RVG29 modified dual-response drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to RVG29 modified dual-response drug-loaded nanoparticles as well as a preparation method and application thereof. According to the RVG29 modified dual-response drug-loading nano-particles, an organic metal framework with a piezoelectric property is taken as a carrier, eslicarbazepine acetate is loaded on the organic metal framework with the piezoelectric property, and the surface of the eslicarbazepine acetate is coated with a lipid layer to obtain the nano-particles; the maleimide group on the lipid layer and the sulfydryl on the targeting RVG29 polypeptide are subjected to a Michael addition reaction, and the targeting RVG29 polypeptide is covalently coupled to the surface of the nanoparticle, so that the RVG29 modified dual-response drug-loaded nanoparticle is obtained. The RVG29 modified dual-response drug-loaded nanoparticles are a safe and efficient anti-epilepsy strategy, and a non-invasive method not only provides a new thought for epilepsy treatment, but also provides an important reference for developing therapy of other nervous system diseases based on electrical stimulation.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to an RVG29-modified dual-response drug-loaded nanoparticle, its preparation method, and its application. Background Technology

[0002] Epilepsy is a common central nervous system disorder, and its pathophysiological changes involve multiple mechanisms, including voltage-gated sodium channel gating dysregulation, imbalance of excitatory and inhibitory neurotransmitters, abnormal synaptic remodeling, and reactive glial proliferation. These disturbances ultimately lead to neuronal overexcitation and abnormal synchronized discharge, manifesting as characteristic epileptic seizures. Existing antiepileptic drugs mainly target ion channels or neurotransmitter systems; for example, carbamazepine, oxcarbazepine, and eslicarbazepine acetate (ESL) exert their effects by inhibiting voltage-gated sodium channels. However, approximately 30% of patients develop drug-resistant epilepsy. A more critical challenge lies in the blood-brain barrier (BBB), which, while protecting the brain from harmful substances, blocks over 98% of small molecule drugs and almost all large molecule therapeutic agents from entering the brain, severely limiting efficacy. Alternative therapies such as surgical resection of epileptogenic foci, neuromodulation, and the ketogenic diet are limited by applicability, invasiveness, or long-term tolerability issues. Therefore, there is an urgent need to develop novel antiepileptic strategies that combine high BBB penetration and low side effects. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides RVG29-modified dual-response drug-loaded nanoparticles, their preparation method, and their applications.

[0004] The specific technical solution of this invention is as follows.

[0005] This invention provides RVG29-modified dual-response drug-loaded nanoparticles, which utilize a piezoelectric organometallic framework as a carrier. Eslicarbazepine acetate is loaded onto the piezoelectric organometallic framework, and a lipid layer is coated on its surface to obtain nanoparticles. Maleimide groups on the lipid layer and thiol groups on the targeted RVG29 peptide are covalently coupled to the surface of the nanoparticles via a Michael addition reaction, resulting in the RVG29-modified dual-response drug-loaded nanoparticles.

[0006] The piezoelectric organometallic framework of this invention serves as an ultrasound-responsive carrier. Its porous structure allows for the loading of esilicarbazepine acetate. An outer lipid layer is encapsulated and coupled with the RVG29 peptide, which has a bilayer structure. The targeted RVG29 peptide can target nicotinic acetylcholine receptors highly expressed on the surface of brain microvascular endothelial cells and neurons, achieving receptor-mediated BBB penetration. The piezoelectric signal generated by the piezoelectric organometallic framework modulates abnormal neuronal activity to achieve immediate epilepsy relief. Under the ultrasonic cavitation effect, it provides long-term release of esilicarbazepine acetate to maintain long-term efficacy, thus achieving a dual ultrasound-piezoelectric response.

[0007] In another preferred embodiment, the particle size of the RVG29-modified dual-response drug-loaded nanoparticles is 194 nm to 202 nm.

[0008] In another preferred embodiment, the dual response refers to both an ultrasonic response and a piezoelectric signal response.

[0009] In another preferred embodiment, the piezoelectric organometallic framework is UIO-66.

[0010] A second aspect of this invention provides a method for preparing the RVG29-modified dual-response drug-loaded nanoparticles, comprising the following steps: UIO-66 and eslicarbazepine acetate were mixed in an alcoholic environment at a mass ratio of 1 to 4:1, stirred in the dark, and centrifuged to obtain UIO-66 loaded with eslicarbazepine acetate. Preferably, in order to better load eslicarbazepine acetate, UIO-66 was further activated before loading eslicarbazepine acetate. The specific activation process was to activate at 150°C to remove water molecules and other impurities present in its pores, so that the subsequent eslicarbazepine acetate could be better loaded onto UIO-66. UIO-66 loaded with eslicarbazepine acetate was hydrated with liposomes in an aqueous environment at a mass ratio of 1:1~2, followed by sonication to obtain a nanoparticle solution. Tris(2-carboxyethyl)phosphine and a targeted RVG29 peptide were added to a nanoparticle solution at a molar ratio of 50:4~10. The mixture was subjected to a Michael addition reaction in the dark, followed by sonication. The precipitate was collected to obtain the RVG29-modified dual-response drug-loaded nanoparticles.

[0011] In another preferred embodiment, the hydration reaction is carried out at a temperature of 37°C to 45°C for a time of 40 min to 60 min. The Michael addition reaction is carried out at a temperature of 20°C to 28°C for 2 to 4 hours. This invention effectively regulates the particle size of nanoparticles and promotes the forward reaction by controlling the time and temperature of the hydration reaction. By controlling the temperature and time of the Michael addition reaction, the RVG29 peptide can be efficiently linked to liposomes, improving the linkage efficiency.

[0012] In another preferred embodiment, the alcohol reagent is anhydrous ethanol.

[0013] A third aspect of the present invention provides the application of the RVG29-modified dual-response drug-loaded nanoparticles in the preparation of antiepileptic drugs.

[0014] In another preferred embodiment, the drug is an injection, and the solvent in the injection is physiological saline.

[0015] The fourth aspect of this invention provides the application of the RVG29-modified dual-response drug-loaded nanoparticles in the preparation of drugs that inhibit neuronal excitability-induced damage.

[0016] In another preferred embodiment, the inhibition of neuronal excitability damage is the inhibition of SH-SY5Y cell neuronal excitability damage.

[0017] The fifth aspect of the present invention provides the application of the RVG29-modified dual-response drug-loaded nanoparticles in the preparation of brain-delivered drugs, wherein the brain-delivered drugs are brain-delivered drugs for the blood-brain barrier.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention loads esilicarbazepine acetate onto a piezoelectric organometallic framework (UMB). The MMB not only serves as a carrier but also exhibits ultrasonic responsiveness. The piezoelectric signals generated by the MMB regulate abnormal neuronal activity, achieving immediate epilepsy relief. Furthermore, under ultrasonic cavitation, it provides long-term release of esilicarbazepine acetate to maintain long-term efficacy. A lipid layer is then coated onto the surface of the esilicarbazepine acetate-loaded MMB. The targeted RVG29 peptide is covalently coupled to the nanoparticle surface via a Michael addition reaction between maleimide groups on the lipid layer and thiol groups on the targeted RVG29 peptide, resulting in RVG29-modified dual-response ultrasonic nanoparticles. The targeted RVG29 peptide can target nicotinic acetylcholine receptors highly expressed on the surface of brain microvascular endothelial cells and neurons, achieving receptor-mediated BBB penetration.

[0019] In this invention, RVG29-modified dual-response drug-loaded nanoparticles, during electro-pharmaceutical synergistic antiepileptic treatment, exhibit a dual effect triggered synchronously by external ultrasound. The piezoelectric signal generated by UIO-66 modulates abnormal neuronal activity to achieve immediate epilepsy relief; the ultrasound cavitation effect releases ESL on demand to maintain long-term efficacy. This synergistic strategy of physical regulation and chemical therapy not only solves the core limitations of traditional antiepileptic drugs, such as poor BBB penetration and drug resistance, but also avoids the invasiveness and infection risks of surgery or neuromodulation, providing a revolutionary solution for epilepsy treatment. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the mechanism of action of antiepileptic treatment.

[0021] Figure 2 Characterization results of UIO-66 piezoelectric nanoparticles. In the figures, a is a transmission electron microscope (TEM) image, b is a scanning electron microscope (SEM) image, c is an XRD pattern depicting the crystal structure of UIO-66, d is an atomic force microscope (AFM) image of UIO-66, e is an AFM phase hysteresis loop diagram, f is an AFM amplitude butterfly loop diagram, g is an analysis of the acoustic-to-electric energy conversion capability of UIO-66 detected by an electrochemical workstation, and h shows the ultrasonic cavitation effect in UIO-66 aqueous solution, displaying the voltage changes before and after ultrasonic irradiation. In the figures, "Before US irradiation" indicates before ultrasonic irradiation, and "After" indicates after ultrasonic irradiation.

[0022] Figure 3 This is the elemental distribution spectrum of UIO-66.

[0023] Figure 4 Thermogravimetric analysis curves for UIO-66.

[0024] Figure 5 The image shows the Fourier transform infrared spectroscopy results of UIO@ES.

[0025] Figure 6 This is a drug release curve of UIO@ESL in PBS solution.

[0026] Figure 7 Transmission electron microscope image of UIO / Lipo@ESL nanoparticles.

[0027] Figure 8 Fourier transform infrared spectrum analysis of UIO / LipoR@ESL.

[0028] Figure 9Figure 1 shows the characterization results of dual-response drug-loaded nanoparticles; a) shows the hydrodynamic particle size distribution of UIO-66, UIO@ESL, UIO / Lipo@ESL, and UIO / LipoR@ESL; b) shows the Zeta potential results; c) shows the PDI results; d) shows the hemolysis experiment results of different concentrations of UIO / LipoR@ESL; e) shows the cytotoxicity results of UIO / LipoR@ESL after co-culturing with somatic cells for 24 hours; f) shows the cytotoxicity of UIO / LipoR@ESL after co-culturing with nerve cells for 24 hours; g) shows the cytotoxic effect of 200 μg / mUIO / LipoR@ESL on key target brain cells under different ultrasound power densities; h) shows the fluorescence microscopy image of calcium ion influx in SH-SY5Y cells after stimulation with 100 mM glutamate for 6 hours; i) shows the neuroprotective effect of UIO / LipoR@ESL stimulated by ultrasound at different glutamate concentrations.

[0029] Figure 10 Figure 1 shows the results of the brain permeability and neuronal uptake characteristics analysis of dual-response drug-loaded nanoparticles; a is a schematic diagram of the in vitro Transwell model; b is the IVIS imaging result of the upper chamber of the Transwell system after 4 hours of treatment with UIO / Lipo@Cy5.5 and UIO / LipoR@ESL nanoparticles; c is the IVIS imaging result of the lower chamber of the Transwell system; d is the quantitative fluorescence analysis of nanoparticle penetration efficiency; 1 and 2 in the figure are images of the hemolysis experiment; e is the fluorescence microscopy image of SH-SY5Y cells after 24 hours of co-culture with UIO / Lipo@Cy5.5 and UIO / LipoR@ESL; f is the quantitative analysis figure of e; g is the in vivo fluorescence imaging of the UIO / Lipo@Cy5.5 group, UIO / Lipo@Cy5.5+US group, UIO / LipoR@Cy5.5 group and UIO / LipoR@Cy5.5+US group in the rat head at different time points; h is the imaging image of the isolated brain tissue of the same treatment group after 24 hours; i is the quantitative fluorescence analysis result of h.

[0030] Figure 11 The first image shows the in vivo therapeutic effect of dual-response drug-loaded nanoparticles on an acute epilepsy model; image a is a schematic diagram of the establishment of a KA-induced acute epilepsy model and ultrasound-responsive UIO / LipoR@ESL treatment. In the image, US represents ultrasound, NPs represent ultrasound-responsive nanoparticles, US opens the BBB means ultrasound is used to open the blood-brain barrier, US therapy means ultrasound therapy, and Openfield test means openfield test; image b shows the EEG signal recording results of different treatment groups within 30 minutes after acute epilepsy induction. The image on the left is the EEG signal image, and the image on the right is the corresponding time-frequency energy image.

[0031] Figure 12 The following are the results of in vivo behavioral evaluation and neuroprotective effects of dual-response drug-loaded nanoparticles on rats with acute epilepsy: a) shows the movement trajectory of rats in different treatment groups in the open field experiment after induction of the acute epilepsy model; b) shows the total movement distance of rats in the treatment group in the open field experiment; c) shows the activity level; d) shows the average velocity; e) shows the Nissl staining results of the hippocampus in different treatment groups; f) shows the immunofluorescence staining results of NeuN and GFAP in the CA3 region of the hippocampus.

[0032] Figure 13 The image shows the H&E staining results of rat tissues after different treatments in an acute epilepsy model. In the image, Heart represents the heart, Liver represents the liver, Spreep represents the spleen, Lung represents the lung, and Kidney represents the kidney. Detailed Implementation

[0033] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0034] In recent years, ultrasound-responsive piezoelectric nanotechnology has emerged as a new direction for the treatment of various diseases due to its non-invasive nature. Its core mechanism lies in the inherent mechano-electric coupling properties of piezoelectric materials: under external ultrasound stimulation, a local electric field can be generated to regulate cellular electrophysiological activities. Furthermore, piezoelectric materials such as metal-organic frameworks (MOFs) can serve as smart carriers to achieve spatiotemporally controlled drug release. Especially when modified with brain-targeting peptides, these nanosystems can bypass the brain-body barrier (BBB) ​​to achieve precise and efficient brain drug delivery. This innovative fusion of physical neuromodulation and chemotherapy provides a groundbreaking electro-chemical synergistic paradigm for epilepsy treatment.

[0035] Based on this, the present invention provides RVG29-modified dual-responsive drug-loaded nanoparticles for electro-drug synergistic antiepileptic therapy. The RVG29-modified dual-responsive drug-loaded nanoparticles are prepared by loading ESL onto UIO-66, encapsulating a lipid bilayer structure on its surface, and then covalently coupling the targeted RVG29 peptide to the nanoparticle surface via a Michael addition reaction between the maleimide groups of the lipid membrane and the thiol groups on the targeted RVG29 peptide, thus obtaining RVG29-modified dual-responsive ultrasonic nanoparticles. The therapeutic principle of the RVG29-modified dual-responsive drug-loaded nanoparticles in the electro-drug synergistic antiepileptic therapy of the present invention is as follows: Figure 1As shown, UIO-66, with its piezoelectric properties, serves as an ultrasound-responsive carrier. Its porous structure loads ESL drugs, and its outer layer is encapsulated in a lipid bilayer coupled with a targeting RVG29 peptide. This targeting RVG29 peptide can target nicotinic acetylcholine receptors (nAChRs) highly expressed on the surface of brain microvascular endothelial cells and neurons, achieving receptor-mediated BBB penetration. During treatment, external ultrasound simultaneously triggers a dual effect: (i) the piezoelectric signal generated by UIO-66 modulates abnormal neuronal activity to achieve immediate epilepsy relief; (ii) the ultrasound cavitation effect releases ESL on demand to maintain long-term efficacy. This synergistic physical-chemical therapy strategy addresses the core limitations of traditional antiepileptic drugs, such as poor BBB penetration and drug resistance, while avoiding the invasiveness and infection risks of surgery or neuromodulation, providing a revolutionary solution for epilepsy treatment.

[0036] The following section provides a detailed description of RVG29-modified dual-response drug-loaded nanoparticles, their preparation methods, and applications.

[0037] RVG29 peptide was provided by Nanjing Genscript Biotech Co., Ltd.; 1,2-distearyl-SN-glycerol-3-phosphorylethanolamine-N-maleimide-polyethylene glycol 2000 (DSPE-PEG2000-MAL) was purchased from Xi'an Haoran Biotechnology Co., Ltd.; cholesterol, penicillin / streptomycin antibiotics, PBS buffer, and egg yolk lecithin (EPC) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; eslicarbazepine acetate (ESL), kaempferol (KA), dichloromethane, benzoic acid, and N,N-dimethylformamide were purchased from Maclean Biotechnology Co., Ltd.; Fluo-4 AM calcium ion fluorescent probe was purchased from Beyotime Biotechnology Co., Ltd.; ultrafiltration centrifuge tubes were purchased from Merck Millipore; Cy5.5-NHS fluorescent labeling agent was purchased from APExBIO; DAPI staining solution was purchased from Med... ChemExpress; chromatographic grade acetonitrile and trifluoroacetic acid were purchased from Thermo Fisher Scientific; anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.; zirconium tetrachloride (ZrCl4) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; 2-aminoterephthalic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and ordinary carbon support membrane was purchased from Beijing Zhongjing Scientific Instruments Technology Co., Ltd.

[0038] 1. Experimental Procedure 1.1 Preparation of RVG29-modified dual-response drug-loaded nanoparticles 139.8 mg of zirconium tetrachloride was dissolved in 15 mL of N,N-dimethylformamide, and 180 mg of p-2-aminoterephthalic acid and 3750 mg of benzoic acid were dissolved in 15 mL of dimethylformamide F. After complete dissolution, both solutions were transferred to 100 mL glass sample vials, thoroughly mixed, and reacted at 120 °C for 16 h. After the reaction was completed, the precipitate was collected by centrifugation at 11000 rpm for 5 min, washed with anhydrous methanol, and centrifuged three times to remove unreacted reagents. Finally, the precipitate was dried overnight at 70 °C in a vacuum drying oven, ground, and UIO-66 was obtained.

[0039] UIO-66 was activated in a 150℃ drying oven for 3 hours to remove moisture and impurities from the pores of UIO-66, so that ESL could be better loaded onto UIO-66 in the future. Then, it was mixed with ESL in anhydrous ethanol at a mass ratio of 2:1 and stirred in the dark at 23℃ for 24 hours. Finally, the nanoparticles were collected by centrifugation and denoted as UIO@ESL.

[0040] Liposomes were prepared using a thin-film hydration method: EPC, cholesterol, and DSPE-PEG2000-MAL were dissolved in dichloromethane at a mass ratio of 6:1.5:1.2. The organic solvent was then removed by rotary evaporation at 37°C to form a lipid membrane. Subsequently, 5 ml of a 1 mg / mL UIO@ESL aqueous solution was added, and the mixture was hydrated at 37°C for 40 minutes. Finally, the mixture was sonicated for 5 minutes to obtain nanoparticles, denoted as UIO / Lipo@ESL.

[0041] Tris(2-carboxyethyl)phosphine and RVG29 peptide were dissolved in a 2 mg / mL UIO / Lipo@ESL solution at a molar ratio of 50:4. After stirring in the dark for 3 h, the mixture was collected by ultrafiltration and centrifugation to obtain RVG29-modified dual-response drug-loaded nanoparticles, denoted as UIO / LipoR@ESL.

[0042] 1.2 Structural Characterization The morphology and microstructure of nanoparticles were observed using transmission electron microscopy and scanning electron microscopy; their piezoelectric properties were analyzed using atomic force microscopy; functional groups were identified using Fourier transform infrared spectroscopy with potassium bromide pellet method; and particle size distribution, zeta potential and aggregation index (PDI) were determined by DLS.

[0043] 1) Drug loading status First, ESL was prepared into standard solutions of different concentrations. The peak area (y) and the corresponding ESL concentration (x) were measured by HPLC and plotted to form the ESL standard curve. Then, UIO@ESL nanoparticles were synthesized using the same method, and the centrifuged liquid was collected. The peak area was measured by HPLC. The ESL concentration in the centrifuged liquid was determined by analyzing the relationship between ESL concentration and peak area in the ESL standard curve. The encapsulation efficiency and drug loading of ESL were calculated using the following formulas:

[0044] Encapsulation ratio = ×100; Drug loading = ×100%.

[0045] Note: m1: mass of ESL in nanoparticles; m2: total amount of ESL administered; m3: mass of nanoparticles.

[0046] 2) Drug release 1 ml of UIO / LipoR@ESL at a concentration of 4 mg / ml was placed in a dialysis bag (3500 MW) and then immersed in a brown glass vial containing 100 mL of neutral buffer. Ultrasonic stimulation (frequency 1 MHz, power density 1.5 W / cm²) was applied for 5 minutes at preset time points, followed by sampling and analysis of the ESL release curve.

[0047] 3) RVG29 peptide coupling efficiency Standard solutions of RVG29 peptide at different concentrations were prepared. The peak area (y) and the corresponding ESL concentration (x) were measured by HPLC and plotted to form the RVG29 peptide standard curve. UIO / LipoR@ESL nanoparticles were synthesized using the above method. The filtrate was collected after ultrafiltration and centrifugation. The peak area was measured by HPLC. The concentration of RVG29 peptide in the filtrate was determined by analyzing the relationship between the RVG29 peptide concentration and peak area in the RVG29 peptide standard curve. The linkage rate of the RVG29 peptide was calculated using the following formula:

[0048] Connection rate = ×100%.

[0049] Note: m4 is the mass of RVG29-Cys in the centrifuged liquid; m5 is the total amount of RVG29-Cys sample added.

[0050] 1.3 Cell Experiments Cell culture: bEnd.3 cells, HUVEC cells, 3T3 cells, BV2 cells and MA-c cells were cultured in DMEM medium containing 1 wt% penicillin-streptomycin and 10 wt% fetal bovine serum in an incubator at 37°C with 5% CO2 in the air.

[0051] SH-SY5Y cells were cultured in DMEM / F-12 medium containing 1 wt% penicillin-streptomycin and 10% FBS in an incubator at 37°C with 5% CO2 in the air.

[0052] Cytotoxicity HUVEC cells, 3T3 cells, BV2 cells, and MA-c cells were cultured at a rate of 8 × 10⁻⁶. 3 Cells were seeded at different densities in 96-well plates. Different concentrations of UIO / LipoR@ESL nanoparticles were added and incubated for 24 hours. Cell viability was assessed using a CCK-8 assay.

[0053] bEnd.3 cells and SH-SY5Y cells were mixed at a rate of 8 × 10⁻⁶. 3 Cells were seeded at a density of 5 wells per group in a cross-hatching pattern in 96-well plates for easy sonication. The same concentration of UIO / LipoR@ESL nanoparticles was added, and the cells were incubated for 24 hours. A 0.5 W / cm² hydrochloric acid solution was used. 2 1W / cm 2 1.5W / cm 2 Cells were stimulated with ultrasound at DE power density. After 1 hour of treatment, cell viability was assessed using a CCK-8 assay. The formula is as follows:

[0054] Cell activity = ×100%.

[0055] Hemolysis test: Blood was extracted from mouse eyeballs. Plasma and platelets were removed by repeated centrifugation. Red blood cells were diluted with PBS and mixed thoroughly. 300 μL of the diluted solution was then mixed with 1 mL of deionized water, PBS, and different concentrations of UIO / LipoR@ESL nanoparticles. After incubation at room temperature for 4 hours, the mixture was centrifuged, and the state of the mixture after centrifugation was photographed.

[0056] Gently transfer the supernatant to another centrifuge tube and centrifuge to precipitate the material. Transfer 200 μL of the supernatant to each well of a 96-well plate. Measure the absorbance at 450 nm using a microplate reader and calculate the hemolysis rate. The formula is as follows:

[0057] Hemolysis rate = [(OD sample - OD negative) / (OD positive - OD negative)] × 100%.

[0058] Note: OD is the absorbance at 450 nm; positive is the deionized water group; negative is the PBS group.

[0059] 1.4 Inhibition of glutamate by UIO / LipoR@ESL 1) UIO / LipoR@ESL inhibits glutamate-induced calcium ion influx SH-SY5Y cells were used at a rate of 1×105 The cells were seeded at a density of 100 cells / well in 6-well plates and treated with UIO / LipoR@ESL containing 100 mM glutamate for 6 hours. After adding Hepes buffer containing 2 μM Fluo-4AM to each well, the calcium ion influx was observed using a fluorescence microscope.

[0060] 2) UIO / LipoR@ESL inhibits Glu-induced excitotoxicity SH-SY5Y cells were used at a rate of 8 × 10 3 Cells were seeded at 96-well plate densities and then co-incubated with Glu at concentrations of 0 mM, 10 mM, 20 mM, 50 mM, and 100 mM. UIO / LipoR@ESL nanoparticles were added and unconditioned stimulation was applied. Cell viability was assessed using a CCK-8 assay.

[0061] 3) Transwell model bEnd.3 cell is divided into 1×10 5 Cells were seeded at a density per well into the upper compartments of a Transwell-6-well plate. Cell barrier integrity was assessed by measuring the permeability of fluorescein sodium, which allows cell penetration. 10 μg / mL of fluorescein sodium was added to the upper compartment, and the substrate was collected after 1 hour. Fluorescence intensity was measured at an excitation wavelength of 460 nm and an emission wavelength of 515 nm. The permeability coefficient for each compartment was calculated using the formula below:

[0062] P test = .

[0063] Note: C1 is the concentration of sodium fluorescein in the upper chamber; V1 is the volume of the upper chamber; t is the incubation time; A is the membrane area of ​​the Transwell; C2 is the concentration of sodium fluorescein in the lower culture well.

[0064] Different Cy5.5-loaded nanoparticles were added and incubated for 4 hours. The chambers and substrates were photographed using IVIS, and their relative fluorescence intensities were calculated.

[0065] 4) Nanoparticle brain penetration In this experiment, rats were administered Cy5.5-labeled nanoparticles via tail vein injection. One mL of a 4 mg / mL nanoparticle solution was injected, and ultrasound stimulation was applied to the rat's head simultaneously. In vivo fluorescence imaging was performed at four predetermined time points: 3, 6, 12, and 24 hours post-injection using an IVIS in vivo imaging system. Twenty-four hours after administration, rats were euthanized, and their brain tissue was completely removed for further in vitro imaging using the IVIS system to assess the brain enrichment effect.

[0066] 5) Epilepsy modeling and evaluation of in vivo anti-epileptic effects Rats were anesthetized with sodium pentobarbital, their head hair was removed, their head skin was cut off, and the fascia, periosteum, and other tissues on the surface of the skull were separated to expose the skull and sutures.

[0067] Electrodes and cannulas were implanted using a stereotaxic instrument with Bregma point as the zero coordinate (AP=-3.6mm, ML=+2.0mm, DV=-3.0mm; AP=-4.2mm, ML=+4.0mm, DV=-3.0mm). The rats were then returned to their cages for a week of post-operative recovery.

[0068] Different drugs were administered via tail vein injection: Rats were anesthetized and placed on a stereotaxic apparatus. 0.5 μL of KA (1 mg / mL) was injected, and the syringe was withdrawn after 5 minutes. The rats' electroencephalogram (EEG) signals were then recorded for 30 minutes using a Cerebus multichannel in vivo neural signal acquisition system. After the experiment, the rats were returned to their cages to rest.

[0069] Before conducting the open field experiment, rats were placed in a dark laboratory environment to acclimatize to the experimental conditions. At the start of the experiment, the rats were placed in the dark chamber. An infrared camera was mounted on the top of the chamber and connected to a small animal video trajectory tracking system. The software recorded the rats' exploration behavior in the dark chamber for 10 minutes, including behavioral data such as the total distance traveled and the movement trajectory of each rat.

[0070] Nissl staining and immunofluorescence staining: After the open field experiment, rats were euthanized. Rat brain tissue was removed and quickly fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned. For Nissl staining, brain sections were treated with Nissl stain for 5 minutes, dehydrated, and mounted. Observation and photography were performed using an optical microscope. For immunofluorescence staining, primary antibodies against NeuN and GFAP were incubated overnight at 4°C. The next day, after washing with PBS, secondary antibodies were added and incubated at room temperature for 1 hour. Anti-fluorescence attenuation mounting medium containing DAPI was added, and coverslips were placed on top. Observation and photography were performed using a fluorescence microscope.

[0071] Statistical analysis The results were analyzed using Excel software, and the significant differences between the groups were assessed using the two-tailed Student's t-test. (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05 were considered statistically significant).

[0072] 2. Results 2.1 Characterization and drug release of UIO / LipoR@ESL The core of the UIO / LipoR@ESL architecture is the piezoelectric organometallic framework UIO-66, which was prepared using a thermal deposition method. Transmission electron microscopy and scanning electron microscopy characterization showed that UIO-66 exhibited a uniform particle structure. Figure 2(a and b in the original text). The synthesized UIO-66 has XRD curves and elemental types similar to those in previous works ( Figure 2 c and Figure 3 This indicates the successful synthesis of UIO-66 (Zr) nanoparticles. To evaluate the piezoelectric properties of the nanoparticles, UIO-66 was suspended on a silicon wafer and observed and analyzed using an atomic force microscope. The 180-degree flip of the phase hysteresis loop indicates that the electrical signal generated on its surface is not derived from electrostatics. Figure 2 The amplitude butterfly circuit in d indicates that UIO-66 exhibits the piezoelectric effect. Figure 2 (e). Meanwhile, the results from the electrochemical workstation showed a strong change in the curve amplitude during the on and off periods, indicating the generation of current and demonstrating that UIO-66 possesses acoustic-electric conversion capabilities. Figure 2 f in the middle.

[0073] To load the drug, UIO-66 was first activated at 150°C to remove water molecules and other impurities from its pores. Thermogravimetric analysis showed that this temperature did not damage the nanoparticles themselves. Figure 4 Furthermore, cavitation experiments demonstrate that UIO-66 exhibits cavitation effects. Figure 2 The g in the figure indicates that nanoparticles can undergo cavitation under ultrasound, which can assist in drug release. Fourier transform infrared (FTIR) results show that ( Figure 5 After loading the antiepileptic drug ESL, the UIO@ESL nanoparticles simultaneously exhibited characteristic peaks of both UIO-66 and ESL, confirming successful ESL loading. The encapsulation efficiency and drug loading were 50.59% and 20.19%, respectively. The effect of ultrasound on ESL drug release was investigated in PBS buffer. When the nanoparticles were exposed to ultrasound, ESL was released rapidly. However, without ultrasound treatment, the ESL release rate was significantly slowed down. Figure 6 This indicates that ultrasound can increase the drug release rate.

[0074] Blood-brain barrier targeted peptide surface modification To achieve blood-brain barrier penetration, a lipid bilayer structure was coated onto the surface of UIO@ESL to prepare UIO / Lipo@ESL. Transmission electron microscopy confirmed that the lipid layer was uniformly coated. Figure 7 Subsequently, the RVG29 peptide was covalently coupled to the nanoparticle surface via a Michael addition reaction between the maleimide groups of the lipid membrane and the thiol groups of the peptide. Fourier transform infrared spectroscopy was performed at 1200 cm⁻¹. - ¹A distinct CS stretching vibration peak was detected nearby ( Figure 8 The study confirmed the formation of a stable thioether bond and the successful coupling of the RVG29 polypeptide, with a coupling efficiency of 86.60% ± 1.29%.

[0075] Physicochemical property characterization of UIO / LipoR@ESL The particle size, surface potential, and dispersion stability of the final prepared UIO / LipoR@ESL were tested. Figure 9 (a~c) Dynamic light scattering (DLS) analysis showed that its hydrodynamic diameter was 198.17±3.65 nm, with uniform distribution. Compared with unmodified UIO-66, the Zeta potential of UIO / LipoR@ESL showed a significant reversal, confirming the successful encapsulation of the lipid layer. After coupling with the RVG29 peptide, the potential of UIO / Lipo@ESL changed from -10.40±0.17 mV to -13.73±0.55 mV, which is consistent with the negatively charged nature of this peptide. The nanoparticles also exhibited excellent dispersion stability, with a polydispersity index (PDI) as low as 0.101±0.016.

[0076] 1.2 Cell Safety To determine whether intravenous administration of nanoparticles would cause cell damage, the safety of UIO / LipoR@ESL was evaluated in vitro. First, a hemolysis assay was performed to assess whether the nanoparticles would cause erythrocyte lysis after intravenous administration. The results showed that even at high concentrations, the nanoparticles did not induce a hemolytic reaction. Figure 9 (d) Subsequently, normal somatic cell lines HUVEC and 3T3, as well as central nervous system cell lines MA-c and BV2, were selected as model cells to evaluate the cytotoxicity of the nanoparticles. When the concentration reached 200 μg / mL, the cells still maintained good viability ( Figure 9 (e and f in the text).

[0077] Nanoparticles deliver electrical stimulation signals and drugs via ultrasound to achieve epilepsy treatment. Therefore, the effect of nanoparticles on cell viability at different ultrasound power densities was evaluated using the CCK-8 assay. bEnd.3 and SH-SY5Y are two key cell lines in epilepsy treatment. Results showed that at 1.5 W / cm²... 2 At an ultrasound power density of 200 μg / mL, cells in UIO / LipoR@ESL still maintained good viability. Figure 9 (g in the text). These results demonstrate that UIO / LipoR@ESL can be safely administered in vivo via intravenous infusion without causing cell damage.

[0078] 1.3 Inhibition of neuronal excitability damage Epilepsy seizures are caused by excessive and abnormal neuronal activity. To simulate epileptic seizures in vitro, SH-SY5Y cells treated with glutamate (Glu) were used as epilepsy model cells. Glu is a key excitatory neurotransmitter in the central nervous system; high concentrations of 100 mM Glu can cause excitotoxicity. The excitotoxicity of Glu is primarily receptor-mediated, as it overactivates N-methyl-D-aspartate receptors, leading to a large influx of calcium ions, which in turn activates a series of biochemical reactions, including mitochondrial dysfunction, oxidative stress, and neuronal damage.

[0079] In this embodiment, SH-SY5Y cells were stimulated with 100 mM Glu, and the intracellular calcium ion concentration was observed using a fluorescence microscope. The results showed ( Figure 9 In the H group, intracellular calcium ion fluorescence significantly increased after Glu stimulation, while it remained significantly within the normal range in the UIO / LipoR@ESL sonication treatment group. Furthermore, although intracellular calcium ion fluorescence decreased slightly in cells treated with ESL alone, it was still lower than in the UIO / LipoR@ESL sonication treatment group. Similarly, despite sonication treatment, intracellular calcium ion fluorescence in the UIO-66 group did not return to normal levels. These results indicate that dual-response drug-loaded nanoparticles, through the synergistic effect of electrical stimulation and ESL, can more effectively address Glu-induced calcium ion influx, thereby protecting SH-SY5Y cells from neuroexcitatory damage.

[0080] The CCK-8 experiment further verified the neuroprotective effect. Figure 9 (i) Exposure of SH-SY5Y to Glu at concentrations of 0 mM, 10 mM, 20 mM, 50 mM, and 100 mM for 6 hours reduced cell viability to 69.85%. After sonication treatment with UIO / LipoR@ESL, cytotoxicity was significantly reduced, and cell viability remained at approximately 90%. The sonication-responsive drug-loaded nanoparticles UIO / LipoR@ESL effectively inhibited Glu-induced excitatory cytotoxicity, demonstrating their significant neuroprotective effect.

[0081] 1.4 In vitro blood-brain barrier permeability study Using bEnd.3 cells to simulate the in vitro blood-brain barrier structure, a Transwell model was constructed to investigate the ability of RVG29-modified dual-response drug-loaded nanoparticles to penetrate the blood-brain barrier via transcytosis. bEnd.3 cells were seeded into the upper chamber of the Transwell insertion well and cultured for 3 days. The integrity of the cell barrier was assessed using a sodium fluorescein permeability test. Cy5.5-labeled nanoparticles UIO / Lipo@Cy5.5 and UIO / LipoR@Cy5.5 were co-incubated with bEnd.3 cells in the upper chamber. The fluorescence signal intensity in the upper and lower chambers was analyzed using a small animal in vivo imaging system. The procedure is as follows: Figure 10 As shown in 'a'.

[0082] The results showed that after 4 hours of incubation, the fluorescence of UIO / Lipo@Cy5.5 was almost exclusively present in the upper chamber, while only a small amount of fluorescence was detected in the lower chamber. Conversely, in the UIO / LipoR@Cy5.5 group, the fluorescence in the lower chamber increased significantly, as shown in the results below. Figure 10 As shown in b, c, and d, this indicates that the RVG29-modified dual-response drug-loaded nanoparticles exhibit significantly enhanced permeability across the endothelial cell barrier in vitro, demonstrating promising potential for brain drug delivery.

[0083] RVG29 peptide enhances neuronal uptake of nanoparticles To verify whether RVG29-modified dual-response drug-loaded nanoparticles could enhance the uptake by SH-SY5Y cells, UIO / Lipo@Cy5.5 and UIO / LipoR@Cy5.5 were incubated with SH-SY5Y cells for 4 hours, and the internalization of the dual-response drug-loaded nanoparticles was analyzed by fluorescence microscopy. The results showed that the RVG29-modified dual-response drug-loaded nanoparticle group exhibited a strong fluorescence signal, which was located in the cytoplasm. Figure 10 As shown in e, the fluorescence signal is approximately 30% higher than that of the UIO / Lipo@Cy5.5 group. Figure 10 As shown in f in the figure. This may be related to the high expression of nAchR on the surface of neurons, and this receptor-mediated recognition promotes the neuronal uptake of nanoparticles.

[0084] In vivo brain targeting with Cy5.5 nanoparticles To evaluate the in vivo brain-targeting ability of dual-response drug-loaded nanoparticles, Cy5.5-loaded nanoparticles were injected via tail vein, and their brain penetration was assessed using IVIS. Ultrasound can penetrate the skull and focus in the brain, causing cavitation effects such as oscillation and expansion of microvesicles in cranial blood vessels, resulting in a temporary opening of the blood-brain barrier. Therefore, ultrasound stimulation was applied simultaneously during the experiment to evaluate the synergistic effect of RVG29 peptide and ultrasound on in vivo brain targeting. Figure 10The g-index showed that the fluorescence accumulation of UIO / Lipo@Cy5.5 in the head was very weak, almost unable to penetrate the brain. After ultrasound application, the fluorescence in the head of the UIO / Lipo@Cy5.5 ultrasound group increased significantly, indicating that ultrasound can increase the brain penetration of nanoparticles. Similarly, the UIO / LipoR@Cy5.5 group modified with RVG29 peptide also showed a stronger fluorescence signal than the UIO / Lipo@Cy5.5 ultrasound group. Among all groups, the UIO / LipoR@Cy5.5 ultrasound group showed the strongest fluorescence accumulation in the brain, significantly better than other groups.

[0085] Twenty-four hours after administration, rats were euthanized, and the retention capacity of the material in the brain under different treatments was investigated by in vitro brain imaging. Figure 10 The h and i values ​​in the figures show that the UIO / LipoR@Cy5.5 ultrasound group exhibited the strongest fluorescence, which is largely consistent with the results of in vivo imaging in rats. To further demonstrate whether the RVG29-modified dual-response drug-loaded nanoparticles, under ultrasound-mediated guidance, entered the brain parenchyma rather than remaining in microvessels or endothelial cells, sagittal sections of rat brain tissue treated with the UIO / LipoR@Cy5.5 ultrasound group for 24 hours were scanned. The results showed that fluorescence was distributed in the brain parenchyma region. These results indicate that ultrasound, in conjunction with the targeting RVG29 peptide, can deliver dual-response drug-loaded nanoparticles into the brain more efficiently.

[0086] Evaluation of the anti-epileptic efficacy of intravenous injection of UIO / LipoR@ESL KA is a potent glutamate analog that induces neuronal hyperexcitability by activating glutamate receptors. To evaluate the in vivo antiepileptic effect of nanoparticles, KA was injected into the hippocampus of rats to establish an acute epilepsy model, and the seizures were observed. Figure 11 As shown in a, 30 minutes of electroencephalography (EEG) was recorded in rats using a Cerebus multichannel in vivo neural signal acquisition system to dynamically monitor seizure activity. Figure 11 Figure b shows that after KA induction, the seizures were prolonged and severe. Tail vein injection provided only slight relief from the seizures, with minimal inhibitory effect. Similarly, the UIO / LipoR@ESL group did not show any improvement in seizure frequency. This indicates that without ultrasound stimulation, drug release is insufficient and no electrical signal is generated, failing to effectively control seizures. In contrast, after ultrasound stimulation, the UIO / LipoR@ESL group exhibited significant anti-epileptic ability, with EEG signals returning to near-normal levels. This is because, after ultrasound stimulation, the dual-response drug-loaded nanoparticles rapidly release electrical signals and drugs, blocking sodium ion channels and reducing neuronal excitability, thereby achieving optimal therapeutic effects.

[0087] As epileptic seizures progressed into the chronic phase, the rats exhibited behavioral abnormalities such as anxiety-like behavior and reduced exploratory behavior. Therefore, an open field experiment was conducted on the rats on the third day. The rats' motor abilities and exploratory behavior were recorded for 10 minutes in the open field to further evaluate the anti-epileptic effect of the nanoparticles. Figure 12 Figures a, b, c, and d show that after KA modeling, the rats only traveled 1856.2 mm ± 991.8 mm, significantly less than the 24209.7 mm ± 1460.2 mm traveled by normal rats. This situation was not significantly improved in the UIO / LipoR@ESL group either. The motor ability of the ESL group rats improved somewhat, but was still lower than that of the normal group rats. In contrast, after receiving ultrasound treatment, the UIO / LipoR@ESL group rats showed a significant increase in mobility, reaching 25537.9 mm ± 1649.8 mm, similar to normal rats. Furthermore, their activity level and walking speed also returned to normal levels.

[0088] Neuroprotective effects and biocompatibility of ultrasound-responsive nanoparticles In a KA-induced epilepsy model, abnormal neuronal firing leads to cell damage and even death. The protective effect of nanoparticles on neurons was assessed using Nissl staining and immunofluorescence staining of rat brain slices. Results showed that the KA-induced model group exhibited loosely arranged neurons, decreased cell density, and significant neuronal loss. The ESL and UIO / LipoR@ESL groups showed improvement compared to the KA group, but neurons still exhibited varying degrees of degeneration and damage. Conversely, Figure 12 As shown in e, the UIO / LipoR@ESL ultrasound group exhibited the most significant neuroprotective effect, with neuronal morphology almost fully restored and neuronal arrangement becoming clear and dense. Similarly, compared to... Figure 12 Immunofluorescence staining, as shown in f, yielded the same conclusion. In the Control group, the CA3 region showed dense NeuN-positive (neuron-specific biomarker) cells, well-organized neurons, almost no neuronal death, and sparse GFAP-positive (glial cell-specific biomarker) cells, indicating a healthy brain tissue condition. In the KA group, NeuN-positive cells were significantly reduced, with loose cell arrangement, showing obvious neuronal loss and death. Simultaneously, GFAP was highly expressed, demonstrating that KA constituted significant neurotoxicity and brain damage. However, these deficiencies were significantly improved in the UIO / LipoR@ESL group. NeuN-positive cells significantly increased, and the number of GFAP-positive cells returned to normal levels. These results indicate that dual-response drug-loaded nanoparticle therapy can significantly prevent neuronal damage caused by epilepsy.

[0089] Finally, histopathological analysis was performed on the heart, liver, spleen, lungs, and kidneys of rats in each group. The biocompatibility of the nanoparticles was assessed using hematoxylin-eosin staining. Results showed that, compared with the Control group, rats in the UIO / LipoR@ESL ultrasound group did not exhibit significant structural changes or tissue damage in any of their organs. Figure 13 As shown in the figure. This indicates that the treatment regimen does not cause toxic side effects in rats and has good safety.

[0090] This invention provides ultrasound-responsive nanoparticles with a dual-mode anti-epileptic mechanism. In vitro experiments showed that RVG29-modified dual-responsive drug-loaded nanoparticles exhibited stronger transmembrane migration in the Transwell model and significantly improved the uptake efficiency of SH-SY5Y cells. These superior brain-targeting properties were further validated in animal experiments, where RVG29 peptide combined with ultrasound stimulation significantly enhanced the brain penetration and retention time of the dual-responsive drug-loaded nanoparticles. This ultrasound-triggered drug release system effectively inhibited glutamate-induced calcium ion influx and reduced related cytotoxicity. In a KA-induced epilepsy rat model, the EEG signal of the UIO / LipoR@ESL combined with ultrasound treatment group recovered to near-normal levels, with significantly better efficacy than the ESL-only group. Behavioral tests confirmed that the ultrasound-activated UIO / LipoR@ESL treatment group rats exhibited stronger motor ability and exploratory activity.

[0091] Nissl staining and immunofluorescence analysis showed that the neurons in the UIO / LipoR@ESL combined with ultrasound therapy group were densely packed, the density of NeuN-positive cells recovered to near normal levels, and no cytotoxicity was detected, fully demonstrating its excellent neuroprotective effect. In summary, the RVG29-modified dual-response drug-loaded nanoparticles of this invention represent a safe and efficient anti-epileptic strategy, successfully overcoming the limitations of traditional therapies. This non-invasive method not only provides a new approach to epilepsy treatment but also offers important reference for the development of other neurological disease therapies based on electrical stimulation.

[0092] It should be noted that when the mass ratio of UIO-66 to esilicarbazepine acetate is 1~4:1, the mass ratio of UIO-66 loaded with esilicarbazepine acetate to liposomes is 1:1~2, and the molar ratio of tris(2-carboxyethyl)phosphine to the targeting RVG29 peptide is any value from 50:4 to 10, such as when the mass ratio of UIO-66 to esilicarbazepine acetate is 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, the molar ratio of UIO-66 loaded with esilicarbazepine acetate to liposomes is 1:1~2, and the molar ratio of tris(2-carboxyethyl)phosphine to the targeting RVG29 peptide is any value from 50:4 to 10, the following conditions apply: When the body mass ratios are 1:1, 1:1.5, and 1:2, and the molar ratios of tris(2-carboxyethyl)phosphine and the targeted RVG29 peptide are 50:4, 50:4.5, 50:5, 50:5.5, 50:6, 50:6.6, 50:6.5, 50:7, 50:7.5, 50:8, 50:8.5, 50:9, 50:9.5, and 50:10, the prepared RVG29-modified dual-response drug-loaded nanoparticles exhibit anti-epileptic, neuronal excitability-inhibiting, and brain drug delivery effects.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An RVG29-modified dual-response drug-loaded nanoparticle, characterized in that, Using a piezoelectric organometallic framework as a carrier, eslicarbazepine acetate is loaded onto the piezoelectric organometallic framework, and a lipid layer is coated on its surface to obtain nanoparticles. The maleimide groups on the lipid layer and the thiol groups on the targeted RVG29 peptide are covalently coupled to the surface of the nanoparticles through a Michael addition reaction, resulting in the RVG29-modified dual-response drug-loaded nanoparticles.

2. The RVG29-modified dual-response drug-loaded nanoparticles according to claim 1, characterized in that, The RVG29-modified dual-response drug-loaded nanoparticles have a particle size of 194 nm to 202 nm.

3. The RVG29-modified dual-response drug-loaded nanoparticles according to claim 2, characterized in that, The dual response refers to both the ultrasonic response and the piezoelectric signal response.

4. The RVG29-modified dual-response drug-loaded nanoparticles according to claim 3, characterized in that, The piezoelectric organometallic framework is UIO-66.

5. A method for preparing RVG29-modified dual-response drug-loaded nanoparticles according to any one of claims 4, characterized in that, Includes the following steps: UIO-66 and eslicarbazepine acetate were mixed in an alcoholic environment at a mass ratio of 1 to 4:1, stirred in the dark, and centrifuged to obtain UIO-66 loaded with eslicarbazepine acetate. UIO-66 loaded with eslicarbazepine acetate was hydrated with liposomes in an aqueous environment at a mass ratio of 1:1~2, followed by sonication to obtain a nanoparticle solution. Tris(2-carboxyethyl)phosphine and a targeted RVG29 peptide were added to a nanoparticle solution at a molar ratio of 50:4~10. The mixture was subjected to a Michael addition reaction in the dark, followed by sonication. The precipitate was collected to obtain the RVG29-modified dual-response drug-loaded nanoparticles.

6. The method for preparing RVG29-modified dual-response drug-loaded nanoparticles according to claim 5, characterized in that, The hydration reaction is carried out at a temperature of 37℃~45℃ for a time of 40min~60min. The Michael addition reaction was carried out at a temperature of 20°C to 28°C for 2 hours to 4 hours.

7. The method for preparing RVG29-modified dual-response drug-loaded nanoparticles according to claim 5, characterized in that, The alcohol reagent is anhydrous ethanol.

8. The use of RVG29-modified dual-response drug-loaded nanoparticles according to any one of claims 1 to 4 in the preparation of antiepileptic drugs, characterized in that, The drug is an injectable preparation, and the solvent in the injectable preparation is physiological saline.

9. The use of the RVG29-modified dual-response drug-loaded nanoparticles according to any one of claims 1 to 4 in the preparation of drugs that inhibit neuronal excitability-induced damage, characterized in that, The inhibition of neuronal excitability damage refers to the inhibition of SH-SY5Y cell neuronal excitability damage.

10. The use of the RVG29-modified dual-response drug-loaded nanoparticles according to any one of claims 1 to 4 in the preparation of brain-delivered drugs, characterized in that, The brain-delivered drug is a brain-delivered drug for use against the blood-brain barrier.

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