A biodegradable vagus nerve stimulation system and methods of making and using the same

Abstract

The application discloses a biodegradable vagus nerve stimulation system and a preparation method and application thereof, and belongs to the field of medical devices. The biodegradable vagus nerve stimulation system comprises an implantable vagus nerve stimulator and an external focused ultrasound emission device used in cooperation with the implantable vagus nerve stimulator. The implantable vagus nerve stimulator comprises an ultrasound-driven degradable friction nanogenerator and a vagus nerve interface lead. The biodegradable vagus nerve stimulation system provided by the application realizes wireless energy supply for the implantable device and vagus nerve stimulation by constructing an ultrasound-driven frictional electric energy conversion mechanism, and fundamentally solves the dependence of traditional implantable devices on built-in batteries.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to a biodegradable vagus nerve stimulation system, its preparation method, and its application. Background Technology

[0002] Cardiovascular diseases (CVDs), particularly atherosclerosis, are the leading cause of death and disability worldwide. The core pathology of atherosclerosis is a chronic inflammatory response in the blood vessel walls, involving lipid deposition, immune cell infiltration, and plaque formation and development. Traditional treatments primarily rely on drug therapy, such as statins for lipid-lowering and antiplatelet drugs, but these methods suffer from side effects, poor patient compliance, and limited efficacy in specific patients. Therefore, developing novel, non-pharmacological treatment strategies is of significant clinical importance.

[0003] In recent years, bioelectronic medicine, as an emerging therapeutic paradigm, employs precise electrical pulses to modulate specific neural circuits, offering unique advantages for the treatment of chronic diseases. Vagus nerve stimulation (VNS), in particular, modulates immune responses and has shown clinical efficacy in inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease.

[0004] However, translating vagus nerve stimulation (VNS) technology from theory into an effective long-term treatment, especially when conducting necessary preclinical studies, faces severe limitations imposed by existing technologies. To evaluate the efficacy of VNS in cardiovascular diseases and explore its underlying biological mechanisms, researchers must rely on animal models, among which mouse models are indispensable tools due to their mature gene-editing technology and short breeding cycle. However, existing VNS systems encounter almost insurmountable challenges when applied to mouse models, which greatly hinder the in-depth development of related research: (1) The fundamental contradiction between size and space: Traditional VNS systems include a large, non-degradable implantable pulse generator (IPG) and battery. The limited physiological space in mice is completely insufficient to accommodate such a large power source and control device. Therefore, conducting long-term, stable chronic neuromodulation experiments in mice becomes extremely difficult, requiring the development of a miniaturized, efficient wireless power supply solution. (2) The enormous challenge of neural interfaces: The vagus nerve in mice is extremely small (usually <150 μm) and exceptionally fragile. Traditional neural electrodes usually require fixation with sutures. This invasive fixation method can easily cause mechanical damage to the mouse vagus nerve, leading to severe inflammation and fibrosis, thus affecting the long-term stability and stimulation effect of the neural interface. Therefore, developing an ultra-flexible, sutureless neural interface strategy is crucial for achieving non-invasive, stable, and long-term coupling with the mouse vagus nerve. (3) The dilemma of long-term implantation and biocompatibility: Conventional VNS systems use bio-inert but non-degradable materials. After completing chronic experiments lasting several weeks or months, how to dispose of these permanent implants becomes a problem. Performing a second surgery on mice to remove the device is not only highly invasive and has a high mortality rate, but also seriously interferes with the detection of experimental endpoints. Therefore, developing a highly biocompatible system that can be completely degraded in vivo is essential for simplifying experimental procedures, improving animal welfare, and ensuring the accuracy of research data.

[0005] In summary, there is a need in this field for a VNS system that can simultaneously meet the three core requirements of miniaturized wireless power supply, ultra-flexible sutureless neural interface, and fully biodegradable materials. This has become a key technical bottleneck for in-depth research on VNS treatment of chronic inflammatory diseases using mouse models. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a biodegradable vagus nerve stimulation system.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A biodegradable vagus nerve stimulation system includes an implantable vagus nerve stimulator (UBVS) and an external focused ultrasound transmitter used in conjunction with the implantable vagus nerve stimulator. The implantable vagus nerve stimulator includes an ultrasound-driven biodegradable triboelectric nanogenerator and a vagus nerve interface lead. The ultrasonically driven biodegradable triboelectric nanogenerator has a layered structure, including a functional layer and an encapsulation layer. The functional layer includes a Mo metal layer, a PCL layer and a Zn metal layer in sequence. The encapsulation layer is wrapped around the functional layer and is a biodegradable self-healing elastomer. The vagus nerve interface lead is a Mo lead, which is electrically connected to the Mo metal layer and the Zn metal layer, respectively.

[0008] Preferably, the thickness of the Mo metal layer is 30-70 μm, the thickness of the PCL layer is 5-15 μm, the thickness of the Zn metal layer is 30-70 μm, the thickness of the biodegradable self-healing elastomer is 60-100 μm, and the width of the Mo lead is 0.3-0.7 mm and the thickness is 30-70 μm.

[0009] Preferably, the operating parameters of the external focused ultrasound transmitting device are: transmission frequency 0.5-2MHz, driving voltage 60-100V, pulse repetition frequency 1-10Hz, number of ultrasound cycles per pulse 1000-3000, and sound intensity range 10-150mW·cm. -2 .

[0010] Preferably, the method for preparing the biodegradable self-healing elastomer (BSHE) includes the following steps: Polytetrahydrofuran and polycaprolactone were dried and dehydrated, and then cooled to obtain a blend. Isophorone diisocyanate and dibutyltin dilaurate were added to N,N-dimethylacetamide and stirred until homogeneous to obtain a mixture. The mixture was then added dropwise to the blend and stirred for reaction. After the reaction was carried out for a preset time, it was cooled to room temperature, and dimethylglyoxime was added to carry out the end-capping reaction. After the end-capping was completed, triethylamine was added and the reaction was carried out at a constant temperature. After the reaction was completed, N,N-dimethylacetamide was added to obtain a polymer solution. Then, the solution was cured and hot-pressed to obtain the final product.

[0011] Preferably, the mass ratio of polytetrahydrofuran to polycaprolactone is 1:1.5-2.5, the amount of isophorone diisocyanate added is 90-100% of the mass of polytetrahydrofuran, the amount of dibutyltin dilaurate added is 0.5-1% of the mass of isophorone diisocyanate, the amount of dimethylglyoxime added is 20-25% of the mass of isophorone diisocyanate, and the amount of triethylamine added is 1-1.5% of the mass of isophorone diisocyanate.

[0012] Preferably, the stirring reaction is carried out at a temperature of 70-75°C for 2-3 hours, the end-capping reaction is carried out at a temperature of 40-45°C for 12-15 hours, the isothermal reaction is carried out at a temperature of 40-45°C for 5-8 hours, the curing reaction is carried out at a temperature of 120-140°C for 10-13 hours, and the hot pressing reaction is carried out at a temperature of 40-50°C for 10-20 minutes.

[0013] This invention also protects a method for preparing a biodegradable vagus nerve stimulation system as described above, comprising the following steps: S1. Preparation of functional layer: Zn metal layer, PCL isolation layer and Mo metal layer are stacked in order from bottom to top to form the functional layer of the ultrasonically driven biodegradable triboelectric nanogenerator. S2. Fabrication and connection of vagus nerve interface leads: Two Mo leads are fabricated by laser cutting of Mo metal sheets, and the two Mo leads are electrically connected to the Mo metal layer and Zn metal layer of the functional layer, respectively. S3. Integrated encapsulation: The functional layer and the connected Mo lead obtained in step S2 are encapsulated using a biodegradable self-healing elastomer, leaving only the end portion of the Mo lead exposed as a nerve stimulation interface, thus obtaining the implantable vagus nerve stimulator.

[0014] The present invention also protects the application of a biodegradable vagus nerve stimulation system as described above in the cardiovascular treatment of a mouse model.

[0015] Preferably, the cardiovascular treatment is for atherosclerosis. By implanting the biodegradable vagus nerve stimulation system around the vagus nerve in the neck of mice and driving it with external focused ultrasound for 30 minutes daily for 6 weeks, at least one of the following therapeutic effects can be achieved: (a) Reduces the burden of atherosclerotic plaques, manifested as a reduction in the area of ​​aortic plaques; (b) Improve the stability of atherosclerotic plaques, manifested by one or more of the following: increased collagen content in the plaque, increased thickness of the fibrous cap in the plaque, and decreased proportion of necrotic core area to total plaque area.

[0016] Preferably, the therapeutic effect is achieved through at least one of the following biological mechanisms: (1) It inhibits vascular and systemic inflammatory responses, manifested as a reduction in the number of inflammatory cells infiltrating local blood vessels and / or a decrease in the levels of TNF-α, IL-6, and IL-1β in plasma; (2) Biological alterations associated with activation of the cholinergic anti-inflammatory pathway, including elevated levels of acetylcholine and / or norepinephrine in peripheral blood or spleen; and / or a decreased spleen weight to body weight ratio.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The biodegradable vagus nerve stimulation system provided by the present invention realizes wireless power supply and vagus nerve stimulation for implantable devices by constructing an ultrasound-driven triboelectric energy conversion mechanism, fundamentally solving the dependence of traditional implantable devices on built-in batteries; the system uses external focused ultrasound as an energy source, which can induce overall micro-vibration inside the implantable device when it propagates in biological tissue. This vibration causes the Mo metal layer and Zn metal layer in the functional layer of the ultrasound-driven biodegradable triboelectric nanogenerator (TENG) to periodically contact and separate. Through the triboelectric effect, opposite static charges are accumulated on the two surfaces, and electrons are driven to flow back and forth in the external circuit (i.e., Mo leads) through electrostatic induction to form a stable alternating current output. This electrical signal can be used to realize electrical stimulation of the vagus nerve, or to obtain a pulse output of a specific mode through a rectification / energy storage circuit to meet different stimulation needs; this solution completely eliminates the bulky and limited-life battery in the traditional solution, which not only greatly reduces the size and invasiveness of the implant, but also avoids the secondary replacement surgery required due to battery depletion, making on-demand and repeatable long-term chronic neuromodulation possible.

[0018] (2) The biodegradable vagus nerve stimulation system provided by the present invention uses a fully degradable material system to construct the entire implantable stimulator, which significantly reduces the foreign body reaction and safety risks of long-term implantation of the device. One of the core innovations of the present invention is that all its key components, including the Mo / Zn metal as the friction layer and interface lead, the PCL as the isolation layer, and the biodegradable self-healing elastomer (BSHE) as the encapsulation layer, are all selected from biomaterials with controllable degradation characteristics. These materials can ensure that the integrity of the device structure and the stability of electrical performance are maintained within the preset treatment window period (e.g., several weeks or several months) to complete the effective nerve stimulation task. After the treatment cycle, these materials will be gradually and safely degraded into non-toxic small molecules and absorbed by the body through hydrolysis, corrosion and other means in the physiological environment of the body. This avoids the problems of chronic inflammation and excessive fibrous tissue encapsulation that may be caused by permanent implants, and also eliminates the need for secondary surgery to remove them, thereby greatly improving the long-term biosafety of the therapy.

[0019] (3) The biodegradable vagus nerve stimulation system provided by this invention, with its flexible structure and self-adhesive encapsulation, forms a neural interface that can achieve stable and non-invasive coupling with tiny, fragile nerves, successfully solving the technical bottleneck of long-term VNS research in small animal models such as mice; In view of the extremely small and vulnerable characteristics of mouse vagus nerves, this invention utilizes the flexibility of the Mo lead itself and the low modulus characteristics of the BSHE encapsulation layer to construct an ultra-flexible interface that can conformally fit onto the surface of curved nerves, effectively reducing contact impedance and mechanical damage caused by tiny movements; More importantly, the self-adhesive properties of the BSHE material surface enable the device to be seamlessly and suturelessly fixed to the nerve by wrapping and pasting. This design ensures that the interface maintains a stable physical and electrical connection during chronic experiments lasting several weeks, thereby enabling reliable and repeatable VNS electrical signal output in atherosclerotic mouse models and ultimately observing clear physiological effects, providing a powerful technical platform for verifying the effectiveness of VNS therapy and exploring its mechanism of action.

[0020] (4) The biodegradable vagus nerve stimulation system provided by the present invention is used in ApoE - / - In animal models of atherosclerosis, such as mice on a high-fat diet, the treatment demonstrated a definite therapeutic effect and excellent biocompatibility, systematically validating the effectiveness and safety of the protocol. In terms of efficacy, 6 weeks of vagal nerve stimulation using the biodegradable vagal nerve stimulation system of this invention significantly reduced the burden of atherosclerotic plaques (manifested as a reduction in aortic plaque area) and effectively improved plaque stability (manifested as increased collagen content, thickened fibrous cap, and reduced proportion of necrotic core area in the plaque). Mechanistically, the experimental results suggest that its therapeutic effect may be related to choline... The activation of anti-inflammatory pathways is closely related, specifically manifested as a reduction in the number of inflammatory cells infiltrating local blood vessels and a significant decrease in the levels of key pro-inflammatory factors such as TNF-α and IL-6 in the blood. In terms of performance and safety, electrical tests show that the device can maintain a stable output that meets the nerve stimulation threshold in vivo. At the same time, histopathological analysis shows that there is no obvious tissue necrosis or severe fibrosis at the implantation site, no significant toxicity is observed in the blood biochemical indicators of major organs, and the device can degrade in vivo according to the preset time pattern, which fully confirms the feasibility and clinical translation potential of this biodegradable vagus nerve stimulation system. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the biodegradable vagus nerve stimulation system of the present invention; Figure 2Characterization of the components of the biodegradable vagus nerve stimulation system of this invention; wherein, (A) scanning electron microscope (SEM) image of the degraded molybdenum (Mo) wire; (B) charge injection capacity (CIC) performance of molybdenum (Mo) and gold (Au) electrodes; (C) long-term electrochemical monitoring results of the molybdenum electrode in PBS at 37°C; (D) Fourier transform infrared spectrum (FT-IR) of BSHE; (E) nuclear magnetic resonance (NMR) spectrum and structural characterization of BSHE; (F) mass change of BSHE film after immersion in 1×PBS at 37°C for 6 weeks; (G) representative tensile stress-strain curve of BSHE; (H) representative load-displacement curves of two self-healing BSHEs; (I) rheological changes of storage modulus G′ and loss modulus G′′ of BSHE, showing that its shear modulus is 1.78 MPa; (J) representative live / dead staining images of PC12 cells co-cultured with UBVS extract for 1, 3, and 5 days; (K) Cell viability of PC12 cells after co-culturing with UBVS extract for 1, 3, and 5 days; Figure 3 This invention evaluates the electrical output performance and biocompatibility of the biodegradable vagus nerve stimulation system (UBVS). The evaluation includes: (A) COMSOL simulation showing the propagation path of focused ultrasound; (B) measuring the sound pressure generated under different voltage drives in water; (C) the open-circuit voltage and short-circuit current of the UBVS under different focused ultrasound transducer drive voltages; (D) the power density of the UBVS under different load resistances; (E) UBVS-mediated vagus nerve stimulation (VNS)-induced complex action potentials (CAPs); (F) the inhibitory effect of the UBVS on LPS-induced systemic inflammation under different focused ultrasound drive voltages; (G) representative immunofluorescence staining images of the vagus nerve cross-section 6 weeks after implantation; (H) representative immunofluorescence staining images of skin tissue 6 weeks after implantation; (I) normalized fluorescence intensities of CD68, S-100, and NF-H in each group; and (J) normalized fluorescence intensities of TNF-α and Caspase-3 in each group. Figure 4This invention evaluates the efficacy of chronic vagal nerve stimulation in electrotherapy for atherosclerosis using the biodegradable vagal nerve stimulation system of this invention. The results include: (A) Experimental timeline: C57 BL / 6 ApoE- / - mice were fed a high-fat diet (HFD) for 12 weeks. At week 6, a UBVS was implanted into the left cervical vagus nerve, stimulating for 30 minutes daily for 6 weeks. Final analysis was performed at week 12. (B) Representative image of the entire aorta stained with Oil Red O, scale bar, 5 mm. (C) Quantification of lipid-stained area normalized to the total vascular area from the surface preparation. (D) Representative staining images of the aortic root transverse section, including Oil Red O, H&E, Masson trichrome staining, and immunohistochemical staining of α-SMA and caspase-3, scale bar, 100 µm. (E) Percentage of necrotic core area to lumen area in aortic sections. (F) Percentage of total plaque area to lumen area. (G) α (H) SMA-positive area as a percentage of plaque area; (I) Collagen content quantified by Masson's trichrome staining, expressed as a percentage of luminal area; (J) Total cholesterol (TC) in plasma lipids; (K) Low-density lipoprotein cholesterol (LDL-C) in plasma lipids; Data are expressed as mean ± SD. P≤0.05, P≤0.01, P≤0.001; NS, not significant; Figure 5 To illustrate the regulatory effect of the biodegradable vagus nerve stimulation system of this invention on the vascular inflammatory response in atherosclerosis, the following data are presented: (A) Representative immunohistochemical staining images of cross-sections of atherosclerotic lesions, detecting indicators including CD68, CD86, CD206, and MCP-1, scale bar, 100 μm; (BE) Quantitative analysis of the markers in Figure A: CD68 (B), CD86 (C), CD206 (D), and MCP-1 (E); (F) TNF-α levels measured by ELISA in the HFD, HFD+UBVS, and ND groups; (G, H) Representative images and quantitative data of VCAM-1 expression detected by immunohistochemistry; (I, J) Detection of circulating factors: sVCAM1 (I) and ET-1 (J); Data are expressed as mean ± SD. P≤0.05, P≤0.01, P≤0.001; NS, not significant. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0024] In this invention, the Mn of the polytetrahydrofuran is approximately 1000, and the Mn of the polycaprolactone is approximately 2000.

[0025] Example 1 A biodegradable vagus nerve stimulation system includes an implantable vagus nerve stimulator and an external focused ultrasound transmitting device used in conjunction with the implantable vagus nerve stimulator. The implantable vagus nerve stimulator includes an ultrasound-driven biodegradable triboelectric nanogenerator and a vagus nerve interface lead. The ultrasonically driven biodegradable triboelectric nanogenerator has a layered structure, including a functional layer and an encapsulation layer. The functional layer includes a Mo metal layer, a PCL layer and a Zn metal layer in sequence. The encapsulation layer is wrapped around the functional layer and is a biodegradable self-healing elastomer. The vagus nerve interface lead is a Mo lead, which is electrically connected to the Mo metal layer and the Zn metal layer, respectively.

[0026] The thickness of the Mo metal layer is 50 μm, the thickness of the PCL layer is 10 μm, and the thickness of the Zn metal layer is 50 μm; the thickness of the biodegradable self-healing elastomer is 80 μm; and the width of the Mo lead is 0.5 mm and the thickness is 50 μm.

[0027] The method for preparing the biodegradable self-healing elastomer includes the following steps: 7.25 g of polytetrahydrofuran and 14.5 g of polycaprolactone were dried and dehydrated, and then cooled to obtain a blend. 6.77 g of isophorone diisocyanate and 0.05 g of dibutyltin dilaurate were added to 10 mL of N,N-dimethylacetamide and stirred until homogeneous to obtain a mixture. The mixture was then added dropwise to the blend and stirred at 70 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, and 1.683 g of dimethylglyoxime was added. The mixture was then capped at 40 °C for 12 h. After the capping was complete, 80 mg of triethylamine was added, and the mixture was kept at 40 °C for 6 h. After the reaction was completed, 30 mL of N,N-dimethylacetamide was added to obtain a polymer solution. The solution was then cured at 130 °C for 12 h and hot-pressed at 40 °C for 10 min to obtain the final product.

[0028] A method for preparing a biodegradable vagus nerve stimulation system includes the following steps: S1. Following the order from bottom to top, Zn metal layer, PCL isolation layer and Mo metal layer are stacked sequentially to form the functional layer of the ultrasonically driven biodegradable triboelectric nanogenerator. S2. Two Mo leads are prepared by laser cutting of Mo metal sheet, and the two Mo leads are electrically connected to the Mo metal layer and Zn metal layer of the functional layer respectively. S3. The functional layer and the connected Mo lead obtained in step S2 are encapsulated using a biodegradable self-healing elastomer, leaving only the end portion of the Mo lead exposed as a nerve stimulation interface, thus obtaining the implantable vagus nerve stimulator.

[0029] Example 2 To test the electrochemical performance of the biodegradable vagus nerve stimulation system proposed in this design, a series of tests were conducted using a three-electrode system in a 1x concentration PBS solution. The Mo electrode served as the working electrode, a 1cm × 1cm platinum sheet as the counter electrode, and an Ag / AgCl electrode as the reference electrode. Under a 0V DC bias, a 5mV AC perturbation was applied from 0.1Hz to 10... 5 Impedance spectra were acquired in the Hz frequency range. Charge injection capability was tested by applying a biphasic pulse (±0.5V, 1.5ms) to a 1x concentration PBS solution, and its charge injection density (Q) was measured. npv ) by cathode charge (Q c ) and anodic charge (Q) a The sum of (A) and (Equation 1) is obtained by dividing the electrode area (A).

[0030] (1); Cyclic voltammetry was used to quantify charge storage capacity (CSC) by scanning at a rate of 100 mV / s within a potential window from -0.5 V to +0.5 V. CSC was calculated by integrating the current (i) over the potential window (from E1 to E2) and then normalizing it to the electrode area (A) (Equation 2).

[0031] (2); To assess long-term stability, we incubated the Mo wire electrode in a 1x concentration PBS solution at 37°C for four weeks. During this period, impedance, Qnpv, and CSC were measured weekly, and surface changes were observed.

[0032] Electrophysiological recordings were performed using a BL-420N biosignal acquisition and analysis system (Chengdu Taimeng Software Co., Ltd.). After surgically exposing the left sciatic nerve, we gently wrapped a molybdenum (Mo) electrode from a triboelectric nanogenerator (TENG) around it. Subsequently, a needle electrode was inserted into the left gastrocnemius muscle and connected to the system's input channel. An external ultrasound transducer was fixed to the superficial skin using medical ultrasound gel, aligned with the implanted neurostimulator, and driven by an external stimulation waveform. We recorded the muscle's action potentials before and after stimulation. Next, we dissected the distal tendon of the left gastrocnemius muscle and sutured it to the system's tension sensor. The same stimulation protocol was then applied, and changes in muscle tension were measured. Finally, we exposed the right vagus nerve, surrounded it with a molybdenum (Mo) stimulation electrode, and stimulated it via an external device, monitoring the nerve's action potentials before and after stimulation.

[0033] Cell compatibility assay: The biocompatibility of the triboelectric nanogenerator (TENG) was assessed on L929 fibroblasts and PC12 neurons using the CCK-8 assay and live / dead cell staining. To prepare the extraction solution, 100 mg of TENG material was incubated in 1 mL of DMEM medium at 37°C for 24 h. In the cell viability assay, L929 and PC12 cells were incubated at 3 × 10⁶ cells per well. 3 2×10 3 and 1.5×10 3 L929 cells were seeded at a density of 1 × 10⁶ cells / well in 24-well plates and cultured at 37°C and 5% CO₂ for 1, 3, and 5 days, followed by CCK-8 assay. For cell morphology evaluation, L929 cells were seeded at a density of 1 × 10⁶ cells / well. 4 Alternatively, PC12 cells can be spaced at 6 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 1, 3, and 5 days under the same conditions. Afterward, live / dead cell staining was performed, and imaging was used to observe cell integrity and viability.

[0034] like Figure 2 As shown in Figure A, scanning electron microscopy (SEM) and gravimetric analysis revealed that Mo filaments exhibited a progressive degradation characteristic in PBS solution. Surface cracks appeared after two weeks of immersion, and by the fourth week, the degradation had evolved into flaking. In electrochemical performance evaluation, the Mo electrode demonstrated significantly superior performance compared to conventional gold (Au) electrodes, reflected in its significantly higher charge injection capability (CIC) (e.g., ...). Figure 2 (As shown in B). Figure 2As shown in Figure C, long-term monitoring over six weeks in PBS at 37°C demonstrated that despite surface degradation of the Mo filament, its key electrochemical parameters (impedance, CSC, and CIC) showed only modest changes, proving its reliable electrochemical properties over the timescales required for chronic neuromodulation. For the encapsulation material, to achieve conformal, sutureless integration with the mouse vagus nerve and ultimately safe absorption, we comprehensively evaluated a biodegradable, self-healing polyurethane elastomer (BSHE). Figure 2 As shown in Figures D and 2E, Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR) confirmed the successful synthesis of BSHE material from a mixture of soft segments of polycaprolactone (PCL) and polytetrahydrofuran (PTHF) with oxime-containing hard segments. This unique molecular design endows the material with high ductility, tissue compatibility, high toughness, and dynamic self-healing capabilities. The introduction of the PCL soft segments, in particular, endows BSHE with crucial biodegradability, such as… Figure 2 As shown in Figure F, after immersion in PBS for six weeks, approximately 85% of its original mass remained, demonstrating a controllable degradation rate. Mechanically, this elastomer can withstand linear strain up to approximately 1250% and possesses a Young's modulus matching that of soft tissue. Figure 2 As shown in G, this material possesses a Young's modulus comparable to that of soft tissue. For example... Figure 2 As shown in H, the repaired interface exhibits extremely strong adhesion. Rheological measurements (such as...) Figure 2 As shown in Figure I, its molecular chains exhibit excellent fluidity. These properties collectively ensure that the material can achieve stable fixation of neural electrodes in a self-adhesive, sutureless manner, while reducing tissue trauma during implantation.

[0035] Finally, regarding biosafety assessment, in order to evaluate the in vitro cytotoxicity of the intact device (UBVS), such as... Figure 2 As shown in Figure J, no significant differences in cell morphology were observed in neural cells (PC12) and fibroblasts (L929) cultured in the device extraction solution for five days, as observed by live / dead cell staining. Ultimately, as... Figure 2 The quantitative analysis shown in K further confirmed that there was no significant difference in relative cell survival rate between the UBVS group and the control group, which strongly demonstrates that the biodegradable vagus nerve stimulator itself does not have any cytotoxic effect.

[0036] Example 3 To verify the propagation pathway of the biodegradable vagus nerve stimulation system and optimize device performance, such as... Figure 3 As shown, we first demonstrated through simulation that most of the acoustic energy is concentrated on the UBVS device ( Figure 3A). To maximize the in-body coupling efficiency, this invention fabricated an acoustic focusing cone and found that the open-circuit voltage and short-circuit current of the device are linearly related to the driving voltage (A). Figure 3 C). Through testing under different loads, it was found that the device reached its peak output power at a load of 10 kΩ, close to the vagal nerve impedance of mice. Figure 3 D). Regarding effectiveness and safety, studies have shown that at a driving voltage of 80V, the UBVS implanted in the vagus nerve successfully elicited compound action potentials (CAPs). Figure 3 E), and can significantly reduce systemic inflammation induced by LPS ( Figure 3 These findings collectively demonstrate the feasibility of using the device for neuromodulation in vivo. Regarding biocompatibility, immunofluorescence analysis of vagus nerve tissue six weeks after implantation showed no significant difference in the expression of macrophages (CD68), Schwann cells (S-100), and neurofilament protein (NF-H) compared to the control group. Figure 3 G, 3I); meanwhile, the skin tissue in contact with the encapsulation material did not show obvious signs of inflammation or apoptosis (G, 3I); Figure 3 These results (H,3J) collectively demonstrate that UBVS has good local biocompatibility.

[0037] Example 4 The biodegradable vagus nerve stimulation system prepared in Example 1 was used in mice, as detailed below.

[0038] Construction of an atherosclerosis (AS) model All animal experimental procedures were approved by the Institutional Animal Protection and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (

[2025] IACUC No.: 4833).

[0039] The experiment used male ApoE - / - Mice (C57BL / 6J background, approximately 18 g, 6 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd., Beijing. After 3 days of acclimatization under specific pathogen-free (SPF) conditions, these mice were fed a high-fat diet (product number D12108C, Research Diets, USA) to induce atherosclerosis. This diet contained 1.25% cholesterol and 17.28% cocoa butter and was fed continuously for 12 weeks. During this period, we monitored the mice's body weight and overall health status weekly. At the experimental endpoint, aortic tissue was collected for pathological verification of plaque formation.

[0040] Implantation of UBVS device for long-term vagus nerve stimulation (VNS): Male ApoE - / -Mice were fed a high-fat diet for 6 weeks prior to the implantation surgery. Under isoflurane anesthesia, the left cervical vagus nerve was exposed, and a UBVS device was implanted. The device was subcutaneously fixed to the back and shoulder region, and the skin was then sutured. Electrical stimulation was performed wirelessly via an external ultrasonic transducer to power the UBVS. Stimulation parameters were: drive voltage 80V, frequency 1MHz, 2000 cycles per pulse, and a 1-second pulse interval. This stimulation was performed for 30 minutes daily for 6 weeks. During each stimulation session, mice were kept anesthetized using an animal anesthesia system (R583S, RWD) with 1-2% isoflurane.

[0041] Histopathological and immunohistochemical examination: Six weeks post-implantation, we carefully dissected the left cervical vagus nerve and surrounding muscle and connective tissue in UBVS-treated and control mice, removing any remaining device components. To assess local biocompatibility, the harvested nerve tissue was fixed in 4% paraformaldehyde, then dehydrated, paraffin-embedded, and processed for H&E staining and immunofluorescence staining. Immunomarkers used included S-100, NFH, CD68, and TNF-α (from Servicebio) to assess nerve integrity and local inflammatory response. To test systemic biocompatibility, we collected major organs including skin, heart, lungs, liver, spleen, and kidneys, and performed H&E staining. Simultaneously, blood samples were analyzed to detect liver and kidney function markers (ALT, AST, BUN, Cr) and immunogenicity markers (IgG).

[0042] After 6 weeks of treatment, we obtained ApoE - / - Aortic root and descending aorta were collected from mice in the model group, UBVS treatment group, and control group. After fixation in 4% paraformaldehyde, the tissues were embedded in paraffin and analyzed by H&E staining and Oil Red O staining to assess plaque burden and morphology. Masson's trichrome staining and α-SMA immunohistochemistry were used to assess fibrosis and vascular remodeling, while Caspase-3 immunohistochemistry was performed to assess intraplaque apoptosis.

[0043] To assess changes in the inflammatory microenvironment, we performed immunohistochemical staining for CD68, MCP-1, CD86, CD206, and VCAM-1 in aortic plaques. We also performed RNA sequencing (RNA-seq) analysis to explore its regulatory pathways. To validate the transcriptomic findings, we used immunofluorescence staining to detect the expression of key functional proteins in aortic tissue, including autophagy markers p62 and Beclin-1, as well as the co-expression of CD68 and MerTK, which are associated with cell burial. We also collected peripheral blood to assess lipid levels, including total cholesterol (TC), free cholesterol (FC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). We used ImageJ software for semi-quantitative analysis of immunohistochemically positive regions and fluorescence signal intensity in all tissue sections.

[0044] like Figure 4 As shown, compared with the control group that only received a high-fat diet, mice treated with UBVS showed a reduction of approximately 70% in the overall aortic plaque area. Figure 4 BC), demonstrating a significant therapeutic effect. Further histological analysis showed that the total lesion area and necrotic core were reduced by approximately 50% and 65%, respectively, in the treatment group. Figure 4 DF), while the content of smooth muscle cells and collagen, which are markers of plaque stability, increased by nearly 120% and 60%, respectively. Figure 4 D, GH), while the level of apoptosis within the plaque also decreased by approximately 65% ​​( Figure 4 These data collectively demonstrate that chronic vagal nerve stimulation not only effectively reduces the overall pathological burden of atherosclerosis, but also promotes the transformation of dangerous arterial plaques into a more stable and lower-risk phenotype by inhibiting inflammation and apoptosis and increasing stabilizing components.

[0045] Since blood lipids are a key factor in atherosclerosis, we further evaluated the effect of UBVS treatment on systemic blood lipids and found that UBVS could reduce total cholesterol (TC) and "bad cholesterol" (LDL-C) by approximately 30% and 45%, respectively. Figure 4 JK) and reduced free cholesterol (FC) by approximately 25% (Figure S25A), but had no significant effect on "good cholesterol" (HDL-C). These improvements in blood lipids may help slow the progression of atherosclerosis. Taken together, these results suggest that UBVS can benefit the treatment of atherosclerosis not only by improving lipid homeostasis but also by reducing systemic inflammatory responses.

[0046] like Figure 5As shown, compared with the control group, UBVS treatment significantly remodeled the macrophage phenotype, with a reduction of approximately 50% in total macrophages (CD68) and 65% in pro-inflammatory M1 macrophages (CD86), while an increase of approximately 100% in anti-inflammatory and reparative M2 macrophages (CD206). Figure 5 BD). Simultaneously, the expression of MCP-1, a key chemokine attracting macrophages, was reduced by nearly 60% in the plaque. Figure 5 E), indicating that it inhibits the recruitment of inflammatory cells at the source. At the systemic level, UBVS treatment also effectively reduced the levels of multiple key pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. Figure 5 Furthermore, plasma levels of norepinephrine and acetylcholine, key neurotransmitters in the cholinergic anti-inflammatory pathway (CAP), were elevated. These results collectively indicate that UBVS-driven vagal stimulation successfully shifted the intraplaque immune environment from a pro-inflammatory to an anti-inflammatory state by activating CAP, thereby effectively inhibiting the progression of atherosclerosis.

[0047] The above description is a further detailed explanation of the present invention in conjunction with specific implementation examples. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the protection scope of the present invention.

[0048] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biodegradable vagus nerve stimulation system, characterized in that, The invention includes an implantable vagus nerve stimulator and an external focused ultrasound transmitting device used in conjunction with the implantable vagus nerve stimulator. The implantable vagus nerve stimulator includes an ultrasound-driven biodegradable triboelectric nanogenerator and a vagus nerve interface lead. The ultrasonically driven biodegradable triboelectric nanogenerator has a layered structure, including a functional layer and an encapsulation layer. The functional layer includes a Mo metal layer, a PCL layer and a Zn metal layer in sequence. The encapsulation layer is wrapped around the functional layer and is a biodegradable self-healing elastomer. The vagus nerve interface lead is a Mo lead, which is electrically connected to the Mo metal layer and the Zn metal layer, respectively.

2. The biodegradable vagus nerve stimulation system according to claim 1, characterized in that, The thickness of the Mo metal layer is 30-70 μm, the thickness of the PCL layer is 5-15 μm, and the thickness of the Zn metal layer is 30-70 μm; the thickness of the biodegradable self-healing elastomer is 60-100 μm; the width of the Mo lead is 0.3-0.7 mm, and the thickness is 30-70 μm.

3. The biodegradable vagus nerve stimulation system according to claim 1, characterized in that, The operating parameters of the external focused ultrasound transmitting device are as follows: transmission frequency 0.5-2MHz, driving voltage 60-100V, pulse repetition frequency 1-10Hz, number of ultrasound cycles per pulse 1000-3000, and sound intensity range 10-150 mW·cm. -2 .

4. The biodegradable vagus nerve stimulation system according to claim 1, characterized in that, The method for preparing the biodegradable self-healing elastomer includes the following steps: Polytetrahydrofuran and polycaprolactone were dried and dehydrated, and then cooled to obtain a blend. Isophorone diisocyanate and dibutyltin dilaurate were added to N,N-dimethylacetamide and stirred until homogeneous to obtain a mixture. The mixture was then added dropwise to the blend and stirred for reaction. After the reaction was carried out for a preset time, it was cooled to room temperature, and dimethylglyoxime was added to carry out the end-capping reaction. After the end-capping was completed, triethylamine was added and the reaction was carried out at a constant temperature. After the reaction was completed, N,N-dimethylacetamide was added to obtain a polymer solution. Then, the solution was cured and hot-pressed to obtain the final product.

5. The biodegradable vagus nerve stimulation system according to claim 4, characterized in that, The mass ratio of polytetrahydrofuran to polycaprolactone is 1:1.5-2.5, the amount of isophorone diisocyanate added is 90-100% of the mass of polytetrahydrofuran, the amount of dibutyltin dilaurate added is 0.5-1% of the mass of isophorone diisocyanate, the amount of dimethylglyoxime added is 20-25% of the mass of isophorone diisocyanate, and the amount of triethylamine added is 1-1.5% of the mass of isophorone diisocyanate.

6. The biodegradable vagus nerve stimulation system according to claim 4, characterized in that, The stirring reaction is carried out at a temperature of 70-75℃ for 2-3 hours, the end-capping reaction is carried out at a temperature of 40-45℃ for 12-15 hours, the isothermal reaction is carried out at a temperature of 40-45℃ for 5-8 hours, the curing reaction is carried out at a temperature of 120-140℃ for 10-13 hours, and the hot pressing reaction is carried out at a temperature of 40-50℃ for 10-20 minutes.

7. A method for preparing a biodegradable vagus nerve stimulation system as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Following the order from bottom to top, Zn metal layer, PCL isolation layer and Mo metal layer are stacked sequentially to form the functional layer of the ultrasonically driven biodegradable triboelectric nanogenerator. S2. Two Mo leads are prepared by laser cutting of Mo metal sheet, and the two Mo leads are electrically connected to the Mo metal layer and Zn metal layer of the functional layer respectively. S3. The functional layer and the connected Mo lead obtained in step S2 are encapsulated using a biodegradable self-healing elastomer, leaving only the end portion of the Mo lead exposed as a nerve stimulation interface, thus obtaining the implantable vagus nerve stimulator.

8. The application of a biodegradable vagus nerve stimulation system as described in any one of claims 1-6 in cardiovascular treatment of a mouse model.

9. The application according to claim 8, characterized in that, The cardiovascular treatment is for atherosclerosis. By implanting the biodegradable vagus nerve stimulation system around the vagus nerve in the neck of mice and driving it with external focused ultrasound for 30 minutes daily for 6 weeks, at least one of the following therapeutic effects can be achieved: (a) Reduces the burden of atherosclerotic plaques, manifested as a reduction in the area of ​​aortic plaques; (b) Improve the stability of atherosclerotic plaques, manifested by one or more of the following: increased collagen content in the plaque, increased thickness of the fibrous cap in the plaque, and decreased proportion of necrotic core area to total plaque area.

10. The application according to claim 9, characterized in that, The therapeutic effect is achieved through at least one of the following biological mechanisms: (1) It inhibits vascular and systemic inflammatory responses, manifested as a reduction in the number of inflammatory cells infiltrating local blood vessels and / or a decrease in the levels of TNF-α, IL-6, and IL-1β in plasma; (2) Biological alterations associated with activation of the cholinergic anti-inflammatory pathway, including elevated levels of acetylcholine and / or norepinephrine in peripheral blood or spleen; and / or a decreased spleen weight to body weight ratio.

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