Modified polyphenol carrier materials, targeted nanodelivery systems, preparation methods and applications

Abstract

The present invention belongs to the field of biomedical materials, and relates to a modified polyphenol carrier material, a targeted nano-delivery system, a preparation method and use thereof, and specifically relates to a preparation method of tea polyphenol modified with hyaluronic acid (hereinafter referred to as HA-EGCG) and a preparation method and application of nanoparticles loaded with microtubule inhibitors thereof, specifically, the active targeting effect of hyaluronic acid (HA) and the biological activity of epigallocatechin gallate (EGCG) synergistically promote the application of spinal cord injury repair and anti-tumor. The modified polyphenol carrier material of the present invention is hereinafter referred to as HA-EGCG carrier, and the structural formula of the HA-EGCG carrier of the present invention is as shown in Formula I. The carrier material prepared by the present invention can also reduce the hemolyticity of EGCG, retain the biological activity of EGCG, and has good biosafety. #imgabs0# In Formula I; n is a positive integer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and relates to a modified polyphenol carrier material, a targeted nano delivery system, a preparation method and uses thereof, and specifically relates to a preparation method of hyaluronic acid-modified tea polyphenol (hereinafter referred to as HA-EGCG) and a preparation method and use of nanoparticles thereof carrying microtubule inhibitors, and specifically relates to the use of synergistically promoting spinal cord injury repair and anti-tumor through the active targeting effect of hyaluronic acid (HA) and the biological activity of epigallocatechin gallate (EGCG). Background Art

[0002] Spinal cord injury (SCI) is a common disabling disease in clinical practice, which often causes sensory, motor and visceral dysfunction below the injury level, leading to varying degrees of hemiplegia or paraplegia, and many patients become disabled for life. Spinal cord injury can be divided into two stages: primary injury and secondary injury. The mechanism of secondary spinal cord injury is not very clear at present, but the generation of reactive oxygen species and lipid peroxidation after spinal cord injury are an important cause of secondary spinal cord injury [1]. Alleviating the oxidative stress state of spinal cord injury tissue is one of the important ways to promote the repair of spinal cord injury. Microtubules are the main components of the cytoskeleton and play an important role in neuronal polarization, axon differentiation and growth [2]. Cytoskeleton reconstruction, such as microtubule assembly, is a key step in the formation and regeneration of growth cones after axonal damage. Maintaining the stability of the microtubule system can prevent axonal atrophy and axonal terminal swelling caused by injury, weaken the inhibitory effect of inhibitory factors such as sphingomyelin-related proteins on axonal growth, and promote growth cone formation and axon regeneration. Combining antioxidant active molecules with microtubule inhibitors for the treatment of spinal cord injury will more efficiently stimulate the regeneration of nerve axons, induce the connection between new axons and their target neurons, and the formation of neural circuits.

[0003] Promoting axon regeneration is a hot topic in spinal cord injury research. Neuronal axon growth, elongation and branching are biological processes mediated by the cytoskeleton. Microtubules are the main components of the cytoskeleton and play an important role in neuronal polarization, axon differentiation and growth [2]. Microtubule inhibitors are a class of small molecules that maintain microtubule stability. After nerve damage, the application of microtubule inhibitors can induce and enhance tubulin polymerization, prevent microtubule depolymerization and breakage, and thus ensure the structural integrity and normal function of the nervous system. Microtubules are involved in the cell division process. Therefore, microtubule-associated proteins have become one of the important targets for the research and development of new anticancer drugs. Microtubule inhibitors interfere with the cell mitosis process by inhibiting tubulin polymerization or promoting tubulin polymerization, thereby exerting an anti-tumor effect.

[0004] Hyaluronic acid, hereinafter referred to as HA, is a natural polysaccharide with good biocompatibility, biodegradability and low immunogenicity. It has specific receptors on the cell membrane surface, including leukocyte differentiation antigen (CD44), liver endothelial cell receptor (LEC) and hyaluronic acid-mediated cell migration receptor (RHAMM). As a carrier material, hyaluronic acid mainly enters tumor cells through receptor mediation, enhancing the drug level in the lesion area, thereby achieving the purpose of targeted therapy. Leukocyte differentiation antigen (CD44) is a transmembrane protein on the cell surface, which is highly expressed on the surface of many tumor cells, including breast cancer, colon cancer, glioma, etc. In addition, when the central nervous system is damaged, the expression of CD44 increases, indicating that CD44 can also serve as a targeted receptor for the repair of central nervous system damage.

[0005] Disulfide bonds are dynamic chemical bonds with strong redox sensitivity, and can respond to both reactive oxygen species (ROS) and glutathione (GSH) that are highly expressed by tumor cells. Generally, disulfide bonds can be reduced to sulfhydryl groups by glutathione and then undergo intramolecular cyclization cleavage, or they can be oxidized to sulfones by reactive oxygen species, which hydrolyze and cleave by enhancing the hydrophilicity of the connecting arm and are then metabolized. It is generally believed that nanoparticles formed by disulfide-bonded compounds are stable in the systemic circulation, and are reduced to sulfhydryl groups under high concentrations of glutathione, thereby increasing the hydrophilicity of the drug system, promoting the hydrolysis of adjacent chemical bonds and the release of drugs. At the same time, the bond angle of the disulfide bond can change the spatial flexibility of the compound, which is beneficial to the formation and stability of nanoparticles, and provides a theoretical basis for the design and construction of nanoformulations.

[0006] Green tea refers to a beverage made from new leaves or buds of tea trees, which are not fermented and are prepared through a series of production processes such as withering, retaining the natural ingredients of fresh leaves. The main pharmacological ingredients in tea include tea polyphenols, caffeine, lipopolysaccharide, theanine, etc. These natural substances have special effects on anti-aging, anti-cancer, sterilization, anti-inflammatory, antioxidant, and anti-thrombotic effects. Epigallocatechin gallate (EGCG) is the main tea polyphenol, accounting for about 40% of the tea polyphenols in green tea; in recent years, a large number of studies have confirmed that epigallocatechin gallate has multiple pharmacological activities, including antioxidant, anti-inflammatory, anti-tumor, and antibacterial effects, but its bioavailability in the body is low, it is not easily absorbed, and it is easily metabolized in the human body through the first-pass effect of the liver and intestine. In addition, high doses of epigallocatechin gallate can cause adverse reactions such as hepatotoxicity, nephrotoxicity, gastrointestinal bleeding, diarrhea and abdominal pain, thereby limiting its clinical application. Therefore, researchers often modify the structure of epigallocatechin gallate to improve the above shortcomings, including PEGylated EGCG[3], chitosanized EGCG[4] and targeted ligand-modified EGCG[5]. How to make epigallocatechin gallate play a more effective biological role will be a key issue to be solved in future research.

[0007] At present, there have been some reports on the preparation of nanoparticles using tea polyphenol derivatives as raw materials: (1) Patent application CN201610649484.1 "EGCG-functionalized chitosan derivatives, magnolia phenol-loaded nanoparticles and preparation methods thereof" reports EGCG-functionalized chitosan derivatives loaded with magnolia phenol nanoparticles and preparation methods of the two. EGCG-modified chitosan derivatives were synthesized through the action of oxidative protection agents. (2) Patent application CN202011018549.5 "A photostable modified curdlan antioxidant and its preparation method and application" grafted EGCG through a chemical coupling method. First, EGCG was succinylated and then reacted with the hydroxyl group of carboxymethyl curdlan to prepare a photostable modified curdlan antioxidant. (3) Patent application CN202011141373.2 "A deep-sea soft hair pine algae moisturizing composition and its preparation method and application" attacks the carbonyl carbon on maleic anhydride through the hydroxyl group on EGCG, and then reacts with the amino group of palmitoyl pentapeptide to modify EGCG and improve the stability of EGCG. The preparation process of the above-mentioned epigallocatechin gallate derivatives is cumbersome, and the preparation of the derivatives requires the use of organic solvents and has a limited scope of application.

[0008] Based on the above technical status quo, the inventors expect to modify epigallocatechin gallate with hyaluronic acid, a natural polysaccharide with good biocompatibility, biodegradability, and low immunogenicity, and develop nanoparticles with active targeting ability and capable of effectively delivering microtubule inhibitors based on this derivative, so as to achieve the treatment of glioma and the repair of spinal cord injury. Summary of the Invention

[0009] The first technical problem to be solved by the present invention is to provide a modified polyphenol carrier material, which can be used to prepare a targeted nano-delivery system for delivering microtubule inhibitors. The modified polyphenol carrier material of the present invention is hereinafter referred to as the HA-EGCG carrier. The structural formula of the HA-EGCG carrier of the present invention is as shown in Formula I.

[0010]

[0011] In Formula I; n is a positive integer.

[0012] Among them, the value of n depends on the molecular weight of hyaluronic acid. If the molecular weight of hyaluronic acid is 3 - 100 kDa, the corresponding value range of n is a positive integer of 8 < n < 267. Another example is that the molecular weight of hyaluronic acid used in the example is about 9000 Da, then n is approximately equal to 24.

[0013] The second technical problem to be solved by the present invention is to provide a preparation method of the modified polyphenol carrier material HA-EGCG carrier of the present invention, including the following steps:

[0014] A. Synthesize the HA-S-S-NH2 intermediate:

[0015] A1. Take hyaluronic acid powder and dissolve it in phosphate buffered saline, then add 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, and stir at room temperature until the hyaluronic acid is completely dissolved, and the solution becomes colorless and transparent;

[0016] A2. Add cystamine dihydrochloride to the solution obtained in step A1 and react;

[0017] A3. Transfer the solution obtained from the reaction in step A2 to a dialysis device for dialysis;

[0018] A4. Collect the dialysis product obtained in step A3 and freeze-dry it to obtain the compound HA-S-S-NH2;

[0019] B. Synthesize the HA-EGCG carrier:

[0020] B1. Dissolve the HA-S-S-NH2 obtained in step A4 in phosphate buffered saline;

[0021] B2. Dissolve epigallocatechin gallate in phosphate buffered saline;

[0022] B3, adding the solution obtained in step B2 to the solution obtained in step B1, and mixing thoroughly;

[0023] B4, adjusting the pH value of the solution obtained in step B3 to 7.0-8.5, and reacting for 3-24 hours;

[0024] B5, adjusting the pH value of the solution obtained in step B4 to 5.0-6.5, and transferring the reaction solution to a dialysis device for dialysis;

[0025] B6. Collect the dialyzed product obtained in step B5, freeze-dry it, and obtain hyaluronic acid-modified epigallocatechin gallate, namely HA-EGCG.

[0026] In the above technical solution, in step A1, the phosphate buffered saline solution has a pH of 4.5-7.4, preferably pH 7.4.

[0027] In the above technical solution, in step A1, the amount of hyaluronic acid powder and phosphate buffered saline solution is such that the hyaluronic acid powder can be dissolved in the phosphate buffered saline solution. In specific operation, the following ratio can be used: 500 mg hyaluronic acid and 50 mL phosphate buffered saline solution.

[0028] In the above technical solution, in step A1, in order to fully activate the -COOH in the HA structure, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (hereinafter referred to as DMTMM) is added. The molar ratio of -COOH of HA to DMTMM is 1 part of -COOH of hyaluronic acid to 1-6 parts of DMTMM. Here, the molar ratio of -COOH of hyaluronic acid to DMTMM is preferably 1 part of -COOH of hyaluronic acid to 2 parts of DMTMM.

[0029] In the above technical solution, in step A1, stirring at room temperature until the hyaluronic acid is completely dissolved and becomes colorless and transparent is performed at 20-37°C for 1-24h; preferably, stirring at room temperature until the hyaluronic acid is completely dissolved is performed at 25°C for 2h.

[0030] In the above technical solution, in step A2, the molar ratio of hyaluronic acid to cystamine dihydrochloride is 1 part of hyaluronic acid to 1-8 parts of cystamine dihydrochloride.

[0031] In the above technical solution, in step A2, the molar ratio of hyaluronic acid to cystamine dihydrochloride is preferably 1 part of hyaluronic acid to 2 parts of cystamine dihydrochloride.

[0032] In the above technical solution, in step A2, the reaction condition is a temperature of 20-37°C, and the preferred reaction condition is room temperature of 25°C.

[0033] In the above technical solution, in step A2, the reaction time is 1-48 hours, and the preferred reaction time is 24 hours.

[0034] In the above technical solution, in step A3, the dialysis device is a dialysis bag. Further preferably, the molecular weight cutoff of the dialysis bag is 1000-20000Da. Most preferably, the molecular weight cutoff of the dialysis bag is 3500Da.

[0035] In the above technical solution, in step A3, the dialysis conditions are: dialysis in 0.1M NaCl solution for 12-48h, followed by dialysis in 25-95% v / v ethanol for 24-48h, and finally dialysis with distilled water for 12-48h. Preferably, the dialysis conditions are: dialysis in 0.1M NaCl solution for 48h, followed by dialysis in 25% v / v ethanol for 24h, and finally dialysis with distilled water for 48h.

[0036] In the above technical solution, in step A4, the freeze-drying conditions are below -60 degrees, the vacuum degree is below 10Pa, and the freeze-drying time is 24-48 hours. The final preferred freeze-drying time is 36 hours.

[0037] In the above technical solution, in step B1, the pH of the phosphate buffered saline solution is 7.4.

[0038] In the above technical scheme, in step B1, HA-SS-NH2 and phosphate buffered saline are prepared according to the following ratio: HA-SS-NH2 250-1000 mg, phosphate buffered saline 10 mM; preferably HA-SS-NH2 500 mg, phosphate buffered saline 10 mM; pH 7.4 buffer, the molar ratio is fixed at 10 mM.

[0039] In the above technical solution, in step B1, the dissolution is assisted by vortexing or ultrasound.

[0040] In the above technical solution, in step B2, the phosphate buffered saline solution is a phosphate buffer with a pH of 7.4.

[0041] In the above technical scheme, in step B2, the molar ratio of epigallocatechin gallate to -COOH in HA is 1-5 parts of epigallocatechin gallate to 1 part of -COOH in HA. The preferred molar ratio is 2 parts of epigallocatechin gallate to 1 part of -COOH in HA.

[0042] In the above technical solution, in step B2, the dissolution is assisted by vortexing or ultrasound.

[0043] In the above technical solution, in step B3, the solution obtained in step B2 is added dropwise to the solution obtained in step B1 so as to be fully mixed.

[0044] In the above technical solution, in step B4, the pH value is adjusted to 7.4.

[0045] In the above technical solution, in step B4, the pH value is adjusted using 1M NaOH solution.

[0046] In the above technical solution, in step B4, the chemical reaction time is 3 hours.

[0047] In the above technical solution, in step B5, the pH value is adjusted to 6.

[0048] In the above technical solution, in step B5, the pH value is adjusted using 1M HCl solution.

[0049] In the above technical solution, in step B5, the dialysis device is a dialysis bag. Further preferably, the molecular weight cutoff of the dialysis bag is 1000-20000Da. Most preferably, the molecular weight cutoff of the dialysis bag is 3500Da.

[0050] In the above technical solution, in step B5, the dialysis conditions are to use anhydrous ethanol, 75%, 50%, 25% ethanol for 1-2 days, and deionized water for 1-2 days. Preferably, 25% ethanol is used for 1 day and deionized water is used for 2 days.

[0051] In the preparation method of the present invention, the intermediate compound HA-SS-NH2 prepared has a structural formula as shown in Formula II.

[0052]

[0053] The third problem solved by the present invention is to provide a nano-delivery system with active targeting, and the targeted nano-delivery system of the present invention is called HA-EGCG blank nanoparticles (Blank NP). The preparation method of HA-EGCG blank nanoparticles of the present invention comprises the following steps:

[0054] (1) dissolving HA-EGCG in deionized water to obtain a HA-EGCG solution as the aqueous phase;

[0055] (2) taking anhydrous ethanol, adding it to the organic solvent, and mixing well to obtain the organic phase;

[0056] (3) adding the HA-EGCG solution obtained in step (1) dropwise to the organic phase obtained in step (2) and stirring; adding the HA-EGCG solution in step 1 dropwise to the organic phase in the above step (2) and stirring;

[0057] (4) evaporating the organic solvent;

[0058] (5) Filter to obtain blank nanoparticles.

[0059] In the above technical solution, in step (1), the concentration of the prepared HA-EGCG solution is 0.5-10 mg / mL; preferably, the concentration of the prepared HA-EGCG solution is 2 mg / mL.

[0060] In the above technical solution, in step (2), the ethanol is anhydrous ethanol.

[0061] In the above technical solution, in step (2), the volume ratio of ethanol to organic solvent is 1-30 parts of ethanol and 10-500 parts of organic solvent. Preferably, in step (2), the volume ratio of ethanol to organic solvent is 1 part of ethanol and 10-300 parts of organic solvent.

[0062] In the above technical solution, in step (2), the volume ratio of ethanol to organic solvent is 1 part of ethanol to 60-300 parts of organic solvent.

[0063] In the above technical solution, in step (2), the organic solvent is at least one of acetone, tetrahydrofuran and dichloromethane. Preferably, acetone and tetrahydrofuran are mixed and used as the organic solvent, and the volume ratio is 1-4 parts of acetone and 1-4 parts of tetrahydrofuran. The most preferred organic solvent is calculated by volume ratio: 1 part of acetone and 2 parts of tetrahydrofuran.

[0064] In the above technical solution, in step (3), the volume ratio of the aqueous phase to the organic phase is 1-3 parts of the aqueous phase and 1-5 parts of the organic phase; preferably, the volume ratio of the aqueous phase to the organic phase is 1 part of the aqueous phase and 3 parts of the organic phase.

[0065] In the above technical solution, in step (3), the stirring is performed by magnetic stirring at room temperature.

[0066] In the above technical solution, in step (3), the magnetic stirring time is 1-24 hours; preferably, the magnetic stirring time is 2 hours.

[0067] In the above technical solution, in step (4), the evaporation of the organic solvent is carried out by using a rotary evaporator to spin dry the organic solvent.

[0068] In the above technical solution, in step (5), the filtration is carried out at 4-25° C. Preferably, the filtration is carried out at 4° C.

[0069] In the above technical solution, in step (5), the filtration is performed by ultrafiltration using a centrifuge.

[0070] In the above technical solution, in step (5), the filtration is carried out by using a device with a molecular weight cut-off of 1000-10000Da. Preferably, the filtration is carried out by using a device with a molecular weight cut-off of 3500Da.

[0071] The targeted nano-delivery system of the present invention can effectively load microtubule inhibitors, and prepare a nano-delivery system with active targeting for clinical application. The microtubule inhibitors that can be loaded into the targeted nano-delivery system of the present invention include docetaxel, paclitaxel and cabazitaxel, etc.; it can be used for the repair of spinal cord injury and anti-tumor. The fourth technical problem solved by the present invention is to provide a targeted nano-delivery system loaded with microtubule inhibitors. The preparation method of the targeted nano-delivery system loaded with microtubule inhibitors of the present invention comprises the following steps:

[0072] (1) dissolving HA-EGCG in deionized water to obtain a HA-EGCG solution as the aqueous phase;

[0073] (2) dissolving a microtubule inhibitor in anhydrous ethanol to obtain a microtubule inhibitor solution, adding the microtubule inhibitor solution to an organic solvent, and mixing to obtain an organic phase;

[0074] (3) adding the HA-EGCG aqueous solution in step (1) dropwise into the organic phase in step (2) and stirring;

[0075] (4) evaporating the organic solvent;

[0076] (5) filtering to remove the unloaded microtubule inhibitors, thereby obtaining a targeted nano-delivery system loaded with microtubule inhibitors.

[0077] In the above technical solution, the microtubule inhibitors are docetaxel, paclitaxel and cabazitaxel.

[0078] In particular, for the treatment of brain glioma, the present invention uses docetaxel as a raw material to prepare a nano delivery system with active targeting, which is called docetaxel / HA-EGCG (Docetaxel / HA-EGCG) nanoparticles, hereinafter referred to as DOC / HA-EGCG NPs. The preparation method of the docetaxel / HA-EGCG nanoparticles of the present invention comprises the following steps:

[0079] (1) dissolving HA-EGCG in deionized water to obtain a HA-EGCG solution as the aqueous phase;

[0080] (2) dissolving docetaxel in anhydrous ethanol to obtain a docetaxel solution, adding the docetaxel solution to an organic solvent, and mixing to obtain an organic phase;

[0081] (3) adding the HA-EGCG aqueous solution in step (1) dropwise into the organic phase in step (2) and stirring;

[0082] (4) evaporating the organic solvent;

[0083] (5) filtering to remove the unloaded docetaxel to obtain docetaxel / HA-EGCG nanoparticles loaded with docetaxel.

[0084] In the above technical solution, in step (1), the concentration of the prepared HA-EGCG solution is 0.5-10 mg / mL; preferably, the concentration of the prepared HA-EGCG solution is 2 mg / mL.

[0085] In the above technical solution, in step (2), the ethanol is anhydrous ethanol.

[0086] In the above technical solution, in step (2), the concentration of the prepared docetaxel solution is 1-10 mg / mL; preferably, the concentration of the prepared docetaxel solution is 5 mg / mL.

[0087] In the above technical solution, in step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1-30 parts of the microtubule inhibitor solution and 10-500 parts of the organic solvent.

[0088] In the above technical solution, preferably, in step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1 part of the microtubule inhibitor solution to 10-300 parts of the organic solvent.

[0089] Most preferably, in step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1 part of the microtubule inhibitor solution to 60-300 parts of the organic solvent.

[0090] Specifically, in step (2), the volume ratio of the docetaxel solution to the organic solvent is 1 part of the docetaxel solution to 300 parts of the organic solvent; or the volume ratio is 2.1 parts of the docetaxel solution to 300 parts of the organic solvent; or the volume ratio is 3.3 parts of the docetaxel solution to 300 parts of the organic solvent; or the volume ratio is 4.5 parts of the docetaxel solution to 300 parts of the organic solvent.

[0091] In the above technical scheme, in step (2), the organic solvent is at least one of acetone, tetrahydrofuran and dichloromethane. Preferably, acetone and tetrahydrofuran are mixed and used as the organic solvent, and the volume ratio is 1-4 parts of acetone and 1-4 parts of tetrahydrofuran. The most preferred organic solvent is 1 part of acetone and 2 parts of tetrahydrofuran by volume.

[0092] In the above technical solution, in step (3), the volume ratio of the aqueous phase to the organic phase is 1-3 parts of the aqueous phase and 1-5 parts of the organic phase; preferably, the volume ratio of the aqueous phase to the organic phase is 1 part of the aqueous phase and 3 parts of the organic phase.

[0093] In the above technical solution, in step (3), the stirring is performed by magnetic stirring at room temperature.

[0094] In the above technical solution, in step (3), the magnetic stirring time is 1-24 hours; preferably, the magnetic stirring time is 2 hours.

[0095] In the above technical solution, in step (4), the evaporation of the organic solvent is carried out by using a rotary evaporator to spin dry the organic solvent.

[0096] In the above technical solution, in step (5), the filtration is carried out at 25-4° C. Preferably, the filtration is carried out at 4° C.

[0097] In the above technical solution, in step (5), the filtration is performed by ultrafiltration using a centrifuge.

[0098] In the above technical solution, in step (5), the filtration is carried out by using a device with a molecular weight cut-off of 1000-10000Da. Preferably, the filtration is carried out by using a device with a molecular weight cut-off of 3500Da.

[0099] In the above preparation method, docetaxel can be replaced by paclitaxel and cabazitaxel to obtain the corresponding drug-loaded HA-EGCG nanoparticles. In this process, the preparation conditions of paclitaxel and cabazitaxel are the same as those of docetaxel and remain unchanged.

[0100] In the present invention, in order to make the nanoparticles better enriched in the tumor site, a targeting material hyaluronic acid is used. In order to make the nanoparticles selectively release drugs at the lesion site, a responsive nanocarrier, GSH response, is used. In order to make the prepared nanoparticles be used for the treatment of various diseases, a compound with multiple pharmacological activities is used, for example, tea polyphenols, microtubule inhibitors (including docetaxel, paclitaxel and cabazitaxel).

[0101] The preparation method of the present invention first uses 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) to activate the hydroxyl groups in hyaluronic acid, and the performance is better than or equivalent to the most popular coupling reagents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC for short) and N-hydroxysuccinimide (NHS for short). Then, cystamine dihydrochloride is used to amino-modify the hyaluronic acid, and then the amino group is used to react with the oxidized quinone structure of epigallocatechin gallate to obtain the carrier material HA-EGCG of the present invention.

[0102] The fifth technical problem solved by the present invention is to provide a new use of the carrier material HA-EGCG of the present invention.

[0103] Specifically, the carrier material HA-EGCG of the present invention is used in the preparation of drugs with antioxidant properties; the carrier material HA-EGCG of the present invention is used in the preparation of drugs loaded with microtubule inhibitors; and the carrier material HA-EGCG of the present invention is used in the preparation of drugs with anti-tumor biological activity.

[0104] This is further reflected in the use of the carrier material HA-EGCG of the present invention in preparing a drug for treating and promoting the repair of spinal cord injury; and the use of the carrier material HA-EGCG of the present invention in preparing a drug for treating brain glioma.

[0105] The sixth technical problem solved by the present invention is to provide a new use of the targeted nano-delivery system loaded with microtubule inhibitors of the present invention.

[0106] Specifically, the use of the targeted nano-delivery system loaded with microtubule inhibitors of the present invention in the preparation of drugs for treating and promoting the repair of spinal cord injuries; and the use of the targeted nano-delivery system loaded with microtubule inhibitors of the present invention in the preparation of drugs with anti-tumor biological activity. In particular, the use of the targeted nano-delivery system loaded with microtubule inhibitors of the present invention in the preparation of drugs for treating brain gliomas.

[0107] The carrier material prepared by the present invention can successfully load microtubule inhibitors through non-covalent bond forces (including hydrophobic interaction and π-π interaction), forming a nano delivery system for promoting spinal cord injury repair and treating brain glioma. The carrier material prepared by the present invention itself has antioxidant and anti-tumor biological activities, as well as the property of actively targeting CD44 receptors, and can enhance the effect of drugs when used for drug delivery.

[0108] The carrier material prepared by the present invention can also reduce the hemolytic property of EGCG, retain the biological activity of EGCG, and has good biological safety.

[0109] Compared with the prior art, the carrier material, blank nanoparticles and drug-loaded nanoparticles of the present invention have the following advantages:

[0110] 1. The carrier material HA-EGCG prepared by the present invention has the property of actively targeting CD44 receptors; meanwhile, it also retains the antioxidant and anti-tumor effects of EGCG.

[0111] 2. The carrier material HA-EGCG prepared by the present invention has a dynamic disulfide bond in its structure, which is the key technical point of the present invention. It is usually stable and will only break when a reducing agent is added. For example, under high levels of glutathione (hereinafter referred to as GSH) and reactive oxygen species (hereinafter referred to as ROS), it can break and release the loaded drug.

[0112] 3. The carrier material HA-EGCG prepared by the present invention can effectively load microtubule inhibitors and has the potential to promote spinal cord injury repair and anti-tumor effects.

[0113] 4. After the carrier material HA-EGCG prepared by the present invention is loaded with a microtubule inhibitor, the formed nanoparticles are dissolved again after freeze-drying, showing good resolubility. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1The structural formula of the carrier material HA-EGCG (A) and 1 H-NMR chart (B).

[0115] Figure 2 XPS graph of the carrier material HA-EGCG.

[0116] Figure 3 Lyophilized powder and resolubility of blank nanoparticles and DOC / HA-EGCG nanoparticles.

[0117] Among them, af represent a: water, b: blank nanoparticles, c: DOC / HA-EGCG nanoparticles (10% theoretical drug loading), d: blank nanoparticle freeze-dried powder, e: DOC / HA-EGCG nano freeze-dried powder (10% theoretical drug loading), and f: DOC / HA-EGCG freeze-dried powder reconstituted solution (10% theoretical drug loading).

[0118] Figure 4 Transmission electron microscopy image of DOC / HA-EGCG nanoparticles (10% theoretical drug loading).

[0119] Figure 5 Hemolysis diagram of carrier material HA-EGCG (A) and hemolysis rate diagram of EGCG and HA-EGCG (B).

[0120] Figure 6 Biosafety of carrier material HA-EGCG: MTT analysis of the toxicity of HA-EGCG to L929 cells.

[0121] Figure 7 DPPH free radical scavenging ability of carrier materials EGCG, HA-EGCG and blank nanoparticles.

[0122] Figure 8 MTT analysis of the antitumor activity of HA-EGCG.

[0123] Fig. 9 Competition experiments with HA.

[0124] Fig.10 Survival of mice bearing orthotopic gliomas.

[0125] Fig.11 Immunohistochemistry of brain glioma tissue sections: CD31 and KI67 immunohistochemistry.

[0126] in: Fig.11 (A) is the immunohistochemistry of CD31 and KI67; Fig.11 (B) is a semi-quantitative analysis statistical graph of CD31 in Figure (A); Fig.11 (C) is a semi-quantitative analysis statistical graph of KI67 in Figure (A);

[0127] Fig.12 BBB scores of SCI rats in each group. DETAILED DESCRIPTION

[0128] The present invention provides a tea polyphenol derivative as a carrier material, and a targeted nano delivery system prepared therefrom. The targeted nano delivery system can be used to prepare drug-loaded nanoparticles for delivering microtubule inhibitors, as well as a technical solution for anti-tumor and promoting spinal cord injury repair.

[0129] The English abbreviations appearing in the following embodiments are interpreted as follows:

[0130] Hyaluronic acid, hereinafter referred to as HA.

[0131] Epigallocatechin gallate, hereinafter referred to as EGCG.

[0132] 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, hereinafter referred to as DMTMM.

[0133] Reactive oxygen species, hereinafter referred to as ROS.

[0134] Glutathione, hereinafter referred to as GSH.

[0135] Leukocyte differentiation antigen 44, hereinafter referred to as CD44.

[0136] Hepatic endothelial cell receptor, hereafter referred to as LEC.

[0137] Receptor for hyaluronan-mediated cell migration, referred to as RHAMM.

[0138] Phosphate buffered saline, hereinafter referred to as PBS.

[0139] Docetaxel, hereinafter referred to as DOC.

[0140] Docetaxel / HA-EGCG nanoparticles, hereafter referred to as DOC / HA-EGCG NPs.

[0141] Photosensitizer dihydrochlorin, hereinafter referred to as Ce6.

[0142] The blank nanoparticles of the present invention are referred to as Blank NPs hereinafter.

[0143] Spinal cord injury, hereinafter referred to as SCI.

[0144] The following are the manufacturers and specifications of the reagents or equipment used in the embodiments and application examples.

[0145] 1. HA (Huaxi Freda Biopharmaceutical Co., Ltd., 97%)

[0146] 2. DMTMM (Aladdin Co., Ltd., 97%)

[0147] 3. Cystamine dihydrochloride (Aladdin Co., Ltd., 97%)

[0148] 4. EGCG (Dalian Meilun Biotechnology Co., Ltd., 95%)

[0149] 5. DOC (Dalian Meilun Biotechnology Co., Ltd., 99%)

[0150] 6. Ce6 dye (Beijing Bailingwei Technology Co., Ltd., 93-98%)

[0151] 7. L929 cells (American Type Culture Collection)

[0152] 8. MTT solution (Shanghai Biotech Biotechnology Co., Ltd.)

[0153] 9. Culture medium of Application Example 2 (Gibco)

[0154] 10. DMSO (Sigma)

[0155] 11. DPPH (Shanghai Biotech Biotechnology Co., Ltd.)

[0156] 12. GL261 glioma cells (American Model Culture Collection)

[0157] 13. Culture medium of Application Example 4 (Gibco)

[0158] 14. 4% paraformaldehyde solution (Savier Biotechnology Co., Ltd.)

[0159] 15. DAPI (Shanghai Biotech Biotechnology Co., Ltd.)

[0160] 16. CLSM (confocal microscopy)

[0161] 17. CD31 (Savier Biotechnology Co., Ltd.)

[0162] 18. KI67 (Savier Biotechnology Co., Ltd.)

[0163] 19. Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Savier Biotechnology Co., Ltd.)

[0164] 20. DAB solution (Savier Biotechnology Co., Ltd.)

[0165] 21. Hematoxylin (Savier Biotechnology Co., Ltd.)

[0166] Example 1

[0167] In this embodiment, the tea polyphenol derivative HA-EGCG is synthesized, and the preparation process thereof is as follows:

[0168] Synthesis of HA-SS-NH2 intermediate: Weigh 500mg HA (MW≈9000Da, 1.31mmol-COOH) powder and dissolve it in 50mL PBS (pH 7.4), then add 727mg DMTMM (2.62mmol) powder, stir at room temperature 25°C for 2h, finally, add 590mg cystamine dihydrochloride (2.62mmol) to the above solution, and react at room temperature. After 24h, transfer the above reaction solution to a dialysis bag (molecular weight cutoff of 3500Da), dialyze in 0.1M NaCl solution for 48h, then dialyze in 25% ethanol for 24h, and finally dialyze with distilled water for 48h. Collect the sample, freeze-dry it, and obtain the compound HA-SS-NH2.

[0169] Synthesis of HA-EGCG compound: Under room temperature, 500 mg HA-SS-NH2 was dissolved in PBS buffer solution (pH 7.4, 10 mM), and ultrasonic dissolution was performed; excess EGCG powder was weighed and dissolved in PBS buffer solution (pH 7.4, 10 mM), appropriately heated and ultrasonically promoted dissolution, and finally slowly added to the above HA-SS-NH2 solution; after it was fully mixed, the pH of the reaction system was adjusted to 7.4 with 1 M NaOH solution, and the reaction was stirred at room temperature for 3 hours, and the pH of the system was adjusted to 6.0 with 1 M HCl solution. The reaction product was transferred to a dialysis bag (MWCO = 3500 Da), and dialyzed with 25% ethanol for one day and deionized water for two days, and the product was collected and freeze-dried to prepare HA-modified EGCG (HA-SS-EGCG, hereinafter referred to as HA-EGCG). Figure 1 As shown, 1 H-NMR showed that the material HA-EGCG was successfully synthesized, indicating that the present invention obtained a compound as shown in Formula I. Figure 2 As shown, the XPS results show that the HA-EGCG structure contains S elements, indicating that the HA-EGCG structure contains disulfide bonds.

[0170]

[0171] In formula I, n is a positive integer.

[0172] In Formula I of the structural formula obtained in this example, since the molecular weight of the hyaluronic acid HA used is about 9000Da (MW≈9000Da), the corresponding n≈24.

[0173] Example 2

[0174] In this embodiment, a DOC drug-loaded nanosystem is prepared, and the preparation process is as follows:

[0175] (1) Accurately weigh 10.000 mg of DOC powder and dissolve it in 2 mL of anhydrous ethanol. Use ultrasonic dissolution to achieve a final DOC concentration of 5 mg / mL.

[0176] (2) Accurately weigh 10.000 mg of HA-EGCG material and dissolve it in 5 mL of deionized water. Ultrasonic dissolution was used to dissolve the HA-EGCG to a final concentration of 2 mg / mL.

[0177] (3) The DOC solution in step (1) is added to a mixed solvent of 3 mL of acetone and tetrahydrofuran in a volume ratio of 1:2 according to the theoretical drug loading (theoretical drug loading = mass of drug / (mass of material + mass of drug) × 100%). After stirring and mixing, 1 mL of HA-EGCG solution in the above step (2) is added dropwise to the acetone. The theoretical drug loading of DOC is 2.5%, 5%, 7.5%, and 10%, respectively. Stir at room temperature for 2 h, remove the organic solvent by rotary evaporation, collect the product, ultrafilter at 4°C for 30 min (molecular weight cutoff 3500Da), collect the precipitate, and resuspend to obtain a DOC / HA-EGCG nanosuspension. After freeze-drying the blank nanoparticles and DOC / HA-EGCG (10% theoretical drug loading) nanoparticles, a freeze-dried powder is obtained, which is then redissolved in water. Figure 3 As shown in Figure 2, both nanoparticles exhibited good resolubility. Transmission electron microscopy was used to observe the morphology of DOC / HA-EGCG (10% theoretical drug loading) nanoparticles. Figure 4 As shown, the nanoparticles are monodispersed and spherical.

[0178] Example 3

[0179] In this example, photosensitizer dihydrochlorin (abbreviated as Ce6 dye) was used instead of DOC to analyze the behavior of nanoparticles in cells; the preparation process is as follows:

[0180] (1) Accurately weigh 10.000 mg of Ce6 powder and dissolve it in 5 mL of anhydrous ethanol. Use ultrasonic dissolution to achieve a final Ce6 concentration of 2 mg / mL.

[0181] (2) Accurately weigh 10.000 mg of HA-EGCG material and dissolve it in 5 mL of deionized water. Ultrasonic dissolution was used to dissolve the HA-EGCG to a final concentration of 2 mg / mL.

[0182] (3) Add 30 μL of Ce6 solution in step (1) to a mixed solvent of 3 mL of acetone and tetrahydrofuran in a volume ratio of 1:2, stir and mix, then add 1 mL of HA-EGCG solution in step (2) dropwise to the mixed solvent of acetone and tetrahydrofuran, and the theoretical drug loading of Ce6 is 5%. Stir at room temperature for 2 h, remove the organic solvent by rotary evaporation, collect the product, ultrafilter at 4 ° C for 30 min (molecular weight cutoff 3500 Da), collect the precipitate, and resuspend to obtain Ce6 / HA-EGCG nanosuspension.

[0183] Application Example 1

[0184] The carrier material HA-EGCG synthesized according to the method of Example 1 was prepared into different concentrations and mixed with 2% rabbit blood for hemolysis test analysis. After incubation at 37°C for 1 hour, the absorbance value of the mixed solution at 540nm was detected by a multifunctional microplate reader. Among them: PBS is phosphate buffer (pH 7.4), which is used as the negative control group, and water is used as the positive control group. Hemolysis test see Figure 5 From the experimental results, it was found that when the concentration of HA-EGCG reached 1000 μg / mL, no hemolysis occurred, indicating that the material HA-EGCG prepared by the present invention has good biosafety.

[0185] Application Example 2

[0186] The effect of the carrier material HA-EGCG synthesized according to the method of Example 1 on the viability of normal L929 cells was detected. L929 cells in the logarithmic growth phase were digested with trypsin, and the cells were collected by centrifugation. 3 The density of cells / well was inoculated in a sterile 96-well plate. After the cells adhered to the wall and grew, gradient concentrations of sterile HA-EGCG solution (diluted with culture medium) were added. Cells without any treatment were used as the control group. After 24 hours of culture, the HA-EGCG solution was removed, and the plates were washed twice with PBS. 100 μL of fresh culture medium was added to each well, followed by 20 μL of MTT solution (5 mg / mL). The plates were cultured in a cell culture incubator for 2.5 hours. The culture medium was carefully removed, and 150 μL of DMSO was added to each well. The plates were placed on a shaker for 15 minutes until the purple crystals at the bottom of the plate were completely dissolved. The absorbance at 570 nm was detected with a multifunctional microplate reader to calculate the survival rate of L929 cells. Figure 6 As shown, when the concentration of HA-EGCG was as high as 1200 μg / mL, the activity of L929 cells was higher than 85%, indicating that within this range, HA-EGCG had no obvious toxicity to normal cells.

[0187] Application Example 3

[0188] Weigh an appropriate amount of DPPH and dissolve it in ethanol to 250 μM; transfer 100 μL of the above DPPH solution to a 96-well plate, and then add 20 μL of gradient concentrations of EGCG, HA-EGCG solution, and Blank NP solution, respectively, with the concentrations set to 10, 20, 50, 100, 200, 300, and 400 μg / mL; after incubation at 25°C for 15 min, use a multifunctional microplate reader to measure the absorption value at 517 nm; set a separate DPPH solution group as the positive control group; calculate the oxygen free radical scavenging efficiency using the formula, where A0 is the absorption value of the positive control group, A t To detect the absorbance value of the sample group.

[0189] Clearance rate = (A0-A t ) / A0×100%

[0190] like Figure 7 As shown in the figure, after HA modification, HA-EGCG still retains the antioxidant effect of EGCG and has a high efficiency in scavenging oxygen free radicals. After HA-EGCG is prepared into nanoparticles, its oxygen free radical scavenging effect is still retained, indicating that the nanocarrier Blank NP prepared using HA-EGCG has a certain antioxidant effect.

[0191] Application Example 4

[0192] GL261 brain glioma cells in the logarithmic growth phase were digested and collected by centrifugation. 3 The density of cells / well was inoculated in a sterile 96-well plate, cultured for 12 hours, and gradient concentrations of HA-EGCG (pre-filtered with a 0.22μm sterile filter membrane) were added. The cells were cultured in a cell culture incubator for 24 hours, the old culture medium was removed, and PBS was washed twice. 100μL of fresh culture medium was added, followed by 20μL of MTT solution (5mg / mL). After incubation for another 2.5 hours, the culture medium was carefully aspirated, and 150μL of DMSO was added to each well. The plates were shaken until the purple crystals at the bottom of the plates were completely dissolved. The absorbance at 570nm was detected by a multifunctional microplate reader to calculate the survival rate of GL261 cells. Figure 8 As shown, after modification with HA, HA-EGCG had an inhibitory effect on tumor growth of GL261 cells, indicating that HA-EGCG retained the anti-tumor activity of EGCG.

[0193] Application Example 5

[0194] GL261 cells in the logarithmic growth phase were cultured at 1×10 5The cells / well were inoculated at a density of 100 μg / mL in a sterile 6-well plate covered with a cell slide. After overnight incubation, 10 mg / mL HA was added to the experimental group, and no HA was added as the control group. After 2 h of incubation, the HA solution of the experimental group was removed, and Ce6 / HA-EGCG NPs prepared in Example 3 were added. After further incubation for 2 h, the cells were washed twice with PBS, fixed with 4% paraformaldehyde solution for 30 min, stained with DAPI for 10 min, washed twice with PBS, and observed with laser confocal CLSM. Fig. 9 As shown, after adding HA to incubate GL261 cells, the uptake of nanoparticles decreased, indicating that the nanoparticles entered the cells through the action mediated by HA-related receptors.

[0195] Application Example 6

[0196] Mice inoculated with GL261 glioma cells were randomly divided into 4 groups (n=5). Normal saline (NS), Blank NP, free DOC (10 mg / kg), and DOC / HA-EGCG (10% of the theoretical drug loading; DOC: 10 mg / kg) were injected into the tail vein on days 10, 12, 14, and 16 after inoculation. The growth of the mice was observed every day, and the death time was recorded. The survival curve was drawn using GraphPadPrism7.0 to evaluate the average survival time of each group. Fig.10 As shown, the survival period of mice in the saline group was 30 days, the survival period of mice in the Blank group was 40 days, the survival period of mice in the free DOC group was 60 days, and the survival period of mice in the DOC / HA-EGCG group was 69 days. Compared with the saline group, the free DOC and DOC / HA-EGCG groups significantly prolonged the survival period of tumor-bearing mice, especially the DOC / HA-EGCG group.

[0197] Application Example 7

[0198] After 20 days of modeling, PBS and 4% paraformaldehyde solution were used for cardiac perfusion, brain tissue was removed, fixed in 4% paraformaldehyde solution for 24 hours, transferred to 15% and 30% sucrose solutions for gradient dehydration, and paraffin sections (6μm). The sections were placed in 60℃ for 2 hours, dewaxed twice with xylene; gradient hydration with ethanol, 3 minutes each, and rinsed with tap water. Incubated at room temperature for 10 minutes with 3% H2O2, and washed three times with PBS. Diluted CD31 and KI67 specific primary antibodies were added separately, incubated at room temperature for 2 hours, and washed three times with PBS, each for 5 minutes. Add polymer enhancer, incubate at room temperature for 20 minutes, and wash three times with PBS. Add horseradish peroxidase-labeled goat anti-rabbit secondary antibody, incubate at room temperature for 30 minutes, and wash three times with PBS. Then add freshly prepared DAB solution, observe under a microscope for 5 minutes, counterstain with hematoxylin, differentiate with 0.1% HCl, rinse with tap water, and return to blue. Finally, the slices were dehydrated and dried using the ethanol gradient corresponding to the above hydration, and transparentized with xylene for 5 min each, and then sealed with neutral gum, dried at room temperature, and observed under a microscope. Fig.11 As shown in the figure, compared with the saline group, the free DOC group and the DOC / HA-EGCG group had statistically significant differences (P<0.001), inhibiting tumor angiogenesis and tumor cell proliferation. In particular, the DOC / HA-EGCG group had a higher inhibition rate.

[0199] Application Example 8

[0200] Rats were anesthetized with chloral hydrate and disinfected with iodine. An incision of about 3 cm was made along the spinous process with T9 as the center. The incision was opened layer by layer to expose the spinous process of T9-T10 and its bilateral transverse processes and lamina. The spinous processes and lamina were bitten off with bone rongeurs to completely expose the spinal cord of this segment. The spinal cord was cut with scissors to establish a complete transection model. After stopping the serum wound, the rats were randomly divided into four groups and the spinal cord was administered in situ: saline group, blank nanoparticle group (hereinafter referred to as Blank NP group), free DOC group (0.4 mg / kg), DOC / HA-EGCG nanoparticle group (10% of the theoretical drug loading; DOC dose 0.4 mg / kg, total volume 10 μL, injection speed 1 μL / min); the muscles, fascia and skin were sutured with sutures, disinfected with iodine, and cefathiamidine was injected intraperitoneally once a day after surgery. The recovery of motor function in rats was evaluated by the BBB motor function score method. At a certain time point, the rats were placed in an open field and their hip, knee, ankle, toe, front and back limb coordination movements were observed for 5 minutes, and two people scored them in a double-blind manner. Fig.12 As shown in Figure 2, the drug promoted nerve repair in rats with spinal cord injury. Fig.12As shown, eight weeks after surgery, the scores of the saline group were 7 points, the BlankNP group was 9 points, the free DOC group was 11 points, and the DOC / HA-EGCG group was 15 points. After the action of DOC and DOC / HA-EGCG, the motor function of the rats recovered significantly, which was statistically different from the saline group (P<0.001), promoting the nerve repair of rats with spinal cord injury. At the same time, the BBB score of the DOC / HA-EGCG group was higher than that of the DOC group, with a statistically significant difference (P<0.05). Therefore, the introduction of HA-EGCG can promote the nerve repair of rats with spinal cord injury.

[0201] In summary, the present invention discloses a carrier material HA-EGCG, a targeted nano-delivery system prepared from the carrier material, and the use of the targeted nano-delivery system to load bioactive ingredients (such as microtubule inhibitors) to achieve medicinal purposes. It has antioxidant and anti-tumor biological activities and can be applied to spinal cord injury repair and brain glioma indications. It provides a new technical idea for clinical application.

[0202] References:

[0203] [1]Ji RR, Xu ZZ, Gao YJ: Emerging targets in neuroinflammation-drivenchronic pain. Nat Rev Drug Discov (2014) 13(7):533-548.

[0204] [2]Hellal F, Hurtado A, Ruschel J, Flynn KC, Laskowski CJ, Umlauf M, Kapitein LC, Strikis D, Lemmon V, Bixby J, Hoogenraad CC et al: Microtubulestabilization reduces scarring and causes axon regeneration after spinal cordinjury. Science (2011) 331 (6019): 928-931.

[0205] [3]Chung JE,Tan S,Gao SJ,Yongvongsoontorn N,Kim SH,Lee JH,Choi HS,Yano H,Zhuo L,Kurisawa M,Ying JY:Self-assembled micellar nanocomplexescomprising green tea catechin derivatives and protein drugs for cancertherapy.Nature nanotechnology(2014)9(11):907-912

[0206] [4].Lei F,Liu F,Yuan F,Gao Y:Impact of chitosan–egcg conjugates onphysicochemical stability ofβ-carotene emulsion.Food Hydrocolloids(2014)39(163-170.

[0207] [5].Lee F,Chung JE,Xu K,Kurisawa M:Injectable degradation-resistanthyaluronic acid hydrogels cross-linked via the oxidative coupling of greentea catechin.ACS Macro Letters(2015)4(9):957-960.。

Claims

1. A targeted nano-delivery system loaded with microtubule inhibitors, characterized in that: It is made from the modified polyphenol carrier material HA-EGCG described in Formula I and a microtubule inhibitor, and the preparation method includes the following steps: (1) Take HA-EGCG and dissolve it in deionized water to obtain an HA-EGCG solution as the aqueous phase; (2) Take the microtubule inhibitor and dissolve it in absolute ethanol to obtain a microtubule inhibitor solution. Take the microtubule inhibitor solution and add it to an organic solvent, and mix well to obtain the organic phase; (3) Slowly add the HA-EGCG aqueous phase solution in step (1) dropwise to the organic phase in step (2), and stir; (4) Evaporate the organic solvent; (5) Filter to remove the unloaded microtubule inhibitor to obtain a targeted nanodelivery system loaded with the microtubule inhibitor; In Formula I; n is a positive integer with a value range of 8 < n < 267.

2. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 1, characterized in that: The preparation method of the modified polyphenol carrier material includes the following steps: A. Synthesize the HA-S-S-NH2 intermediate: A1. Take hyaluronic acid powder and dissolve it in phosphate buffer solution, then add 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, and stir at room temperature until the hyaluronic acid is completely dissolved and the solution is colorless and transparent; A2. Add cystamine dihydrochloride to the solution obtained in step A1 and react; A3. Transfer the solution obtained from the reaction in step A2 to a dialysis device for dialysis; A4. Collect the dialysis product obtained in step A3 and freeze-dry it to obtain the compound HA-S-S-NH2; B. Synthesize the HA-EGCG carrier: B1. Dissolve the HA-S-S-NH2 obtained in step A4 in phosphate buffer solution; B2. Dissolve epigallocatechin gallate in phosphate buffer solution; B3. Add the solution obtained in step B2 to the solution obtained in step B1 and mix well; B4. Adjust the pH value of the solution obtained in step B3 to 7.0 - 8.5 and react for 3 - 24 hours; B5. Adjust the pH value of the solution obtained in step B4 to 5.0 - 6.5, and transfer the reaction solution to a dialysis device for dialysis; B6. Collect the dialysis product obtained in step B5 and freeze-dry it to obtain epigallocatechin gallate modified by hyaluronic acid, that is, HA-EGCG.

3. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 2, characterized in that: At least meet any one of the following: In step A1, the phosphate buffer solution has a pH of 4.5 - 7.4; In step A1, the dosage of hyaluronic acid powder and phosphate buffer solution is prepared so that the hyaluronic acid powder can be dissolved in the phosphate buffer solution; In step A1, the molar ratio of -COOH of hyaluronic acid to DMTMM is 1 part of -COOH of hyaluronic acid and 1 - 6 parts of DMTMM; In step A1, the condition for stirring at room temperature until the hyaluronic acid is completely dissolved and in a colorless and transparent state is to stir at 20 - 37°C for 1 - 24 h; In step A2, the molar ratio of hyaluronic acid to cystamine dihydrochloride is 1 part of hyaluronic acid and 1 - 8 parts of cystamine dihydrochloride; In step A2, the reaction condition is a temperature of 20 - 37°C; In step A2, the reaction time is 1 - 48 hours; In step A3, the dialysis device is a dialysis bag; In step A3, the dialysis conditions are: dialysis in 0.1M NaCl solution for 12-48 hours, followed by dialysis in 25-95% v / v ethanol for 24-48 hours, and finally dialysis with distilled water for 12-48 hours; In step A4, the freeze-drying conditions are below -60 degrees, the vacuum degree is below 10Pa, and the freeze-drying time is 24-48 hours; In step B1, the phosphate buffered saline solution has a pH of 7.4; In step B1, the HA-SS-NH2 and phosphate buffered saline are prepared according to the following ratio: HA-SS-NH2 250-1000 mg, phosphate buffered saline 10 mM; In step B1, the dissolution is vortexed or ultrasonically assisted; In step B2, the phosphate buffered saline solution is a phosphate buffer with a pH of 7.4; In step B2, the molar ratio of epigallocatechin gallate to -COOH in HA is 1-5 parts of epigallocatechin gallate to 1 part of -COOH in HA; In step B2, the dissolution is assisted by vortexing or ultrasound; In step B3, the solution obtained in step B2 is added dropwise to the solution obtained in step B1 and mixed thoroughly; In step B4, the pH value is adjusted to 7.4; In step B4, the pH value is adjusted using 1M NaOH solution; In step B4, the chemical reaction time is 3 h; In step B5, the pH value is adjusted to 6; In step B5, the pH value is adjusted using 1M HCl solution; In step B5, the dialysis device is a dialysis bag; In step B5, the dialysis conditions are: dialysis with anhydrous ethanol, 75%, 50% or 25% ethanol for 1-2 days, and dialysis with deionized water for 1-2 days.

4. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 3, characterized in that: At least one of the following is met: In step A1, the phosphate buffered saline solution has a pH of 7.4; In step A1, the hyaluronic acid powder and phosphate buffered saline solution are prepared according to the following ratio: 500 mg hyaluronic acid and 50 ml phosphate buffered saline solution; In step A1, the molar ratio of -COOH of hyaluronic acid to DMTMM is 1 part of -COOH of hyaluronic acid to 2 parts of DMTMM; In step A1, stirring at room temperature until the hyaluronic acid is completely dissolved is stirring at 25° C. for 2 h; In step A2, the molar ratio of hyaluronic acid to cystamine dihydrochloride is 1 part of hyaluronic acid to 2 parts of cystamine dihydrochloride; In step A2, the reaction condition is room temperature 25°C; In step A2, the reaction time is 24 hours; In step A3, the molecular weight cut-off of the dialysis bag is 1000-20000Da; In step A3, the dialysis conditions are: dialysis in 0.1 M NaCl solution for 48 h, followed by dialysis in 25% v / v ethanol for 24 h, and finally dialysis in distilled water for 48 h; In step A4, the freeze-drying time is 36 hours; In step B1, the HA-SS-NH2 and phosphate buffered saline are prepared according to the following ratio: HA-SS-NH2 500 mg, phosphate buffered saline 10 mM; In step B2, the molar ratio of epigallocatechin gallate to -COOH in HA is 2 parts of epigallocatechin gallate to 1 part of -COOH in HA; In step B5, the molecular weight cut-off of the dialysis bag is 1000-20000Da; In step B5, the dialysis conditions are: dialysis with 25% ethanol for 1 day and dialysis with deionized water for 2 days.

5. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 4, characterized in that: At least one of the following is met: In step A3, the molecular weight cut-off of the dialysis bag is 3500Da; In step B5, the molecular weight cut-off of the dialysis bag is 3500Da.

6. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 1, characterized in that: At least one of the following is met: In step (1), the concentration of the prepared HA-EGCG solution is 0.5-10 mg / mL; In step (2), the concentration of the prepared microtubule inhibitor solution is 1-10 mg / mL; In step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1-30 parts of the microtubule inhibitor solution to 10-500 parts of the organic solvent; In step (2), the organic solvent is at least one of acetone, tetrahydrofuran, and dichloromethane; In step (3), the volume ratio of aqueous phase to organic phase is 1-3 parts aqueous phase and 1-5 parts organic phase; In step (3), the stirring is performed by magnetic stirring at room temperature; In step (3), the magnetic stirring time is 1-24 hours; In step (4), the step of volatilizing the organic solvent is to use a rotary evaporator to spin dry the organic solvent; In step (5), the filtration is performed at 4-25°C; In step (5), the filtration is performed by ultrafiltration using a centrifuge; In step (5), the filtration is performed using a device with a molecular weight cutoff of 1000-10000Da.

7. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 6, characterized in that: At least one of the following is met: In step (1), the concentration of the prepared HA-EGCG solution is 2 mg / mL; In step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1 part of the microtubule inhibitor solution to 10-300 parts of the organic solvent; In step (2), the organic solvent is a mixture of acetone and tetrahydrofuran, with a volume ratio of 1-4 parts of acetone to 1-4 parts of tetrahydrofuran; In step (2), the concentration of the prepared microtubule inhibitor is 5 mg / mL; In step (3), the volume ratio of aqueous phase to organic phase is 1 part aqueous phase to 3 parts organic phase; In step (3), the stirring is magnetic stirring, and the stirring time is 2 hours; In step (5), the filtration is performed at 4°C; In step (5), the filtration is performed using a device with a molecular weight cutoff of 3500Da.

8. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 7, characterized in that: At least one of the following is met: In step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1 part of the microtubule inhibitor solution to 60-300 parts of the organic solvent; In step (2), the organic solvent is a mixture of acetone and tetrahydrofuran, with a volume ratio of 1 part acetone to 2 parts tetrahydrofuran.

9. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 8, characterized in that: In step (2), the volume ratio of the microtubule inhibitor solution to the organic solvent is 1 part of the microtubule inhibitor solution to 300 parts of the organic solvent; or the volume ratio is 2.1 parts of the microtubule inhibitor solution to 300 parts of the organic solvent; or the volume ratio is 3.3 parts of the microtubule inhibitor solution to 300 parts of the organic solvent; or the volume ratio is 4.5 parts of the microtubule inhibitor solution to 300 parts of the organic solvent.

10. The targeted nano-delivery system loaded with microtubule inhibitors according to claim 1, characterized in that: The microtubule inhibitor is docetaxel, paclitaxel or cabazitaxel.

11. Use of the targeted nano-delivery system loaded with microtubule inhibitors according to claim 1 in the preparation of drugs with antioxidant, therapeutic and promotion of spinal cord injury repair or anti-tumor biological activities.

12. The use according to claim 11, characterized in that: The anti-tumor biological activity is the treatment of brain glioma.

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