Minimally invasive flexible piezoelectric-triboelectric hybrid nanometer nerve stimulation device and application thereof

Abstract

The invention discloses a minimally invasive flexible piezoelectric-triboelectric hybrid nanometer nerve stimulation device and application thereof.The middle layer of the nerve stimulation device is a PVDF / TMCM-MnCl3 composite nanofiber membrane, Au electrodes are evaporated on the two faces of the PVDF / TMCM-MnCl3 composite nanofiber membrane, two gold wire or copper wire electrodes are connected with the Au electrodes respectively, and the two gold wire or copper wire electrodes are connected with the Au electrodes respectively. The surface of the Au electrode is subjected to primary packaging by adopting a PU waterproof film, and the surface of the PU waterproof film is subjected to secondary packaging by adopting PDMS. The nerve stimulation device is used for conventional treatment of SIRS. The vagus nerve stimulator can generate an adjustable current up to 0.85 mA under programmable ultrasonic driving, and can be directly used for VNS. The PTNG is ultrasonically driven in vitro to assist the VNS in treating lipopolysaccharide-induced SIRS rats, release of inflammatory factors such as TNF-alpha, IFN-gamma and IL-1beta can be effectively reduced, cytokine storm is relieved, and multi-organ tissue damage and dysfunction are relieved, so that the survival hope is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of piezoelectric materials, and in particular relates to a minimally invasive flexible piezoelectric-triboelectric hybrid nano-nerve stimulation device and its application. Background Art

[0002] Systemic inflammatory response syndrome (SIRS) is an overreaction of the body to stress factors, which can lead to multiple organ dysfunction and even death. It is usually caused by infection, trauma, burns, acute pancreatitis and other reasons. The mortality rate of SIRS varies depending on the cause and individual differences, and can be between 5% and 50%. In the SIRS state, the neuro-immune interaction system is unbalanced, manifested by the sympathetic nervous system enhancing the activity of immune cells, such as increasing neutrophil migration and the release of inflammatory factors, while the vagus nerve's regulatory function is insufficient, resulting in an uncontrolled inflammatory response.

[0003] As an important part of the neuroendocrine-immune coordination network, the regulatory effect of the vagus nerve on systemic and local inflammation has received widespread attention in recent years. Several clinical studies have shown that vagus nerve stimulation (VNS) (through an electrical stimulation device) can restore or enhance vagus nerve activity, thereby reducing the release of proinflammatory cytokines and improving control of inflammatory responses. In lethal endotoxemia, direct electrical stimulation of the vagus nerve can significantly alleviate septic shock. Commercial neurostimulators using direct current pulses can effectively activate neuroimmune interactions and achieve efficient anti-inflammatory treatment of sepsis. Therefore, it is necessary to provide a degradable piezoelectric material with good biocompatibility for application in the field of biomedical materials.

[0004] VNS devices are mainly implemented by using external implantable devices that provide controlled electrical pulse stimulation. However, current VNS devices face some challenges, such as the bulky size of neurostimulators and limited battery capacity. These limitations require repeated surgical interventions, which increase the risk of complications such as bleeding and infection, hindering the continued application of long-term VNS. Therefore, it is crucial to study new, long-term, controllable, and minimally invasive wireless power implantation strategies. Summary of the invention

[0005] The purpose of the present invention is to provide a minimally invasive flexible piezoelectric-triboelectric hybrid nano-nerve stimulation device and its application.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a minimally invasive flexible piezoelectric-triboelectric hybrid nano-neurostimulation device, wherein the middle layer of the neurostimulation device is a PVDF / TMCM-MnCl3 composite nanofiber membrane, Au electrodes are evaporated on both sides of the PVDF / TMCM-MnCl3 composite nanofiber membrane, two gold wire or copper wire electrodes are respectively connected to the Au electrodes, the surface of the Au electrode is encapsulated once with a PU waterproof film, and the surface of the PU waterproof film is encapsulated twice with PDMS.

[0008] In the above technical solution, the preparation method of the PVDF / TMCM-MnCl3 composite nanofiber membrane comprises the following steps:

[0009] S11, Synthesis of TMCM-MnCl3 molecular piezoelectric crystal, the specific synthesis method is:

[0010] S111: equimolar amounts of trimethylamine-30% aqueous solution and dichloromethane are mixed in an acetonitrile solution to react at room temperature to synthesize chloromethyltrimethylammonium chloride;

[0011] S112: adding the chloromethyltrimethylammonium chloride and anhydrous manganese chloride obtained in S111 into a methanol solution and stirring and dissolving them, and the solution is allowed to stand at room temperature to evaporate, thereby obtaining white block crystals, namely the TMCM-MnCl3 molecular piezoelectric crystal;

[0012] S12, synthesis of PVDF piezoelectric nanofiber membrane, the specific synthesis method is:

[0013] S121: PVDF powder was dissolved in dimethylformamide and acetone and magnetically stirred at room temperature to form a homogeneous solution;

[0014] S122: preparing the PVDF piezoelectric nanofiber membrane by using an electrospinning device on the solution;

[0015] S13, synthesis of PVDF / TMCM-MnCl3 composite nanofiber membrane, the specific synthesis method is:

[0016] S131: treating the PVDF piezoelectric nanofiber membrane prepared in S122 with oxygen plasma to obtain a hydrophilic surface;

[0017] S132: dissolving the TMCM-MnCl3 molecular piezoelectric crystals obtained in S112 in a solvent, loading them into an electrospray device, and spraying them quantitatively and uniformly onto the hydrophilic surface of the PVDF piezoelectric nanofiber membrane. The solvent evaporates slowly to obtain the PVDF / TMCM-MnCl3 composite nanofiber membrane.

[0018] In the above technical solution, the preparation method of the nerve stimulation device comprises the following steps:

[0019] S1, preparation of the PVDF / TMCM-MnCl3 composite nanofiber membrane;

[0020] S2, using a thermal evaporation coating machine to deposit Au electrodes on both sides of the PVDF / TMCM-MnCl3 composite nanofiber membrane obtained in S1 to obtain an Au / PVDF / TMCM-MnCl3 / Au composite film;

[0021] S3, use two gold or copper wire electrodes to connect the

[0022] Au electrodes on the upper and lower surfaces of Au / PVDF / TMCM-MnCl3 / Au composite film;

[0023] S4, use PU waterproof film to connect the gold wire or copper wire electrode in S3

[0024] The two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film were encapsulated once to obtain the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film;

[0025] S5, using PDMS to perform secondary packaging on both sides of the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film prepared in S4 to obtain a PDMS / PU / Au / PVDF / TMCM-MnCl3 / Au / PU / PDMS composite film, i.e., the neural stimulation device.

[0026] In the above technical scheme, in S4, the specific method of using a PU waterproof film to encapsulate the two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film connected to the gold wire or copper wire electrodes is: use a PU waterproof film to stick the two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film to form the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film.

[0027] In the above technical scheme, in S112, the chloromethyltrimethylammonium chloride, anhydrous manganese chloride and methanol solution are added in a ratio of (5 mmol): (5 mmol): (10 mL).

[0028] In the above technical solution, in S121, the ratio of the PVDF powder, dimethylformamide and acetone is 0.75 g:2.5 mL:2.5 mL.

[0029] In the above technical solution, in S131, the oxygen plasma treatment uses a radio frequency plasma cleaning machine, and its parameters are set to 118W, 0.1mbar, a gas flow rate of 50sccm O2, and lasts for 300s.

[0030] In a second aspect, the present invention provides the use of the above-mentioned nerve stimulation device in the treatment of inflammatory diseases.

[0031] In the above technical solution, after being implanted in the body, the nerve stimulation device converts the received ultrasonic stimulation into electrical signals to stimulate the vagus nerve, thereby reducing inflammation.

[0032] The beneficial effects of the present invention are as follows: the present invention uses a PVDF / TMCM-MnCl3 piezoelectric / triboelectric hybrid thin film nano-power generation device. MnCl3 is a piezoelectric nano-material with good biocompatibility and high conductivity, TMCM can be used as an auxiliary agent and stabilizer to enhance the performance of the material, PVDF is a flexible polymer with excellent chemical tolerance, PU and PDMS are chemically resistant polymers, and good biocompatibility and insulation make the material not reduce the vitality of cells. PTNG generates electrical signals that stimulate the vagus nerve, thereby regulating the immune cells of SIRS rats to achieve a therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The schematic diagram and the physical picture of the manufacturing process of the PVDF / TMCM-MnCl3 nanofiber membrane according to the embodiment of the present invention;

[0034] Figure 2 This is a scanning electron microscope image of PVDF according to an embodiment of the present invention;

[0035] Figure 3 This is a scanning electron microscope image of the PVDF / TMCM-MnCl3 nanofiber membrane of an embodiment of the present invention;

[0036] Figure 4 This is a scanning electron microscope EDX element mapping diagram of the PVDF / TMCM-MnCl3 nanofiber membrane of an embodiment of the present invention;

[0037] Figure 5 This is a transmission electron microscope image of the PVDF / TMCM-MnCl3 nanofiber membrane of an embodiment of the present invention;

[0038] Figure 6 X-ray diffraction characterization of PVDF, TMCM-MnCl3 and PVDF / TMCM-MnCl3 nanofiber films of the embodiments of the present invention;

[0039] Figure 7 This is a scanning electron microscope image of a cross section of a PVDF / TMCM-MnCl3 nanofiber membrane according to an embodiment of the present invention;

[0040] Figure 8 This is a scanning electron microscope image of a cross section of PTNG according to an embodiment of the present invention;

[0041] Fig. 9 The voltage output of PTNG under ultrasonic stimulation of different powers according to the embodiment of the present invention;

[0042] Fig.10 The current output of the PTNG under ultrasonic stimulation of different powers according to the embodiment of the present invention;

[0043] Fig.11 This is a schematic diagram of PTNG implanted in vivo according to an embodiment of the present invention;

[0044] Fig.12 The PTNG of the present invention is at 20mW / cm 2 Changes in compound action potentials caused by ultrasound stimulation of the vagus nerve;

[0045] Fig.13 Comparison of live and dead cell staining and CCK8 experiment after culturing PC12 (rat adrenal pheochromocytoma cell line, with neuronal cell characteristics) and L929 (mouse fibroblasts) with PTNG culture medium extract of the present invention for 1, 3, and 5 days;

[0046] Fig.14 This is a comparison chart of the body weight, heart, lung, liver, kidney and spleen HE staining and organ coefficients of SD rats injected intraperitoneally with PTNG physiological saline extract according to the embodiment of the present invention;

[0047] Fig.15 HE staining and immunofluorescence images of the implantation site of PTNG implanted in SD rats 4 weeks after implantation;

[0048] Fig.16 HE images of important organs 4 weeks after PTNG implantation in an embodiment of the present invention;

[0049] Fig.17 This is an electrocardiogram 4 weeks after PTNG implantation in an embodiment of the present invention;

[0050] Fig.18 This is a graph showing changes in clinical symptoms of SIRS rats after PTNG stimulated the vagus nerve for 20 minutes according to an embodiment of the present invention;

[0051] Fig.19 This is a graph showing changes in cytokines in SIRS rats after 20 minutes of vagus nerve stimulation with PTNG in an embodiment of the present invention. DETAILED DESCRIPTION

[0052] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0053] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The terms "comprise", "include", etc. used herein indicate the existence of features, steps, operations and / or components, but do not exclude the existence or addition of one or more other features, steps, operations or components.

[0054] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification, and should not be interpreted in an idealized or overly rigid manner.

[0055] In the case of using expressions such as "at least one of A, B, and C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, or C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.).

[0056] Some of the terms and abbreviations of the present invention are explained as follows:

[0057] SIRS: Systemic Inflammatory Response Syndrome

[0058] VNS: Vagus nerve stimulation

[0059] PTNG: Minimally invasive flexible piezoelectric-triboelectric hybrid nano-neurostimulation device

[0060] PVDF: Polyvinylidene fluoride

[0061] PU: Polyurethane

[0062] PDMS: polydimethylsiloxane

[0063] TNF: Tumor necrosis factor

[0064] IFN: interferon

[0065] IL: interleukin

[0066] CCL: chemotactic cytokine ligand

[0067] Ethanol: 75% alcohol

[0068] HCHO: 0.5% formaldehyde

[0069] Ultrasound has the characteristics of high-frequency mechanical vibration, which can effectively transmit energy to subcutaneous tissue with minimal attenuation and large penetration depth, thereby directly affecting subcutaneous structures. The combination of ultrasound and nanogenerators has promoted innovative methods for ultrasound-driven wireless energy supply. Nanogenerators can act as neurostimulators, converting ultrasound energy into electrical energy, which is then used to stimulate neural tissue. This approach does not require additional battery power and integrated circuit control modules, greatly reducing the size of the stimulation device and improving its biocompatibility. In order to treat systemic inflammatory response syndrome in small animal models by ultrasonic wireless vagus nerve electrical stimulation, it is necessary to develop nanogenerators with enhanced conversion efficiency. Piezoelectric materials have the ability to self-generate electricity under mechanical stress and can be widely used as self-powered electrical stimulators in various biomedical applications, including implantable sensors, ultrasonic transducers, and actuators. In the field of tissue engineering, piezoelectric materials have demonstrated their ability in bone, cartilage, skeletal muscle, skin, and nerve regeneration.

[0070] Piezoelectric materials are smart materials that generate electrical signal energy under the action of external forces. They can be used as self-powered scaffolds for neural tissue engineering, and can stimulate nerves with current by in situ stimulation of human movement or external mechanical vibration. In order to enhance the piezoelectric properties of molecular piezoelectric materials, the integration of piezoelectric / triboelectric composite materials can be used. These composites synergistically combine two energy exchange mechanisms, thereby improving the efficiency of mechanical energy conversion. It is worth noting that polyvinylidene fluoride (PVDF) fiber membranes prepared by electrospinning process exhibit excellent piezoelectric properties. Studies have shown that PVDF nanofiber membrane structures also exhibit piezoelectric triboelectric effects, which greatly improves the application potential of electrospun PVDF fiber materials in various electromechanical devices.

[0071] As a piezoelectric material, PVDF has weaker piezoelectric properties than traditional inorganic piezoelectric materials, which limits the further application of PVDF in the field of energy collection. Inorganic piezoelectric nanomaterials have good piezoelectric properties, but the bulk ceramics are hard and the processing and thinning process is complicated, which is not suitable for the preparation of flexible electronic devices. In order to solve the shortcomings of organic piezoelectric polymers and inorganic piezoelectric materials in the performance of flexible piezoelectric materials, it was found in the process of realizing the concept of the present invention that the synthesis of PVDF nanofibers using molecular calcium manganese ferroelectrics and electrospinning technology provides a simple and scalable method for preparing flexible molecular piezoelectric / triboelectric hybrid thin film nano-power generation devices.

[0072] The present invention provides a minimally invasive flexible piezoelectric-triboelectric hybrid nano-nerve stimulation device and its application in inflammatory diseases. The operation of promoting the proliferation and differentiation of nerve cells includes:

[0073] The nerve stimulation device (PTNG) of the present invention was implanted into SIRS rats. PTNG stimulated the vagus nerve by converting the received ultrasonic stimulation into electrical signals, thereby alleviating inflammation.

[0074] The present invention adds inorganic perovskite TMCM-MnCl3 molecular piezoelectric crystals as additives to the surface of PVDF nanofibers through in-situ growth, constructing a simple and efficient minimally invasive flexible piezoelectric-triboelectric hybrid nano-neurostimulation device (PTNG), which can achieve stable piezoelectric / triboelectric properties within a certain period of time, thereby regulating the neural differentiation of neural stem cells at the cellular level, thereby achieving the purpose of treating and repairing injuries.

[0075] The PTNG of the present invention can stimulate the vagus nerve, and the ultrasonic stimulation is converted into an electrical signal to stimulate the nerve to achieve the effect of treating SIRS rats.

[0076] The ultrasonic stimulation of the present invention can be provided by an ultrasonic transducer, for example, the ultrasonic transducer can be driven to generate constant ultrasonic waves to trigger PTNG to generate electrical signals. According to an embodiment of the present invention, PTNG was implanted into SIRS rats for 20 minutes of vagus nerve stimulation; lipopolysaccharide was intravenously injected to induce SIRS in rats.

[0077] Lipopolysaccharide was injected into the femoral vein of SD rats. Lipopolysaccharide is the main component of the cell wall of Gram-negative bacteria and can induce a strong immune response. It can directly enter the circulatory system through femoral vein injection and quickly affect the systemic immune response.

[0078] The middle layer of the nerve stimulation device (PTNG) of the present invention is a PVDF / TMCM-MnCl3 composite nanofiber membrane, Au electrodes are evaporated on both sides of the PVDF / TMCM-MnCl3 composite nanofiber membrane, two gold wires or copper wire electrodes are respectively connected to the Au electrodes, the surface of the Au electrode is encapsulated with a PU waterproof film for a primary package, and the surface of the PU waterproof film is encapsulated with PDMS for a secondary package. The specific preparation method includes the following steps:

[0079] S1, preparation of PVDF / TMCM-MnCl3 composite nanofiber membrane;

[0080] S11, Synthesis of TMCM-MnCl3 molecular piezoelectric crystal, the specific synthesis method is:

[0081] S111: equimolar amounts of trimethylamine-30% aqueous solution and dichloromethane are mixed in an acetonitrile solution to react at room temperature to synthesize chloromethyltrimethylammonium chloride;

[0082] S112: adding chloromethyltrimethylammonium chloride and anhydrous manganese chloride prepared in S111 into methanol solution and stirring and dissolving them thoroughly, and the solution is allowed to stand at room temperature for evaporation to obtain white block crystals, namely TMCM-MnCl3 molecular piezoelectric crystals;

[0083] The ratio of chloromethyltrimethylammonium chloride, anhydrous manganese chloride and methanol solution added is (5 mmol): (5 mmol): (10 mL).

[0084] S12, synthesis of PVDF piezoelectric nanofiber membrane, the specific synthesis method is:

[0085] S121: PVDF powder was dissolved in dimethylformamide and acetone and magnetically stirred at room temperature to form a homogeneous solution;

[0086] The ratio of PVDF powder, dimethylformamide and acetone is 0.75 g:2.5 mL:2.5 mL.

[0087] S122: preparing the solution into a PVDF piezoelectric nanofiber membrane by using an electrospinning device;

[0088] S13, synthesis of PVDF / TMCM-MnCl3 composite nanofiber membrane, the specific synthesis method is:

[0089] S131: treating the PVDF piezoelectric nanofiber membrane prepared in S122 with oxygen plasma to obtain a hydrophilic surface;

[0090] The oxygen plasma treatment uses a radio frequency plasma cleaning machine with parameters set to 118W, 0.1mbar, a gas flow rate of 50sccm O2, and lasts for 300s. The membrane surface treated with these parameters has hydrophilicity.

[0091] S132: dissolving the TMCM-MnCl3 molecular piezoelectric crystal prepared in S112 in a solvent, loading it into an electrospray device, and spraying it quantitatively and uniformly onto the hydrophilic surface of the PVDF piezoelectric nanofiber membrane, and slowly evaporating the solvent to obtain a PVDF / TMCM-MnCl3 composite nanofiber membrane. Preferably, the solvent is deionized water or methanol.

[0092] S2, using a thermal evaporation coating machine to deposit Au electrodes on both sides of the PVDF / TMCM-MnCl3 composite nanofiber membrane obtained in S1, to obtain an Au / PVDF / TMCM-MnCl3 / Au composite film. As one embodiment, the specific steps are:

[0093] The PVDF / TMCM-MnCl3 composite nanofiber membrane was attached to the surface of the metal plate and located just above the water cooling device in the vacuum chamber. A gold block was placed in the evaporation heating chamber and vacuumed with a mechanical pump and a molecular pump. -5 Pa, the gold block is subjected to a series of heating processes, including roasting, pre-melting and evaporation. The gold block is melted, evaporated, and then uniformly deposited on the surface of the PVDF / TMCM-MnCl3 composite nanofiber membrane; when the deposition thickness reaches 50-100nm, the evaporation source is turned off, and then the molecular pump and the mechanical pump are gradually turned off; after completing these steps, the exhaust switch is turned on until the pressure in the evaporation chamber returns to the atmospheric level; the chamber door is opened to recover the PVDF / TMCM-MnCl3 composite nanofiber membrane with Au on the surface; the above steps are repeated on the recovered PVDF / TMCM-MnCl3 composite nanofiber membrane, and a layer of Au is continued to be evaporated on the back to obtain the Au / PVDF / TMCM-MnCl3 / Au composite film.

[0094] S3, using two gold or copper wire electrodes to connect the upper and lower Au electrodes of the Au / PVDF / TMCM-MnCl3 / Au composite film prepared in S2;

[0095] S4, using a PU waterproof film to encapsulate both sides of the Au / PVDF / TMCM-MnCl3 / Au composite film connected to the gold wire or copper wire electrodes in S3, to obtain a PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film;

[0096] As one of the embodiments, the specific method of using a PU waterproof film to encapsulate the two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film connected to the gold wire or copper wire electrodes is: use a PU waterproof film to stick the two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film to form a PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film.

[0097] S5, using PDMS to perform secondary packaging on both sides of the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film prepared in S4 to prepare a PDMS / PU / Au / PVDF / TMCM-MnCl3 / Au / PU / PDMS composite film, i.e., a neural stimulation device.

[0098] As one of the embodiments, the specific steps are: PDMS and PDMS coagulant are prepared in a ratio of 10:1, and after being fully stirred, they are placed in a vacuum drying oven for exhaust treatment. Then the glass sheet is placed in a glass substrate instrument, and then the exhausted PDMS is coated on the glass sheet. The coating apparatus is turned on, and a uniform and dense PDMS film can be obtained after drying. Before the PDMS film is dry, the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film is placed on the PDMS film. The PDMS film will stick to the composite film. The same operation is repeated on both sides to complete the secondary packaging of the composite film.

[0099] Example 1 Preparation of PTNG

[0100] As one embodiment, the preparation method of PTNG includes the following steps:

[0101] At room temperature, equimolar amounts of trimethylamine (30% aqueous solution) and dichloromethane were mixed in an acetonitrile solution and reacted for 24 hours to synthesize chloromethyltrimethylammonium chloride, and the obtained colorless solid was stored in a vacuum desiccator. The above chloromethyltrimethylammonium chloride (5mmol) and anhydrous manganese chloride (5mmol) were added to a methanol solution (10mL) and stirred to dissolve. The solution was kept still in a beaker covered with a porous filter paper at room temperature, and white block crystals Me3NCH2ClMnCl3 (TMCM-MnCl3 molecular piezoelectric crystals) were obtained by solvent evaporation.

[0102] The preparation of PVDF piezoelectric nanofiber membrane involves the use of commercial electrospinning equipment and a high-speed collection device. First, 0.75 g of PVDF powder was dissolved in 2.5 mL of dimethylformamide and acetone and magnetically stirred at room temperature to form a homogeneous solution. The solution was then loaded into a 5 mL syringe with a 25-gauge needle, and the electrospinning process was started by adjusting the voltage to produce the PVDF piezoelectric nanofiber membrane.

[0103] The PVDF piezoelectric nanofiber membrane was treated with oxygen plasma to obtain a hydrophilic surface. The RF plasma cleaning machine was operated at 118W, 0.1mbar, and a gas flow rate of 50sccm O2 for 300s. The TMCM-MnCl3 molecular piezoelectric crystal was dissolved in a solvent (such as deionized water or methanol), loaded into an electrospray device, and quantitatively and uniformly sprayed onto the surface of the PVDF piezoelectric nanofiber membrane treated with oxygen plasma. The solvent slowly evaporated to obtain a PVDF / TMCM-MnCl3 composite nanofiber membrane. The thermal evaporation coating machine evaporated about 50 to 100nm of Au electrodes on both sides of the film. Two gold or copper wire electrodes were used to connect the Au electrodes respectively.

[0104] The Au / PVDF / TMCM-MnCl3 / Au composite film is encapsulated once with a commercial PU waterproof film, and then the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film is encapsulated twice with PDMS using a spin coating process. The two encapsulations play a role of protection and insulation, thereby obtaining the neural stimulation device (PTNG) of the present invention.

[0105] Preferably, the nerve stimulation device (PTNG) has a length×width×thickness=15 mm×15 mm×200 μm.

[0106] In the present invention, inorganic perovskite TMCM-MnCl3 molecular piezoelectric crystals are added as additives to the surface of PVDF piezoelectric nanofiber membranes through in-situ growth to construct a simple and efficient minimally invasive flexible piezoelectric-triboelectric hybrid nano-neurostimulator (PTNG) ( Figure 1 AB). The core component of the PTNG device is the PVDF / TMCM-MnCl3 composite nanofiber membrane located in the middle layer, with Au electrodes evaporated on both sides, two gold or copper wire electrodes connected to the Au electrodes respectively, the surface of the Au electrode is encapsulated with a PU waterproof film for the first time, and the surface of the PU waterproof film is encapsulated with PDMS for the second time ( Figure 1 A). The diameter of PVDF nanofibers produced by electrospinning is reduced and the specific surface area is increased, thereby enhancing the contact interface between TMCM-MnCl3 crystals and PVDF ( Figure 2 The surface morphology of the nanofibers was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 5 This indicates that TMCM-MnCl3 crystallizes on the PVDF surface. Compared with the PVDF core, the surface morphology of the PVDF / TMCM-MnCl3 composite is rougher. X-ray diffraction (XRD) analysis further confirms the crystallinity of the TMCM-MnCl3 coating. Figure 6As shown in Figure 2, the diffraction peaks of PVDF / TMCM-MnCl3 are more obvious than those of pure TMCM-MnCl3, indicating that there are a large number of TMCM-MnCl3 crystals on the surface of PVDF, which can effectively fill the gaps between nanofibers. The surface morphology of the composite membrane was examined by elemental analysis using scanning electron microscopy, such as Figure 3 As shown. TMCM-MnCl3 is evenly distributed on PVDF nanofibers. The elemental composition analysis of PVDF / TMCM-MnCl3 membrane shows that C, F, Cl and Mn elements are evenly distributed on PVDF nanofibers, as shown Figure 4 In addition, the cross-sectional scanning electron micrograph of PVDF / TMCM-MnCl3 (as shown in Figure 7 The sample thickness is about 130 μm.

[0107] Example 2 PTNG output experiment under ultrasonic drive

[0108] To investigate the performance of PTNG under ultrasound, we fabricated a PTNG consisting of a PVDF / TMCM-MnCl3 nanofiber film, gold electrodes, and PDMS encapsulation. Gold electrodes with a thickness of approximately 50-100 nm were deposited on both sides of the film using a thermal evaporation coater. Commercial polyurethane (PU) waterproof film was used as the main encapsulation material for the PVDF / TMCM-MnCl3 PTNG. Subsequently, an external encapsulation layer (denoted as PDMS) was prepared by a spin coating process to provide protection and insulation for the PTNG. By encapsulating the device with wire electrodes, a flexible device with a size of approximately 200 μm ( Figure 8 ). To evaluate the electrical output characteristics of PTNG under high-frequency vibration, an oscilloscope (with a probe internal impedance of 10 MΩ) was used to measure the voltage response of PTNG under corresponding sound pressure. A current amplifier connected to the oscilloscope was used to measure the current generated by PTNG in response to ultrasonic waves ( Fig.10 .according to( Figure 10-11 ) shows that as the transducer power increases, the Vpp (peak-to-peak output voltage) and Ipp (peak-to-peak output current) of the PTNG both show an upward trend. It is worth noting that when the transducer power reaches 20mW / cm 2 When the PTNG Vpp reaches 19V, the output Ipp is 0.85mA. Ultrasound provides programmable pulsed mechanical wave stimulation to the subcutaneously implanted PTNG device through the skin and muscle tissue, and with the help of ultrasound coupling gel ( Figure 1 B) PTNG responds to pulsed ultrasonic excitation to generate controllable electrical pulses ( Fig.11 We stimulated the vagus nerve with electrical pulses generated by ultrasound-driven PTNG and recorded the action potentials induced by the stimulation ( Fig.12). This indicates that the electrical pulses generated by ultrasound-driven PTNG can directly stimulate the vagus nerve without the need for additional auxiliary circuits. Therefore, the wireless vagus nerve stimulation system we developed can be successfully applied to vagus nerve stimulation in animal models.

[0109] Example 3 PTNG biocompatibility test

[0110] The cell compatibility of PTNG was studied using DMEM medium as a negative control and 75% alcohol as a positive control:

[0111] Prepare PTNG complete medium for cell culture. Soak PTNG in 75% ethanol (Sinopharm, China) for 24 hours, then aspirate, replace with PBS (Chreagen, China) and irradiate with ultraviolet light for 24 hours, repeat twice, obtain sterilized device and soak in high-glucose DMEM medium (Hyclone, USA) at 37°C for 24 hours. Take out the device and add 10% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin-streptomycin solution (PS) (Hyclone, USA) and mix.

[0112] Cell culture. PC12 cells were divided into three groups. The control group was cultured with high-glucose DMEM complete medium, the PTNG group was cultured with PTNG complete medium, and the ethanol group was the same as the control group but was incubated with 50% ethanol at 37°C for 30 seconds before detection as a positive control.

[0113] Calcein-AM / PI staining. Add 20 μL of Calcein-AM (Biyuntian, China) and 30 μL of PI (Biyuntian, China) restored to room temperature into 10 mL of PBS, mix well, and store in the dark for later use. Carefully discard the culture medium in the multi-well plate, add 1 mL of PBS, let it stand for a few seconds, then gently remove it, add 200 μL of staining solution, culture in a cell culture incubator in the dark for 15 minutes, carefully discard the staining solution, add 500 μL of PBS to wash once, and observe under a fluorescence microscope.

[0114] CCK-8 kit detection. On the 1st, 2nd, 3rd, 4th and 5th days of cell culture, 1 mL of trypsin was used to digest the cells for 2 min. After all the cells floated up under the microscope, 2 mL of 1200 rpm was added and centrifuged for 3 min. The cells were collected and resuspended in the culture medium, counted and diluted to 50,000 cells / mL. 100 μL of cell suspension was added in sequence, followed by 10 μL of CCK-8 solution (Biyuntian, China), and the cells were placed in a cell culture incubator for 2 h before measuring the OD value with an enzyme marker.

[0115] After being fully disinfected and sterilized with PTNG, the cells were immersed in the basal medium for 24 hours to obtain the extraction medium, which was used to prepare the complete medium for culturing PC12 (rat adrenal pheochromocytoma cell line with neuronal cell characteristics) and L929 (mouse fibroblasts). The cells in the control group (DMEM complete medium) and the PTNG extract group grew over the entire visual field on the 5th day and stacked into different layers. The cells stained with Calcein-AM / PI had normal morphology and all showed green fluorescence, while the positive control group (Ethanol group) treated with 75% ethanol showed red fluorescence in the entire visual field ( Fig.13 A) CCK-8 was used to qualitatively analyze cell proliferation and cytotoxicity. Fig.13 As shown in BC, there was no statistical difference in the OD values ​​between the control group and the PTNG group, but both were higher than that of the positive control group, indicating that PTNG had no effect on cell activity and cell proliferation.

[0116] The in vivo compatibility of PTNG was studied using physiological saline as invisible control and 0.5% formaldehyde as positive control:

[0117] PTNG sterilized with 75% alcohol and ultraviolet light was soaked in physiological saline (Sinopharm Group, China) at 37°C for 24 hours to prepare PTNG saline extract for acute toxicity test. SD rats were randomly divided into three groups and injected with different fluids at 10 mL / kg intraperitoneally: the control group was given physiological saline, the PTNG group was given PTNG saline extract, and the HCHO group was given 0.5% formaldehyde solution (Servicebio, China) as a positive control. SD rats were weighed, marked, and intraperitoneally injected according to the group. After 3 days, the young rats were weighed, anesthetized, and killed. The heart, lungs, liver, spleen, and kidneys were removed, rinsed with sterile PBS, and dried with absorbent paper. The organ coefficients were weighed and calculated, and the organs were immersed in 4% paraformaldehyde fixative for more than 24 hours for HE staining.

[0118] After the VNS device was disinfected and sterilized, it was soaked in saline for 24 hours to obtain the extract, which was intraperitoneally injected at a dose of 10 mL / kg. The animals were dissected 72 hours later. The body weight and organ weight of Sprague-Dawley (SD) rats were recorded, and the organ coefficient was calculated: organ coefficient = organ weight / body weight. The animals in the control group (intraperitoneal injection of equal doses of saline) and the PNTG group (intraperitoneal injection of device extract) were in positive spirits, had shiny and thick skin, and gained weight, while the rats in the HCHO group (intraperitoneal injection of equal doses of formaldehyde) had rough and messy hair, gained weight, and had diarrhea ( Fig.14 A). Compared with the normal shape, size and color of the organs of rats in the Control group and PTNG group, the organs (heart, lung, liver, kidney and spleen) of rats in the formaldehyde group were obviously congested and edematous, which was consistent with the conclusion reflected by the organ coefficient ( Fig.14BF). HE staining showed that the structures of the heart, lung, liver, kidney, and spleen of rats in the Control group and PTNG group were regular and clear, the cells were neatly arranged, and the morphology was normal ( Fig.14 G). In the HCHO group, cell debris, myocardial fiber rupture and cavitation were observed in various organs, alveolar septa were destroyed and filled with inflammatory exudates, hepatocytes had focal necrosis, renal tubular epithelial cells had scattered necrosis, accompanied by glomerular necrosis and atrophy, unclear boundaries between the white and red pulp of the spleen, and increased and enlarged periarterial lymphoid sheaths (PALS), lymph nodes and germinal centers.

[0119] Both cell experiments and animal experiments have proved that PTNG has a good interaction with the body and does not cause adverse immune or toxic reactions such as inflammation, allergies, tissue damage, etc. when in contact with biological tissues. On the contrary, it can coexist harmoniously with the body and does not interfere with normal biological functions. This is the premise that we can safely apply PTNG to SIRS treatment.

[0120] Example 4 PTNG biological long-term implantation experiment

[0121] PTNG insertion surgery: The rats were fixed under isoflurane anesthesia, the neck was depilated, iodine was used for disinfection, and 75% ethanol was used for deiodination. The skin was cut from the sternum upward along the midline of the neck, the incision was about 1 cm long, the subcutaneous fascia and skin were bluntly separated, and the superficial muscles of the neck were exposed. The neck muscles were separated, and the carotid sheath was found on both sides of the sternohyoid muscle and below the sternocleidomastoid muscle according to the carotid artery pulsation. The vagus nerve was carefully separated. The flexible electrode was wrapped around the vagus nerve, and the PTNG was placed under the back skin. The incision was sutured with 4-0 silk sutures (Medtronic, USA) and warmed for resuscitation.

[0122] Minimally invasive implantation of PTNG was performed to observe whether the material would cause adverse reactions such as PTNG failure, chronic inflammation, and infection 4 weeks after implantation. Electrocardiogram (ECG) recorded the potential of ultrasound-driven PTNG to stimulate the vagus nerve, which was more obvious on lead I ( Fig.17 ). Electrocardiograms showed that after 4 weeks of tissue fluid infiltration and tissue friction in the body, PTNG could stably generate sufficient current to stimulate the vagus nerve under ultrasound stimulation. Fig.15 In A, we can see intuitively that compared with the control group, there is no obvious inflammatory infiltration and skin necrosis in the PTNG group. To further determine whether long-term implantation of PTNG will lead to infection or necrosis, we used immunofluorescence staining ( Fig.15 B) and normalized fluorescence intensity analysis ( Fig.15CD) detected tumor necrosis factor (TNF-α) and caspase-3 at the implantation site. These results clearly showed that the PTNG group, like the control group, did not show obvious inflammatory response and tissue abnormalities. In addition, we also observed the effects of long-term implantation of PTNG neurostimulators on other organs. Similar to the conclusions of the acute toxicity experiment, no abnormalities were found in the important organs of the PTNG group and the control group ( Fig.16 ). The myocardial fibers are arranged regularly and tightly, and the intercalated discs are clearly visible. The alveolar septa are uniform and there is no inflammatory exudate. The radially arranged hepatic sinusoids flow into the central vein of the liver, and the hepatocytes are arranged neatly. The renal tubules are arranged normally, and the nearly spherical kidney body is of reasonable size and shape. Scattered white pulp and red pulp can be seen in the spleen parenchyma, and PLAs and lymph nodes can be seen in the white pulp. The boundary between the white pulp and the marginal zone is obvious. It is proved that the PTNG neurostimulator has no obvious immune response and tissue damage in the body, and has good biocompatibility.

[0123] Example 5 Experiment on ultrasound-driven PTNG stimulation of vagus nerve for SIRS treatment

[0124] Ultrasound-driven PTNG for VNS treatment of SIRS. Rats were randomly divided into 3 groups. The control group underwent PTNG implantation surgery and 10 mg / kg saline was injected through the femoral vein. LPS (Sigma-Aldrich, Germany) was used to replace saline in the SIRS group, and the rest of the operations were exactly the same. The VNS group was immediately given 20 minutes of VNS on the basis of the SIRS group. 4 hours after intravenous injection, 2 mL of whole blood was collected from EDTA anticoagulation tubes (BD, USA) and sent to blood routine (animal peripheral blood). The collected 2 mL whole blood samples were placed in serum separation tubes, placed at 4°C overnight, centrifuged at 1000×g for 20 minutes, and the supernatant was taken for biochemical examination. The rats were dissected, and the heart, lungs, liver, kidneys, intestines, thymus, and spleen were taken for HE staining. Finally, the rat was euthanized.

[0125] like Fig.18 As shown in B, LPS successfully induced SIRS rats, which had faster breathing, heart rate, and body temperature. The number of peripheral blood leukocytes in SIRS rats was significantly higher than that in normal rats ( Fig.18 C). After VNS treatment, the above systemic infection symptoms were alleviated, but still more severe than normal rats. The present invention also found that rats with SIRS induced by a lethal dose of LPS often had multiple organ dysfunction and tissue damage, and most died 4 to 12 hours after LPS injection without intervention. The mortality rate of rats in the VNS group was significantly reduced ( Fig.18 A). 20 min VNS treatment can significantly reduce SIRS-induced myocarditis, as shown by a significant decrease in creatine kinase (CK) in peripheral blood ( Fig.18D). It was confirmed that the severe liver dysfunction of SIRS rats was also improved after VNS, and serum alanine aminotransferase (ALT) ( Fig.18 E). Similarly, similar trends were observed in the evaluation of renal function using blood urea nitrogen (BUN) as an indicator ( Fig.18 F). These fully demonstrate that SIRS rats have systemic inflammatory response and multiple organ failure, which is verified by the tissue damage shown by HE staining of multiple organs ( Fig.18 G). The vasodilation, congestion, inflammatory cell infiltration and myocardial punctate necrosis in the VNS group were milder than those in the LPS group, and no obvious myocardial fiber rupture was observed. However, the myocardial fiber arrangement of the rats in the VNS group was not as regular and tight as that in the control group; compared with the aggravated alveolar fusion and increased inflammatory exudates in SIRS rats, the widening of the alveolar septum in the VNS group was not obvious, and the number of immune cells was small; SIRS rats had necrosis and inflammatory infiltration of liver and kidney tissue cells, and the intestinal villi were broken and detached, and VNS could alleviate the above symptoms; in addition, VNS could restore the abnormalities of immune organs caused by LPS to a certain extent, including a significant increase in the ratio of thymic cortex to medulla, and significant expansion of spleen PALS and lymph nodes.

[0126] The present invention found that VNS inhibited the synthesis and release of multiple inflammatory mediators TNF-α, IFN-γ, IL-1β, IL-6, IL-18, CCL3 and CCL5 in the peripheral blood of SIRS rats, and promoted the expression of anti-inflammatory biological molecule IL-10, with statistically significant differences ( Fig.19 ). These factors work together to regulate and coordinate the immune response and use their advantages to balance pro-inflammatory and anti-inflammatory mechanisms. It is worth noting that although the expression patterns of cytokines in the three groups were similar at the same time, the expression trends of cytokines in each group over time were not completely consistent. The concentrations of serum TNF-α and macrophage inflammatory protein CCL3 in SIRS rats gradually decreased over time, but in the VNS group, their expression pattern turned into a peak type and reached a peak at 2 hours ( Fig.19 A and F). In contrast, IFN-γ, IL-1β, and IL-6 increased over time in SIRS rats and the VNS group ( Fig.19 BD). IL-18 and IL-10 increased first and then decreased, reaching peak values ​​at 3 hours and 2 hours after LPS injection, respectively. After VNS treatment, the peripheral blood IL-18 of rats showed a peak trend, similar to that of the SIRS group, while IL-10 continued to subside ( Fig.19 E, H); The CCL5 level in the SIRS group reached a peak at the 2nd hour and then maintained a relatively high level, while the CCL5 level in the VNS group fluctuated slightly at a low level ( Fig.19 G)

[0127] Obviously, the symptoms of fever, shortness of breath, elevated white blood cell count, elevated myocardial enzymes, and liver and kidney dysfunction in the SIRS model we constructed with a lethal dose of LPS are very close to clinical symptoms, with significantly increased levels of peripheral inflammatory cytokines and decreased levels of anti-inflammatory factors. It is worth emphasizing that VNS effectively reduced tissue damage and inflammatory responses in multiple organs of multiple systems, including the heart, lungs, liver, kidneys, intestines, thymus, and spleen in SIRS rats, which may be the reason why the rats in the VNS group had lower systemic inflammation and limited multiple organ failure.

[0128] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A minimally invasive flexible piezoelectric-triboelectric hybrid nano-nerve stimulation device, characterized in that: The middle layer of the nerve stimulation device is a PVDF / TMCM-MnCl3 composite nanofiber membrane, Au electrodes are evaporated on both sides of the PVDF / TMCM-MnCl3 composite nanofiber membrane, two gold wire or copper wire electrodes are respectively connected to the Au electrodes, the surface of the Au electrode is encapsulated once with a PU waterproof film, and the surface of the PU waterproof film is encapsulated twice with PDMS.

2. The nerve stimulation device according to claim 1, characterized in that: The preparation method of the PVDF / TMCM-MnCl3 composite nanofiber membrane comprises the following steps: S11, Synthesis of TMCM-MnCl3 molecular piezoelectric crystal, the specific synthesis method is: S111: equimolar amounts of trimethylamine-30% aqueous solution and dichloromethane are mixed in an acetonitrile solution to react at room temperature to synthesize chloromethyltrimethylammonium chloride; S112: adding the chloromethyltrimethylammonium chloride and anhydrous manganese chloride obtained in S111 into a methanol solution and stirring and dissolving them, and the solution is allowed to stand at room temperature to evaporate, thereby obtaining white block crystals, namely the TMCM-MnCl3 molecular piezoelectric crystal; S12, synthesis of PVDF piezoelectric nanofiber membrane, the specific synthesis method is: S121: PVDF powder was dissolved in dimethylformamide and acetone and magnetically stirred at room temperature to form a homogeneous solution; S122: preparing the PVDF piezoelectric nanofiber membrane by using an electrospinning device on the solution; S13, synthesis of PVDF / TMCM-MnCl3 composite nanofiber membrane, the specific synthesis method is: S131: treating the PVDF piezoelectric nanofiber membrane prepared in S122 with oxygen plasma to obtain a hydrophilic surface; S132: dissolving the TMCM-MnCl3 molecular piezoelectric crystals obtained in S112 in a solvent, loading them into an electrospray device, and spraying them quantitatively and uniformly onto the hydrophilic surface of the PVDF piezoelectric nanofiber membrane. The solvent evaporates slowly to obtain the PVDF / TMCM-MnCl3 composite nanofiber membrane.

3. The nerve stimulation device according to claim 2, characterized in that: The preparation method of the nerve stimulation device comprises the following steps: S1, preparation of the PVDF / TMCM-MnCl3 composite nanofiber membrane; S2, using a thermal evaporation coating machine to deposit Au electrodes on both sides of the PVDF / TMCM-MnCl3 composite nanofiber membrane obtained in S1 to obtain an Au / PVDF / TMCM-MnCl3 / Au composite film; S3, using two gold wire or copper wire electrodes to respectively connect the upper and lower Au electrodes of the Au / PVDF / TMCM-MnCl3 / Au composite film prepared in S2; S4, using a PU waterproof film to encapsulate both sides of the Au / PVDF / TMCM-MnCl3 / Au composite film connected to the gold wire or copper wire electrodes in S3, to obtain a PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film; S5, using PDMS to perform secondary packaging on both sides of the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film prepared in S4 to obtain a PDMS / PU / Au / PVDF / TMCM-MnCl3 / Au / PU / PDMS composite film, i.e., the neural stimulation device.

4. The nerve stimulation device according to claim 3, characterized in that: In S4, the specific method of using a PU waterproof film to encapsulate the two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film connected to the gold wire or copper wire electrodes is: using a PU waterproof film to stick the two sides of the Au / PVDF / TMCM-MnCl3 / Au composite film to form the PU / Au / PVDF / TMCM-MnCl3 / Au / PU composite film.

5. The nerve stimulation device according to claim 2, characterized in that: In S112, the chloromethyltrimethylammonium chloride, anhydrous manganese chloride and methanol solution are added in a ratio of (5 mmol): (5 mmol): (10 mL).

6. The nerve stimulation device according to claim 2, characterized in that: In S121, the ratio of the PVDF powder, dimethylformamide and acetone is 0.75 g:2.5 mL:2.5 mL.

7. The nerve stimulation device according to claim 2, characterized in that: In S131, the oxygen plasma treatment uses a radio frequency plasma cleaning machine, and its parameters are set to 118W, 0.1mbar, a gas flow rate of 50sccm O2, and lasts for 300s.

8. Use of the nerve stimulation device according to any one of claims 1 to 7 in the treatment of inflammatory diseases.

9. According to the use of claim 8, after being implanted in the body, the nerve stimulation device converts the received ultrasonic stimulation into electrical signals to stimulate the vagus nerve, thereby reducing inflammation.

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