEI and PEG-PEI can be used alone or mixed in any proportion;

Dendrimer PAMAM and polyethylene glycol-PAMAM copolymer (PEG-PAMAM), wherein: PAMAM molecules are divided into G3, G4, G5, G6, G7, G8, G9 generation, preferably G5-G7 generation; PEG molecular weight is 1000-20000Da, the preferred molecular weight is 2000-5000Da; the polyethylene glycol part of the above-mentioned PEG-PAMAM can be monomethoxypolyethylene glycol, or it can be polyethylene glycol with methoxy at one end and other active groups at the other end. Diol derivatives, PAMAM and PEG-PAMAM can be used alone or mixed in any proportion;

Other compounds and their polyethylene glycol copolymers have a high density of positive charges on the surface under physiological conditions.

The above active group is one of maleimide group, sulfhydryl group, amide group, amino group, carboxyl group, biotin or avidin.

The present invention uses benzamide analogs as brain targeting molecules, preferably containing benzamide, benzoic acid, p-hydroxybenzoic acid, o-hydroxybenzoic acid and derivatives thereof.

In the drug delivery system of the present invention, the drug loading method is encapsulation or electrostatic adsorption.

The drug delivered in the present invention may be one or more of diagnostic drugs and gene drugs.

The diagnostic drugs include: nuclear medicine diagnostic drugs such as radioisotope iodine, technetium or indium, and magnetic resonance diagnostic drugs such as metal ion gadolinium. Genetic medicines refer to plasmid DNA containing therapeutic genes, including: brain-derived neurotrophic factor genes for the treatment of neurodegenerative diseases, stroke and brain trauma; tyrosine hydroxylase and aromatic amino acid decarboxylase genes for the treatment of Parkinson's disease; β-glucuronidase gene; hexosaminidase A gene; tumor apoptosis gene; herpes simplex virus thymidine kinase gene; or antisense RNA encoding epidermal growth factor receptor gene; encoding acquired Antisense RNA for immunodeficiency syndrome genes. Plasmid DNA also includes DNA sequences before and after the therapeutic gene, which can be promoters, enhancers, mRNA protein translation and stable DNA sequences that promote the transcription of therapeutic genes, DNA sequences that enable episomes to replicate in transfected cells.

The benzamide analogue-polyethyleneimine intracerebral drug delivery system of the present invention uses new indocyanine green, rhodamine fluorescent labeling and tracking technology, and radioactive ¹²⁵ I labeling and tracking technology to perform in vivo and isolated brain targeting characterization, And the pharmacodynamics evaluation of in vivo transfection in mice was carried out by encapsulating green fluorescent protein (EGFP) reporter gene with benzamide analogue-polyethyleneimine.

1. Fluorescent labeling of p-hydroxybenzoic acid-polyethyleneimine

The p-hydroxybenzoic acid and polyethyleneimine were reacted at room temperature, and the product was purified by gel chromatography.

6-aminocaproic acid and new indocyanine green (IR820) were reacted at 85°C under alkaline conditions, and the product was purified by flash chromatography.

The p-hydroxybenzoic acid-polyethyleneimine and 6-aminocaproic acid-IR820 were reacted overnight at room temperature under alkaline conditions, and the product was purified by gel chromatography.

2. ¹²⁵ I labeling of p-hydroxybenzoic acid-polyethyleneimine

The p-hydroxybenzoic acid-polyethyleneimine was reacted with Na ¹²⁵ I solution at 40°C for 5 minutes, and the product was purified by gel chromatography.

3. Preparation of p-hydroxybenzoic acid-polyethyleneimine/EGFP nanoparticles

p-hydroxybenzoic acid-polyethyleneimine and EGFP were mixed in different proportions to prepare p-hydroxybenzoic acid-polyethyleneimine/EGFP nanoparticles.

4. Animal tissue distribution test

1) p-hydroxybenzoic acid-polyethyleneimine-IR820

Using a small animal in vivo imaging system, observe the distribution of the IR820-labeled drug delivery system in normal mice, and observe the fluorescence distribution in various organs after the mice are sacrificed. Whole brain frozen sections of mice were used to observe the distribution of the drug delivery system in the brain.

2) ¹²⁵ I labeling of p-hydroxybenzoic acid-polyethyleneimine

The distribution of ¹²⁵ I-labeled p-hydroxybenzoic acid-polyethyleneimine in mice was observed by γ-scintillation counter.

5. Pharmacodynamic evaluation of p-hydroxybenzoic acid-polyethyleneimine/EGFP nanoparticles

Using a confocal microscope, observe the in vivo transfection of p-hydroxybenzoic acid-polyethyleneimine-encapsulated EGFP reporter gene in the mouse brain.

The results show the distribution of the above substances in normal mice, suggesting that the p-hydroxybenzoic acid-polyethyleneimine drug delivery system has a good effect of penetrating the blood-brain barrier and brain aggregation, and can realize the EGFP reporter gene in mice. live transfection.

The present invention realizes the function of drug delivery into the brain by means of intravenous injection, which can avoid the potential risk of invasive drug administration and the complicated drug administration process; Simple, patient compliance and other advantages.

Compared with protein polypeptide brain-targeting molecules such as monoclonal antibodies, transferrin, lactoferrin, cationic albumin, and RVG that currently have brain-targeting functions, the p-hydroxybenzoic acid used in the present invention has small molecular weight, no It has the advantages of immunogenicity, simple preparation method, low price and easy availability.

Description of drawings:

Figure 1: Fluorescence distribution of p-HA-PEI-IR820, PEI-IR820, IR820 mouse living tissue,

It shows, 24 hours after mice were injected with PEI-IR820, p-HA-PEI-IR820, and IR820 respectively into tail vein, the in vivo fluorescence imaging pictures after 10% chloral hydrate anesthesia. 1A is PEI-IR820, 1B is IR820 dye, and 1C is p-HA-PEI-IR820. It can be seen from the fluorescence distribution diagram that p-HA-PEI-IR820 has obvious fluorescence distribution in the mouse brain, while IR820 and PEI -IR820 has no fluorescence distribution in the brain, suggesting that p-HA-PEI-IR820 can penetrate the blood-brain barrier and enter the brain through p-hydroxybenzoic acid.

Figure 2: Fluorescence distribution of isolated tissues of p-HA-PEI-IR820 mice,

It shows that mice were injected with p-HA-PEI-IR820 into tail vein at different time points, anesthetized with 10% chloral hydrate, and the fluorescence distribution diagrams of isolated organs of mice were perfused with normal saline heart. 2A is 0.5 hours, 2B is 2 hours, 2C is 12 hours, and 2D is 24 hours. It can be seen from the fluorescence distribution that p-HA-PEI-IR820 has obvious fluorescence at various time points in the mouse brain Distribution, and there is a tendency to gradually aggregate within a certain time point, suggesting that p-HA-PEI-IR820 can penetrate the blood-brain barrier mediated by p-hydroxybenzoic acid and distribute in the brain.

Figure 3: Tissue distribution of ¹²⁵ Ip-HA-PEI mice

It shows that the mice were injected with ¹²⁵ Ip-HA-PEI solution through the tail vein, and the mice were anesthetized with 10% chloral hydrate at 0.5, 1, 2 and 4 hours after the administration, and the heart was perfused with normal saline, and the heart, lungs, and liver were taken. Organs or tissues such as spleen, kidney and brain were weighed, radioactive counts were performed, and the percentage of tissue per unit weight in the total injected radioactive counts (ID%/g) was calculated. The results of tissue distribution suggested that ¹²⁵ Ip-HA-PEI was enriched in the brain to a certain extent.

Figure 4: Confocal image of in vivo transfection in the brain of p-HA-PEI-encapsulated EGFP reporter gene mice

It shows that the mice were injected with p-HA-PEI/EGFP nanoparticles through the tail vein for 4 consecutive days, anesthetized with 10% chloral hydrate, perfused with normal saline and 4% paraformaldehyde (PA), and the brain tissue was injected with 20% and 30% The green fluorescence distribution map of sucrose PA solution after dehydration, fixation, and frozen section. The results showed that EGFP was expressed in the cortex, hippocampus, striatum and other parts, suggesting that p-HA-PEI can carry gene drugs through the blood-brain barrier mediated by p-hydroxybenzoic acid and enter the brain and express.

Detailed ways

The description of the following examples will help to further understand the present invention, but the present invention is not limited to the scope of the following description.

Example 1

Preparation of p-hydroxybenzoic acid-polyethyleneimine-IR820 (p-HA-PEI-IR820)

1. Preparation of p-hydroxybenzoic acid-polyethyleneimine (p-HA-PEI)

Weigh 0.130g of PEI and dissolve in 3ml of DMF, dissolve 7.5mg of p-hydroxybenzoic acid, 9.5mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 1ml of DMF, and stir It was added dropwise into the DMF solution of PEI, and stirred overnight at room temperature to obtain a white suspension. Discard the precipitate after centrifugation, add 100ml of cold ether to the supernatant for precipitation treatment, discard the supernatant after centrifugation, and wash the precipitate with cold ether 3 times, 10ml each time. After vacuum drying for 24 hours, the product was dissolved in a small amount of double-distilled water (dH2O), eluted with dH2O on a G-25 gel column, and the corresponding components were collected and freeze-dried to obtain p-HA-PEI. The results of proton nuclear magnetic spectrum showed that the compound had the characteristic peak of polyethyleneimine at 2.5-3.0ppm, and the characteristic peak of p-hydroxybenzoic acid appeared at 6.8-7.8ppm, p-HA-PEI was confirmed.

2. Preparation of 6-aminocaproic acid-new indocyanine green (IR820)

Weigh 100mg of IR820 and dissolve it in 5ml of DMF, add 46.4mg of 6-aminocaproic acid and 10μl of triethylamine, and react at 85°C for 3 hours under the protection of nitrogen, the solution turns from green to blue. The reaction solution was purified by flash chromatography with gradient elution of ethyl acetate/methanol (70/30 to 0/100), and the corresponding fractions were collected. After vacuum drying for 24 hours, the product was dissolved with a small amount of dH ₂ O, and freeze-dried to obtain 6-aminocaproic acid-IR820.

3. Preparation of p-hydroxybenzoic acid-polyethyleneimine-IR820 (p-HA-PEI-IR820)

Weigh 13 mg of EDC·HCL, add it to 2 ml of anhydrous DMF solution containing 30 mg of 6-aminocaproic acid-IR820, and add it dropwise to 170 mg of p-HA-PEI anhydrous DMF (5 ml) under nitrogen protection. React overnight in the dark, carefully suck off the supernatant, and wash the precipitate with glacial ether three times, 10ml each time. After vacuum drying for 24 hours, the product was dissolved with a small amount of dH ₂ O, and eluted with dH ₂ O on a G-25 gel column. The corresponding fractions were collected and freeze-dried to obtain p-HA-PEI-IR820. The results of H NMR spectroscopy show that the compound has the characteristic peak of polyethyleneimine at 2.5-3.0ppm, the characteristic peak of p-hydroxybenzoic acid at 6.8-7.8ppm, and the characteristic peak of IR820 at 2.1-2.2ppm. p-HA-PEI-IR820 was confirmed.

4. Preparation of polyethyleneimine-IR820 (PEI-IR820)

The preparation method of polyethyleneimine-IR820 (PEI-IR820) is the same as that of p-hydroxybenzoic acid-polyethyleneimine-IR820 (p-HA-PEI-IR820).

Example 2

Animal Test of p-Hydroxybenzoic Acid-Polyethyleneimine-IR820 (p-HA-PEI-IR820) Intracerebral Drug Delivery System

1. Distribution of p-hydroxybenzoic acid-polyethyleneimine-IR820 mouse living tissue

Weigh the appropriate amount of p-HA-PEI-IR820, PEI-IR820 and IR820 respectively, dissolve them with normal saline to prepare a 1 mg/ml solution, and inject 100 μl/mouse into the tail vein, respectively, at 0.5, 2, 4, The mice were anesthetized with 10% chloral hydrate at 12 and 24 hours, and the fluorescence distribution in the mice was observed in the live animal imaging system. It can be seen from the distribution diagram that p-HA-PEI-IR820 has obvious fluorescence distribution in the mouse brain, while IR820 and PEI-IR820 have no fluorescence distribution in the brain, suggesting that p-HA-PEI-IR820 can pass through p-hydroxybenzene Formic acid mediates penetration of the blood-brain barrier into the brain (Figure 1).

2. Distribution of p-hydroxybenzoic acid-polyethyleneimine-IR820 mouse tissue in vitro

Weigh an appropriate amount of p-HA-PEI-IR820, dissolve it with physiological saline to prepare a 1mg/ml solution, inject 100μl/mouse into the tail vein, and use 10% The mice were anesthetized with chloral hydrate, the heart was perfused with 100ml of normal saline, and the heart, lung, liver, spleen, kidney and brain were taken out and placed in the living animal imaging system for observation. The results of fluorescence distribution in various organs show that p-HA-PEI-IR820 has obvious fluorescence distribution in the mouse brain, suggesting that p-HA-PEI-IR820 can penetrate the blood-brain barrier mediated by p-hydroxybenzoic acid and distribute in the brain (figure 2).

3. Distribution in the brain of p-hydroxybenzoic acid-polyethyleneimine-IR820 mice

Weigh an appropriate amount of p-HA-PEI-IR820, dissolve it with physiological saline to prepare a 1mg/ml solution, inject 100μl/mouse into the tail vein, and use 10% Mice were anesthetized with chloral hydrate, followed by saline (100ml/mouse), 4% paraformaldehyde (4% PA 100ml/mouse) cardiac perfusion, the whole brain was taken, rinsed with PBS, placed in 20% and 30% sucrose PA successively Dehydrated in solution, fixed, frozen section, sealed with phosphate glycerol, placed in live animal imaging system to observe the fluorescence distribution of various parts of the brain. The results showed that there were fluorescence distributions in the cerebellum, midbrain, and brain, which further confirmed the ability of p-HA-PEI-IR820 to penetrate the blood-brain barrier and enter the brain.

Example 3

Distribution Test of p-Hydroxybenzoic Acid-Polyethyleneimine ¹²⁵ I Labeled in Mice

1. ¹²⁵ I labeling of p-hydroxybenzoic acid-polyethyleneimine ( ¹²⁵ Ip-HA-PEI)

Add 50μl of p-HA-PEI (65μg/μl), add Na ¹²⁵ I 0.702mCi, react in water bath at 40℃ for 5 minutes, purify on G-25 gel column, elute with gradient HPLC, detect on C-18 chromatographic column, the labeling rate is 100 %, the product activity is 0.636mCi.

2. In vivo distribution test of ¹²⁵ I-labeled p-hydroxybenzoic acid-polyethyleneimine mice

Mice were injected with 100 μl/mouse of ¹²⁵ Ip-HA-PEI solution (0.199 μCi/μl, 1.0 μg/μL) into the tail vein, and were anesthetized with 10% chloral hydrate at 0.5, 1, 2 and 4 hours after the administration, respectively. Perfuse the heart with 100ml of normal saline, take the heart, lung, liver, spleen, kidney, brain and other organs or tissues, weigh them, measure the radioactive count, and calculate the percentage of tissue per unit weight in the total injected radioactive count (ID%/g ). The results of tissue distribution showed that ¹²⁵ Ip-HA-PEI was enriched to a certain extent in the brain (Fig. 3).

Example 4

Pharmacodynamic evaluation of p-hydroxybenzoic acid-polyethyleneimine-encapsulated EGFP reporter gene in vivo transfection in mouse brain

1. Preparation of p-hydroxybenzoic acid-polyethyleneimine/EGFP nanoparticles

Prepare p-HA-PEI and EGFP into 1mg/ml solutions respectively, accurately absorb p-HA-PEI and EGFP respectively according to N/P=12, mix drop by drop, and blow evenly to prepare p-HA-PEI/EGFP nanoparticles.

2. Pharmacodynamic evaluation of in vivo transfection of p-hydroxybenzoic acid-polyethyleneimine/EGFP nanoparticles in mouse brain

Mice were injected with p-HA-PEI/EGFP nanoparticles 100 μl/only (containing EGFP 40 μg) into the tail vein for 4 consecutive days, and 24 hours after the last administration, the mice were anesthetized with 10% chloral hydrate, followed by normal saline ( 100ml/only), 4% PA (100ml/only) heart perfusion, take the whole brain, rinse with PBS, place in 20% and 30% sucrose PA solution successively, dehydrate, fix, cryosection, cover with phosphoglycerol, place in Confocal microscopy was used to observe the expression of green fluorescent protein in various parts of the brain. The results showed that EGFP was expressed in cortex, hippocampus, striatum and other parts (Fig. 4).

Example 5

Preparation of gadolinium-labeled p-hydroxybenzoic acid-polyethyleneimine

1. Preparation of p-hydroxybenzoic acid-polyethyleneimine-diethylenetriaminepentaacetic acid (p-HA-PEI-DTPA)

Weigh 181mg of p-HA-PEI and dissolve it in 2ml of DMF, add dropwise to 20mg of diethylenetriaminepentaacetic acid (SCN-DTPA) substituted by isothiocyanate in 2ml of DMF solution, stir for 3 hours, and the milky white reaction The solution was centrifuged, and the precipitate was kept for use. The supernatant was precipitated by adding ether. The two precipitates were combined, dried in vacuo to remove the ether, and an appropriate amount of water was added to freeze-dry.

2. Preparation of gadolinium-labeled p-hydroxybenzoic acid-polyethyleneimine (p-HA-PEI-DTPA-Gd)

Weigh 100 mg of p-HA-PEI-DTPA and dissolve it in an appropriate amount of water, add 14.5 mg of gadolinium trioxide, adjust the pH (3-4) with dilute hydrochloric acid, stir and react for 30 minutes, put the reaction solution on a G-25 gel column, and purify , to obtain Gd-HA-PEI.

Example 6

Preparation of fluorescein isothiocyanate-labeled p-hydroxybenzoic acid-polyethyleneimine (p-HA-PEI-FITC)

Weigh 250 mg of p-HA-PEI and dissolve it in 3 ml of 0.01M PBS, add dropwise to a dimethyl sulfoxide (DMSO) solution containing 4 mg of fluorescein isothiocyanate (FITC) under stirring, and stir for 2 hours to react. G-25 gel column purification to obtain p-HA-PEI-FITC.

Preparation of p-hydroxybenzoic acid-polyethyleneimine (p-HA-PEI-RITC) labeled with rhodamine isothiocyanate

Weigh 250mg of p-HA-PEI and dissolve it in 3ml 0.01M PBS, add dropwise to the DMSO solution containing 4mg fluorescein isothiocyanate (RITC) under stirring, stir and react for 2 hours, and purify the reaction solution on G-25 gel column , yielding p-HA-PEI-RITC.

Example 7

Preparation of p-hydroxybenzoic acid-PAMAM-(p-HA-PAMAM)

Weigh 0.5g of PAMAM (G5) and dissolve in 5ml of DMF, and dissolve 7.5mg of p-hydroxybenzoic acid and 9.5mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 1ml of DMF , was added dropwise into the DMF solution of PAMAM under stirring, and reacted overnight with magnetic stirring at room temperature to obtain a white suspension. Discard the precipitate after centrifugation, add 100ml of cold ether to the supernatant for precipitation treatment, discard the supernatant after centrifugation, and wash the precipitate with cold ether 3 times, 10ml each time. After vacuum drying for 24 hours, the product was dissolved in a small amount of double distilled water (dH ₂ O), and eluted with dH ₂ O on a G-25 gel column. The corresponding fractions were collected and freeze-dried to obtain p-HA-PAMAM.

Claims (13)

1. a brain-targeted drug delivery system mediated by benzamide analogues, characterized in that it comprises mediator molecules, carriers and medicines, wherein the mediator molecules are benzamide analogues, and the carrier It is a polycationic macromolecule, and the two are covalently combined to form a drug-loading system, and the drug is loaded by entrapment or adsorption. 2. The intracerebral drug delivery system according to claim 1, wherein said benzamide analog is selected from benzamide, benzoic acid, p-hydroxybenzoic acid or o-hydroxybenzoic acid. 3. The intracerebral drug delivery system according to claim 1, wherein said polycationic macromolecules are selected from polyethyleneimine compounds and derivatives thereof, polyethyleneimine-polyethylene glycol copolymers Derivatives, dendrimer PAMAM and its derivatives or derivatives of PAMAM-polyethylene glycol copolymer. 4. The intracerebral drug delivery system according to claim 3, characterized in that the polyethyleneimine compound and its derivatives are linear or dendritic. 5. according to the described intracerebral drug delivery system of claim 3, it is characterized in that the derivative of described polyethyleneimine-polyethylene glycol copolymer, the polyethylene glycol in the derivative of PAMAM-polyethylene glycol copolymer The glycol moiety is monomethoxyether polyethylene glycol, or polyethylene glycol derivatives with other functional groups. 6. The intracerebral drug delivery system according to claim 5, wherein said functional group is one of maleimide, sulfhydryl, amido, amino, carboxyl, biotin or avidin kind. 7. The intracerebral drug delivery system according to claim 3, characterized in that the derivative of the polyethyleneimine-polyethylene glycol copolymer is diethylenetriaminepentaacetic acid-polyethyleneimine-polyethylene Glycol copolymers; derivatives of PAMAM-polyethylene glycol copolymers are diethylenetriaminepentaacetic acid-PAMAM-polyethylene glycol copolymers. 8. The intracerebral drug delivery system according to claim 1, characterized in that the drug delivery system is made of nanoparticles or micelles with a particle size of 10-500nm. 9. The intracerebral drug delivery system according to claim 1, characterized in that the drug contained or adsorbed in the drug delivery system is a gene drug or a diagnostic drug. 10. The intracerebral drug delivery system according to claim 9, wherein the gene drug is selected from brain-derived neurotrophic factor, glial cell-derived neurotrophic factor or tumor necrosis factor-related apoptosis-inducing ligands. body gene. 11. The intracerebral drug delivery system according to claim 9, wherein the diagnostic drug is selected from metal ion gadolinium, radioisotope iodine, technetium or indium, fluorescein isothiocyanate or rhodamine isothiocyanate . 12. The intracerebral drug delivery system according to claim 1, characterized in that the covalent bonding in the drug delivery system is direct covalent bonding or polyethylene glycol bridged covalent bonding. 13. The intracerebral drug delivery system according to claim 2 or 3, characterized in that the benzamide analogs in the drug delivery system are covalently combined with polycationic macromolecular carriers in any pair.

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