WO2023125404A1 - Spinal cord injury targeted drug, polymer-hydrophobic compound micelle, and preparation method for spinal cord injury targeted drug - Google Patents

Abstract

A polymer-hydrophobic compound water-soluble micelle, which can assist in the dissolution of poorly soluble compounds. When the micelle is made into an aqueous solution, the micelle has a targeted enrichment effect and the effect of promoting the penetration of a blood-spinal cord barrier at a spinal cord injury site, can help to enrich poorly soluble neuromodulatory drugs in spinal cord injury tissues, and can be used to prepare targeted drugs for spinal cord injuries. Also provided is a modification method for improving the targeting of poorly soluble neuromodulatory drugs. An ROS-responsive polymer is synthesized by using controlled polymerization, neurotransmitter modifications of a prodrug are used, and a drug is loaded by means of self-assembly to prepare a targeted nano drug for a spinal cord injury.

Description

Spinal cord injury targeting drug, polymer-hydrophobic compound micelle and preparation method thereof technical field

The invention relates to a polymer-hydrophobic compound micelle, in particular to a targeted drug for spinal cord injury, and also to a preparation method for a polymer-hydrophobic compound micelle and a targeted drug for spinal cord injury.

Background technique

There are more than 300,000 to 500,000 new spinal cord injury patients in the world every year, and the number of spinal cord injury patients in China has exceeded 5 million. Clinically, more than 90% of spinal cord injury patients are not completely transected anatomically, and some residual nerve fibers still connect the two ends of the damaged spinal cord after injury, but more than half of the patients are completely functionally lost. At present, functional recovery after spinal cord injury is a major unsolved medical problem in the world. The means and curative effect of clinical treatment are very limited. The root cause of this situation is that the mechanism that hinders functional recovery after spinal cord injury is unclear.

At present, the development of new treatment options from neuroprotection is one of the main research directions in the field of spinal cord injury. However, few drugs have been reported to improve neuronal function after spinal cord injury. The research group of He Zhigang of Harvard Medical School and Gu Xiaosong of Nantong University suggested a therapy (see Non-Patent Document 1), mentioning KCC2 agonists as a promising treatment to promote functional recovery after spinal cord injury, which used (neuron Agonists of specific K+-Cl cotransporters (KCC2 agonists, eg CLP290, CLP257).

Non-Patent Document 1: Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations Cell[J].Volume 174,ISSUE 3,P521-535.e13,July 26,2018

However, known common KCC2 agonists such as CLP290 and CLP257 have poor water solubility, which limits their application. These potential drugs are mainly neuronal ion channel protein regulators, and the actual animal experiments are also very small. In addition, they are ubiquitous, have poor selectivity, and cannot be targeted to the affected area (damaged spinal cord tissue) of patients with spinal cord injury. It is inevitable that the large amount of medication and low delivery efficiency will cause poor functional recovery and large side effects. safety and other shortcomings. In addition, in the existing literature reports (such as the above-mentioned non-patent literature 1), there is still a huge distance between intraperitoneal injection for animal experiments and actual clinical medication.

For this reason, how to solve the above-mentioned problems of neurological drugs (such as CLPs) and increase the possibility of clinical application is a very realistic technical problem.

Contents of the invention

In view of this, the inventors first used the small molecular neurokines secreted by nerve cells to modify the structure of existing nerve drugs (such as CLP) for spinal cord injury to increase the selectivity of drugs for specific nerve cells. Experiments using CLP have shown that the compounds obtained through the existing structural modification can be selectively absorbed by nerve cells, thereby greatly reducing the dosage.

However, there is still poor solubility in water, so this class of drugs needs to overcome the difficulty of solubility if they want to be used clinically. In addition, the spinal cord is different from other organs, and the drug delivery after spinal cord injury also involves a special barrier of the spinal cord-vascular barrier, so it is also crucial to improve the drug through the barrier. Nanomedicines are known to improve drug availability and drug delivery efficiency. Passive targeted delivery and active targeting of specific neurons for spinal cord injury can greatly improve drug efficacy and reduce possible side effects. Therefore, the synthesis of targeted nanomedicine for spinal cord injury needs to be developed urgently

In order to solve the problems of solubility and delivery efficiency of spinal nerve repair drugs, the inventors of the present invention have successfully developed a polymer-hydrophobic compound water-soluble micelle, which can assist the dissolution of insoluble compounds, and the micelle When the bundle itself is made into an aqueous solution, it has a targeted enrichment effect on the spinal cord injury site, and can assist insoluble nerve repair drugs to be enriched in the spinal cord injury tissue, and can be used to prepare targeted drugs for spinal cord injury.

Specifically, the present invention provides a targeted drug for spinal cord injury, which includes amphoteric polymer micelles and hydrophobic nerve repair drugs, and the hydrophobic nerve repair drugs are encapsulated by amphoteric polymer micelles to form water-soluble particles. Micelles are formed by the association of amphiphilic polymers containing hydrophilic segments and hydrophobic segments.

In a preferred embodiment of the present invention, the chemical structure of the hydrophobic neurorestoration drug contains a fragment of small molecule neurokines secreted by neurons. These fragments form chemical bonds through chemical reactions and connect to hydrophobic nerve repair drugs. The small molecule nerve factor fragments secreted by nerve cells have their own transport mechanism in nerve cells. Using these molecular guidance, the effect of hydrophobic nerve repair drugs on nerves can be realized. The active selectivity of cells, the so-called active selectivity, means that the drug itself improves the transport and entry ability in nerve cells through structural modification. After the factor fragment is connected to the hydrophobic nerve repair drug, it can still be removed through cell metabolism after entering the cell, so that the hydrophobic nerve repair drug can play a role in the cell.

In a preferred embodiment of the present invention, the hydrophobic nerve repairing drug is not particularly limited, as long as it is used for nerve repairing and neurotrophic drugs, and it is a drug that is poorly soluble in water, it can be used in the present invention, and can be selected as Any drug from CLP257, CLP290, baclofen, bumetanide, NMDA receptor antagonist CP101606, 8-OHDPAT, quinazine, 4-AP, coupled with the small molecular nerve factors secreted by nerve cells through chemical bonds The resulting hydrophobic nerve repair drug.

In the examples of the present invention, it is confirmed that GABA and DOPA can assist hydrophobic nerve repair drugs to improve the so-called active selectivity. It is speculated that small molecule nerve factors secreted by other nerve cells also have similar effects. In a preferred embodiment of the invention, the nerve Small-molecule neurokines secreted by cells include, but are not limited to, the following small-molecule neurokines secreted by nerve cells.

The so-called modification by these compounds refers to the formation of chemical bonds between the hydrophobic drug molecules and the above-mentioned molecules, and the above-mentioned molecules are connected to the hydrophobic molecules, so that the nerve cells can improve the hydrophobicity through the recognition and transport of the above-mentioned molecules. Transport efficiency of drug molecules into nerve cells.

In a preferred embodiment of the present invention, the hydrophobic nerve repair drug can be selected from CLP257, CLP290, baclofen, bumetanide, NMDA receptor antagonist CP101606, 8-OHDPAT, Any one of quinozine and 4-AP, but not limited to these.

On the other hand, neuroprotection is known to be fundamental to functional recovery after spinal cord injury, so it is also important to provide appropriate post-injury protection. High-intensity ROS after injury is one of the main factors causing sustained spinal cord injury. For this reason, we proposed a method for regulating the ROS environment after injury, the method is to link the ROS sacrificial agent on the amphoteric polymer micelle of the targeting drug for spinal cord injury, that is, in the preferred embodiment of the present invention, the Active oxygen sacrificial groups capable of reacting with active oxygen to consume active oxygen are attached to the amphoteric polymer micelle chain. Furthermore, the active oxygen sacrificial group is preferably a group selected from the following groups,

Wherein, R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkane; group, hydroxyl group, C1-C5 alkyl ether group, C1-C5 alkyl sulfide group.

As long as it is a micelle formed by amphiphilic polymer polymerization, the purpose of the present invention can be achieved, but in order to better encapsulate the hydrophobic nerve restoration drug, the present invention also develops a specific micelle, which is a preferred embodiment of the present invention . Specifically, in a preferred embodiment of the present invention, the amphoteric polymer micelles in the targeted drug for spinal cord injury are micelles formed by the association of amphoteric polymer compounds represented by formula (1),

Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group; ^R1 and ^R2 are each independently selected from hydrogen, C1～C5 alkyl group, cyano group, and R ¹ and R ² are not cyano at the same time; E represents C1~C5 alkylene, C6~C10 arylene, C1~C5 alkylene-C6~C10 arylene, C6~C10 arylene Aryl-C1~C5 alkylene, or E may not exist; R ³ represents C1~C5 alkyl, hydroxyl, COOR ⁵ , R ⁵ represents hydrogen, C1~C5 alkyl, N-succinyl imines, PEG residues;

R ⁴ and R ⁷ are each independently hydrogen or methyl;

R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

R ⁶ represents hydrogen, amino, carboxyl, hydroxyl, C1-C5 alkyl, N-succinimide, PEG residue;

o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

X is one of the groups of the following formulas (2) to (5):

The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl, C1~C5 alkyl ether group, C1~C5 alkyl sulfide group,

In the micelles, the hydrophilic residues in ( ) _p in the compound represented by formula (1) form the outer hydrophilic layer, providing the water solubility of the micelles, and the remaining parts form the inner hydrophobic inner core, and the hydrophobic inner core Hydrophobic nerve restoration drugs are encapsulated, and the diameter of the micelles is 10nm-300nm.

Further preferably, the amphoteric polymer compound shown in formula (1) is the amphoteric polymer compound shown in formula (1-1),

Among them, Z, R ¹ , E, R ³ , R ⁵ , R ⁴ and R ⁷ , R ⁶ , o, p, X, R ⁸ , R ⁹ , and R ¹⁰ have the same meanings as in formula (1), q is an integer of 6-20, Preferably it is an integer of 7-12.

In the present invention, the expression of Ca~Cb means that the number of carbon atoms of the group is a~b, and unless otherwise specified, generally speaking, the number of carbon atoms does not include the number of carbon atoms of the substituent. In the present invention, the expressions of chemical elements generally include the concept of isotopes with the same chemical properties, such as the expression "hydrogen", and also include the concepts of "deuterium" and "tritium" with the same chemical properties, unless otherwise specified.

Therefore, the so-called C1-C15 alkyl group includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl base, n-pentyl, sec-pentyl, neopentyl, n-hexyl, neohexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, dodecyl, etc., but not Among these, C1-C5 alkyl groups are preferred, and preferred groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl , 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, etc.

The C1-C15 alkylthio group refers to the above-mentioned C1-C15 alkyl-S group, and among them, dodecylthio group and the like are preferable.

The term "alkylene group" refers to a divalent group obtained by removing one hydrogen atom from the above-mentioned alkyl group, for example, methylene group, ethylene group and the like.

The so-called C6-C10 aryl group includes phenyl, naphthyl, anthracenyl, phenanthrenyl and the like, among which phenyl is preferred.

The C6-C10 arylene group includes divalent groups obtained by removing one hydrogen atom from the above-mentioned C6-C10 aryl group, among which phenylene and naphthylene are preferable.

In a preferred embodiment of the present invention, Z is preferably methyl, phenyl or dodecylthio; ^R and ^R are preferably hydrogen, methyl, cyano; E is preferably methylene, ethylene, methylene Phenyl, methylene-phenylene, phenylene-methylene,; ^R3 is preferably methyl, ethyl, hydroxyl, ^COOR5 , ^R5 is preferably hydrogen, methyl, ethyl, N-succinate imide, PEG residues;

R ⁴ and R ⁷ are each independently preferably hydrogen or methyl; R ⁶ represents hydrogen or methyl; o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35; q is 6 An integer of -20, preferably 8-12.

The above-mentioned C1～C5 alkylene group-C6～C10 arylene group, C6～C10 arylene group-C1～C5 alkylene group refer to C1～C5 alkylene group and C6～C10 arylene group Specific examples of divalent groups formed by linking groups can refer to the above-mentioned specific examples of C1-C5 alkylene groups and specific examples of C6-C10 arylene groups.

The so-called PEG residues refer to

The group is a residue that is capped with a polyethylene glycol-like structure.

Based on the above micelles, the present invention can provide a targeted drug for spinal cord injury with excellent performance.

The present invention also provides an injection for spinal cord injury, which contains the above-mentioned targeted drug for spinal cord injury of the present invention and pharmaceutically acceptable auxiliary materials.

The invention provides a new drug synthesis method, which has high water solubility, can passively target the spinal cord injury area and can actively target specific neurons, and can promote the recovery of motor function when applied to rehabilitation after spinal cord injury. Specifically, in the preparation method of the targeted drug for spinal cord injury of the present invention, the amphiphilic polymer compound and the hydrophobic nerve repair drug to be encapsulated are dissolved in an organic solvent, and then water is slowly injected into it, stirred slowly, and passed through the Self-assembled to form micelles, then the micelles solution was transferred to a dialysis bag, and deionized water was used for dialysis for 12-72 hours, and the nano micelles solution was freeze-dried,

The organic solvent is selected from DMSO, DMF, tetrahydrofuran, halogenated hydrocarbon solvents, C1-C6 alkanol solvents, ester solvents, benzene, toluene, pyridine and the like. The so-called halogenated hydrocarbon solvent refers to a saturated or unsaturated chlorinated hydrocarbon with 1 or 2 carbon atoms, usually selected from dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane alkane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1, 2,2-tetrachloroethane, pentachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene and tetrachloroethylene. More preferred are dichloromethane, chloroform, 1,1,1-trichloroethane, trichloroethylene, and tetrachloroethylene, and further preferred are dichloromethane and chloroform. As the so-called C1-C6 alkanol solvents, commonly used methanol, ethanol, isopropanol, n-butanol, cyclohexanol, etc. can be used, but not limited to these solvents. Examples of ester solvents include solvents such as ethyl acetate, methyl acetate, and ethyl formate, but are not limited to these solvents.

In a preferred embodiment of the present invention, the organic solvent is selected from DMSO, DMF, THF, halogenated hydrocarbons, C1-C6 alkanol solvents, ester solvents, benzene, toluene, and pyridine.

In a preferred embodiment of the present invention, in the preparation method of polymer-hydrophobic compound water-soluble micelles, the ratio between the amphoteric polymer compound and the hydrophobic compound that needs to be encapsulated is: in terms of mass ratio, the amphoteric polymer compound: Hydrophobic compound to be encapsulated = 3-20:1.

In the preparation method of the polymer-hydrophobic compound water-soluble micelles of the present invention, after the amphoteric polymer is mixed with the hydrophobic compound to form micelles, no further chemical reaction or chemical modification is required, and the formed water-soluble polymer micelles It can be directly used in injection preparations without further chemical reaction or modification, so that the preparation method of the invention is simple and the quality controllability is further improved. Of course, those skilled in the art can perform other chemical modifications after forming micelles according to needs, which is well known.

The targeted drug for spinal cord injury of the present invention is a polymer-hydrophobic compound micelle (hereinafter also referred to as the micelle of the present invention) is a class of nanomicelle, composed of a hydrophilic layer and a hydrophobic inner core, and the hydrophilic layer is composed of block The brush-like PEG segment of the polymer provides the water solubility of the nanoparticles; the hydrophobic core is composed of a hydrophobic layer and a hydrophobic drug, and the hydrophobic layer is composed of phenylboronic acid groups of block polymers and other similar segments. The hydrophobic compound can pass through The self-assembly process is loaded within the hydrophobic layer. The overall size is uniform in appearance, spherical in shape, and has good monodispersity. The micelles of the present invention are particularly suitable for encapsulating hydrophobic drugs.

The above micelles of the present invention have good water solubility and can be made into injections. Therefore, the micelles of the present invention can be used for injection administration of poorly soluble drugs.

In a preferred embodiment of the present invention, in the preparation method of the targeted drug for spinal cord injury of the present invention, the amphoteric polymer compound used for self-assembly is preferably a compound represented by the above formula (1), more preferably the above formula (1- 1) Compounds shown.

It has been confirmed by animal experiments that the micelles of the present invention have a very obvious enrichment effect in the damaged spinal cord tissue after being administered by injection. Since the micelle of the present invention not only solves the water-solubility problem of poorly soluble drugs, but also has an enrichment effect in damaged spinal cord tissue, it greatly improves the delivery efficiency of therapeutic drugs after spinal cord injury.

In a preferred embodiment of the present invention, a targeted drug for spinal cord injury is provided, which comprises the polymer-hydrophobic compound water-soluble micelle according to claim 1, and the hydrophobic compound is a hydrophobic drug.

In a preferred embodiment of the present invention, X is preferably a group represented by formula (2), which has excellent encapsulation efficiency, is easier to prepare, and has less toxicity.

The principle of the present invention is shown in Figure 1. The targeted nanomedicine is mainly composed of two parts, one of which is a block amphiphilic polymer, in which the hydrophobic part of the chain segment has the ability to react with active oxygen to provide ROS sacrificial agents and response functions. The water chain segment provides nanometer water solubility; the second is targeting small molecule drugs, the main drug components of which are known neuron-related ionic protein agonists/inhibitors, and the targeting end is composed of neurotransmitters and their derivatives.

In a preferred embodiment of the present invention, the hydrophobic drug is preferably GABA-modified CLP257, which has higher binding efficiency and delivery efficiency in the micelle delivery system of the present invention, and has significantly improved water solubility.

The present invention also provides a method for constructing active targeting drugs for neurons. The so-called active targeting refers to increasing the uptake rate of drugs by neuron cells through structural modification. In contrast to the passive targeting effect, the aggregation effect of the micelles of the present invention in the spinal cord injury tissue can be understood as a passive targeting effect. If the passive targeting effect of the micelles of the present invention is combined with the neuron active targeting drug, the most ideal technical effect of the present invention can be realized.

The invention realizes the construction of the neuron-targeted drug by linking the specific neurotransmitter with the drug precursor. Taking GABAergic neurons as the target and neuronal excitability regulator CLP-257 as the prodrug as an example, a brief introduction to its construction method is as follows (it should be noted that the following example is only for a clearer explanation , the synthesis method of the present invention is not limited thereto): dissolving CLP-257 in dimethyl sulfoxide (DMSO); then adding N, N'-carbonyldiimidazole, stirring slowly for 30 minutes; then adding γ-aminobutyric acid (GABA), reacted overnight. The mixture was precipitated in deionized water and filtered to obtain a pale yellow solid. The resulting solid was then repeatedly washed with a large amount of methanol, and vacuum-dried to obtain the drug GABA-CLP that finally actively targets GABAergic neurons.

In another embodiment of the present invention, it provides a polymer-hydrophobic compound water-soluble micelle, which is formed by encapsulating the hydrophobic compound in the micelle formed by the association of amphoteric polymer compounds represented by the above formula (1). The amphoteric polymer compound represented by the formula (1) of the present invention can not only be used in the present invention, but also can be used for the coating of other hydrophobic compounds to form water-soluble micelles, thereby improving the clinical use efficiency of poorly soluble drugs. At the same time, the present invention also provides a method for solubilizing insoluble drugs or insoluble compounds. The so-called solubilization refers to increasing water solubility. Poorly soluble drugs or poorly soluble compounds.

In another embodiment of the present invention, an amphoteric polymer compound represented by the above formula (1) is provided, which can be used to form water-soluble micelles and can be used to solubilize poorly soluble substances. There are a large number of active oxygen sacrificial groups on its surface, which is especially suitable for spinal cord injury drug delivery system, used for solubilization and drug delivery of poorly soluble drugs, not only increasing solubility, but also increasing tissue selectivity for spinal cord injury, It can also improve the therapeutic effect through active oxygen sacrificial groups, and has various excellent technical effects.

In another embodiment of the present invention, a method for preparing an amphoteric polymer compound represented by formula (1) is provided, which comprises the following steps in sequence,

Hydrophilic segment construction step S1, using the chain transfer catalyst shown in formula (6), in the presence of a free radical initiator, the compound shown in formula (7) undergoes a free radical polymerization reaction, by controlling the formula (6) The equivalent ratio of the shown chain transfer catalyst and the compound shown in formula (7) controls the degree of polymerization to be 20～40 to obtain the compound shown in formula (8),

The hydrophobic segment is combined with step S2 to make the compound represented by the formula (8) react with the compound of the formula (9) by free radical polymerization, and control the degree of polymerization to be 2 to 4 by controlling the equivalent ratio and reaction time to obtain the compound represented by the formula (1). Represents amphoteric polymer compounds,

Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group, preferably methyl, phenyl, dodecylthio group; ^R1 and ^R2 are each independently Selected from hydrogen, C1-C5 alkyl, cyano, and R ¹ and R ² are not cyano at the same time; E represents C1-C5 alkylene, C6-C10 arylene, C1-C5 alkylene Group-C6～C10 arylene group, C6～C10 arylene group-C1～C5 alkylene group, or E may not exist; R ³ represents C1～C5 alkyl group, hydroxyl group, COOR ⁵ , R ⁵ represents hydrogen, C1-C5 alkyl, N-succinimide, PEG residue;

R ⁴ and R ⁷ are each independently hydrogen or methyl; R ⁶ represents hydrogen, C1-C5 alkyl, hydroxyl;

R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

X is one of the groups of the following formulas (2) to (5):

The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl group, C1-C5 alkyl ether group, C1-C5 alkyl sulfide group.

In the preparation method of the preferred amphoteric polymer compound of the present invention, the compound of formula (7) is the compound of following formula (7-1), the compound of formula (9) is the compound of following formula (9-1),

The synthesis method of the amphoteric polymer compound represented by the above formula (1) of the present invention is essentially to self-assemble the hydrophilic-lipophilic block polymer prepared by controllable polymerization in aqueous solution to form nanocarriers. First prepare the water-soluble polymer with controllable chain segment length by controlled polymerization method, preferred polymer can be POEGMA (methacrylic acid polyethylene glycol monomethyl ether, namely in above-mentioned formula (7), R Be ^hydrogen , R ⁴ is methyl).

The chain transfer catalyst shown in formula (6) can be synthesized by oneself, also can obtain commercially available product, for example can obtain following available chain transfer catalyst from Merck reagent company: 4-cyano group-4-(phenyl thioformyl Thio)valeric acid, 4-cyano-4-[(phenylthiomethyl)thio]-2,5-dioxo-1-pyrrolidinylvaleric acid (Cas No.864066-74- 0), 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (Cas No.461642-78-4), 2-cyano-2-propyldodecyl Trithiocarbonate (Cas No.870196-83-1), 2-cyano-2-propyl-4-cyanobenzenedithiocarbonate (Cas No.851729-48-1), polyethylene glycol Alcohol-4-cyano-4-(phenylcarbonylthio)pentanoate PEG CTA, 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (Cas No.461642-78- 4). As the chain transfer catalyst represented by the formula (6), 4-cyano-4-(phenylthioformylthio)pentanoic acid is preferably used.

The radical polymerization initiator used in the above method of the present invention is not particularly limited, and azo compounds and peroxides can be used. As an azo free radical polymerization initiator, it is a compound containing an azo group -N=N- in the molecular structure and connected with two alkyl groups (R, R'). The general formula is R—N=NR’, which can decompose under the action of light and heat to release nitrogen and generate free radicals, such as azobisisobutyronitrile (AIBN), azobisisoheptanonitrile and azo Dimethyl diisobutyrate initiator, etc. Azo compounds with hydrophilic groups such as carboxyl and sulfonic acid groups are suitable for aqueous solution polymerization, and the water-soluble ones include azodiisobutylamidine hydrochloride (V-50 initiated agent), suitable for initiating decomposition reactions at moderate temperatures.

As a peroxygen compound, it is a kind of compound containing peroxy group (—O—O—). After being heated, the—O—O—bond is broken and split into two corresponding free radicals, thereby initiating the polymerization of monomers, which is called peroxide. oxide initiator. There are two types of inorganic peroxides and organic peroxides. Inorganic peroxide initiators include hydrogen peroxide, ammonium persulfate or potassium persulfate, etc., which are soluble in water and used as initiators for aqueous solution polymerization and emulsion polymerization; organic peroxide initiators include benzoyl peroxide , benzoyl tert-butyl peroxide, methyl ethyl ketone peroxide, etc. Preferably used in the present invention are azos, particularly preferably AIBN.

Taking the polymer POEGMA as an example, the general synthesis process is as follows. It should be noted that the following examples are only for clearer explanation, and the synthesis method of the present invention is not limited thereto.

The first step is to dissolve polyethylene glycol monomethyl ether methacrylate, controllable chain transfer agent (chain transfer catalyst shown in formula (6), 2,2-azobisisobutyronitrile (AIBN) in 1, 4 dioxane; after it is fully dissolved, add the mixed solution to the Schlenk tube, connect it with nitrogen, and fill it with nitrogen; then the mixed solution is frozen by liquid nitrogen and vacuum pumped for 5-10 minutes, and the After thawing with nitrogen filling, repeat freezing and pumping three times; then transfer the thawed mixed solution to a 70°C oil bath, continue filling nitrogen, and stir slowly for 12-24 hours; then remove the solvent in the mixed solution by rotary evaporation, and dry it in anhydrous Settled three times in ether; finally obtained a red oily polymer POEGMA.

The second step is also to synthesize a hydrophobic ROS-responsive segment after the obtained water-soluble polymer through a controlled polymerization method (the so-called ROS-responsive segment refers to that the phenylboronic acid in the phenylboronic acid segment can react with active oxygen and consume Active oxygen generates phenolic groups and produces phenylboronic acid which is removed from the polymer main chain, which affects the stability of micelles), such as polymerizing POEGMA and continuing to polymerize 3-acrylamidophenylboronic acid (BAA) to obtain POEGMA-BAA , and its synthesis process is as follows:

Dissolve the polymers POEGMA, AIBN, and BAA in NN dimethylformamide; after they are fully dissolved, add the mixed solution to the Schlenk tube, connect nitrogen gas, and fill with nitrogen for 2 minutes; then the mixed solution is frozen by liquid nitrogen Finally, vacuum pump for 5-10 minutes, and then repeat the operation three times after filling with nitrogen at normal pressure; then, transfer the thawed mixture to an oil bath at 70°C, continue filling with nitrogen, and stir slowly for 6-24 hours; then remove it by rotary evaporation solvent in the mixed solution, and settled twice in anhydrous ether, then dissolved in water and dialyzed for 24-72 hours to obtain an aqueous solution which is the nanocarrier. The yellow oil obtained by freeze-drying is the polymer POEGMA-BAA.

The present invention cleverly designs the amphoteric polymer compound shown in formula (1), so that hydrophobic molecules, especially hydrophobic drugs, can be conveniently self-assembled with it into nano-micelle particles. Still taking the above-mentioned POEGMA-BAA and the drug GABA-CLP as examples, the operation method of self-assembly will be introduced in detail. First, POEGMA-BAA and GABA-CLP were dissolved in DMSO, then slowly injected into ionized water and stirred slowly to form nanoparticles by self-assembly. Then the nanoparticle solution was transferred to a dialysis bag and dialyzed in deionized water for 24-72 hours; finally, the solution was lyophilized to obtain the nanomedicine GABA-Nano and stored in a refrigerator at 4°C.

The invention utilizes controllable polymerization to synthesize ROS-responsive polymers, utilizes the neurotransmitter modification of drug prodrugs, and loads drugs through self-assembly to prepare targeted nano-medicines for spinal cord injuries.

The present invention provides targeted nano-medicine for spinal cord injury and its synthesis method. Compared with the prior art, it has the following significant advantages:

1. The resulting nano-drugs are far superior to ordinary small-molecule drugs in water solubility, and have no obvious cytotoxicity and in vivo side effects;

2. The obtained nanomedicine can be efficiently enriched to the injured spinal cord, and can effectively target specific neurons to improve the efficacy of the drug;

3. The obtained drug can be easily injected into the tail vein, which is different from the more dangerous drug methods such as intrathecal injection of the spinal cord and secondary surgery, and the safety and possible clinical risks are greatly reduced;

4. The obtained drug has a long metabolism time in the body, and due to the targeting effect, etc., its dosage can be greatly reduced to reduce the side effects of the drug.

5. Experimental verification of rat contusion spinal cord injury model: the obtained targeted nanomedicine can effectively protect residual cells after injury, target and activate the function of dormant neurons, and significantly improve the level of functional recovery after spinal cord injury.

Description of drawings:

Figure 1 is a schematic diagram of the synthesis method of targeted nano-medicine for spinal cord injury;

Figure 2 is a diagram representing the synthesis and characterization of nanomedicines;

Figure 3 is a diagram of the characterization of targeted drugs;

Fig. 4 is the figure of the cell safety and antioxidant characterization of nanomedicine;

Figure 5 is a diagram of the spinal cord enrichment and targeting capabilities of nanomedicines;

Fig. 6 is the graph of the breakthrough spinal cord-vascular barrier ability of nanomedicine;

Figure 7 is a graph of the in vivo safety of nanomedicines;

Fig. 8 is the figure that nano-medicine is to the evaluation of spinal cord tissue protection ability;

Fig. 9 is a figure of functional recovery evaluation under ROS NANO and PBS treatment after spinal cord injury;

Figure 10 is a graph showing the functional recovery evaluation of nano-medicines GABA Nano and DOPA Nano after spinal cord injury.

Detailed ways:

The present invention will be described in further detail below in conjunction with embodiment and accompanying drawing, but embodiment of the present invention is not limited thereto:

The abbreviation meaning used in the present invention:

BAA: 3-acrylamidophenylboronic acid;

GABA: gamma-aminobutyric acid;

ROS: reactive oxygen species;

ROS NANO: active oxygen sacrificial agent nanoparticles;

PBS: Phosphate Buffered Saline;

CCK-8: Cell Viability Detection Kit;

LPS: lipopolysaccharide;

DOPA: dopamine;

SDS PAGE: Sodium Lauryl Sulfate – Polyacrylamide;

PBST: phosphate buffered saline containing Tween-20;

iNOS/IBA1: Activated immune cell specific antibody/microglial cell specific antibody;

GFAP/NeuN: Astrocyte-specific antibody/Neuron cell-specific antibody.

Example 1:

The preparation process of targeted nano drug GABA Nano:

In the first step, the process of synthesizing POEGMA ₃₀ is as follows:

Polyethylene glycol monomethyl ether methacrylate (Mn=475, 1.9g, 40mmol, 40equ.), 4-cyano-4-(phenylthioformylthio)valeric acid (28mg, 0.1mmol , 1equ.), AIBN (1.6mg, 0.01mmol, 0.1equ.) were dissolved in 1,4-dioxane (3mL); after it was fully dissolved, the mixture was added to the Schlank tube, and nitrogen was connected , filled with nitrogen for 2 minutes; then the mixed solution was frozen with liquid nitrogen and then vacuum pumped for 5-10 minutes, after being thawed with nitrogen at normal pressure, repeated freezing and pumping for three times; then the thawed mixed solution was transferred to a 70°C oil bath , continue to fill with nitrogen, stir slowly for 12 hours; then remove the solvent in the mixed solution by rotary evaporation, and settle in anhydrous ether three times; finally obtain a red oily substance that is the polymer POEGMA. According to NMR measurement (as shown in Figure 2.B), the degree of polymer is 30, that is, the polymer POEGMA ₃₀ is obtained.

The second step, followed by the synthesis of POEGMA ₃₀ -BAA ₂ , the synthesis process is as follows:

Polymer POEGMA ₃₀ (1.41g, ~0.1mmol, 1equ.), AIBN (1.6mg, 0.01mmol, 0.1equ.), BAA (12mg, 0.062mmol) were dissolved in N,N-dimethylformamide (1.5 mL); after it is fully dissolved, add the mixed solution to the Schlenk tube, connect it with nitrogen, and fill it with nitrogen for 2 minutes; then the mixed solution is frozen in liquid nitrogen, vacuum pumped for 5-10 minutes, and then thawed with nitrogen at normal pressure Then, repeat the operation three times; then, transfer the thawed mixed solution to an oil bath at 70°C, continuously fill with nitrogen, and stir slowly for 12 hours; then remove the solvent in the mixed solution by rotary evaporation, and settle twice in anhydrous ether, and then Dissolved in water and dialyzed for 24 hours to obtain an aqueous solution which is the nanocarrier. The yellow oil obtained by freeze-drying is the polymer POEGMA ₃₀ -BAA ₂ .

The characterization results are shown in Figure 2. The specific meanings of each part in Figure 2 are: (a, b, c) the synthesis route of a typical polymer and its H NMR characterization; (d) the size characterization of nanomicelles formed by different polymers; ( e) The data table of nanomicelle size and drug-loading capacity obtained by the synthesis of several polymers with different ratios; (f, g, h) TEM images of typical three types of drug nanomicelles ROS Nano, GABA Nano and DOPA Nano.

The third step is the targeted modification of the drug, which is the synthesis of GABA-CLP. The synthesis process is as follows:

First, CLP-257 (30.7 mg, 0.1 mmol, 1 equ.) was dissolved in DMSO (2 mL); then N, N'-carbonyldiimidazole (20 mg, ~0.12 mmol, 1.2 equ.) was added, and the reaction was stirred slowly for 30 minutes ; Add GABA (11 mg, 0.1 mmol, 1 equ.) to the mixture and react overnight. The mixture was precipitated in deionized water several times, and filtered to obtain a light yellow solid. Subsequently, the resulting solid was repeatedly washed with a large amount of methanol; vacuum-dried to finally obtain the drug GABA-CLP that actively targets GABAergic neurons. Figure 3 (a, b) shows the H NMR spectra of KCC2 agonists GABA-CLP and DOPA-CLP modified by γ-aminobutyric acid and dopamine, respectively.

The fourth step is the loading of targeted nano-drugs, the process is as follows:

First, POEGMA ₃₀ -BAA ₂ (10 mg) and GABA-CLP (5 mg) were weighed and dissolved in DMSO (10 mL), then slowly injected into ionized water (5 mL) and stirred slowly to form nanoparticles by self-assembly. Then the nanoparticle solution was transferred to a dialysis bag and dialyzed in deionized water for 24 hours; finally, the solution was lyophilized to obtain the nanomedicine GABA-Nano and stored in a refrigerator at 4 °C.

Example 2 The drug efficacy testing process of targeted nanomedicine GABA Nano:

2.1. Cell safety and in vivo safety testing;

2.1.1 Polymer-hydrophobic compound water-soluble micelles have good cell safety

In order to determine the cell safety of polymer-hydrophobic compound water-soluble micelles (sometimes referred to as micelles for short in the present invention). GABA Nano and PC12 cells were co-cultured, and their cytotoxicity was observed. The results are shown in Figure 5. GABA Nano showed high cell safety, and it still did not show obvious cytotoxicity at a concentration of 0.5mg/mL. Specifically, the specific meanings of each part in Figure 4 are, (a, b) the life-and-death fluorescence photos and related CCK8 statistics after co-culture of different concentrations of ROS Nano and PC12 cells for 24 hours;

2.1.2 Polymer-hydrophobic compound water-soluble micelles have good in vivo safety;

Through the intravenous injection experiment of GABA-Nano (Cy5.5) (see above for the steps), its in vivo distribution results are shown in Figure 6. It can be concluded that GABA Nano can circulate in the body for a long time and can fully enter the injured spinal cord. Its acute metabolism is mainly through liver and kidney metabolism, and long-term metabolism is mainly through kidney metabolism, showing high safety. Through the H&E staining sections of the main organs in rats after one week of intravenous injection of GABA-Nano, it can be seen that it has no adverse effects on internal organs and has good in vivo safety. The results are summarized in Figures 7-10. Figure 7 shows the in vivo safety of nano-drugs; the specific meanings of each part are: (a, b) the metabolic distribution of main organs in the nano-drug; (c) H&E tissue sections of main organs and normal organs in the body after administration Compare;

2.2. Spinal cord enrichment and targeting effect test;

To evaluate the targeting effect and biodistribution of the nanoparticles, GABA Nano@cy5.5 and DOPA Nano@cy5.5 were injected through the tail vein 3 hours after injury. At regular intervals (3, 6 and 24 hours after injection), rats were deeply anesthetized with sodium pentobarbital (0.5 ml/100 g) and intracardiacly perfused with 4% paraformaldehyde. The heart, liver, spleen, lung, kidney, brain and spinal cord were collected and observed with an in vivo fluorescence imaging system (CRi Company, MK50101-EX, USA). To test the window of micelles across the blood-brain barrier, intact animals and SCI animals were injected with GABA Nano@cy5.5 at 4, 7, and 14 days after injury. Spinal cords were dissected 6 hours after injection and tested with an in vivo fluorescence imaging system and immunofluorescent histology.

The details are as follows: in the first step, GABA-Nano (Cy5.5) is obtained through the above synthesis process by using the fluorescent agent Cy5.5 as a fluorescent label instead of the drug CLP257; in the second step, GABA-Nano (Cy5.5) is injected through the tail vein The method is to enter the spinal cord injury model rats; the third step is to take rats at different time periods (3h, 6h, 24h) to observe the spinal cord enrichment and GABAergic neuron targeting effect of GABA-Nano (Cy5.5). The characterization results are shown in Figure 4. GABA-Nano (Cy5.5) exhibits high enrichment of the spinal cord injury area and good targeting effect. Specifically, the specific meanings of each part in Figure 5 are: (a, b) Nanomedicine Schematic diagram of the experimental process of timely delivery and fluorescent labeling after spinal cord injury; (c, d, e) fluorescent photos of the spinal cord in vivo and related statistics of nanomedicines; (f, g, h.i) photos of cell targeting of nanomedicines in the spinal cord and related statistics; (j) Targeting efficiency of nanomedicines GABA Nano and DOPA Nano. The specific meanings of each part in Figure 6 are: (a, b) schematic diagram of the delivery of nano-drugs breaking through the spinal cord vascular barrier and the design of related animal experiments; (c, d) fluorescent photos of the spinal cord in vivo of nano-drugs and related statistics; (e) nano-drugs Fluorescent photographs of drugs breaking through the spinal cord-vascular barrier.

2.3. Drug efficacy evaluation for spinal cord injury;

2.3.1 Polymer-hydrophobic compound water-soluble micelles protect cells from ROS damage;

In order to determine whether polymer-hydrophobic compound water-soluble micelles (sometimes simply referred to as micelles in the present invention) can protect cells from ROS-induced apoptosis. Medium containing H ₂ O ₂ (100 μM) was used in cell culture. ROS Nano, GABA Nano, and DOPA Nano were also added to the medium at concentrations ranging from 0 to 1 mg/ml, respectively. After 24 hours, cells were washed twice with PBS and detected with CCK-8.

To observe ROS production, we used LPS-containing medium to mimic the ROS microenvironment after tissue injury. Cells were cultured with medium containing LPS (100ng/mL), and treated with ROS Nano, GABA Nano and DOPA Nano (250μg/ml) for 6 hours. Then, 10 μM of DCFH-DA was added, and DCFH-DA detection was performed at 37°C for 20 min, followed by observation with an inverted fluorescence microscope.

Specifically, Figure 4(c) Quantitative statistics of dead and alive CCK8 after three drug nanomicelles ROS Nano, GABA Nano and DOPA Nano were co-cultured with PC12 cells treated with H ₂ O ₂ (100 μM) for 24 hours at different concentrations; (d, e) Fluorescent photographs and statistics of the ROS intensity comparison in PC12 cells treated with three drug nanomicelles ROS Nano, GABA Nano and DOPA Nano and lipopolysaccharide LPS (100ng/mL). In addition, nanoparticles at a concentration of only 0.25 mg/mL can also significantly increase the survival rate of cells in the presence of hydrogen peroxide at a concentration of 100 μM.

2.3.2 Animals and surgical procedures;

The weekly intravenous injection of GABA Nano was used to observe the functional recovery within 9 weeks on the rat spinal cord injury model. The specific operation method is as follows:

All animal protocols were approved by Institutional Animal Care and used in accordance with the regulations of the Animal Experiment Committee of Zhejiang University (ZJU202010110). Sprague-Dawley rats (200-250 g, female) were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences (Hangzhou, China). All rats were used in the experiment, grouped as follows: PBS group; ROS Nano group; GABA Nano group; DOPA Nano group.

The spinal cord contusion model was established by an infinite vertical impactor (68099, China RWD Life Science Company). To expose the dorsal side of the spinal cord, a laminectomy was performed at the level of the 10th thoracic vertebra (T10-11), while the rat was anesthetized with sodium pentobarbital (0.5 ml/100 g). Then, the tip of the 68099 impactor was lowered until it just touched the exposed spinal cord. Spinal cord contusion was performed by hitting the spinal cord with a 3 mm diameter cylinder at a speed of 2.5 m/s. After the operation, the muscles and skin were sutured, and the rats were placed on an electric heating pad to maintain body temperature at 32°C until they woke up. Provide bladder care twice daily until spontaneous voiding resumes. During the first week after SCI, 200 μL of nanosolution (10 mg/mL) or PBS was injected via the tail vein every 48 h. In the following weeks, inject 200 μL of the nanosolution (10 mg/mL) via the tail vein once a week. For forward tracing of corticospinal axons, rats underwent dorsal resection at the level of the eighth thoracic vertebra (T7-8). Then, AAV2-9-mCherry was injected into the spinal cord of rats 6 weeks after SCI according to the reported method (R). Eight weeks after injury, histological evaluation was performed at the injury site. Rats were bred for 14 days after receiving tracer injections.

2.3.3 Histology and three-dimensional reconstruction;

For the rigorous collection of injured spinal cord tissue, rats were deeply anesthetized with sodium pentobarbital (0.5 ml/100 g) and perfused intracardiacly with 4% paraformaldehyde at 9 weeks after injury. Before embedding, the fixed spinal cords were soaked in the prepared 30% and 15% sucrose solutions. Spinal cord blocks were sectioned using a cryostat (CryoStar NX50; Thermo, USA) and thawed and mounted on Super Frost Plus slides (Fisher Scientific, USA). These sections were then processed for immunohistochemistry as described above. After blocking with 5% donkey serum and 0.3% Triton X-100 in PBS, the spinal cord tissue sections were incubated in the primary antibody. Then, sections were rinsed three times with PBS and incubated in appropriate secondary antibodies. Finally, the sections were observed with a confocal laser scanning microscope (A1Ti, Nikon, Japan).

Quantitative analysis of the volume of the injured spinal cord tissue cavity was analyzed in SCI rats as previously described (R). In brief, they were stained with hematoxylin and eosin (H&E) and imaged by a virtual digital slide scanning system (VS120, Olympus, Japan) for 3D reconstruction. Three-dimensional images were created by Amira software, and white matter, gray matter, cystic cavities, and pathological tissues were quantified separately. The detailed results are shown in Figure 8, which shows the evaluation of the ability of nanomedicine to protect spinal cord tissue. The specific meanings of each section are: (a) Schematic diagram of the experimental process and design; (b, c) WB characterization and statistics of inflammatory and apoptotic factors in the spinal cord treated with ROS NANO and PBS after spinal cord injury; (d) After spinal cord injury Cross-sectional iNOS/IBA1 and GFAP/NeuN immunofluorescence photos of the injury center treated with ROS NANO and PBS to observe the inflammation and cell survival; (e, f, g) Optical photos, H&E staining and 3D reconstruction photos between the two groups; (h) Statistics of cavities and residual tissues after injury; (i-n) Histomorphological photographs and quantitative differences between the two groups.

2.3.4 The anti-apoptotic and anti-inflammatory functions of ROS nanoparticles in vivo;

To evaluate the in vivo biocompatibility of ROS nanoparticles, animals treated with ROS nanoparticles and PBS were anesthetized and perfused with 4% paraformaldehyde 7 days after injury. Sections of heart, liver, spleen, lung, kidney, and brain were collected and subjected to H&E staining to observe tissue morphology. To test anti-inflammatory function, 1–2 mm spinal cord segments were dissected from the site of injury distally and caudally. The tissue was then homogenized with lysis buffer and centrifuged at 12000g, 4°C for 10 minutes to obtain a supernatant. The total protein content was determined with BCA protein assay kit. 40 μg/sample protein was loaded onto a 10% polyacrylamide gel, separated by SDS PAGE, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk in PBST for 1 hour at room temperature, the membrane was moved to primary antibodies (rabbit anti-TGF-β, rabbit anti-Bcl-2, rabbit anti-Bax, diluted 1:1000 in 5% BSA) and incubated in overnight at 4°C. After washing 3 times with PBST, the membrane was incubated in secondary antibody (goat anti-rabbit HRP) for 1 hour at room temperature. The protein signal was then visualized with an ECL kit and measured with Image Lab software provided by Bio-Rad. The results show that as shown in Figure 8(b,c), the nanomedicine has good anti-apoptosis and anti-inflammation effects. 2.3.5 Behavioral assessment and electromyography (EMG) recording

Rats were behaviorally assessed weekly in the open environment according to the original report from the BBB. For detailed hindlimb kinematic analysis, follow the reported procedure. The hindlimb movements of different groups of rats were recorded using MotoRater (Vicon Motion Systems, UK). Movements of stick views and rotational angles of the hindlimbs were performed by MATLAB in a blinded manner.

Eight weeks after contusion, implantation of bipolar electrodes was performed as reported. Briefly, the electrode (AS632, Connor wire) was drawn out from a 5-gauge needle and inserted into the mid-abdomen of the medial gastrocnemius (GS) and tibialis anterior (TA) muscles of the hindlimb of the rats, while the rats were deeply anesthetized. Insert a common ground wire subcutaneously in the Achilles tendon region of the hindlimb. The wires run subcutaneously through the back to a small percutaneous connector firmly secured to the rat's skull. EMG signals were acquired using a differential neuronal signal amplifier (BTAM01L, Braintech, China), filtered at 30–2000 Hz, sampled at 30 kHz using a Neurostudio system (Braintech, China), and analyzed by a custom MATLAB code.

Figure 9 shows the evaluation of functional recovery under the treatment of ROS NANO and PBS after spinal cord injury; the specific meanings of each part are: (a) BBB score statistics between the two groups; (b) schematic diagram of the rat hindlimb movement process; (c, d , e) Comparison of hindlimb gait, movement angle, muscle signal of normal group, ROS NANO and PBS group.

Figure 10 shows the evaluation of the functional recovery of the nano-medicines GABA Nano and DOPA Nano after spinal cord injury; the specific meanings of each part are: (a) BBB score statistics among the four groups; (b) the specific recovery degree distribution within the four groups; (c) , d, e) Comparison of hindlimb gait, movement angle, and muscle signals of normal group, GABA Nano and DOPA Nano groups; (f, g) Statistics of hindlimb muscle signals of GABA Nano and DOPA Nano groups; (h) four groups Statistical arrangement of differences in motor function between

The research results show that GABA Nano can effectively improve the functional recovery effect of rats. Compared with the PBS group, the behavioral BBB score of the rats has increased by nearly 4 points. Moreover, it can effectively improve the movement state of the rat's hind limbs, increase the amplitude of the joint activity of the rat's hind limbs, improve the control of the muscles by the spinal cord, and significantly enhance the muscle signals during the movement of the hind limbs.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, etc., shall be included in the protection scope of the present invention.

Claims (16)

1. A targeted drug for spinal cord injury, characterized in that it comprises amphoteric polymer micelles and hydrophobic neurorestorative drugs, the hydrophobic neurorestorative drugs are wrapped by amphoteric polymer micelles to form water-soluble particles, and the amphoteric polymer micelles The bundle is formed by the association of amphoteric polymers containing hydrophilic segments and hydrophobic segments,

   The amphoteric polymer micelles are micelles formed by the association of amphoteric polymer compounds shown in formula (1),

   

   Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group; ^R1 and ^R2 are each independently selected from hydrogen, C1～C5 alkyl group, cyano group, and R ¹ and R ² are not cyano at the same time; E represents C1~C5 alkylene, C6~C10 arylene, C1~C5 alkylene-C6~C10 arylene, C6~C10 arylene Aryl-C1~C5 alkylene, or E may not exist; R ³ represents C1~C5 alkyl, hydroxyl, COOR ⁵ , R ⁵ represents hydrogen, C1~C5 alkyl, N-succinyl imines, PEG residues;

   R ⁴ and R ⁷ are each independently hydrogen or methyl;

   R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

   R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

   R ⁶ represents hydrogen, amino, carboxyl, hydroxyl, C1-C5 alkyl, N-succinimide, PEG residue;

   o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

   X is one of the groups of the following formulas (2) to (5):

   

   

   The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

   R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl, C1~C5 alkyl ether group, C1~C5 alkyl sulfide group,

   In the micelles, the hydrophilic residues in () _p in the compound represented by formula (1) form the outer hydrophilic layer, which provides the water solubility of the micellar particles, and the rest constitute the inner hydrophobic core, and the hydrophobic inner core Hydrophobic nerve restoration drugs are encapsulated, and the diameter of the micelles is 10nm-300nm.
2. The targeted drug for spinal cord injury according to claim 1, characterized in that, the chemical structure of the hydrophobic neurorestoration drug includes a small molecule nerve factor fragment secreted by nerve cells,

   The hydrophobic nerve restoration drug is any drug selected from CLP257, CLP290, baclofen, bumetanide, NMDA receptor antagonist CP101606, 8-OHDPAT, quinazine, and 4-AP, and is secreted by nerve cells. Hydrophobic neurorestoration drug obtained by coupling small molecular neurofactors through chemical bonds.
3. The targeted drug for spinal cord injury according to claim 2, wherein the small molecule nerve factors secreted by the nerve cells include the following small molecule nerve factors secreted by the nerve cells:

   
4. The targeted drug for spinal cord injury according to claim 3, wherein the hydrophobic neurorestorative drug is selected from the group consisting of CLP257, CLP290, baclofen, bumetanide, NMDA receptor antagonist modified by gamma aminobutyric acid (GABA). Any of CP101606, 8-OHDPAT, quinazine, and 4-AP.
5. The targeted drug for spinal cord injury according to claim 1, wherein the amphoteric macromolecular compound shown in formula (1) is the amphoteric macromolecular compound shown in formula (1-1),

   

   Among them, Z, R ¹ , E, R ³ , R ⁵ , R ⁴ and R ⁷ , R ⁶ , o, p, X, R ⁸ , R ⁹ , and R ¹⁰ have the same meanings as in formula (1), q is an integer of 6-20, Preferably it is an integer of 7-12.
6. The targeted drug for spinal cord injury according to claim 1, wherein,

   X is a group represented by formula (2).
7. An injection for spinal cord injury, which contains the targeted drug for spinal cord injury according to claims 1-6 and pharmaceutically acceptable auxiliary materials.
8. A method for preparing a targeted drug for spinal cord injury according to claim 1, characterized in that the amphoteric polymer compound shown in formula (1) and the hydrophobic nerve repair drug that needs to be encapsulated are dissolved in an organic solvent, Then slowly inject water into it, stir slowly, make it self-assemble to form micelle particles, then transfer the micelle particle solution to a dialysis bag, use deionized water to carry out dialysis for 12-72 hours, and freeze-dry the nano-micelle particle solution ,

   The organic solvent is selected from DMSO, DMF, THF, halogenated hydrocarbons, C1-C6 alkanol solvents, ester solvents, benzene, toluene, and pyridine.

   

   Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group; ^R1 and ^R2 are each independently selected from hydrogen, C1～C5 alkyl group, cyano group, and R ¹ and R ² are not cyano at the same time; E represents C1~C5 alkylene, C6~C10 arylene, C1~C5 alkylene-C6~C10 arylene, C6~C10 arylene Aryl-C1~C5 alkylene, or E may not exist; R ³ represents C1~C5 alkyl, hydroxyl, COOR ⁵ , R ⁵ represents hydrogen, C1~C5 alkyl, N-succinyl imines, PEG residues;

   R ⁴ and R ⁷ are each independently hydrogen or methyl;

   R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

   R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

   R ⁶ represents hydrogen, amino, carboxyl, hydroxyl, C1-C5 alkyl, N-succinimide, PEG residue;

   o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

   X is one of the groups of the following formulas (2) to (5):

   

   

   The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

   R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl group, C1-C5 alkyl ether group, C1-C5 alkyl sulfide group.
9. The preparation method of the targeted drug for spinal cord injury according to claim 8,

   Wherein, the amphoteric polymer compound shown in formula (1) is the amphoteric polymer compound shown in formula (1-1),

   

   Among them, Z, R ¹ , E, R ³ , R ⁵ , R ⁴ and R ⁷ , R ⁶ , o, p, X, R ⁸ , R ⁹ , and R ¹⁰ have the same meanings as in formula (1), q is an integer of 6-20, Preferably it is an integer of 7-12.
10. According to the preparation method of the targeted drug of spinal cord injury according to claim 9, wherein, the ratio between the amphiphilic polymer compound shown in formula (1) and the hydrophobic nerve repair drug that needs to be encapsulated is: in mass ratio, formula ( 1) The amphoteric polymer compound shown: the hydrophobic neurorestorative drug to be encapsulated = 3-20:1.
11. A polymer-hydrophobic compound water-soluble micelle is formed by encapsulating a hydrophobic compound in a micelle formed by the association of an amphoteric polymer compound shown in formula (1),

    

    Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group; ^R1 and ^R2 are each independently selected from hydrogen, C1～C5 alkyl group, cyano group, and R ¹ and R ² are not cyano at the same time; E represents C1~C5 alkylene, C6~C10 arylene, C1~C5 alkylene-C6~C10 arylene, C6~C10 arylene Aryl-C1~C5 alkylene, or E may not exist; R ³ represents C1~C5 alkyl, hydroxyl, COOR ⁵ , R ⁵ represents hydrogen, C1~C5 alkyl, N-succinyl imines, PEG residues;

    R ⁴ and R ⁷ are each independently hydrogen or methyl;

    R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

    R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

    R ⁶ represents hydrogen, amino, carboxyl, hydroxyl, C1-C5 alkyl, N-succinimide, PEG residue;

    o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

    X is one of the groups of the following formulas (2) to (5):

    

    

    The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

    R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl, C1~C5 alkyl ether group, C1~C5 alkyl sulfide group,

    In the micelle, the hydrophilic residue part in ()p in the compound represented by formula (1) constitutes the outer hydrophilic layer, which provides the water solubility of the micelle particles, and the remaining part constitutes the inner hydrophobic inner core, and the hydrophobic inner core contains Hydrophobic nerve repair medicine is encapsulated, and the diameter of the micelles is 10nm-300nm.
12. The polymer-hydrophobic compound water-soluble micelle according to claim 11, the amphoteric polymer compound shown in formula (1) is the amphoteric polymer compound shown in formula (1-1),

    

    Among them, Z, R ¹ , E, R ³ , R ⁵ , R ⁴ and R ⁷ , R ⁶ , o, p, X, R ⁸ , R ⁹ , and R ¹⁰ have the same meanings as in formula (1), q is an integer of 6 to 20, preferably an integer of 7 to 12,

    X is a group represented by formula (2).
13. An amphoteric polymer compound shown in formula (1),

    

    Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group; ^R1 and ^R2 are each independently selected from hydrogen, C1～C5 alkyl group, cyano group, and R ¹ and R ² are not cyano at the same time; E represents C1~C5 alkylene, C6~C10 arylene, C1~C5 alkylene-C6~C10 arylene, C6~C10 arylene Aryl-C1~C5 alkylene, or E may not exist; R ³ represents C1~C5 alkyl, hydroxyl, COOR ⁵ , R ⁵ represents hydrogen, C1~C5 alkyl, N-succinyl imines, PEG residues;

    R ⁴ and R ⁷ are each independently hydrogen or methyl;

    R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

    R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

    R ⁶ represents hydrogen, amino, carboxyl, hydroxyl, C1-C5 alkyl, N-succinimide, PEG residue;

    o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

    X is one of the groups of the following formulas (2) to (5):

    

    The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

    R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl group, C1-C5 alkyl ether group, C1-C5 alkyl sulfide group.
14. The amphoteric polymer compound according to claim 13, wherein the amphoteric polymer compound shown in formula (1) is the amphoteric polymer compound shown in formula (1-1),

    

    Among them, Z, R ¹ , E, R ³ , R ⁵ , R ⁴ and R ⁷ , R ⁶ , o, p, X, R ⁸ , R ⁹ , and R ¹⁰ have the same meanings as in formula (1), q is an integer of 6 to 20, preferably an integer of 7 to 12,

    X is a group represented by formula (2).
15. A method for increasing the solubility of a hydrophobic compound in water, characterized in that the hydrophobic compound is encapsulated by the micelle formed by the amphoteric polymer compound shown in claim 13.
16. A preparation method of the amphoteric polymer compound shown in claim 13, which comprises the following steps in sequence,

    Hydrophilic segment construction step S1, using the chain transfer catalyst shown in formula (6), in the presence of a free radical initiator, the compound shown in formula (7) undergoes a free radical polymerization reaction, by controlling the formula (6) The equivalent ratio of the shown chain transfer catalyst and the compound shown in formula (7) controls the degree of polymerization to be 20～40 to obtain the compound shown in formula (8),

    The hydrophobic segment is combined with step S2 to make the compound represented by the formula (8) react with the compound of the formula (9) by free radical polymerization, and control the degree of polymerization to be 2 to 4 by controlling the equivalent ratio and reaction time to obtain the compound represented by the formula (1). Represents amphoteric polymer compounds,

    

    

    Wherein, Z represents C1～C15 alkyl group, C1～C15 alkylthio group, C6～C10 aryl group, preferably methyl, phenyl, dodecylthio group; ^R1 and ^R2 are each independently Selected from hydrogen, C1-C5 alkyl, cyano, and R ¹ and R ² are not cyano at the same time; E represents C1-C5 alkylene, C6-C10 arylene, C1-C5 alkylene Group-C6～C10 arylene group, C6～C10 arylene group-C1～C5 alkylene group, or E may not exist; R ³ represents C1～C5 alkyl group, hydroxyl group, COOR ⁵ , R ⁵ represents hydrogen, C1-C5 alkyl, N-succinimide, PEG residue;

    R ⁴ and R ⁷ are each independently hydrogen or methyl; R ⁶ represents hydrogen, C1-C5 alkyl, hydroxyl;

    R ^x represents a divalent group selected from phenylene substituted by phenyl, -ph-COO-, ph-CONH-, -COO-, -CONH-;

    R ^y represents absence, or a divalent group selected from C1-C5 alkyl, N-succinimide, and PEG residues;

    o is an integer of 2 to 4; p is an integer of 20 to 40, preferably an integer of 25 to 35;

    X is one of the groups of the following formulas (2) to (5):

    

    The wavy line indicates the connection position, "—" is connected in the middle of the aromatic ring, which means that it can be connected to any possible position of the aromatic ring,

    R ⁸ and R ⁹ are each independently hydrogen, C1-C5 alkyl, C6-C10 aryl, C1-C5 alkyl sulfide group; R ¹⁰ are each independently hydrogen, C1-C5 alkyl, Hydroxyl, C1~C5 alkyl ether group, C1~C5 alkyl sulfide group,

    Preferably the compound of formula (7) is the compound of following formula (7-1), the compound of formula (9) is the compound of following formula (9-1),

    

    

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