Glucosyl ros response type brain targeting nanodisc and preparation method and application thereof

Abstract

The application provides a glucose-based ROS-responsive brain-targeting nanodisc, raw materials for preparing the glucose-based ROS-responsive brain-targeting nanodisc include apolipoprotein and liposomes, and raw materials for preparing the liposomes include DSPE-PEG-TK-PHT, phospholipid, DSPE-PEG-Glucose, DMG-PEG and cholesterol. The application also provides a preparation method of the glucose-based ROS-responsive brain-targeting nanodisc and application of the glucose-based ROS-responsive brain-targeting nanodisc in preparation of a medicine for treating epilepsy. The nanodisc guarantees specific release and play of the medicine at a lesion site, reduces non-specific uptake of healthy tissues, and thus realizes efficient and low-toxicity treatment of epilepsy.

Description

Technical Field

[0001] This invention patent relates to the biomedical field, and in particular to a glucose-based ROS-responsive brain-targeting nanodisk, its preparation method, and its application. Background Technology

[0002] Epilepsy is a chronic neurological disorder caused by abnormal synchronized electrical discharges in the brain, characterized by rapid onset, long course, and frequent relapses. While some patients can control their epilepsy with medication, drug-resistant epilepsy persists due to the difficulty of most antiepileptic drugs crossing the blood-brain barrier and insufficient drug concentration at the epileptic focus. Furthermore, the peripheral distribution of antiepileptic drugs often causes intolerable side effects, significantly reducing patient adherence and leading to frequent and difficult-to-control seizures. Currently, most nanodelivery systems for epilepsy treatment utilize surface-modified peptides or antibodies to target corresponding receptors on the blood-brain barrier. These delivery systems typically have particle sizes exceeding 100 nm, resulting in insufficient efficiency in crossing the blood-brain barrier and inadequate drug accumulation in the brain. Therefore, it is necessary to find more suitable targeted ligands to achieve highly efficient targeted drug delivery.

[0003] Epilepsy foci are typically located in specific areas of the brain, and accurate drug delivery to and release into these lesions is crucial for effective treatment. In epilepsy treatment, balancing the local drug concentration and release rate in brain tissue is particularly important; release that is too rapid or too slow can lead to poor therapeutic efficacy or drug overdose. However, most existing strategies lack the ability to control release, resulting in low drug release efficiency, poor release rates, widespread peripheral distribution, and potential peripheral toxicity. Studies have reported that epilepsy foci often exhibit high levels of oxidative stress, with a large distribution of reactive oxygen species (ROS) at the lesion site. Therefore, designing a rational nanomedicine delivery system targeting this characteristic holds promise for achieving precise targeting and on-demand release.

[0004] Phenytoin is currently a first-line drug for the treatment of epilepsy, but due to its poor solubility, it is difficult to cross the blood-brain barrier and accumulate in the brain region. It is often necessary to increase the drug concentration to achieve the effective dose. This results in its wide peripheral distribution, narrow therapeutic window, strong peripheral toxicity, and poor patient compliance, which limits its clinical application. Summary of the Invention

[0005] The purpose of this invention is to provide a glucose-based ROS-responsive brain-targeting nanodisk for epilepsy targeted therapy, thereby addressing the problems existing in the prior art. This glucose-based ROS-responsive brain-targeting nanodisk not only achieves efficient brain delivery by targeting glucose transporter receptor 1 (GLUT1) on the blood-brain barrier with glucose ligands on its surface, but also achieves ROS-responsive release and accumulation of drugs in the epileptic focus area, reducing non-specific uptake by healthy tissues, thus achieving highly efficient and low-toxicity epilepsy treatment, and exhibiting good biocompatibility.

[0006] This invention utilizes the characteristic of GLUT1 overexpression at the blood-brain barrier to design a glucose ligand, DSPE-PEG, capable of inserting into nanodiscs. 2000 -Glucose; Secondly, taking advantage of the high concentration of ROS specific to epileptic lesions, a glucose-based ROS-responsive phenytoin prodrug, DSPE-PEG, was obtained by introducing ketothiolate bonds (TK) for prodrug derivatization. 2000 -TK-PHT; then the prodrug DSPE-PEG 2000 -TK-PHT with dioleoyl lecithin (DOPC) and DSPE-PEG 2000 -Glucose, 1,2-Dimyristic-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) 2000 The mixture of cholesterol and MSP1E3D1 protein was incubated together and purified by exclusion method to obtain a glucose-based ROS-responsive brain-targeting nanodisk GLNT-TP.

[0007] First, this invention utilizes the interaction between glucose ligands on the surface of the nanodiscs and GLUT1 transporters widely present on the blood-brain barrier to achieve brain-targeted delivery. Simultaneously, by combining a glycemic regulation strategy of fasting and glucose rehydration, the ability of the nanodiscs to cross the blood-brain barrier is further enhanced. Furthermore, under the influence of ROS, the TK bonds in the derivatized prodrug break in response, releasing the pharmacokinetic antiepileptic drug PHT, which exerts its anti-abnormal discharge effect at the epileptic focus. Therefore, the nanodiscs prepared in this invention possess both GLUT1 binding and transport capabilities and ROS-stimulated responsiveness, overcoming the shortcomings of existing antiepileptic drug therapies, significantly increasing drug content at the epileptic focus, releasing drugs on demand, and reducing peripheral toxicity.

[0008] Therefore, in a first aspect of the present invention, the present invention provides a glucose-based ROS-responsive brain-targeting nanodisk, wherein the raw materials for preparing the glucose-based ROS-responsive brain-targeting nanodisk include apolipoproteins and liposomes, and the raw materials for preparing the liposomes include DSPE-PEG-TK-PHT, phospholipids, DSPE-PEG-Glucose, DMG-PEG and cholesterol.

[0009] The structure of the DSPE-PEG-TK-PHT is shown below:

[0010]

[0011] Among them, 22 ≤ n1 ≤ 117;

[0012] Preferably, the DSPE-PEG-TK-PHT is DSPE-PEG. 2000 -TK-PHT, the structure is as follows:

[0013]

[0014] The structural formula of DSPE-PEG-Glucose is shown below:

[0015]

[0016] Among them, 22 ≤ n2 ≤ 117.

[0017] Preferably, the DSPE-PEG-Glucose is DSPE-PEG. 2000 -Glucose, the structure is shown below:

[0018]

[0019] In one or more of the technical solutions of the present invention, the molar ratio of the apolipoprotein to the liposome is 1:(25-100), preferably 1:60.

[0020] In one or more of the technical solutions of the present invention, the apolipoprotein is a membrane scaffold protein (MSP), which is selected from at least one of MSP1E3D1, MSP1D1, MSP1D1-dH5 and MSP2N2, preferably MSP1E3D1; the phospholipid is selected from at least one of dioleoyl lecithin (DOPC), dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylserine (DOPS), preferably dioleoyl lecithin (DOPC).

[0021] Preferably, the particle size of the glucose-based ROS-responsive brain-targeting nanodiscs is 5-40 nm.

[0022] In one or more of the technical solutions of the present invention, the DSPE-PEG-TK-PHT is prepared from phenytoin, and the reaction formula for preparing DSPE-PEG-TK-PHT from phenytoin is shown below:

[0023]

[0024] The preparation of DSPE-PEG-TK-PHT from phenytoin includes the following steps:

[0025] S1: Add phenytoin, formalin, and potassium carbonate to water to make a suspension, stir, filter, wash, and dry to obtain PHT-OH;

[0026] S2: Dissolve COOH-TK-COOH in the first organic solvent, add PHT-OH obtained in step S1, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide, stir, react, and evaporate the solvent to obtain PHT-TK;

[0027] S3: PHT-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide are dissolved in a second organic solvent, stirred under inert gas protection, and then DSPE-PEG-NH2 solution is added. The reaction is carried out and purified to obtain the DSPE-PEG-TK-PHT.

[0028] The structural formula of COOH-TK-COOH is as follows:

[0029] In one or more technical solutions of the present invention, in step S1, the ratio of phenytoin, formalin, potassium carbonate and water is 1g:2-8mL:4.5-5.5mg:32-40mL, preferably 1g:2mL:5mg:36mL; in step S2, the second organic solvent is dichloromethane, and the mass ratio of COOH-TK-COOH, PHT-OH, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide is 32:28-32:1.8-2.2:40-42, preferably 32.0:29.2:2.0:41.6; in step S3, the mass ratio of PHT-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is 20:6-8:5-6:55-65, preferably 20:7.4:5.5:60.

[0030] Preferably, in step S1, the detergent used for washing is a 3wt% formaldehyde solution.

[0031] Preferably, in step S3, the second organic solvent is N,N-dimethylformamide.

[0032] Preferably, in step S3, the purification is dialysis purification, which includes: placing the reaction solution in a dialysis bag with a molecular weight cutoff of 1000 Da, dialyzing with N,N-dimethylformamide for 10-14 h, dialyzing with purified water for 10-14 h, and then lyophilizing.

[0033] In one or more technical solutions of the present invention, in step S2, COOH-TK-COOH is prepared by acetone and 3-mercaptopropionic acid under the action of anhydrous concentrated hydrochloric acid. The reaction formula for preparing COOH-TK-COOH is as follows:

[0034]

[0035] The ratio of acetone, 3-mercaptopropionic acid and anhydrous concentrated hydrochloric acid is 5.8g:5-6g:8-12mL, preferably 5.8g:5.2g:10mL.

[0036] Preferably, the acetone is anhydrous acetone, and the dehydrating agent for the anhydrous acetone and anhydrous concentrated hydrochloric acid is anhydrous sodium sulfate.

[0037] Preferably, the reaction of acetone and 3-mercaptopropionic acid in anhydrous concentrated hydrochloric acid further includes washing with n-hexane.

[0038] In one or more technical solutions of the present invention, the DSPE-PEG-Glucose is prepared from DSPE-PEG-COOH (distearylphosphatidylethanolamine-polyethylene glycol-carboxyl) and D-glucosamine. The reaction formula for preparing DSPE-PEG-Glucose is shown below:

[0039]

[0040] The preparation of DSPE-PEG-Glucose includes: dissolving DSPE-PEG-COOH (distearylphosphatidylethanolamine-polyethylene glycol-carboxyl), 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and D-glucosamine in a third organic solvent, reacting them under the action of triethylamine to obtain the initial product, purifying it, and obtaining the DSPE-PEG-Glucose.

[0041] In one or more technical solutions of the present invention, the third organic solvent is N,N-dimethylformamide, and the ratio of DSPE-PEG-COOH (distearate-phosphatidylethanolamine-polyethylene glycol-carboxyl), 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and D-glucosamine to triethylamine is 50mg:10-11mg:6-7mg:12-13mg:70-80μl, preferably 50mg:10.4mg:6.3mg:12.5mg:75μl.

[0042] Preferably, the purification is dialysis purification, which includes: placing the primary product in a dialysis bag with a molecular weight cutoff of 1000 Da, dialyzing with purified water for 10-14 hours, and then lyophilizing.

[0043] In a second aspect, the present invention provides a method for preparing the above-mentioned glucose-based ROS-responsive brain-targeting nanodiscs, comprising the following steps:

[0044] Step 1): Dissolve the liposomes in chloroform to obtain a liposome mixture;

[0045] Step 2): Place the liposome mixture obtained in Step 1) into a nitrogen blow-off tube and blow it dry with chloroform to form a lipid film;

[0046] Step 3): Dissolve the lipid film obtained in Step 2) in a nanodisk buffer containing n-octyl-β-D-glucopyranoside, remove the n-octyl-β-D-glucopyranoside from the nanodisk buffer, incubate, separate, concentrate, and obtain the glucosyl ROS-responsive brain-targeting nanodisk.

[0047] Preferably, the nanodisc buffer solution is a nanodisc buffer solution containing 1 wt% n-octyl-β-D-glucopyranoside.

[0048] In a third aspect, the present invention provides a pharmaceutical formulation comprising the glucose-based ROS-responsive brain-targeting nanodiscs described in the first aspect and / or the glucose-based ROS-responsive brain-targeting nanodiscs prepared by the preparation method described in the second aspect.

[0049] In a fourth aspect, the present invention provides the use of the glucose-based ROS-responsive brain-targeting nanodisk described in the first aspect and / or the glucose-based ROS-responsive brain-targeting nanodisk prepared by the preparation method described in the second aspect and / or the pharmaceutical preparation described in the third aspect in the preparation of a drug for treating epilepsy.

[0050] The beneficial effects of this invention are as follows:

[0051] 1. The glucose-based ROS-responsive brain-targeting nanodiscs prepared in this invention have ultra-small particle size, which is beneficial for crossing the blood-brain barrier; in addition, the nanodiscs have uniform morphology, a small amount of negative charge on the surface, are mainly metabolized by the liver, and have good biocompatibility.

[0052] 2. The glucose-based ROS-responsive brain-targeting nanodisc prepared by this invention can be used as a nanomedicine for the treatment of epilepsy and is suitable for intravascular injection. The glucose-based ROS-responsive brain-targeting nanodisc can target glucose transporter 1 on the microvascular epithelial cells of the brain with its surface glucose ligand, and efficiently cross the blood-brain barrier under the action of blood glucose regulation, and accumulate in the epileptic lesion.

[0053] 3. The glucose-based ROS-responsive brain-targeting nanodiscs prepared in this invention can release the anti-epileptic drug phenytoin under the stimulation of high concentrations of reactive oxygen species in epileptic lesions, ensuring the specific release and effect of the drug at the lesion site, reducing non-specific uptake by healthy tissues, thereby achieving highly efficient and low-toxicity epilepsy treatment, and providing a brand-new technical solution for the application of phenytoin. Attached Figure Description

[0054] Figure 1 For DSPE-PEG 2000 -Glucose 1 HNMR spectrum;

[0055] Figure 2 For COOH-TK-COOH 1 HNMR spectrum;

[0056] Figure 3 For PHT-OH 1 HNMR spectrum;

[0057] Figure 4 For DSPE-PEG 2000 -TK-PHT 1 HNMR spectrum;

[0058] Figure 5 A schematic diagram of the preparation process of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisk;

[0059] Figure 6 Transmission electron microscopy morphology and particle size distribution of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisk.

[0060] Figure 7 Dynamic light scattering particle size and potential distribution of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisk;

[0061] Figure 8 For DSPE-PEG 2000 - Schematic diagram of the reaction mechanism of TK-PHT decomposition by reactive oxygen species;

[0062] Figure 9The release curve of phenytoin after ROS stimulation of the glucose-based ROS-responsive brain-targeting nanodisc GLND-TP;

[0063] Figure 10 The results of cytotoxicity of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisc, in SH-SY5Y cells;

[0064] Figure 11 Results of hemolysis experiments for GLND-TP, a brain-targeting nanodisk with different concentrations of glucose-based ROS-responsive properties;

[0065] Figure 12 The graph shows the effect of different groups (Saline, PHT-10, GLND-TP-0.1) on the latency of status epilepticus.

[0066] Figure 13 The figure shows the effect of different groups (Saline, PHT-10, GLND-TP-0.1) on the latency of generalized seizures. Detailed Implementation

[0067] The present invention will be further described below with reference to specific embodiments. However, these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0068] Example 1

[0069] A method for preparing a glucose-based ROS-responsive brain-targeting nanodisk GLND-TP loaded with a phenytoin prodrug includes the following steps:

[0070] (1) Glucose ligand DSPE-PEG 2000 -Glucose synthesis

[0071] Glucose ligand DSPE-PEG 2000 -Glucose is a DSPE-PEG 2000 The mixture was prepared by reacting COOH, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and D-glucosamine in an organic solvent under the action of triethylamine.

[0072] The reaction formula is shown below:

[0073]

[0074] The main process is as follows:

[0075] 50mg DSPE-PEG 2000 -COOH was dissolved in 2.5 ml of anhydrous N,N-dimethylformamide (DMF). While stirring, 10.35 mg of EDC and 6.25 mg of NHS dissolved in 0.5 ml of ultra-dry DMF were added sequentially to the above solution. The reaction was allowed to proceed for 1 h to obtain DSPE-PEG. 2000 -NHS solution: 12.5 mg D-glucosamine was dissolved in 2 ml of anhydrous DMF and mixed with 2 ml of anhydrous DMF containing 75 μl of triethylamine to prepare deprotonated glucosamine. This solution was then added dropwise to the above DSPE-PEG. 2000 The reaction was carried out overnight at room temperature in an NHS solution. The reaction solution was dialyzed with purified water in a 1000 Da dialysis bag for 12 h with frequent water changes, and then lyophilized to obtain a white solid product, DSPE-PEG. 2000 -Glucose.

[0076] Figure 1 For DSPE-PEG 2000 -Glucose 1 HNMR spectrum 1 H NMR (600MHz, CDCl3) δ3.64 (s, 180H), 2.91 (s, 1H), 1.25 (s, 64H), 0.88 (t, J = 7.0Hz, 6H).

[0077] (2) Synthesis of ROS-responsive group COOH-TK-COOH

[0078] The ROS responsive group COOH-TK-COOH is prepared by reacting anhydrous acetone with 3-mercaptopropionic acid in the presence of anhydrous concentrated hydrochloric acid.

[0079] The reaction formula is shown below:

[0080]

[0081] The main process is as follows:

[0082] Acetone and concentrated hydrochloric acid were dehydrated overnight using anhydrous sodium sulfate. 5.2 g of 3-mercaptopropionic acid was mixed with 5.8 g of anhydrous acetone and stirred at room temperature for 1 hour under nitrogen protection. Then, 10 mL of anhydrous concentrated hydrochloric acid was added to the reaction mixture, and the reaction was allowed to proceed at room temperature for 12 hours. When a white precipitate formed and could not be stirred, more anhydrous concentrated hydrochloric acid was added. The reaction was stopped when no more white solid precipitated. The reaction mixture was then precipitated with a large amount of pre-cooled n-hexane, followed by filtration. The product was washed three times with pre-cooled n-hexane and then three times with purified water. Finally, it was lyophilized to obtain a white solid product, COOH-TK-COOH.

[0083] Figure 2 For COOH-TK-COOH 1 HNMR spectrum 1 HNMR (600MHz, CDCl3) δ2.91 (t, J = 7.4Hz, 4H), 2.68 (t, J = 7.4Hz, 4H), 1.60 (s, 6H).

[0084] (3) ROS-responsive prodrug DSPE-PEG 2000 Synthesis of -TK-PHT

[0085] ROS-responsive prodrug DSPE-PEG 2000 The synthesis of -TK-PHT involves three steps: obtaining PHT-OH, PHT-TK, and the final product, which is ultimately obtained by reacting PHT-TK with DSPE-PEG. 2000 -NH2 is prepared by an amide reaction under the action of EDC and NHS.

[0086] The reaction formula is shown below:

[0087]

[0088] The main process is as follows:

[0089] Synthesis of PHT-OH: Take 1g phenytoin (PHT), 4mL formalin (37wt.%, aq.), 5.0mg potassium carbonate, add 36mL water to make a suspension, stir at room temperature for 24h, then filter, wash three times with 3% formaldehyde solution, and dry in a fume hood for 24h to obtain white solid product PHT-OH.

[0090] Figure 3 For PHT-OH 1 HNMR spectrum 1 H NMR (600MHz, DMSO-d6) δ9.63 (s, 1H), 7.42-7.34 (m, 10H), 6.41 (d, J = 7.1Hz, 1H), 4.82 (d, J = 7.1Hz, 2H).

[0091] Synthesis of PHT-TK: 32.0 mg COOH-TK-COOH was dissolved in 1 mL of anhydrous dichloromethane (DCM) and cooled to 0°C under nitrogen protection. Then, 29.2 mg PHT-OH (dissolved in 4 mL of anhydrous DCM), 2.0 mg 4-dimethylaminopyridine (DMAP, dissolved in 0.5 mL of anhydrous DCM), and 41.6 mg N,N'-dicyclohexylcarbodiimide (DCC, dissolved in 1.5 mL of anhydrous DCM) were added sequentially. The mixture was stirred at 0°C for 1 h, followed by reaction at room temperature for 24 h. After the reaction, the solvent was evaporated to dryness, the product was reconstituted with ethyl acetate, washed three times with 0.1 M hydrochloric acid solution, washed once with saturated sodium bicarbonate solution, and then washed twice with saturated brine. Anhydrous sodium sulfate was added and the mixture was allowed to stand for 45 min to remove water. After filtration, the filtrate was retained, and the solvent was evaporated to dryness to obtain the crude white solid product PHT-TK. This product was not purified and proceeded directly to the next reaction.

[0092] ROS-responsive prodrug DSPE-PEG 2000 Synthesis of -TK-PHT: 20 mg of PHT-TK, 7.4 mg of EDC, and 5.5 mg of NHS were dissolved in 2 mL of anhydrous DMF. The solution was stirred at room temperature for 1 h under nitrogen protection to activate the carboxyl group. Subsequently, 60 mg of distearate-phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG) was added. 2000 -NH2) was dissolved in 3 mL of anhydrous DMF and added dropwise to the aforementioned solution. After reacting at room temperature for 24 h, the mixture was purified by dialyzing with DMF and deionized water using a 1000 Da dialysis bag for 48 h. Subsequently, it was lyophilized to obtain a white powder, DSPE-PEG. 2000 -TK-PHT.

[0093] Figure 4 For DSPE-PEG 2000 -TK-PHT 1 HNMR spectrum 1 H NMR (600MHz, CDCl3) δ7.39-7.34 (m, 10H), 3.64 (s, 180H), 2.71-2.60 (m, 8H), 1.57-1.49 (m, 6H), 1.25 (s, 64H), 0.87 (d, J = 7.1Hz, 6H).

[0094] (4) Preparation of GLND-TP, a glycosyl ROS-responsive brain-targeting nanodisk

[0095] DOPC, DSPE-PEG 2000 -Glucose, DSPE-PEG 2000 -TK-PHT, DMG-PEG 2000Cholesterol was dissolved in chloroform, mixed well, and then a certain amount of chloroform was added to make up the volume to 1 mL, so that the total amount of lipids in the system was 3000 nmol, thus obtaining a liposome mixture; DOPC and DSPE-PEG were also present. 2000 -Glucose, DSPE-PEG 2000 -TK-PHT, DMG-PEG 2000 The mass fractions of cholesterol were 69%, 15%, 10%, 1%, and 5%.

[0096] Take 100 μL of the prepared liposome mixture and place it in a nitrogen blow-dry tube. Use a nitrogen blow-dryer to dry the lipids, forming a blue lipid film.

[0097] The lipid membrane and MSP1E3D1 protein were dissolved separately in nanodisk buffer (nanodisk buffer containing 1% n-octyl-β-D-glucopyranoside (OG)); then the two were mixed to obtain a mixed system; the molar ratio of lipid membrane to MSP1E3D1 protein was 60:1, and the amount of MSP1E3D1 protein in the system was 5 nmol. The amphiphilicity of MSP1E3D1 protein can constrain the phospholipids to form nanodisks; then the final volume was made up to 300 μL with nanodisk buffer solution.

[0098] Vortex oscillation dissolves the lipid film components into the nanodisc buffer solution. After complete dissolution, the mixture is transferred to 300 mg of SM2 Bio-Beads wetted with ultrapure water to remove OG from the system.

[0099] Nanodiscs were formed by incubating overnight in a shaker at 4°C.

[0100] After incubation overnight, the nanomaterials were removed and centrifuged at 10,000 rpm for 5 min using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to remove unbound lipids.

[0101] To further purify the nanomaterials, the protein-containing nanomaterials were separated using Superdex 200 molecular sieves;

[0102] The glucose-based ROS-responsive brain-targeting nanodisk GLND-TP was finally obtained by concentrating the nanodisk using an ultrafiltration tube with a molecular weight cutoff of 30 kDa and centrifugation at 10,000 rpm for 5 min.

[0103] Figure 5 A schematic diagram of the preparation process of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisk; the process is as follows: first, DOPC, DPG, DPTP, and DMG-PEG dissolved in chloroform are... 2000The cholesterol mixture was dried with nitrogen, then reconstituted with ND Buffer containing 1% OG and mixed with MSP1E3D1 protein. Pre-wetted SM2-Bio Beads were then added to remove OG from the solution to facilitate the formation of nanodiscs. Finally, the nanodiscs were purified using Superdex 200 molecular sieves.

[0104] The following tests were performed on the above-mentioned glucose-based ROS-responsive brain-targeting nanodisk GLND-TP:

[0105] Figure 6 Transmission electron microscopy (TEM) images and particle size distribution of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisc, are shown. The nanoparticles are nearly disk-shaped with uniform morphology and a particle size ranging from 10 to 35 nm, with the largest concentrated around 20 nm.

[0106] Figure 7 The dynamic light scattering particle size and potential distribution of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisc, are shown. The nanodisc has a particle size of 23.79±0.87 nm and a concentrated peak distribution. The surface carries a trace amount of negative charge, and the Zeta potential is -8.63±0.86 mV.

[0107] Figure 8 For DSPE-PEG 2000 -TK-PHT decomposes in response to ROS stimulation. In a high ROS environment, DSPE-PEG... 2000 The ketithiothiol group in -TK-PHT is specifically oxidized to a sulfonated derivative of phenytoin, which is then hydrolyzed to generate the PHT-OH intermediate. Since PHT-OH is unstable in water, it rapidly hydrolyzes to the amino form of PHT.

[0108] Hydrogen peroxide is one of the most typical reactive oxygen species in living organisms. Therefore, a hydrogen peroxide solution at a concentration of 10 mM was selected to simulate reactive oxygen species and co-incubated with GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisk, at 37 °C. The ROS release efficiency of the GLND-TP nanodisk over time was then measured. The results are as follows: Figure 9 As shown, Figure 9 The release curve of phenytoin after ROS stimulation of the glucose-based ROS-responsive brain-targeting nanodisc GLND-TP; by Figure 9 It can be seen that, over time, the nanodiscs produced more and more phenytoin under hydrogen peroxide stimulation, reaching a peak of 91.57±1.55% at 48h; while without hydrogen peroxide stimulation, the nanodiscs released almost no phenytoin, with a release of only 14.93±0.69% at 48h, which was significantly lower than the former, indicating that the glucose-based ROS-responsive brain-targeting nanodisc GLND-TP has good ROS-responsive release performance.

[0109] To evaluate the effects of GLND-TP, a glucose-based ROS-responsive brain-targeting nanodisk, on cell growth and function, and to determine its biocompatibility and potential toxicity, a CCK8 assay was conducted to investigate whether GLND-TP at concentrations ranging from 0 to 15 μg / mL would affect the normal growth of SH-SY5Y cells. The concentration referred to was the phenytoin content. Figure 10 The results of the cytotoxicity experiment of the glucose-based ROS-responsive brain-targeting nanodisk GLND-TP in SH-SY5Y cells are shown. It can be seen that the survival rate of SH-SY5Y cells was above 90% after 24 h of incubation with the glucose-based ROS-responsive brain-targeting nanodisk GLND-TP, indicating that within this concentration range, the glucose-based ROS-responsive brain-targeting nanodisk GLND-TP did not produce significant cytotoxicity and has good cell compatibility.

[0110] To ensure that the glucose-based ROS-responsive brain-targeting nanodisk GLND-TP has no risk of hemolysis and is suitable for subsequent tail vein injection, the blood compatibility of the glucose-based ROS-responsive brain-targeting nanodisk GLND-TP in the concentration range of 2.5-40 μg / mL was investigated through in vitro hemolysis experiments before formal animal experiments were conducted. Figure 11 The results showed the hemolytic activity of different concentrations of GLND-TP (glucosyl ROS-responsive brain-targeting nanodisk). No hemolysis was observed in either the experimental group or the negative control group. Quantitative results indicated that the hemolysis rate of GLND-TP was less than 5% at all concentrations. At a concentration of 40 μg / mL, the hemolysis rate of GLND-TP was 1.65 ± 0.79%, further demonstrating that GLND-TP is non-hemolytic, has good blood safety, and can be used as a safe nanomedicine for tail vein injection in mice.

[0111] Example 2

[0112] Application of a glucose-based ROS-responsive brain-targeting nanodisc loaded with phenytoin prodrug in a lithium-pilocarpine-induced status epilepticus model

[0113] Balb / c mice (female, 8 weeks old) were randomly divided into 3 groups of 8 mice each. First, mice were injected intraperitoneally with 127 mg / kg lithium chloride. One day later, mice were injected intraperitoneally with 1 mg / kg atropine; 30 minutes later, mice were injected intraperitoneally with 30 mg / kg pilocarpine hydrochloride to establish a status epilepticus (SE) model. Then, mice were injected intravenously with saline, 10 mg / kg phenytoin sodium, and 0.1 mg / kg of the glucose-based ROS-responsive brain-targeting nanodisk GLND-TP prepared in Example 1. The mice were then subjected to 60 minutes of behavioral observation to monitor seizure activity. Finally, the seizure latency (GS) and SE latency of each group were statistically analyzed to evaluate the efficacy of the positive drug treatment.

[0114] Figure 12 To investigate the effects of different drug groups on the latency of SE attacks in a lithium-pilocarpine-induced SE model (Note: Figure 12 In the figure, Saline is normal saline, PHT-10 is 10 mg / kg phenytoin sodium, and GLND-TP-0.1 is 0.1 mg / kg phenytoin-containing glucose-based ROS-responsive brain-targeting nanodisk (GLND-TP). As shown in the figure, the GLND-TP-0.1 group can significantly prolong the latency of SE attacks and achieve a significantly better therapeutic effect than the positive drug phenytoin sodium group.

[0115] Figure 13 To investigate the effects of different drug groups on the seizure latency of generalized seizures (GS) in a lithium-pilocarpine-induced SE model (Note: Figure 13 In the figure, Saline is physiological saline, PHT-10 is 10 mg / kg phenytoin sodium, and GLND-TP-0.1 is 0.1 mg / kg phenytoin glucose-based ROS-responsive brain-targeting nanodisk (GLND-TP). As shown in the figure, the GLND-TP-0.1 group can significantly prolong the seizure latency of GS in epileptic mice, and its effect is significantly better than that of phenytoin sodium.

[0116] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, all of which should be included within the protection scope of the present invention.

Claims

1. A glucose-based ROS-responsive brain-targeting nanodisc, characterized in that, The raw materials for preparing the glucose-based ROS-responsive brain-targeting nanodiscs include apolipoproteins and liposomes. The raw materials for preparing the liposomes include DSPE-PEG-TK-PHT, phospholipids, DSPE-PEG-Glucose, DMG-PEG, and cholesterol. The structure of the DSPE-PEG-TK-PHT is shown below: Among them, 22 ≤ n1 ≤ 117; The structural formula of DSPE-PEG-Glucose is shown below: Among them, 22 ≤ n2 ≤ 117.

2. The glucosyl ROS-responsive brain-targeting nanodisc according to claim 1, characterized in that, The molar ratio of apolipoprotein to liposomes is 1:25-100; the apolipoprotein is a membrane scaffold protein, selected from at least one of MSP1E3D1, MSP1D1, MSP1D1-dH5, and MSP2N2; the phospholipid is selected from at least one of dioleoyl lecithin, dioleoyl phosphatidylethanolamine, and dioleoyl phosphatidylserine; the particle size of the glucosyl ROS-responsive brain-targeting nanodiscs is 5-40 nm.

3. The glucosyl ROS-responsive brain-targeting nanodisc according to claim 1, characterized in that, The DSPE-PEG-TK-PHT was prepared from phenytoin, and the reaction formula for the preparation of DSPE-PEG-TK-PHT from phenytoin is shown below: The preparation of DSPE-PEG-TK-PHT from phenytoin includes the following steps: S1: Add phenytoin, formalin, and potassium carbonate to water to make a suspension, stir, filter, wash, and dry to obtain PHT-OH; S2: Dissolve COOH-TK-COOH in the first organic solvent, add PHT-OH obtained in step S1, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide, stir, react, and evaporate the solvent to obtain PHT-TK; S3: PHT-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide are dissolved in a second organic solvent, stirred under inert gas protection, and then DSPE-PEG-NH2 solution is added. The reaction is carried out and purified to obtain the DSPE-PEG-TK-PHT. The structural formula of COOH-TK-COOH is as follows:

4. The glucosyl ROS-responsive brain-targeting nanodisc according to claim 3, characterized in that, In step S1, the ratio of phenytoin, formalin, potassium carbonate, and water is 1g:2-8mL:4.5-5.5mg:32-40mL; in step S2, the second organic solvent is dichloromethane, and the mass ratio of COOH-TK-COOH, PHT-OH, 4-dimethylaminopyridine, and N,N'-dicyclohexylcarbodiimide is 32.0:28-32:1.8-2.2:40-42; in step S3, the mass ratio of PHT-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide is 20:6-8:5-6:55-65.

5. The glucosyl ROS-responsive brain-targeting nanodisc according to claim 3, characterized in that, In step S2, COOH-TK-COOH is prepared from acetone and 3-mercaptopropionic acid under the action of anhydrous concentrated hydrochloric acid. The reaction formula for preparing COOH-TK-COOH is shown below: The ratio of acetone, 3-mercaptopropionic acid and anhydrous concentrated hydrochloric acid is 5.8g:5-6g:8-12mL.

6. The glucosyl ROS-responsive brain-targeting nanodisc according to claim 1, characterized in that, The DSPE-PEG-Glucose was prepared from DSPE-PEG-COOH and D-glucosamine. The reaction formula for preparing DSPE-PEG-Glucose is shown below: The preparation of DSPE-PEG-Glucose includes: dissolving DSPE-PEG-COOH, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and D-glucosamine in a third organic solvent, reacting them under the action of triethylamine to obtain a primary product, purifying it, and obtaining the DSPE-PEG-Glucose.

7. The glucosyl ROS-responsive brain-targeting nanodisc according to claim 6, characterized in that, The third organic solvent is N,N-dimethylformamide, and the ratio of DSPE-PEG-COOH, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, D-glucosamine to triethylamine is 50 mg: 10-11 mg: 6-7 mg: 12-13 mg: 70-80 μl.

8. A method for preparing a glucose-based ROS-responsive brain-targeting nanodisc according to any one of claims 1-7, characterized in that, Includes the following steps: Step 1): Dissolve the liposomes in chloroform to obtain a liposome mixture; Step 2): Place the liposome mixture obtained in Step 1) into a nitrogen blow-off tube and blow it dry with chloroform to form a lipid film; Step 3): Dissolve the lipid film obtained in Step 2) in a nanodisk buffer containing n-octyl-β-D-glucopyranoside, remove the n-octyl-β-D-glucopyranoside from the nanodisk buffer, incubate, separate, concentrate, and obtain the glucosyl ROS-responsive brain-targeting nanodisk.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the glucose-based ROS-responsive brain-targeting nanodisk as described in any one of claims 1-7 and / or the glucose-based ROS-responsive brain-targeting nanodisk prepared by the preparation method of claim 8.

10. The use of a glucose-based ROS-responsive brain-targeting nanodisk according to any one of claims 1-7 and / or a glucose-based ROS-responsive brain-targeting nanodisk prepared by the preparation method of claim 8 and / or a pharmaceutical preparation according to claim 9 in the preparation of a drug for treating epilepsy.

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