Nanometer responder for enhancing magnetoacoustic coupling stimulation and preparation method and application thereof

Abstract

The invention relates to a nano responder for enhancing magnetoacoustic coupling stimulation and a preparation method and application thereof. The nano responder comprises a magnetic nano bubble, the magnetic nano bubble comprises a core and a shell, the core comprises perfluoropropane, the shell comprises lipid, magnetic nano particles are encapsulated in the shell, and the surface of the shell is connected with phosphatidylserine and an anti-Piezo1 antibody. The targeted microglial cell Piezo1 magnetic nano bubble prepared by the invention can be used as a TMAS magnetic actuator, and can amplify the effect of TMAS and position the TMAS to a specific cell of a required brain area, so as to improve the stimulation effect and accuracy of TMAS.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a nanoresponder for enhancing magnetoacoustic coupling stimulation and a preparation method and application thereof, in particular to a magnetic nanobubble targeting Piezo1 of microglia. Background Art

[0002] Neuromodulation is a biomedical engineering technique that uses physical or chemical means, such as electricity, magnetism, light, and sound, to manipulate neural activity. It provides a critical and unique tool for interpreting brain signals and regulating brain function. Common neuromodulation techniques currently include deep brain stimulation (DBS), transcranial direct current stimulation (tDCS), and transcranial magnetic stimulation (TMS). DBS directly implants electrodes into specific brain regions. While this method offers a precise and deep stimulation target, it carries the risk of bleeding and infection, a significant invasive procedure. The rapid decay of current in non-invasive tDCS limits its ability to precisely target and stimulate specific areas, hindering its application in the treatment of precise brain disorders. TMS utilizes a varying magnetic field to induce induced currents, achieving reversible and repeatable brain neurostimulation. However, TMS struggles to achieve millimeter-scale magnetic or electric field focusing, and its stimulation depth is limited to the cerebral cortex, making it difficult to directly stimulate deep brain regions. Although several alternative approaches have been developed, such as optogenetics, photothermal, and magnetothermal stimulation, nonspecific gene delivery or overheating damage to nonspecific tissues are considered key challenges to be overcome. Therefore, a precise, non-invasive, and highly focused neuromodulation method is urgently needed to treat brain diseases.

[0003] Transcranial magneto-acoustic stimulation (TMAS) is a composite neuromodulation technology that combines the magneto-acoustic coupling effect of an electric field and an ultrasound stimulation (transcranial ultrasound stimulation, TUS) field. It has the advantages of high penetration depth (can specifically act on deep brain regions) and high focusing (resolution can reach the millimeter level). While stimulating, it can take into account both the depth of stimulation within the brain and the localization of brain function. It has important scientific research value and development prospects for areas such as brain regions and deep cranial brain stimulation that require high stimulation specificity. This method is different from previous methods that directly use changes in electric and magnetic fields to induce electrical stimulation. Instead, it is based on the magneto-acoustic coupling effect of conductive tissue and utilizes the high focusing characteristics of ultrasound. In the presence of a static magnetic field, it generates a focused electric field with the same spatial and temporal distribution as the ultrasound field, achieving non-invasive electrical stimulation with high spatial resolution. Compared with TMS, TMAS can achieve millimeter-level focused stimulation of the entire brain, including deep brain areas, and the focused ultrasound spot can reach about 2mm; compared with TUS, TMAS has neuroelectrophysiology as a carrier, and the mechanism by which electrical stimulation exerts physiological effects is clearer, making it more suitable for clinical verification and implementation.

[0004] However, non-invasive stimulation devices also face challenges in achieving cellular precision and targeting, which can significantly reduce the stimulation effect of neuromodulation technology. In addition, the heterogeneity of different skulls and unpredictable individual activities may significantly reduce the stimulation focus of TMAS. The brain has a certain inherent sensitivity to ultrasonic mechanical forces and electric fields. This sensitivity is unevenly distributed across various brain regions through mechanisms such as mechanical or voltage-sensitive ion channels, cytoskeleton, and cell-ECM interactions. This may lead to low mechanical or electrical sensitivity in certain areas of the brain, which may reduce the effectiveness of TMAS. Therefore, there is an urgent need to develop improved TMAS protocols. Summary of the Invention

[0005] In response to the deficiencies of the existing technology and actual needs, the present invention provides a nanoresponder for enhancing magnetoacoustic coupling stimulation, as well as its preparation method and application. Magnetic nanobubbles targeting microglia Piezo1 are prepared and can be used as TMAS magnetic actuators. They can amplify the effects of TMAS and target specific cells in the desired brain area, thereby improving the stimulation effect and accuracy of TMAS.

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

[0007] In a first aspect, the present invention provides a nanoresponder for enhancing magnetoacoustic coupling stimulation, wherein the nanoresponder comprises a magnetic nanobubble, wherein the magnetic nanobubble comprises a core and an outer shell, wherein the core comprises a fluorinated carbon gas and / or an inert gas, wherein the fluorinated carbon gas comprises perfluoropropane (C3F8) or perfluorobutane (C4F 10 ) etc., the inert gas includes nitrogen (N2) etc., the shell includes lipids, magnetic nanoparticles are encapsulated in the shell, and the surface of the shell is connected to phosphatidylserine (PS, used to target microglia) and anti-Piezo1 antibodies.

[0008] In the present invention, the nanoresponder designed to enhance magnetoacoustic coupling stimulation is a magnetic nanobubble that can specifically bind / target and regulate Piezo1 of microglia. After responding to the magnetoacoustic physical field of TMAS, it binds to the extracellular domain of the Piezo1 channel of microglia, thereby opening the Piezo1 channel and allowing the Piezo1 ion channel to play the role of a TMAS electromechanical sensor, converting the mechanical and electrical stimulation generated by TMAS into intracellular signals, improving the response efficiency of tissue cells to the acoustic field and magnetoacoustic electric field in TMAS, improving the stimulation effect of TMAS, enabling TMAS to exert a stronger and more targeted neuromodulatory effect in deep brain areas, and improving the spatial resolution and accuracy of TMAS stimulation.

[0009] In the present invention, the piezoelectric mechanosensitive ion channel component 1 (Piezo1) protein is a member of the mechanically gated cation channel family.

[0010] Preferably, the magnetic nanoparticles include Fe3O4 particles.

[0011] Preferably, the lipid comprises a phospholipid.

[0012] Preferably, the outer shell comprises a single layer of phospholipids (DPPC).

[0013] Preferably, the mass percentage of phosphatidylserine in the magnetic nanobubbles is 17% to 19% (such as 18%), the mass percentage of anti-Piezo1 antibodies is 63% to 65% (such as 64%), the mass percentage of perfluoropropane is 1% to 3% (such as 2%), the mass percentage of magnetic nanoparticles is 5% to 7% (such as 6%), and the mass percentage of lipids is 9% to 11% (such as 10%).

[0014] Preferably, the preparation method comprises:

[0015] The core and the magnetic nanoparticles are encapsulated by the shell, and phosphatidylserine and anti-Piezo1 antibodies are connected to the surface of the shell.

[0016] In a second aspect, the present invention provides a method for preparing the nanoresponder for enhancing magnetoacoustic coupling stimulation according to the first aspect, the preparation method specifically comprising the following steps:

[0017] (1) 2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine are mixed with a solvent to form a lipid mixture;

[0018] (2) mixing the lipid mixture with a magnetic nanoparticle solution, performing ultrasonic treatment and dialysis treatment to obtain a nanoparticle dispersion;

[0019] (3) mixing the nanoparticle dispersion with glycerol and 1,2-propylene glycol, and contacting with perchloropropane;

[0020] (4) The product of step (3) is linked to anti-Piezo1 antibody via streptavidin-biotin to obtain magnetic nanobubbles.

[0021] In a third aspect, the present invention provides an application of the nanoresponder for enhancing magnetoacoustic coupling stimulation described in the first aspect in transcranial magnetoacoustic coupling stimulation.

[0022] In a fourth aspect, the present invention provides a transcranial magnetoacoustic coupling stimulation system, which includes an ultrasonic coupling effect electric field module, an ultrasonic stimulation field module, and the nanoresponder for enhancing magnetoacoustic coupling stimulation described in the first aspect.

[0023] In a fifth aspect, the present invention provides a neuromodulation method, comprising:

[0024] Transcranial magnetic-acoustic coupling stimulation of the target area;

[0025] The transcranial magnetoacoustic coupling stimulation process uses the nanoresponder for enhancing magnetoacoustic coupling stimulation described in the first aspect, or uses the transcranial magnetoacoustic coupling stimulation system described in the fourth aspect to perform transcranial magnetoacoustic coupling stimulation.

[0026] Preferably, the nanoresponder is used by injecting it into a target site.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] The present invention designs a nanoresponder for enhancing magnetoacoustic coupled stimulation. Magnetic nanobubbles (PT-MNBs) targeting microglial Piezo1 are prepared. These nanoresponders can serve as actuators, further improving the efficacy and precision of acoustic and magnetic field stimulation. The acoustic and magnetoacoustic-electric field energies of the TMAS are concentrated near microglia, enabling the PT-MNBs to generate an endogenous physical field that couples with the exogenous TMAS physical field, achieving magnetoacoustic response and improving the efficiency of tissue cells responding to the acoustic and magnetoacoustic-electric fields within the TMAS. This achieves targeted and precise TMAS stimulation and regulation of microglial Piezo1, converting it into localized secondary stimulation at the nanomaterial-neuron interface, further enhancing the stimulatory effect of TMAS and enabling TMAS to exert a stronger and more targeted neuromodulatory effect in deep brain regions. The presence of PT-MNBs can further lower the threshold for TMAS to effectively regulate cellular function by mediating the secondary local transfer of mechanical and electrical energy. Furthermore, because only cells near the PT-MNBs are stimulated by TMAS, the spatial resolution of TMAS stimulation can be improved, even when the focus is difficult to control. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure and working principle of PT-MNBs.

[0030] Figure 2 Transmission electron microscopy results of PT-MNBs.

[0031] Figure 3 This is the particle size distribution result of PT-MNBs.

[0032] Figure 4 This is the zeta potential result diagram of PT-MNBs.

[0033] Figure 5 Figure 5 is the element distribution diagram of PT-MNBs, where Figure a represents the TEM electron microscope image of PT-MNBs, Figure b represents the combination diagram of each element in PT-MNBs, Figure c represents the C element in PT-MNBs, Figure d represents the Fe element in PT-MNBs, Figure e represents the P element in PT-MNBs, Figure f represents the O element in PT-MNBs, and Figure g represents the N element in PT-MNBs.

[0034] Figure 6 This is the X-ray photoelectron spectroscopy analysis result of PT-MNBs.

[0035] Figure 7 The thermogravimetric analysis results of PT-MNBs are shown in Figure 2.

[0036] Figure 8 This is the hysteresis curve analysis result of PT-MNBs.

[0037] Figure 9 Figure 2 shows the results of the magnetic attraction experiment of PT-MNBs.

[0038] Figure 10 Figure 2 is the echogenicity analysis result of PT-MNBs.

[0039] Figure 11 Figure 3 is a graph showing the results of Cy5.5-labeled PT-MNBs targeting microglia, where Figure a is a fluorescence micrograph of Cy5.5-labeled PT-MNBs binding to microglia (red is Cy5.5-labeled PT-MNBs, blue is the nucleus of microglia), Figure b is a Cy5.5-labeled PT-MNBs, and Figure c is a bright-field micrograph of PT-MNBs binding to microglia.

[0040] Figure 12 Figure 2 shows the results of recording piezo1-mediated currents in microglia under TMAS stimulation with or without PT-MNBs.

[0041] Figure 13 This is the TMAS sound pressure result diagram of the mouse hippocampus.

[0042] Figure 14 This is the TMAS electric field result of the mouse hippocampus.

[0043] Figure 15 Figure 2 shows the results of TMAS-driven PT-MNBs enhancing synaptic plasticity in hippocampal neurons of model mice. Figure a is a schematic diagram of the implantation positions of stimulating and recording electrodes in in vivo electrophysiological experiments, and Figure b is a graph of the excitatory postsynaptic potential (fEPSP) curves before and after long-term potentiation (LTP) induction. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0045] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] The magnitude of the electric field in transcranial magnetoacoustic stimulation (TMAS) is proportional to the focused ultrasound pressure and the magnetic induction intensity of the magnetic field. High-frequency TMAS can be used to produce a smaller stimulation focus, but the ultrasound pressure must meet the safety limits of the human body and cannot be increased arbitrarily. Therefore, TMAS has a weak ability to penetrate the skull, and the heterogeneity of different skull bones may seriously affect the TMAS stimulation focus. In addition, as a non-invasive stimulation method, TMAS also faces challenges in achieving cellular-level precision and targeting.

[0047] To address the above problems, the present invention develops a nanoresponder for enhancing magnetoacoustic coupling stimulation, which can also be called a nanomagnetic actuator that responds to magnetoacoustic coupling stimulation. Specifically, it is a magnetic nanobubble (MNBs). The schematic diagram of its exemplary structure and working principle is shown in the following figure. Figure 1 As shown, the nanoresponder includes a core and a shell, wherein the core can be selected from perfluoropropane (C3F8), and the shell can be selected from lipids, Fe3O4 particles are encapsulated in the shell, and phosphatidylserine and anti-Piezo1 antibodies are connected to the surface of the shell to form Piezo1 magnetic nanobubbles (PT-MNBs) targeting microglia. After responding to the magnetoacoustic physical field of TMAS, the nanoresponder binds to the extracellular domain of the Piezo1 channel of microglia, thereby opening the Piezo1 channel and allowing the Piezo1 ion channel to play the role of a TMAS electromechanical sensor, converting the mechanical and electrical stimulation generated by TMAS into intracellular signals, and can be used as an actuator to further improve the stimulation efficacy and accuracy of the acoustic field and magnetic field, and gather the acoustic field and magnetoacoustic electric field energy of TMAS near the microglia, so that the PT-MNBs generate endogenous physical fields that couple with the exogenous TMAS physical field to achieve magnetoacoustic response, thereby improving the tissue cell response to TMAS. It can improve the response efficiency of the acoustic field and magnetoacoustic electric field in the brain; achieve targeted and precise TMAS stimulation and regulation of Piezo1 of microglia, and convert it into local secondary stimulation at the nanomaterial-neuron interface, further improving the stimulation effect of TMAS, so that TMAS can play a stronger and more targeted neuromodulatory role in the deep brain area; it can also further lower the threshold of TMAS to effectively regulate cell function by mediating the secondary local transmission of mechanical and electrical energy, and because only cells near PT-MNBs are stimulated by TMAS, this can improve the spatial resolution of TMAS stimulation, even when the focus is not easy to control.

[0048] Example 1

[0049] This example provides magnetic nanobubbles PT-MNBs that target Piezo1 in microglia.

[0050] PT-MNBs consist of a perfluoropropane core and a liposome shell; Fe3O4 particles are encapsulated inside the shell; and the outer side of the shell is also loaded with phosphatidylserine PS and anti-Piezo1 antibodies targeting microglia.

[0051] The preparation method is as follows:

[0052] (1) 2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were dissolved in ethanol and mixed together to form a lipid mixture with a molar ratio of 50%:40%:5%:5%.

[0053] (2) The lipid mixture was injected into ultrapure water in which Fe3O4 was dispersed, and then subjected to 40W water bath ultrasonic treatment at 60°C for 5 min. The solution was then dialyzed with a membrane with a cutoff of 8000-14000Da to completely remove the organic solvent, thereby obtaining an aqueous dispersion of phosphatidylserine-coated superparamagnetic iron oxide nanoparticles.

[0054] (3) Glycerol and 1,2-propylene glycol were mixed with the above dispersion at a volume ratio of 10%:10%:80% in a 3 mL glass bottle.

[0055] (4) Finally, the vial was filled with perchloropropane and then mechanically stirred on a shaker for 45 s to obtain phosphatidylserine (PS)-modified magnetic nanobubbles (PS-MNBs), namely, magnetic nanobubbles (PS-MNBs) targeting microglia.

[0056] To prepare MNBs, a lipid mixture without DPPS (i.e., DSPC: cholesterol: DSPE-PEG2000 = 50%: 45%: 5%, DPPS removed and the cholesterol ratio adjusted to keep the total lipid ratio unchanged) was taken, and a lipid-coated Fe3O4 nanoparticle aqueous dispersion was prepared according to the same method (steps 1-3). Subsequently, the dispersion was mixed with glycerol and 1,2-propylene glycol (10%: 10%: 80%) and filled with perchloropropane in a 3 mL glass bottle. The mixture was stirred on a mechanical shaker for 45 s to obtain magnetic nanobubbles (MNBs).

[0057] (5) Magnetic nanobubbles targeting microglia Piezo1 (PT-MNBs) were prepared by linking the microglia-targeted magnetic nanobubbles with avidin-biotin. Briefly, the cells were washed twice with PBS solution at 400 g for 4 min in a bucket rotor centrifuge to remove excess unbound lipids. 830 μg of avidin was added to the washed magnetic nanobubble dispersion and incubated at room temperature for 30 min. The dispersion was washed twice to remove unreacted avidin and then incubated with 3 μg of biotinylated anti-Piezo1 antibody (Thermo Fisher Scientific, MA5-32876) at room temperature for 30 min. The free antibody was then washed with PBS to remove the free antibody.

[0058] Example 2

[0059] This example verifies the PT-MNBs prepared in Example 1.

[0060] Transmission electron microscopy (TEM) was performed. Method: PT-MNBs were evenly dispersed in PBS solvent and ultrasonicated to help disperse and avoid agglomeration. A microgrid was picked up with tweezers and placed on the surface of the PT-MNB solution. The PT-MNBs in the solution were adsorbed on the microgrid and then TEM (TF20, FEI, US) was performed. The results are shown below. Figure 2 As shown, PT-MNBs are round vesicles.

[0061] Particle size and zeta potential analysis were performed. Method: The size and zeta potential of PT-MNBs were measured by Zetasizer NanoZS instrument (NanoBrook 90plus PALS, Brookhaven, US). The results are shown in Figure 2. Figure 3 and Figure 4 As shown, the average diameter and zeta potential of PT-MNBs were 127.84±10.28 nm and -25.78±0.90 mV, respectively.

[0062] Element distribution analysis was performed by uniformly dispersing PT-MNBs on a carbon film, drying the sample, and then examining it on a TEM (TF20, FEI, US). Element analysis was performed using an energy dispersive X-ray spectrometer (EDS) connected to the TEM. Figure 5 As shown in the figure, Figure a represents the TEM electron microscope image of PT-MNBs, Figure b represents the combination diagram of each element in PT-MNBs, Figure c represents the C element in PT-MNBs, Figure d represents the Fe element in PT-MNBs, Figure e represents the P element in PT-MNBs, Figure f represents the O element in PT-MNBs, and Figure g represents the N element in PT-MNBs, indicating that PT-MNBs contain a symmetrical distribution of C, Fe, P, O and N elements.

[0063] X-ray photoelectron spectroscopy analysis was performed. Method: The functional groups of PT-MNBs were analyzed using a photoelectron spectrometer (Thermo Scientific NexsaG, K-Alpha, US). PT-MNBs were placed in the vacuum chamber of the XPS instrument, and the sample surface was irradiated with X-rays, causing electrons in the sample to escape and form photoelectrons. The energy and intensity of these photoelectrons were measured by an energy analyzer to obtain an XPS spectrum. X-ray photoelectron spectroscopy (XPS) showed the characteristic P 2p peak (about 133 eV) and Fe 2p peak (about 713 eV) in PT-MNBs, indicating that phosphatidylserine (PS) and Fe3O4 were successfully loaded on the magnetic nanobubbles ( Figure 6 ).

[0064] Thermogravimetric analysis (TGA) was performed using a thermogravimetric analyzer (TG 209F3 Tarsus, Netzsch, Germany). The PT-MNBs sample was placed in a crucible in the thermogravimetric analyzer, which was then placed in a heating furnace. The thermogravimetric analyzer was started and the temperature was raised. During the test, the instrument recorded the change in mass of the PT-MNBs with temperature in real time, generating a thermogravimetric curve (TGA curve). The results are shown in Figure 2. Figure 7 As shown, phosphatidylserine and anti-Piezo1 antibody accounted for 18% and 64% of the total weight of PT-MNBs, respectively.

[0065] TMAS uses a 0.5 MHz focused ultrasound probe with parameters set as follows: pulse repetition frequency of 1 Hz, pulses of 10 ms, total duration of 120 s, peak positive acoustic pressure of 0.5 MPa, static magnetic field of 0.3 T, and duration of 5 minutes. PT-MNBs can be microinjected into specific brain regions, facilitating precise neuromodulation deep within the brain while also further improving the spatial resolution and targeted stimulation of TMAS.

[0066] A vibrating sample magnetometer (PPMS-9T, Quantum Design, US) was used to obtain the hysteresis curve of PT-MNBs to analyze their magnetic properties. The method was as follows: the sample holder with the PT-MNBs fixed was placed in the measurement chamber of the vibrating sample magnetometer. The instrument applied a periodically changing magnetic field to the sample and measured the changes in the magnetic moment of the sample at different magnetic field strengths. As the external magnetic field strength gradually increased and decreased, the changes in the magnetic moment of the sample were recorded, thereby obtaining the hysteresis curve data. The results of the hysteresis curve showed that the remanence and coercive force of the PT-MNBs were almost zero, indicating that there was no residual magnetization after the external magnetic force was removed ( Figure 8In addition, magnetic attraction experiments were also conducted to further verify the magnetic properties of PT-MNBs. The method was as follows: a container containing PT-MNB suspension was placed near a magnetic field source, and the movement and aggregation of PT-MNBs under the action of the magnetic field were observed. The results are shown in Figure 2. Figure 9 As shown in the figure, PT-MNBs obviously gathered near the magnetic field source after 30 minutes. To further verify the acoustic performance of PT-MNBs, echogenicity analysis was performed. The method was as follows: PT-MNB suspension was placed in an acoustic test tank containing an ultrasonic probe, the ultrasonic equipment was turned on, ultrasonic pulses (5MHz) were emitted, and the echo images generated by PT-MNBs were received. The results are shown in the figure. Figure 10 As shown in the figure, compared with PBS solution, PT-MNBs produce nonlinear vibration under ultrasound, which also confirms that the prepared PT-MNBs have a good response to ultrasonic vibration.

[0067] Example 3

[0068] This embodiment tests the PT-MNBs function.

[0069] TMAS uses a 0.5 MHz focused ultrasound probe with the following parameters: pulse repetition frequency 1 Hz, pulse 10 ms, total duration 120 s, peak positive sound pressure 0.5 MPa, static magnetic field 0.3 T, and duration 5 min.

[0070] Before TMAS, PT-MNBs were labeled with Cy5.5. The method was as follows: PT-MNBs were dispersed in PBS buffer solution, the pH value of the solution was adjusted to make the surface of the nanoparticles positively charged, and then Cy5.5 solution was added and stirred evenly to allow Cy5.5 to be electrostatically adsorbed to the surface of PT-MNBs. Cy5.5-labeled PT-MNBs were added to the microglial cell culture medium, and it was found that PT-MNBs could clearly target the microglial cell membrane ( Figure 11 , among which, Figure a is a fluorescence micrograph of Cy5.5-labeled PT-MNBs binding to microglia (red is Cy5.5-labeled PT-MNBs, blue is the nucleus of microglia), Figure b is Cy5.5-labeled PT-MNBs, and Figure c is a bright field micrograph of PT-MNBs binding to microglia.

[0071] The CON group consisted of untreated microglia (control group); the TMAS group consisted of microglia stimulated with TMAS (stimulation parameters were set as follows: pulse repetition frequency 1 Hz, pulse 10 ms, total duration 120 s, peak positive acoustic pressure 0.5 MPa, static magnetic field 0.3 T, duration 5 min); the TMAS+PT-MNBs group consisted of 200 μL PT-MNBs added to the cell culture dish and incubated with microglia for 2 hours before TMAS stimulation; the TMAS+PT-MNBs+siPiezo1 group consisted of siPizo1 (GenePharma) used to inhibit the Piezo1 ion channel in microglia. After 24 hours of transfection of microglia with 50 nM siPizo1, 200 μL PT-MNBs were added and incubated with microglia for 2 hours before TMAS stimulation. The results are shown in Table 1. Figure 12 As shown in the results, PT-MNBs significantly induced Piezo1 current under the action of TMAS, which produced a stronger effect than TMAS alone, while inhibition of microglial Piezo1 significantly inhibited the Piezo1 current induced by TMAS+PT-MNBs.

[0072] The PT-MNBs (2 mg / mL, 1 μL) prepared in Example 1 were microinjected into the bilateral hippocampus of mice (AP: 2.0 mm, ML: ±1.4 mm, DV: 1.5 mm) and then TMAS stimulation was given (parameters set as: pulse repetition frequency 1 Hz, pulse 10 ms, total duration 120 s, peak positive sound pressure 0.5 MPa, static magnetic field 0.3 T, duration 5 min). It was found that the mouse hippocampus could generate a sound pressure of 0.5 MPa ( Figure 13 ), and simultaneously generates an electric field of 0.2V / m ( Figure 14 ).

[0073] Further in vivo electrophysiological experiments were conducted to verify the effect of TMAS+PT-MNBs on brain neuroplasticity. The control group consisted of mice without any treatment; the TMAS group consisted of mice that received TMAS stimulation (stimulation parameters as described above); and the TMAS+PT-MNBs group consisted of mice that were injected with PT-MNBs into the hippocampus and then underwent TMAS stimulation 3 weeks later.

[0074] like Figure 15 As shown in the figure, Figure a is a schematic diagram of the implantation position of the stimulating electrode and the recording electrode in the in vivo electrophysiological experiment, and Figure b is the excitatory postsynaptic potential (fEPSP) curve before and after long-term potentiation (LTP) induction. The slope of the excitatory postsynaptic potential (fEPSP) curve increased significantly after stimulation, indicating that TMAS+PT-MNBs can enhance the neural plasticity of mice.

[0075] In summary, the present invention designs magnetic nanobubbles PT-MNBs that target Piezo1 in microglia. These PT-MNBs have anti-Piezo1 antibodies and phosphatidylserine modifications on their surfaces, enabling them to specifically bind to / target and regulate Piezo1 in microglia, thereby enabling the Piezo1 ion channel to function as an electromechanical sensor, converting the mechanical and electrical stimulation from TMAS-driven PT-MNBs into intracellular signals. PT-MNBs can serve as nanomagnetic actuators that respond to magnetoacoustic coupling stimulation, promoting the guidance and amplification of TMAS energy without the need for genetic modification. They can also mediate the response of endogenous physical fields of cells to exogenous TMAS physical field energy, thereby enhancing the sensitivity of cells to the mechanical and electrical microenvironment of the brain and improving the accuracy of TMAS. PT-MNBs can also be used to non-invasively stimulate specific cells in deep brain regions, achieving stronger and more targeted neural regulation in deep brain regions.

[0076] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A nanoresponder for enhancing magnetoacoustic coupling stimulation, characterized in that: The nanoresponder includes a magnetic nanobubble, which includes a core and a shell. The core includes a fluorinated carbon gas and / or an inert gas, the fluorinated carbon gas includes perfluoropropane and / or perfluorobutane, the inert gas includes nitrogen, the shell includes lipids, magnetic nanoparticles are encapsulated in the shell, and the surface of the shell is connected to phosphatidylserine and anti-Piezo1 antibodies.

2. The nanoresponder for enhancing magnetoacoustic coupling stimulation according to claim 1, characterized in that: The magnetic nanoparticles include superparamagnetic Fe3O4 particles.

3. The nanoresponder for enhancing magnetoacoustic coupling stimulation according to claim 1 or 2, characterized in that: The lipids include phospholipids.

4. The nanoresponder for enhancing magnetoacoustic coupling stimulation according to any one of claims 1 to 3, characterized in that: The mass percentage of phosphatidylserine in the magnetic nanobubbles is 17% to 19%, the mass percentage of anti-Piezo1 antibodies is 63% to 65%, the mass percentage of perfluoropropane is 1% to 3%, the mass percentage of magnetic nanoparticles is 5% to 7%, and the mass percentage of lipids is 9% to 11%.

5. The method for preparing a nanoresponder for enhancing magnetoacoustic coupling stimulation according to any one of claims 1 to 4, characterized in that: The preparation method comprises: The core and the magnetic nanoparticles are encapsulated by the shell, and phosphatidylserine and anti-Piezo1 antibodies are connected to the surface of the shell.

6. The method for preparing a nanoresponder for enhancing magnetoacoustic coupling stimulation according to claim 5, characterized in that: The preparation method specifically comprises the following steps: (1) 2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine are mixed with a solvent to form a lipid mixture; (2) mixing the lipid mixture with a magnetic nanoparticle solution, performing ultrasonic treatment and dialysis treatment to obtain a nanoparticle dispersion; (3) mixing the nanoparticle dispersion with glycerol and 1,2-propylene glycol, and contacting with perchloropropane; (4) The product of step (3) is linked to anti-Piezo1 antibody via streptavidin-biotin to obtain magnetic nanobubbles.

7. Use of the nanoresponder for enhancing magnetoacoustic coupling stimulation according to any one of claims 1 to 5 in transcranial magnetoacoustic coupling stimulation.

8. A transcranial magnetic-acoustic coupling stimulation system, characterized in that: The transcranial magnetoacoustic coupling stimulation system includes an ultrasonic coupling effect electric field module, an ultrasonic stimulation field module, and a nanoresponder for enhancing magnetoacoustic coupling stimulation according to any one of claims 1 to 5.

9. A neural regulation method, characterized in that: The neuromodulation method comprises: Transcranial magnetic-acoustic coupling stimulation of the target area; The transcranial magnetoacoustic coupling stimulation process uses the nanoresponder for enhancing magnetoacoustic coupling stimulation described in any one of claims 1 to 5, or uses the transcranial magnetoacoustic coupling stimulation system described in claim 8 to perform transcranial magnetoacoustic coupling stimulation.

10. The neural regulation method according to claim 9, characterized in that: The method of using the nano-responder is to inject it into the target site.

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