A nerve-targeted quercetin nanocrystal and its preparation method and application

Abstract

The present invention provides a nerve-targeted quercetin nanocrystal, which belongs to the field of targeted drug technology. The present invention uses a nanocrystal structure formed by carboxylated Pluronic F127 to encapsulate loaded quercetin, which has good transmembrane ability, can overcome the limitation that quercetin is difficult to absorb, and improve the bioavailability of quercetin. The present invention uses the neurotropic virus-derived peptide RVG29 to modify the quercetin nanocrystal, which can give the quercetin nanocrystal neural targeting, accurately deliver the quercetin nanocrystal to damaged central nervous system damaged tissue, and improve its therapeutic effect. The present invention encapsulates a water-quenched fluorescent probe in the nanocrystal, which can play a tracing role. When the quercetin nanocrystal is absorbed and decomposed by the damaged tissue, the probe is quenched, which can timely display the decomposition and absorption of the drug, and provide support for the molecular mechanism research of the therapeutic effect of quercetin.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted drugs, and in particular to a nerve-targeting quercetin nanocrystal and a preparation method and application thereof. Background Art

[0002] Quercetin (QUE) is a polyhydroxyflavonol compound widely found in nature. It is not only rich in traditional Chinese medicines such as Bupleurum, Morus alba, and Ginkgo biloba, but also found in apples, broccoli, and cabbage. Pharmacological studies have shown that quercetin has antioxidant, free radical scavenging, anti-tumor, anti-inflammatory, and antibacterial effects. However, quercetin is extremely insoluble in water and difficult to absorb as an oral preparation. This results in low bioavailability of quercetin, making its clinical application highly limited.

[0003] Currently, existing technologies for improving quercetin's bioavailability use polymeric micelles (PMs) as drug carriers to achieve quercetin delivery and release. However, the low encapsulation efficiency and slow diffusion rate of quercetin in polymeric micelles can reduce quercetin's efficacy. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a nerve-targeted quercetin nanocrystal and its preparation method and application. The nerve-targeted quercetin nanocrystal provided by the present invention has good bioavailability and good therapeutic effect on central nervous system damage.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a nerve-targeted quercetin nanocrystal, comprising a quercetin nanocrystal and a neurotropic virus-derived peptide RVG29 modified on the surface of the quercetin nanocrystal;

[0007] The quercetin nanocrystal has a core-shell structure, wherein the shell layer of the core-shell structure is carboxylated Pluronic F127, and the core is a water-quenched fluorescent probe and quercetin.

[0008] Preferably, the water-quenched fluorescent probe is an ACQ-P2 fluorescent probe.

[0009] Preferably, the particle size of the nerve-targeting quercetin nanocrystals is 60 to 500 nm.

[0010] Preferably, the mass ratio of quercetin to carboxylated Pluronic F127 is 1:0.2-1;

[0011] The mass ratio of quercetin to the water-quenched fluorescent probe is 10000:2-5;

[0012] The mass ratio of the quercetin to the neurotropic virus-derived peptide RVG29 is 1:1-2.

[0013] The present invention provides a method for preparing the above-mentioned nerve-targeting quercetin nanocrystals, comprising the following steps:

[0014] (1) mixing Pluronic F127, an acid anhydride compound, an organic base, a catalyst, and an organic solvent to perform a carboxylation reaction to obtain carboxylated Pluronic F127;

[0015] (2) mixing quercetin, a water-quenched fluorescent probe, and an alcohol solvent to obtain an alcohol mixture;

[0016] The alcohol mixture is mixed with an aqueous solution of carboxylated Pluronic F127, and co-precipitated under stirring to obtain quercetin nanocrystals;

[0017] Alternatively, quercetin, a water-quenched fluorescent probe, carboxylated Pluronic F127, and water are mixed and subjected to medium grinding to obtain quercetin nanocrystals;

[0018] (3) The quercetin nanocrystals are mixed with a carboxyl activator, maleimide, a neurotropic virus-derived peptide RVG29 and a polar solvent to carry out a coupling reaction to obtain nerve-targeted quercetin nanocrystals.

[0019] Preferably, the acid anhydride compound in step (1) is succinic anhydride; the organic base is triethylamine; and the catalyst is 4-dimethylaminopyridine.

[0020] Preferably, the stirring rate in step (2) is 800 to 1200 rpm; the coprecipitation temperature is -20 to -10°C, and the coprecipitation time is 8 to 12 minutes.

[0021] Preferably, the medium grinding rate in step (2) is 1000-1200 rpm, and the time is 4-8 hours.

[0022] Preferably, the carboxyl activator is N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;

[0023] The coupling reaction time is 4 to 12 hours.

[0024] The present invention provides the use of the above-mentioned nerve-targeting quercetin nanocrystals in preparing a medicine for treating central nervous system damage.

[0025] The present invention provides a nerve-targeted quercetin nanocrystal, comprising a quercetin nanocrystal surface-modified with a neurotropic virus-derived peptide, RVG29. The quercetin nanocrystal has a core-shell structure, wherein the shell layer is carboxylated Pluronic F127, and the core is a water-quenched fluorescent probe and quercetin. Quercetin has excellent anti-inflammatory and antioxidant properties and can improve inflammatory edema following nerve injury. The present invention utilizes quercetin for the treatment of central nervous system injuries, making it more suitable. The nanocrystal structure formed by carboxylated Pluronic F127 is used to encapsulate and load quercetin, which has excellent transmembrane ability, overcomes the limitation of quercetin's poor absorption, and improves its bioavailability. Modifying the quercetin nanocrystal with the neurotropic virus-derived peptide RVG29 imparts neural targeting to the quercetin nanocrystal, enabling precise delivery of the quercetin nanocrystal to damaged central nervous system tissue, thereby enhancing its therapeutic efficacy. The present invention encapsulates a water-quenched fluorescent probe within the nanocrystal, which acts as a tracer. When the quercetin nanocrystal is absorbed and decomposed by damaged tissue, the probe is quenched, providing timely visualization of the drug's decomposition and absorption, providing support for research into the molecular mechanisms underlying quercetin's therapeutic effects. Results from the examples demonstrate that the nerve-targeted quercetin nanocrystals provided by the present invention can treat spinal cord injury by inhibiting lipid peroxidation, without significant toxicity to the liver or kidneys.

[0026] The present invention provides a method for preparing the above-mentioned nerve-targeting quercetin nanocrystals. The preparation method provided by the present invention is simple to operate, low in cost, and easy to achieve industrialized mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a flow chart for the preparation of nerve-targeted quercetin nanocrystals;

[0028] Figure 2 The microscopic morphology of quercetin raw material and quercetin nanocrystals;

[0029] Figure 3 The particle size distribution of quercetin nanocrystals obtained at a rotation speed of 1200 rpm;

[0030] Figure 4 It is the color development result of reactive oxygen free radical ROS detection;

[0031] Figure 5 The results of cell activity detection using CCK-8 kit;

[0032] Figure 6 The size of the cavity in the spinal cord injury area after HE staining;

[0033] Figure 7 The results of immunofluorescence staining of rat spinal cord neurons 6 weeks after injury;

[0034] Figure 8 Catwalk footprint analysis results after spinal cord contusion in rats;

[0035] Figure 9 This is the BBB score result after spinal cord contusion in rats;

[0036] Figure 10 This is the Western-blot test result of lipid peroxidation inhibitory protein in the spinal cord 2 days after contusion;

[0037] Figure 11 The results of liver and kidney morphology staining in rats after spinal cord contusion. DETAILED DESCRIPTION

[0038] The present invention provides a nerve-targeted quercetin nanocrystal, comprising a quercetin nanocrystal and a neurotropic virus-derived peptide RVG29 modified on the surface of the quercetin nanocrystal;

[0039] The quercetin nanocrystal has a core-shell structure, wherein the shell layer of the core-shell structure is carboxylated Pluronic F127, and the core is a water-quenched fluorescent probe and quercetin.

[0040] In the present invention, the water-quenched fluorescent probe is preferably an ACQ-P2 fluorescent probe. In the present invention, the ACQ-P2 fluorescent probe has a structure shown in Formula I:

[0041]

[0042] In the present invention, the mass ratio of quercetin to the water-quenched fluorescent probe is preferably 10000:2-5, more preferably 10000:3-4.

[0043] In the present invention, the carboxylated Pluronic F127 is a nonionic surfactant, a polyoxyethylene polyoxypropylene ether triblock copolymer. In the present invention, the mass ratio of quercetin to carboxylated Pluronic F127 is preferably 1:0.2-1, more preferably 1:0.5-0.8.

[0044] In the present invention, the sequence of the neurotropic virus-derived peptide RVG29 is shown in SEQ ID NO. 1, specifically YTIWMPENPRPGTPCDIFTNSRGKRASNGC from N-terminus to C-terminus. In the present invention, the mass ratio of quercetin to neurotropic virus-derived peptide RVG29 is preferably 1:1-2, more preferably 1:2.

[0045] In the present invention, the particle size of the nerve-targeting quercetin nanocrystals is preferably 60 to 500 nm, more preferably 120 to 400 nm, and even more preferably 150 to 200 nm.

[0046] The present invention provides a method for preparing the above-mentioned nerve-targeting quercetin nanocrystals, comprising the following steps:

[0047] (1) mixing Pluronic F127, an acid anhydride compound, an organic base, a catalyst, and an organic solvent to perform a carboxylation reaction to obtain carboxylated Pluronic F127;

[0048] (2) mixing quercetin, a water-quenched fluorescent probe, and an alcohol solvent to obtain an alcohol mixture;

[0049] The alcohol mixture is mixed with an aqueous solution of carboxylated Pluronic F127, and co-precipitated under stirring to obtain quercetin nanocrystals;

[0050] Alternatively, quercetin, a water-quenched fluorescent probe, carboxylated Pluronic F127, and water are mixed and subjected to medium grinding to obtain quercetin nanocrystals;

[0051] (3) The quercetin nanocrystals are mixed with a carboxyl activator, maleimide, a neurotropic virus-derived peptide RVG29 and a polar solvent to carry out a coupling reaction to obtain nerve-targeted quercetin nanocrystals.

[0052] The present invention mixes Pluronic F127, an acid anhydride compound, an organic base, a catalyst and an organic solvent, and performs a carboxylation reaction to obtain carboxylated Pluronic F127, which is recorded as F127-COOH.

[0053] In the present invention, the acid anhydride compound is preferably succinic anhydride; the organic base is preferably triethylamine; the catalyst is preferably 4-dimethylaminopyridine; and the organic solvent is preferably tetrahydrofuran, 1,4-dioxane or diethyl ether.

[0054] In the present invention, the mass ratio of Pluronic F127 to the anhydride compound is preferably 12-15:1-1.5, more preferably 13-14:1-1.5. The mass ratio of Pluronic F127 to the organic base is preferably 12-15 g:135-140 μL. In the present invention, the mass ratio of Pluronic F127 to the catalyst is preferably 12-15:0.12.

[0055] The present invention has no special requirements for the mixing method, and any mixing method well known to those skilled in the art can be used, such as stirring mixing.

[0056] In the present invention, the temperature of the carboxylation reaction is preferably room temperature, and the time is preferably 24 to 48 hours, more preferably 30 to 36 hours.

[0057] After the carboxylation reaction, the present invention preferably performs post-treatment on the obtained carboxylation reaction solution, and the post-treatment preferably comprises the following steps:

[0058] The organic solvent of the carboxylation reaction solution was removed, and the remaining components were mixed with trichloroethane. The obtained mixture was filtered and recrystallized to obtain pure carboxylated Pluronic F127.

[0059] In the present invention, the method for removing the organic solvent is preferably evaporation using a rotary evaporator. In the present invention, the filtration is preferably performed using a 0.45 μm filter. In the present invention, the reagents used for the recrystallization are preferably anhydrous ether and ethanol, in that order.

[0060] After obtaining the carboxylated Pluronic F127, the present invention mixes quercetin, a water-quenched fluorescent probe and an alcohol solvent to obtain an alcohol mixture.

[0061] In the present invention, the alcohol solvent is preferably ethanol. In the present invention, the mass ratio of quercetin to water-quenched fluorescent probe is preferably 10000:2-5, more preferably 10000:3-4.

[0062] In the present invention, the mixing method is preferably stirring and mixing. In the present invention, the mixing temperature is preferably 60°C.

[0063] In the present invention, the alcohol mixture is mixed with an aqueous solution of carboxylated Pluronic F127 and co-precipitated under stirring to obtain quercetin nanocrystals, designated F-Qu-NCs. In the present invention, the mass concentration of carboxylated Pluronic F127 in the aqueous solution is preferably 1-2%, more preferably 1.5%. In the present invention, the temperature of the aqueous solution of carboxylated Pluronic F127 is preferably 20°C.

[0064] In the present invention, the stirring rate is preferably 800 to 1200 rpm, more preferably 1000 to 1200 rpm. In the present invention, the coprecipitation temperature is preferably -20 to -10°C, more preferably -15°C; the coprecipitation time is preferably 8 to 12 minutes, more preferably 10 minutes.

[0065] In the present invention, after the coprecipitation, the coprecipitation product is subjected to post-treatment, and the post-treatment preferably comprises the following steps:

[0066] The coprecipitated product is filtered and washed to obtain pure quercetin nanocrystals.

[0067] In the present invention, the filtration is preferably cake filtration. In the present invention, the washing detergent is preferably double distilled water. The present invention removes free probes that may be adsorbed on the surface of the nanocrystals through the washing.

[0068] Alternatively, after obtaining the carboxylated Pluronic F127, the present invention mixes quercetin, a water-quenched fluorescent probe, carboxylated Pluronic F127, and water and performs medium milling to obtain quercetin nanocrystals. In the present invention, the mass ratio of quercetin, water-quenched fluorescent probe, and carboxylated Pluronic F127 is preferably 100-120:20-40:100-120. In the present invention, the temperature of the water is preferably 20°C.

[0069] The present invention has no special requirements for the mixing method, and any mixing method well known to those skilled in the art can be used, such as stirring mixing.

[0070] In the present invention, the media for media grinding are preferably zirconium oxide grinding beads or zirconium silicate beads.

[0071] In the present invention, the media grinding rate is preferably 1000-1200 rpm, and the grinding time is preferably 4-8 hours, more preferably 5-6 hours.

[0072] After the media grinding, the grinding product is preferably filtered. The filtration is preferably performed using a 0.2 μm filter.

[0073] After obtaining the quercetin nanocrystals, the present invention preferably disperses the quercetin nanocrystals in an aqueous solution of carboxylated Pluronic F127, wherein the concentration of the aqueous solution of carboxylated Pluronic F127 is preferably 1 to 2 wt %. In the present invention, the dispersion method is preferably ultrasonic dispersion, and the ultrasonic dispersion power is preferably 250 W, the frequency is preferably 40 kHz, and the time is preferably 15 minutes.

[0074] After obtaining the quercetin nanocrystals, the present invention mixes the quercetin nanocrystals with a carboxyl activator, maleimide, a neurotropic virus-derived peptide RVG29, and a polar solvent to perform a coupling reaction to obtain nerve-targeted quercetin nanocrystals, designated as FR-Qu-NCs. In the present invention, the carboxyl activator is preferably N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and the mass ratio of NHS to EDC is preferably 1:1.

[0075] In the present invention, the mass ratio of the quercetin nanocrystals to the carboxyl activator is preferably 1:2-4, more preferably 1:3; the mass ratio of the quercetin nanocrystals to maleimide is preferably 1:1-2, more preferably 1:1.5.

[0076] In the present invention, the polar solvent is preferably a mixture of DMF and diethyl ether, and the volume ratio of DMF to diethyl ether is preferably 2 to 4:1, more preferably 3:1. In the present invention, the role of the DMF is to ensure the dissolution of all targeting peptides, and the role of diethyl ether is to prevent the crystallized quercetin nanocrystal structure from dissolving again during the coupling process with the targeting peptide.

[0077] The present invention has no special requirements for the mixing method, and any mixing method well known to those skilled in the art can be used, such as stirring mixing.

[0078] In the present invention, the coupling reaction temperature is preferably room temperature, and the reaction time is preferably 4 to 12 hours, more preferably 8 to 10 hours.

[0079] In the present invention, during the coupling reaction, the carboxyl group on the surface of the quercetin nanocrystal undergoes an amidation reaction with maleimide, and the thiol group of the neurotropic virus-derived peptide RVG29 undergoes a reverse addition reaction with the double bond of maleimide, thereby achieving the modification of the neurotropic virus-derived peptide RVG29 on the surface of the quercetin nanocrystal.

[0080] In the present invention, the preparation flow chart of the nerve-targeting quercetin nanocrystals is as follows: Figure 1 shown.

[0081] The present invention provides a nerve-targeted quercetin nanocrystal or an application of the nerve-targeted quercetin nanocrystal prepared by the above-mentioned preparation method in preparing a drug for central nervous system injury.

[0082] In the present invention, the central nervous system is preferably the spinal cord.

[0083] The nerve-targeting quercetin nanocrystals, preparation methods, and applications of the present invention are described in detail below with reference to the following examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0084] Example 1 Preparation of nerve-targeted quercetin nanocrystals by coprecipitation

[0085] (1) Carboxylation of Pluronic F127

[0086] 12g of Pluronic F127 was dissolved in 55mL of tetrahydrofuran (THF), followed by the addition of 122.5mg of 4-dimethylaminopyridine (DMAP), 135μL of triethylamine (TEA), and 1g of succinic anhydride. The reaction was stirred at room temperature for 24h, and then the solvent was evaporated using a rotary evaporator and dissolved in 75mL of chloroform. Excess succinic anhydride was removed through a 0.45μm filter, and the mixture was precipitated with anhydrous ether for 48h. The mixture was then redissolved in ethanol, filtered, and dried to obtain the carboxylated Pluronic F127, designated F127-COOH.

[0087] (2) Preparation of quercetin nanocrystals

[0088] 100 mg of quercetin and 20 μg of ACQ-P2 fluorescent probe were mixed with 6 mL of ethanol and completely dissolved at 60°C. The mixture was then quickly poured into 100 mL of F127 (1%, w / v) aqueous solution that had been pre-chilled at -20°C for 20 minutes. The mixture was stirred at 800 rpm for 8 hours. The precipitate was collected by filtration, and the filter cake was rinsed with 10 mL of double-distilled water. Finally, the filter cake was dispersed in 5 mL of F127 (1%, w / v) aqueous solution under 250 W ultrasonication to obtain quercetin nanocrystals.

[0089] The particle size of the obtained quercetin nanocrystals was tested under the magnetic stirring speed conditions of 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, and 1200 rpm, respectively. The results are shown in Table 1.

[0090] Table 1 Particle size of quercetin nanocrystals at different rotation speeds

[0091] Stirring speed (rpm) Particle size (nm) 0(API) 2000～10000 800 200～500 900 150～400 1000 120～200 1100 80～150 1200 60～120

[0092] Among them, the microscopic morphology of quercetin original drug, quercetin nanocrystals obtained at 800 rpm, 1000 rpm and 1200 rpm is as follows Figure 2 shown. Figure 2 In the figure, the scale of quercetin original drug is 20μm, and the scale of quercetin nanocrystal is 1μm. Among them, the particle size distribution of quercetin nanocrystals obtained at a speed of 1200rpm is shown in the figure below. Figure 3 shown.

[0093] From Table 1, Figure 2 and Figure 3 It can be seen that the particle size of the obtained quercetin nanocrystals is significantly smaller than that of the raw material drug, and with the increase of the stirring speed, the particle size of the nanocrystals will gradually decrease, and the particle size of the quercetin nanocrystals obtained at a speed of 1200 rpm is concentrated in the range of 60 to 120 nm.

[0094] (3) Coupling of RVG29

[0095] 100 mg of quercetin nanocrystals were dispersed in a DMF aqueous solution with 200 mg of N-hydroxysuccinimide (NHS), 200 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 100 mg of maleimide, and 100 mg of a neurotropic virus-derived peptide RVG29. The mixture was coupled at 20°C for 8 h to obtain nerve-targeted quercetin nanocrystals.

[0096] Example 2 Preparation of Nerve-Targeted Quercetin Nanocrystals by Media Grinding

[0097] (1) Prepare carboxylated Pluronic F127 in the manner of Example 1

[0098] (2) 120 mg of quercetin, 40 μg of ACQ probe, and 120 mg of F127-COOH were mixed in a deionized water solution pre-chilled at -20°C for 20 min, while using magnetic stirring at 1200 rpm for 12 h. The nanocrystal suspension was collected by filtration through a 0.2 μm filter. Finally, the suspension was dispersed in 5 mL of F127 (2%, w / v) aqueous solution under 250 W ultrasonication to obtain quercetin nanocrystals. The obtained quercetin nanocrystals were tested to have a particle size of 60 to 120 nm.

[0099] (3) 100 mg of quercetin nanocrystals were dispersed in a DMF aqueous solution with 400 mg of N-hydroxysuccinimide (NHS), 400 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 200 mg of maleimide, and 200 mg of a neurotropic virus-derived peptide RVG29. 12 g of the mixture was coupled at 20°C to obtain nerve-targeted quercetin nanocrystals.

[0100] Test Example 1 Inhibitory Effect of Neuro-Targeted Quercetin Nanocrystals on ROS

[0101] The VSC4.1 cell line was induced to oxidative stress by the inducer Erastin, and the ROS content under different conditions was tested (cells were seeded in 24-well plates, with a cell number of ≈50,000 / well and grouped into the simple culture medium ctl group, simple quercetin nanocrystal Qu group, Erastin-induced group Era, and post-induction drug administration group Qu+Era group. The drug administration concentrations were: Erastin 8 μmol / mL, nerve-targeted quercetin nanocrystals 10 μmol / mL). The ROS detection kit was used for testing, and the color development results of different groups are shown in the figure below. Figure 4 As shown, Figure 4 The scale bar is 100 μm. Figure 4 It can be seen that the ROS in the Era group increased significantly, while the ROS in the Qu+Era group was similar to that in the ctl group, indicating that quercetin nanocrystals can reduce oxidative stress by inhibiting reactive oxygen free radicals.

[0102] Test Example 2 Inhibitory Effect of Nerve-Targeted Quercetin Nanocrystals on Lipid Peroxidation

[0103] The VSC4.1 cell line was induced to oxidative stress by the inducer Erastin, and different concentrations of quercetin nanocrystals were administered (simple culture medium control group, lipid peroxidation inhibitor Fer-1 group, Erastin induced group Erastin, and Qu group after induction). The cell activity was detected by CCK-8 kit. The results are shown in the figure. Figure 5 As shown. Figure 5 It can be seen that the cell activity was significantly improved compared with the Erastin-induced group starting from the concentration of 8 μM, indicating that quercetin nanocrystals can improve cell activity by inhibiting lipid peroxidation.

[0104] Test Example 3 Therapeutic Effect of Nerve-Targeted Quercetin Nanocrystals on Central Nervous System Injury

[0105] Rats 6 weeks after spinal cord contusion were divided into a sham operation group (Sham), a simple spinal cord injury group (SCI), and a nerve-targeted quercetin nanocrystal administration group (Qu). The nerve-targeted quercetin nanocrystals were administered by intraperitoneal injection into rats, starting at 0 h after spinal cord injury and administered once every 24 h for 2 weeks. The solvent was normal saline at a concentration of 20 mg / mL, and 1 mL was injected each time.

[0106] The size of the cavitation in the spinal cord injury area was observed by HE staining. Figure 6 As shown, Figure 6 Scale bar = 500 μm. Figure 6 It can be seen that the cavity area of ​​the nerve-targeted quercetin nanocrystals administration group was significantly reduced.

[0107] Immunofluorescence staining was used to observe the recovery of spinal cord neurons in rats 6 weeks after injury. Figure 7 As shown, Figure 7 Scale bar = 500 μm. Figure 7 It can be seen that the neuronal marker Neun in the Qu group was significantly more than that in the SCI group, indicating that neuron-targeted quercetin nanocrystals have a good therapeutic effect on central nervous system injury.

[0108] Test Example 4: Animal Behavior Analysis Test

[0109] Eight weeks after spinal cord contusion in rats, the rat footprints were recorded and analyzed using the Catwalk footprint analysis system. Figure 8 Sham is the sham operation group, SCI is the simple spinal cord injury group, and Qu is the post-injury medication group. The medication method is the same as that in Test Example 3.

[0110] The BBB score was scored weekly from the first day to the sixth week after spinal cord contusion in rats. The BBB score was observed in a double-blind experiment to test the activity and coordination of the hind limbs of rats 1 week to 6 weeks after spinal cord injury. The results were as follows: Figure 9 As shown. Figure 9 It can be seen that the BBB score of the post-injury drug administration group was higher than that of the simple spinal cord injury group, indicating that the hind limb motor ability of the rats recovered better.

[0111] Test Example 5: Neuro-targeted Quercetin Nanocrystals Inhibit Post-Injury Lipid Peroxidation to Treat Spinal Cord Injury

[0112] The spinal cords were taken out 2 days after contusion (sham group, spinal cord injury group SCI, and Qu group after injury, with the administration method being the same as that in Test Example 3) and Western-blotted to detect the lipid peroxidation inhibitory proteins XCT and GPX4. The results are as follows: Figure 10 As shown. Figure 10 It can be seen that XCT and GPX4 in the Qu group were significantly upregulated compared with the injury group. Figure 6 The HE staining results of the cavities in the spinal cord injury area showed that quercetin nanocrystals can treat spinal cord injury by inhibiting lipid peroxidation.

[0113] Test Example 6 Toxicity of Nerve-Targeted Quercetin Nanocrystals

[0114] The morphology of liver and kidney of rats after spinal cord contusion was observed by HE staining. Figure 11 As shown, Figure 11 Middle scale bar = 200 μm. Sham is the sham operation group, SCI is the simple spinal cord injury group, and Qu is the post-injury drug administration group. The drug administration method is the same as that in Test Example 3.

[0115] Depend on Figure 11 It can be seen that there are no obvious histomorphological changes (cavities, necrosis, etc.) among the three groups, indicating that quercetin nanocrystals have no obvious drug toxicity to these organs.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nerve-targeting quercetin nanocrystal, characterized in that: The invention comprises quercetin nanocrystals and a neurotropic virus-derived peptide RVG29 modified on the surface of the quercetin nanocrystals; The quercetin nanocrystals have a core-shell structure, wherein the shell layer of the core-shell structure is carboxylated Pluronic F127, and the core is a water-quenched fluorescent probe and quercetin; the particle size of the quercetin nanocrystals is 60 to 120 nm; the water-quenched fluorescent probe is an ACQ-P2 fluorescent probe; The mass ratio of quercetin to carboxylated Pluronic F127 is 1:0.2-1; The mass ratio of quercetin to water-quenched fluorescent probe is 10000:2-5; The mass ratio of quercetin to the neurotropic virus-derived peptide RVG29 is 1:1-2; The method for preparing the nerve-targeting quercetin nanocrystals comprises the following steps: (1) mixing Pluronic F127, an acid anhydride compound, an organic base, a catalyst, and an organic solvent to perform a carboxylation reaction to obtain carboxylated Pluronic F127; the acid anhydride compound is succinic anhydride; the organic base is triethylamine; and the catalyst is 4-dimethylaminopyridine; (2) mixing quercetin, a water-quenched fluorescent probe, and an alcohol solvent to obtain an alcohol mixture; mixing the alcohol mixture with an aqueous solution of carboxylated Pluronic F127, and co-precipitating under stirring conditions to obtain quercetin nanocrystals; the stirring rate is 1200 rpm; Alternatively, quercetin, a water-quenched fluorescent probe, carboxylated Pluronic F127 and water are mixed and subjected to medium milling to obtain quercetin nanocrystals; the medium milling speed is 1200 rpm and the time is 4 to 8 hours; (3) The quercetin nanocrystals are mixed with a carboxyl activator, maleimide, a neurotropic virus-derived peptide RVG29, and a polar solvent to perform a coupling reaction to obtain nerve-targeted quercetin nanocrystals; the carboxyl activator is N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; and the coupling reaction time is 4 to 12 hours.

2. The method for preparing the nerve-targeting quercetin nanocrystals according to claim 1, characterized in that: The following steps are involved: (1) mixing Pluronic F127, an acid anhydride compound, an organic base, a catalyst, and an organic solvent to perform a carboxylation reaction to obtain carboxylated Pluronic F127; the acid anhydride compound is succinic anhydride; the organic base is triethylamine; and the catalyst is 4-dimethylaminopyridine; (2) mixing quercetin, a water-quenched fluorescent probe, and an alcohol solvent to obtain an alcohol mixture; mixing the alcohol mixture with an aqueous solution of carboxylated Pluronic F127, and co-precipitating under stirring conditions to obtain quercetin nanocrystals; the stirring rate is 1200 rpm; Alternatively, quercetin, a water-quenched fluorescent probe, carboxylated Pluronic F127 and water are mixed and subjected to medium milling to obtain quercetin nanocrystals; the medium milling speed is 1200 rpm and the time is 4 to 8 hours; (3) The quercetin nanocrystals are mixed with a carboxyl activator, maleimide, a neurotropic virus-derived peptide RVG29, and a polar solvent to perform a coupling reaction to obtain nerve-targeted quercetin nanocrystals; the carboxyl activator is N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; and the coupling reaction time is 4 to 12 hours.

3. The preparation method according to claim 2, characterized in that The co-precipitation temperature in step (2) is -20 to -10°C, and the co-precipitation time is 8 to 12 minutes.

4. Use of the nerve-targeted quercetin nanocrystal according to claim 1 or the nerve-targeted quercetin nanocrystal prepared by the preparation method according to any one of claims 2 to 3 in the preparation of a drug for treating central nervous system damage.

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