Ultrasound-driven nerve regulation nano-device for noninvasive treatment of epilepsy and preparation method and application of ultrasound-driven nerve regulation nano-device

Abstract

The invention relates to an ultrasound-driven nerve regulation nano device for non-invasive treatment of epilepsy as well as a preparation method and application of the ultrasound-driven nerve regulation nano device. The ultrasound-driven nerve regulation nano device comprises a microenvironment response module, an ultrasound response module and a biological targeting module, the microenvironment response module is platinum nanoparticles, the ultrasonic response module is piezoelectric nanoparticles UIO-66-NH2, and the biological targeting module is brain targeting peptide; wherein the platinum nanoparticles are dispersed and embedded in a UIO-66-NH2 structure, and the brain-targeted peptide is modified on the surface of the UIO-66-NH2 structure. The nano device can effectively decompose hydrogen peroxide in an epilepsy focus area and reduce the oxidative stress level; the mechanical stress can be converted into an electric signal under the ultrasonic action to trigger neuron electrophysiological reaction, so that noninvasive precise regulation and control of an epilepsy reactive neural circuit are realized; meanwhile, the blood brain barrier can be efficiently traversed, and the cumulant of the epilepsy focus part in the brain is obviously enhanced through the synergistic effect of the external field ultrasound and the biological targeting module.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to an ultrasound-driven nerve regulation nanodevice for non-invasive treatment of epilepsy, as well as a preparation method and application thereof. Background Art

[0002] Epilepsy is the second most common disease of the nervous system, and its main feature is temporary central nervous system dysfunction caused by abnormal discharges of brain neurons. About 20%-30% of epilepsy patients are diagnosed with refractory epilepsy. It is difficult for such patients to completely control their condition with conventional anti-epileptic drugs, and their mortality rate is higher than that of ordinary epilepsy patients. At present, the treatment methods for refractory epilepsy mainly include the combination of multiple drugs or surgical implantation of neuromodulatory devices. However, the high risk of surgery and the possible side effects of implanted devices have become the main difficulties faced by patients. Therefore, how to effectively regulate epileptic neural signals and repair neural pathways in a non-invasive way has become a key challenge in the current treatment of refractory epilepsy.

[0003] Piezoelectric nanomaterials can convert mechanical stress into electrical signals under the action of ultrasound, triggering electrophysiological responses of neurons, such as calcium ion transients and neuroplasticity regulation. However, the existence of the blood-brain barrier is one of the important factors limiting the therapeutic effect. As a natural barrier between blood and brain tissue, the blood-brain barrier is composed of brain microvascular endothelial cells, pericytes and astrocytes, which only allow specific molecules to pass through. Most neuroprotective substances have difficulty reaching brain lesions. In addition, the abnormal characteristics of the epileptic microenvironment further increase the difficulty of treatment. Studies have shown that epileptic lesions are often accompanied by increased oxidative stress, neuroinflammation and local microenvironment imbalance. These factors not only promote abnormal discharge of neurons, but may also weaken the accuracy and sustainability of external field regulation. Therefore, it is difficult to achieve effective intervention in epileptic neural circuits by relying solely on external field stimulation.

[0004] How to integrate microenvironmental regulation with noninvasive technologies to enhance responses to external field signals and improve neurological function in the affected area has become a key area that urgently needs breakthroughs. To address this situation, the development of noninvasive neuromodulatory smart nanodevices that can cross the blood-brain barrier and possess microenvironmental regulation capabilities has become an important direction for improving the treatment of intractable epilepsy. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention aims to provide an ultrasound-driven neuromodulatory nanodevice for the noninvasive treatment of epilepsy, as well as its preparation method and application. This nanodevice can cross the blood-brain barrier and simultaneously couple with ultrasound external field signals to produce enhanced electrical stimulation locally within the epileptic lesion, thereby enabling precise intervention in neural circuits. Furthermore, this nanodevice can respond to abnormal characteristics of the epileptic microenvironment, such as oxidative stress, by regulating the local microenvironment to alleviate abnormal neuronal excitation and repair damaged neural networks, thereby achieving synergistic, noninvasive treatment for epilepsy.

[0006] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an ultrasound-driven neuromodulation nanodevice for non-invasive treatment of epilepsy. The ultrasound-driven neuromodulation nanodevice includes a microenvironment-responsive module, an ultrasound-responsive module, and a biotargeting module; the microenvironment-responsive module is platinum nanoparticles, the ultrasound-responsive module is piezoelectric nanoparticles UIO-66-NH2, and the biotargeting module is a brain-targeting peptide; wherein the platinum nanoparticles are dispersed and embedded in the UIO-66-NH2 structure, and the brain-targeting peptide is modified on the surface of the UIO-66-NH2 structure.

[0008] The ultrasound-driven neuromodulation nanodevice involved in the present invention creatively combines platinum nanoparticles (Pt NPs) with free radical scavenging ability, UIO-66-NH2 nanoparticles that can convert mechanical force into electrical signals, and a brain-targeting peptide with specific blood-brain barrier targeting ability; this nanodevice has excellent catalytic activity and antioxidant properties, can effectively decompose hydrogen peroxide in the epileptic focus area, reduce the level of oxidative stress, and improve the abnormal microenvironment; under the action of ultrasound, this nanodevice can convert mechanical stress into electrical signals, trigger neuronal electrophysiological responses, and achieve non-invasive and precise regulation of epileptic reactive neural circuits; at the same time, this nanodevice can efficiently cross the blood-brain barrier, and through the synergistic effect of external field ultrasound and the biotargeting module, significantly enhance the accumulation amount in the brain epileptic focus area. The nanodevice provided by the present invention has strong ability to regulate neural circuits, high regulation precision, excellent brain entry efficiency and has microenvironment-responsive characteristics, and can be used for highly efficient non-invasive treatment of refractory epilepsy.

[0009] Preferably, the platinum nanoparticles are in a cubic structure, with a particle size of 2-6 nm, such as 2 nm, 2.2 nm, 2.4 nm, 2.6 nm, 2.8 nm, 3.0 nm, 3.5 nm, 3.8 nm, 4.0 nm, 5.0 nm, 6.0 nm, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0010] Preferably, the piezoelectric nanoparticles UIO-66-NH2 are in an octahedral structure, with a particle size of 100-160 nm, such as 100 nm, 105 nm, 110 nm, 112 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 150 nm, 160 nm, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0011] Preferably, the brain-targeting peptide is Angiopep-2 peptide, and its sequence includes SEQ ID NO.1: TFFYGGSRGKRNNFKTEEY.

[0012] Preferably, the particle size of the ultrasonic-driven nerve regulation nanodevice is 100-160 nm, such as 100 nm, 105 nm, 110 nm, 112 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 150 nm, 160 nm, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0013] Preferably, the mass ratio of the platinum nanoparticles to the piezoelectric nanoparticles UIO-66-NH2 is 1:(10-60); the specific point values of (10-60) can be independently selected as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0014] Preferably, the mass ratio of the brain-targeting peptide to the piezoelectric nanoparticles UIO-66-NH2 is 1:(1.0-1.5), and the specific point values of (1.0-1.5) can be independently selected as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0015] In a second aspect, the present invention provides a preparation method of the ultrasonic-driven nerve regulation nanodevice according to the first aspect, and the preparation method includes the following steps:

[0016] (1) Hafnium tetrachloride and 2-aminoterephthalic acid are dispersed in an organic solvent, mixed with platinum nanoparticles for thermal reaction, and after the reaction is completed, the product is collected by centrifugation, washed and dried to obtain Pt@UIO-66-NH2 nanomaterials;

[0017] (2) The activated brain-targeting peptide is mixed with the Pt@UIO-66-NH2 nanomaterials for amide reaction to obtain the ultrasonic-driven nerve regulation nanodevice.

[0018] The present invention synthesizes the ultrasonic-driven nerve regulation nanodevice by a simple method, with simple experimental operations and a clear synthesis route, which is suitable for the highly efficient non-invasive treatment of epilepsy.

[0019] Preferably, the molar ratio of hafnium tetrachloride to 2-aminoterephthalic acid in step (1) is 1:(1.2-1.8), and the specific point values of (1.2-1.8) can be independently selected as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0020] Preferably, the organic solvent in step (1) includes N,N-dimethylformamide.

[0021] Preferably, benzoic acid is also dispersed in the organic solvent in step (1).

[0022] Preferably, the molar ratio of benzoic acid to hafnium tetrachloride is (40 - 60):1. Specific point values within (40 - 60) can be independently selected as 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0023] Preferably, the dispersion method in step (1) includes ultrasonic treatment.

[0024] Preferably, the thermal reaction in step (1) is carried out at 115 - 125 °C (such as 115 °C, 116 °C, 117 °C, 118 °C, 119 °C, 120 °C, 121 °C, 122 °C, 123 °C, 124 °C, 125 °C, etc.) for 14 - 18 h (such as 14 h, 14.5 h, 15 h, 15.5 h, 16 h, 16.5 h, 17 h, 17.5 h, 18 h, etc.).

[0025] Preferably, the rotation speed of centrifugation in step (1) is 9000 - 13000 rpm (such as 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, 12500 rpm, 13000 rpm, etc.), and the centrifugation time is 3 - 8 min (such as 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, etc.).

[0026] Preferably, the activated brain - targeting peptide in step (2) is obtained by mixing the brain - targeting peptide with N - hydroxysuccinimide and 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride and activating at 35 - 40 °C (such as 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, etc.) for 0.5 - 2 h (such as 0.5 h, 1 h, 1.5 h, 2 h, etc.).

[0027] Preferably, the amide reaction in step (2) is carried out at 35 - 40 °C (such as 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, etc.) for 12 - 16 h (such as 12 h, 13 h, 14 h, 15 h, 16 h, etc.).

[0028] Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0029] In the present invention, the preparation method of the platinum nanoparticles includes chemical reduction method, colloid method, electrochemical method, biological method, physical method or thermal decomposition method.

[0030] Among them, the chemical reduction method uses a reducing agent to reduce platinum salts (such as H2PtCl6, K2PtCl4) to platinum atoms, which nucleate and grow into nanoparticles; the reducing agents include strong reducing agents (NaBH4), mild reducing agents (sodium citrate, ascorbic acid, etc.), and polyols (ethylene glycol or polyvinylpyrrolidone as both solvent and reducing agent).

[0031] Among them, the colloid method is to mix platinum salts (such as H2PtCl6, K2PtCl4) with a stabilizer (such as PVP) in a solvent, heat or add a reducing agent (such as NaBH4) to initiate reduction, and purify the particles by centrifugation and washing.

[0032] Among them, the electrochemical method is to reduce and deposit platinum ions on the electrode surface by adjusting the voltage / current in an electrolyte containing platinum salts.

[0033] Among them, the physical method is to bombard a platinum target with high-energy particles, sputter platinum atoms to deposit on the substrate as nanoparticles; or irradiate a platinum target with a laser in a liquid to form colloidal nanoparticles.

[0034] Among them, the biological method is to use plant extracts (such as green tea polyphenols), microorganisms or enzymes to reduce platinum salts.

[0035] Among them, the thermal decomposition method is obtained by decomposing platinum acetylacetonate in a high-temperature organic solvent.

[0036] In a third aspect, the present invention provides the use of the ultrasound-driven neuromodulation nanodevice according to the first aspect in the preparation of products related to non-invasive treatment of epilepsy.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The ultrasound-driven neuromodulation nanodevice involved in the present invention creatively combines platinum nanoparticles (Pt NPs) with the ability to scavenge free radicals, UIO-66-NH2 nanoparticles that can convert mechanical force into electrical signals, and a brain-targeting peptide with blood-brain barrier specific targeting ability; this nanodevice has excellent catalytic activity and antioxidant properties, can effectively decompose hydrogen peroxide in the epileptic focus area, reduce the level of oxidative stress, and improve the abnormal microenvironment; under the action of ultrasound, this nanodevice can convert mechanical stress into electrical signals, trigger neuronal electrophysiological responses, and achieve non-invasive and precise regulation of epileptic reactive neural circuits; at the same time, this nanodevice can efficiently cross the blood-brain barrier, and through the synergistic effect of external field ultrasound and the biological targeting module, significantly enhance the accumulation amount in the epileptic focus area in the brain. The nanodevice provided by the present invention has strong neural circuit regulation ability, high regulation precision, excellent brain entry efficiency and has microenvironment response characteristics, and can be used for the efficient non-invasive treatment of refractory epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the TEM morphology image of the Pt NPs prepared in the preparation example;

[0040] Figure 2 It is the TEM morphology image of the Pt@UIO-66-NH2 nanomaterial prepared in Examples 1-3;

[0041] Figure 3 It is the TEM morphology image of the PUANPs prepared in Example 1;

[0042] Figure 4 It is the TEM morphology image of the PUANPs prepared in Example 2;

[0043] Figure 5 It is the particle size distribution map and zeta potential map of the PUNPs prepared in Example 1;

[0044] Figure 6 It is the particle size distribution map and zeta potential map of the PUANPs prepared in Example 1;

[0045] Figure 7 It is the elemental mapping image of the PUNPs prepared in Example 1;

[0046] Figure 8 It is the piezoelectric performance test image of UIO-66-NH2, where a is the phase hysteresis loop measured by piezoresponse force microscopy and b is the amplitude butterfly loop;

[0047] Figure 9 It is the piezoelectric performance test image of the PUNPs prepared in Example 1, where a is the phase hysteresis loop measured by piezoresponse force microscopy and b is the amplitude butterfly loop;

[0048] Figure 10 It is the cytotoxicity result image of the PUANPs prepared in Example 1 against various cells;

[0049] Figure 11 It is the in vitro blood-brain barrier penetration test image of PUNPs and PUANPs, where a is the fluorescence image and b is the fluorescence intensity image;

[0050] Figure 12 It is the in vivo blood-brain barrier penetration test image of PUNPs and PUANPs, where a is the fluorescence image and b is the fluorescence intensity image;

[0051] Figure 13 It is the test image of the decomposition ability of hydrogen peroxide by different concentrations of PUANPs;

[0052] Figure 14 It is the confocal image of the antioxidant ability test of PUANPs at the cellular level, where a cell neuroinflammation model is induced by lipopolysaccharide;

[0053] Figure 15 It is a confocal image of the in vitro anti-epileptic ability test of PUANPs, in which a glutamate-induced epileptic cell model is used. Specific implementation manners

[0054] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0055] The hexachloroplatinic acid hexahydrate involved in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China); ethylene glycol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China); polyvinylpyrrolidone was purchased from Shanghai Macklin Biochemical Co., Ltd. (China); hafnium tetrachloride was purchased from Shanghai Merck Chemical Technology Co., Ltd. (China); 2-aminoterephthalic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China); benzoic acid was purchased from Shanghai Macklin Biochemical Co., Ltd. (China); anhydrous methanol was purchased from Beijing Innochem Science & Technology Co., Ltd.; N-hydroxysuccinimide was from Shanghai Macklin Biochemical Co., Ltd. (China); 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was from Shanghai Merck Chemical Technology Co., Ltd. (China). The Angiopep-2 peptide was purchased from Guoping Pharmaceutical Co., Ltd. (China), and its amino acid sequence is TFFYGGSRGKRNNFKTEEY.

[0056] Preparation example

[0057] This preparation example provides platinum nanoparticles (Pt NPs), and its preparation method is as follows:

[0058] Dissolve hexachloroplatinic acid hexahydrate (50 mM, 2 mL) in 18 mL of ethylene glycol, and add the resulting solution to a round-bottom flask containing 222 mg of polyvinylpyrrolidone (molecular weight 58000). Subsequently, the reaction is allowed to stand at 180 °C in an oil bath for 10 min. After the reaction is completed, the resulting platinum nanoparticles are precipitated by adding 40 mL of acetone and collected by centrifugation (11000 rpm, 5 min). Then, the platinum nanoparticles are washed with acetone and n-hexane (volume ratio 1:1) to remove the excess free polyvinylpyrrolidone, and finally redispersed in 10 mL of N,N-dimethylformamide solvent to prepare a 1.0 mg / mL platinum nanoparticle solution.

[0059] Example 1

[0060] This example provides an ultrasound-driven neuromodulation nanodevice for non-invasive treatment of epilepsy (abbreviated as PUANPs), and its preparation method is as follows:

[0061] (1) A 15 mL N,N-dimethylformamide solution of hafnium tetrachloride (0.6 mmol) and a 15 mL N,N-dimethylformamide solution of 2-aminoterephthalic acid (1 mmol) were added to a round-bottom flask containing 30 mmol benzoic acid, and the mixture was sonicated to disperse evenly. Subsequently, 1 mL of the platinum nanoparticle solution prepared in the preparation example was added to the mixed system. After uniform dispersion, the mixture was allowed to stand and react at 120 °C in an oil bath for 16 h. After the reaction, the product was collected by centrifugation (11000 rpm, 5 min), washed three times by centrifugation with methanol, and dried in vacuo at 70 °C to obtain the Pt@UIO-66-NH2 nanomaterial (abbreviated as PUNPs);

[0062] (2) 1.5 μmol of the brain-targeting peptide Angiopep-2 was dispersed in 3 mL of ultrapure water, 4.5 μmol of N-hydroxysuccinimide (NHS) and 4.5 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added, and the mixture was shaken and activated in a shaker at 37 °C for 1 h. After activation, 2 mg of the PUNPs synthesized in step (1) was added thereto, and the mixture was stirred and reacted at 37 °C for 14 h to prepare an ultrasound-driven nerve regulation nanodevice modified with Angiopep-2 (abbreviated as PUANPs).

[0063] Example 2

[0064] This example provides an ultrasound-driven nerve regulation nanodevice (abbreviated as PUANPs) for non-invasive treatment of epilepsy, and its preparation method is as follows:

[0065] (1) A 30 mL N,N-dimethylformamide solution of hafnium tetrachloride (1.2 mmol) and a 30 mL N,N-dimethylformamide solution of 2-aminoterephthalic acid (2 mmol) were added to a round-bottom flask containing 60 mmol benzoic acid, and the mixture was sonicated to disperse evenly. Subsequently, 1 mL of the platinum nanoparticle solution prepared in the preparation example was added to the mixed system. After uniform dispersion, the mixture was allowed to stand and react at 115 °C in an oil bath for 18 h. After the reaction, the product was collected by centrifugation (13000 rpm, 3 min), washed three times by centrifugation with methanol, and dried in vacuo at 70 °C to obtain the Pt@UIO-66-NH2 nanomaterial (abbreviated as PUNPs);

[0066] (2) Disperse 1.5 μmol of the brain-targeting peptide Angiopep-2 in 3 mL of ultrapure water, add 4.5 μmol of N-hydroxysuccinimide (NHS) and 4.5 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and place it in a shaker at 35 °C for 2 h of oscillation activation. After the activation is completed, add 3 mg of the PUNPs synthesized in step (1) thereto, and stir and react at 35 °C for 16 h to obtain an Angiopep-2-modified ultrasound-driven neuromodulation nanodevice (abbreviated as PUANPs).

[0067] Example 3

[0068] This example provides an ultrasound-driven neuromodulation nanodevice (abbreviated as PUANPs) for non-invasive treatment of epilepsy, and its preparation method is as follows:

[0069] (1) Add a 30 mL N,N-dimethylformamide solution of hafnium tetrachloride (1.4 mmol) and a 30 mL N,N-dimethylformamide solution of 2-aminoterephthalic acid (1.986 mmol) to a round-bottom flask containing 61.416 mmol of benzoic acid, and disperse them evenly by ultrasonic treatment. Subsequently, add 0.5 mL of the platinum nanoparticle solution prepared in the preparation example to the mixed system. After dispersing evenly, let it stand and react in an oil bath at 125 °C for 14 h. After the reaction is completed, collect the product by centrifugation (9000 rpm, 8 min), and then wash it three times by centrifugation with methanol. After drying in vacuo at 70 °C, obtain the Pt@UIO-66-NH2 nanomaterial (abbreviated as PUNPs);

[0070] (2) Disperse 1.5 μmol of the brain-targeting peptide Angiopep-2 in 3 mL of ultrapure water, add 4.5 μmol of N-hydroxysuccinimide (NHS) and 4.5 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and place it in a shaker at 38 °C for 1 h of oscillation activation. After the activation is completed, add 1.5 mg of the PUNPs synthesized in step (1) thereto, and stir and react at 38 °C for 12 h to obtain an Angiopep-2-modified ultrasound-driven neuromodulation nanodevice (abbreviated as PUANPs).

[0071] Test Example 1

[0072] TEM Morphology Observation:

[0073] After the suspension of the platinum nanoparticles (Pt NPs) prepared in the preparation example is ultrasonically dispersed evenly, take 10 μL and drop it on a common carbon support film. After air drying naturally, observe the morphology with TEM, as Figure 1 shown.

[0074] After the Pt@UIO-66-NH2 nanomaterials (PUNPs) suspension prepared in Examples 1-3 was ultrasonically dispersed uniformly, 10 μL was taken and dropped on a common carbon support film. After natural drying, the morphology was observed by TEM, as Figure 2 shown (a, b, and c in the figure correspond to PUNPs prepared in Examples 1-3 respectively).

[0075] After the ultrasonic-driven neuromodulation nanodevices (PUANPs) suspension prepared in Examples 1-2 was ultrasonically dispersed uniformly, 10 μL was taken and dropped on a common carbon support film. After natural drying, the morphology was observed by TEM, as Figure 3 and Figure 4 shown ( Figure 3 and Figure 4 correspond to PUANPs prepared in Example 1 and Example 2 respectively).

[0076] Test Example 2

[0077] Particle size distribution and Zeta potential characterization:

[0078] An appropriate amount of PUNPs or PUANPs nanoparticles prepared in Example 1 was dispersed in deionized water. Through ultrasonic treatment, it was fully dispersed to form a stable suspension. Subsequently, an appropriate amount of the sample was placed in a DLS instrument to measure the particle size distribution and Zeta potential. The results are as Figure 5 and Figure 6 shown ( Figure 5 is the result of the PUNPs sample, Figure 6 is the result of the PUANPs sample).

[0079] Test Example 3

[0080] Elemental analysis experiment:

[0081] After the PUNPs suspension prepared in Example 1 was ultrasonically dispersed uniformly, 10 μL was taken and dropped on an ultrathin carbon support film. After natural drying, it was observed by high-resolution TEM. The results are as Figure 7 shown. It can be seen from the figure that PUNPs contain C, N, O, Hf, and Pt elements, proving the successful synthesis of PUNPs and the effective loading of Pt NPs in the structure of PUNPs.

[0082] Test Example 4

[0083] Piezoelectric property test:

[0084] (1) Preparation of UIO-66-NH2 nanomaterials: A 15 mL N,N-dimethylformamide solution of hafnium tetrachloride (0.6 mmol) and a 15 mL N,N-dimethylformamide solution of 2-aminoterephthalic acid (1 mmol) were added to a round-bottom flask containing 30 mmol benzoic acid. After ultrasonic treatment to disperse evenly, the mixture was allowed to react statically at 120 °C in an oil bath for 16 h. After the reaction, the product was collected by centrifugation (11000 rpm, 5 min), washed three times by centrifugation with methanol, and dried in vacuo at 70 °C to obtain UIO-66-NH2 nanomaterials (abbreviated as UNPs);

[0085] (2) The prepared UNPs and PUNPs obtained in Example 1 were respectively and uniformly dispersed in ethanol, and a stable suspension was formed by ultrasonic treatment. Subsequently, a small amount of the dispersion was drop-coated on the surface of a clean conductive silicon wafer, and a thin film sample was formed after natural drying. The prepared sample was placed in a piezoresponse force microscopy (PFM) device, and an alternating electric field was applied in the probe scanning mode to record the local piezoelectric response of UNPs or PUNPs at different voltages. During the test, by adjusting the contact force between the probe and the sample surface, the electrical signal output of UNPs or PUNPs under mechanical stress was observed, and their phase and amplitude signals were obtained to characterize the directionality and intensity of the piezoelectric effect.

[0086] The test results are shown in Figure 8 (results of the UNPs sample, where a is the phase hysteresis loop and b is the amplitude butterfly loop) and Figure 9 (results of the PUNPs sample, where a is the phase hysteresis loop and b is the amplitude butterfly loop). From Figure 8 and Figure 9 the result comparison, it can be seen that the phase hysteresis loop shows that both UNPs and PUNPs can undergo a 180° change under a DC voltage of -10 V to +10 V, proving that both UNPs and PUNPs have piezoelectric responsiveness, while the amplitude butterfly loop proves that the introduction of Pt NPs enhances the piezoelectric performance of UNPs.

[0087] Test Example 5

[0088] Cytotoxicity test:

[0089] bEND.3, BV2, MA-c, and Neuron cells were seeded in 96-well plates (1×10 4(The cells were seeded at a density of 实验组 cells / well) and cultured in an incubator for 24 h. After removing the spent medium, 100 μL of medium containing different concentrations of the PUANPs prepared in Example 1 (3.125, 6.25, 12.5, 25, 50, 100 μg / mL) was added, and the cells were incubated for another 24 h. Each group had 3 replicate wells. Then, the spent medium was removed, and 100 μL of medium containing the CCK-8 reagent was added to each well and incubated in the incubator for 2 h. Subsequently, the optical density values of each well at a wavelength of 450 nm were measured using a microplate reader, and the cell viability (%) was calculated according to the following formula: Cell viability (%) = (OD 实验组 - OD 空白组 ) / (OD 对照组 - OD 空白组 )

[0090] The results of cell viability are shown in Figure 10 . It can be seen that the ultrasound-driven neuromodulation nanodevice involved in the present invention has good biosafety and basically does not show cytotoxicity within the concentration range of 100 μg / mL.

[0091] Test Example 6

[0092] In vitro blood-brain barrier permeability test:

[0093] (1) bEND.3 cells (1×10 4 cells / well) were seeded into the upper chamber of a Transwell-6 well plate. The medium was DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum, and it was cultured in an incubator at 37 °C and 5% CO2 for 48 h.

[0094] (2) The integrity of the cell barrier was verified by the permeability of sodium fluorescein (Na-F). First, Na-F standard solutions with concentrations of 0.5, 1, 5, 10, 20, 40, and 400 μg / mL were prepared, and their fluorescence intensities (excitation wavelength 460 nm, emission wavelength 515 nm) were measured using a fluorescence microplate reader. Then, a standard curve was plotted based on the fluorescence intensity (y) and concentration (x). Subsequently, the medium in the upper chamber and the basolateral medium of the Transwell were replaced with Hepes buffer. 10 μg / mL Na-F was added to the well, and after incubation for 60 min, 100 μL of buffer was collected from the bottom and the fluorescence intensity was measured. The concentration of Na-F in the bottom was calculated according to the standard curve, and the permeability coefficient was calculated according to the following formula:

[0095] P 测 = C1 × V1 / (tAC2) (1)

[0096] 1 / P model = 1 / P 测 - 1 / P blank (2)

[0097] C1 is the concentration of Na-F in the chamber; V1 is the volume of the chamber; t is the incubation time; A is the membrane area; C2 is the concentration of Na-F in the substrate; P model is the permeability coefficient of the BBB model; P 测 is the permeability coefficient of the chamber with cells; P blank is the permeability coefficient of the cell-free chamber.

[0098] (3) Divide the constructed complete cell barrier chamber into PUNPs group and PUANPs group, with 3 parallel samples set in each group. After removing the chamber medium, add cell culture media containing Cy5.5-labeled PUNPs and PUANPs prepared in Example 1 respectively, and incubate for 4 h. Subsequently, use a small animal in vivo imaging system (IVIS) to image the chamber and the substrate, as Figure 11 shown in a of Figure 11 and calculate the relative fluorescence intensity, as

[0099] Test Example 7

[0100] In vivo blood-brain barrier crossing ability test:

[0101] Randomly divide female BALB / c mice into 9 groups, namely Ctrl group (intravenous injection of PBS in the tail vein), PUNPs group (intravenous injection of 20 mg / kg PUNPs in the tail vein), PUNPs+US-0.5W·cm -2 group (intravenous injection of 20 mg / kg PUNPs in the tail vein, apply ultrasound to the brain 2 h later, parameters are 1 MHz, 0.5W·cm -2 , 50% duty cycle, 5 min), PUNPs+US-1.0W·cm -2 group (intravenous injection of 20 mg / kg PUNPs in the tail vein, apply ultrasound to the brain 2 h later, parameters are 1 MHz, 1.0W·cm -2 , 50% duty cycle, 5 min), PUNPs+US-2.0W·cm -2 group (intravenous injection of 20 mg / kg PUNPs in the tail vein, apply ultrasound to the brain 2 h later, parameters are 1 MHz, 2.0W·cm -2 , 50% duty cycle, 5 min), PUANPs group (intravenous injection of 20 mg / kg PUANPs in the tail vein), PUANPs+US-0.5W·cm -2 group (intravenous injection of 20 mg / kg PUANPs in the tail vein, apply ultrasound to the brain 2 h later, parameters are 1 MHz, 0.5W·cm -2 , 50% duty cycle, 5 min), PUANPs+US-1.0W·cm-2 Group (intravenous injection of 20 mg / kg PUANPs via the tail vein, ultrasound was applied to the brain 2 h later, parameters were 1 MHz, 1.0 W·cm -2 , 50% duty cycle, 5 min), PUANPs + US - 2.0 W cm -2 Group (intravenous injection of 20 mg / kg PAUNPs via the tail vein, ultrasound was applied to the brain 2 h later, parameters were 1 MHz, 2.0 W·cm -2 , 50% duty cycle, 5 min). There were 3 mice in each group. The injected PUNPs and PUANPs were both nanoparticles labeled with Cy5.5 (prepared in Example 1). 24 h after intravenous injection, the mice were sacrificed and the brain tissues were quickly removed, rinsed with PBS to remove residual blood, and then the brain tissues were placed in a small animal in vivo imaging system (IVIS) for fluorescence imaging, as Figure 12 shown in a, and the fluorescence intensity was recorded and statistically analyzed, as Figure 12 shown in b. It was shown that PUANPs modified with brain-targeting peptides exhibited better blood-brain barrier crossing ability, and the ultrasound effect increased the accumulation amount of the nanodevices in the brain.

[0102] Test Example 8

[0103] Hydrogen peroxide decomposition ability test:

[0104] The hydrogen peroxide decomposition ability of the PUANPs prepared in Example 1 was detected using a Leici JPSJ-606L dissolved oxygen meter. Different concentrations of PUANPs (12.5, 25, 50, 100 μg / mL) were dispersed in an aqueous solution containing 10 mM hydrogen peroxide, and the oxygen content was recorded every 30 s for a total of 10 min. The specific results are as Figure 13 shown, indicating that the ultrasound-driven neuromodulation nanodevices involved in the present invention can effectively catalyze the decomposition of hydrogen peroxide and have a concentration dependence.

[0105] Test Example 9

[0106] Antioxidant ability test at the cell level:

[0107] An in vitro neuroinflammation model was induced using lipopolysaccharide (LPS). SH-SY5Y cells were seeded at 1×10 per well 5Cells were inoculated at a certain concentration in a confocal dish and cultured overnight in a cell incubator. The used culture medium was aspirated and the cells were washed with PBS. Subsequently, the confocal dish was divided into 4 groups, namely: Ctrl group (fresh culture medium), PUANPs group (culture medium containing 100 μg / mL PUANPs), LPS group (culture medium containing 5 μg / mL LPS), and LPS+PUANPs group (culture medium containing 5 μg / mL LPS and 100 μg / mL PUANPs), and then cultured for another 24 h. Subsequently, the culture medium was discarded and the cells were washed three times with PBS buffer. Then, the cells were stained with Hoechst and DCFH-DA respectively. Hoechst represents the cell nucleus and DCFH-DA represents the ROS signal. After staining, confocal imaging was performed using a Zeiss LSM 880 confocal laser scanning microscope.

[0108] The specific results are shown in Figure 14 As shown, it can be seen that the ultrasound-driven neural regulation nanodevice involved in the present invention can effectively relieve the symptoms of neuritis, thereby restoring the microenvironment homeostasis.

[0109] Test Example 10

[0110] Anti-epileptic ability test:

[0111] Abnormal Ca 2+ Influx is considered to be the cause of neuronal excitability in epilepsy. Therefore, an epilepsy model was induced in vitro using glutamate (Glu) and the intracellular Ca 2+ levels were observed using a confocal laser scanning microscope. The primary neurons grown on cell culture slides were randomly divided into 5 groups, namely Ctrl group, Glu group, Glu+PUANPs group, Glu+US group, and Glu+PUANPs+US group. 5 μM Glu was added to the culture medium of the Glu group and cultured for another 24 h. After 24 h, the culture medium containing Glu was removed and 50 μg / mL PUANPs was added to the culture medium of the PUANPs group and cultured for another 12 h. After 12 h, ultrasound (parameters: 1 MHz, 1 W·cm -2 , 50% duty cycle, 1 min) was applied to the groups containing US. Subsequently, the culture medium was discarded and the cells were washed three times with Hepes buffer. Then, the cells of each group were stained with the fluorescent calcium marker Fluo-4 AM. After staining, confocal imaging was performed using a Zeiss LSM 880 confocal laser scanning microscope to observe the calcium signal.

[0112] The specific results are shown in Figure 15 As shown, it can be seen that Glu significantly increased the intracellular Ca 2+levels, while the ultrasonic-driven neural regulation nano-device involved in the present invention effectively reduces Glu-induced Ca 2+ influx under the action of ultrasound, alleviates epileptic symptoms, and treats epilepsy.

[0113] The applicant declares that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0115] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination ways.

Claims

1. An ultrasound-driven neuromodulation nanodevice for non-invasive treatment of epilepsy, characterized in that, The ultrasonic-driven neural regulation nanodevice includes a microenvironment-responsive module, an ultrasonic-responsive module, and a biological targeting module; the microenvironment-responsive module is platinum nanoparticles, the ultrasonic-responsive module is piezoelectric nanoparticles UIO-66-NH2, and the biological targeting module is a brain-targeting peptide; wherein the platinum nanoparticles are dispersedly embedded in the UIO-66-NH2 structure, and the brain-targeting peptide is modified on the surface of the UIO-66-NH2 structure.

2. The ultrasonic-driven neural regulation nanodevice according to claim 1, wherein The platinum nanoparticles have a cubic structure with a particle size of 2-6 nm; Preferably, the piezoelectric nanoparticles UIO-66-NH2 have an octahedral structure with a particle size of 100-160 nm.

3. The ultrasonic-driven neural regulation nano-device according to claim 1 or 2, characterized in that The brain-targeting peptide is Angiopep-2 peptide, and its sequence includes TFFYGGSRGKRNNFKTEEY; Preferably, the particle size of the ultrasonic-driven neural regulation nanodevice is 100-160 nm.

4. The ultrasonic-driven neural regulation nano-device according to any one of claims 1-3, characterized in that The mass ratio of the platinum nanoparticles to the piezoelectric nanoparticles UIO-66-NH2 is 1:(10-60); Preferably, the mass ratio of the brain-targeting peptide to the piezoelectric nanoparticles UIO-66-NH2 is 1:(1.0-1.5).

5. The preparation method of the ultrasonic-driven neuromodulation nanodevice according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Hafnium tetrachloride and 2-aminoterephthalic acid are dispersed in an organic solvent, mixed with platinum nanoparticles for thermal reaction, and after the reaction is completed, the product is collected by centrifugation, washed and dried to obtain Pt@UIO-66-NH2 nanomaterials; (2) The activated brain-targeting peptide is mixed with the Pt@UIO-66-NH2 nanomaterials for amide reaction to obtain an ultrasonic-driven neural regulation nanodevice.

6. The preparation method of the ultrasonic-driven nerve regulation nano-device according to claim 5, wherein, In step (1), the molar ratio of hafnium tetrachloride to 2-aminoterephthalic acid is 1:(1.2-1.8); Preferably, the organic solvent in step (1) includes N,N-dimethylformamide; Preferably, benzoic acid is also dispersed in the organic solvent in step (1); Preferably, the molar ratio of benzoic acid to hafnium tetrachloride is (40-60):

1.

7. The preparation method of the ultrasonic-driven neuromodulation nanodevice according to claim 5, characterized in that, The dispersion method in step (1) includes ultrasonic treatment; Preferably, the thermal reaction in step (1) is carried out at 115-125 °C for 14-18 h; Preferably, the rotation speed of the centrifugation in step (1) is 9000-13000 rpm, and the centrifugation time is 3-8 min.

8. The preparation method of the ultrasonic-driven nerve regulation nanodevice according to claim 5, characterized in that, The activated brain-targeting peptide in step (2) is obtained by mixing the brain-targeting peptide with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and activating at 35-40 °C for 0.5-2 h; Preferably, the amide reaction in step (2) is carried out at 35-40 °C for 12-16 h.

9. The preparation method of the ultrasonic-driven nerve regulation nano-device according to claim 5, characterized in that, The preparation method of the platinum nanoparticles includes chemical reduction method, colloid method, electrochemical method, biological method, template method or thermal decomposition method.

10. Use of the ultrasonic-driven neural regulation nanodevice according to any one of claims 1-4 in the preparation of products related to non-invasive treatment of epilepsy.

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