A mast cell targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation and a preparation method and application thereof

Abstract

This invention provides a mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation, its preparation method, and its application, belonging to the technical field of biomedical materials and acupuncture-assisted treatment devices. The invention first synthesizes Fe₃O₄@BaTiO₃–FcεRIA (FBTFA) nanoparticles to achieve mast cell targeting and synergistic stimulation of cells by "mechanical force-electrical energy" under magnetic field induction. Then, it is compounded with a two-component hydrogel. The hydrogel loaded with FBTFA nanoparticles is then loaded into the groove of a threaded needle. After insertion into the local acupoint, the needle is suspended against the thread, achieving precise delivery and local retention of the hydrogel at the acupuncture site, and continuous release of nanoparticles to target subcutaneous mast cells at the acupoint. This invention generates controllable electro-mechanical stimulation under the action of an external magnetic field, inducing degranulation of rat basophilic leukemia granulocytes (RBL-2H3 cells), and achieving a sustained analgesic effect at the rat acupoint through immune-neuro-endocrine circuit regulation.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical materials and acupuncture auxiliary treatment devices, specifically relating to a mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation, its preparation method, and its application. Background Technology

[0002] Pain is a subjective experience resulting from the combined effects of various physiological, psychological, and environmental factors. Its etiology is complex and its manifestations diverse, making it one of the most common clinical symptoms. Long-term or chronic pain not only significantly reduces patients' quality of life but can also induce various pathological states such as anxiety, depression, and immune dysfunction, becoming one of the major public health problems worldwide. Current analgesics mainly rely on local anesthetics, nonsteroidal anti-inflammatory drugs (NSAIDs), topical preparations, and physical therapy; however, their effects are limited in duration and often accompanied by skin irritation, drug resistance, or systemic side effects, making it difficult to achieve precise and sustained analgesia.

[0003] Acupuncture, as a mature minimally invasive intervention, has been used clinically for thousands of years. Its safety and efficacy are recognized by the World Health Organization (WHO) and it is considered a globally recognized non-pharmacological analgesic method. Acupuncture analgesia typically involves selecting specific acupoints (such as Zusanli (ST-36) and Hegu (LI-4)) or directly at the site of pain. Anatomical studies show that acupoints have dense nerve endings, rich blood vessels and microcirculation, and an enrichment of mast cells (MCs) can be observed in the immune cell spectrum. These cells highly express mechanosensitive ion channels such as transient receptor potential vanilloid 1 (TRPV1) on their membranes, exhibiting high sensitivity to various external physical stimuli. The mechanical traction generated by acupuncture can induce Ca2+ through channels such as TRPV1. 2+ Influx triggers rapid degranulation of mesenchymal stem cells (MCs), releasing various active mediators such as histamine, serotonin (5-HT), and adenosine triphosphate (ATP). These mediators can both inhibit pain signal transduction at peripheral sensory nerve endings and regulate the local immune microenvironment, synergistically producing anti-inflammatory and neuromodulatory effects to achieve peripheral-central analgesic effects.

[0004] In clinical practice, acupuncture is widely used to treat various acute and chronic pains, but its efficacy is highly dependent on the practitioner's experience. The intensity and duration of stimulation are difficult to control precisely, resulting in insufficient stability of the effect. Electroacupuncture quantifies the intensity of stimulation by superimposing electrical pulses, but it requires an external power source, has a wide range of effects, and may trigger activation of non-target tissues, which also limits patient compliance. Therefore, the existing acupuncture analgesia techniques still have the following disadvantages in clinical application: (1) The intensity of stimulation is difficult to control precisely, the degree of acupuncture quantification is low, and the efficacy is significantly affected by the operator's experience and individual differences; (2) The duration of analgesia is insufficient. Traditional acupuncture mainly produces instantaneous mechanical stimulation, which is difficult to achieve long-term or controllable analgesic effects; (3) There is a lack of precise regulation of specific pathways. The analgesic effect is mainly limited to macroscopic neural regulation, which is difficult to target key cells (such as mast cells) and their mediated immune-neural signaling pathways, thus affecting the stability and specificity of the efficacy.

[0005] In recent years, functionalized magnetic nanoparticles (MNPs) and piezoelectric materials have been widely used for cell regulation due to their unique physical properties. MNPs can apply mechanical stress to the cell membrane under an external magnetic field, activating mechanosensitive channels and regulating cell signal transduction. Piezoelectric materials, due to their unique antisymmetric crystal structure, can generate an endogenous electric field under mechanical stress, achieving electrical stimulation without an external power source. Among them, barium titanate (BaTiO3) possesses excellent piezoelectric response and good biocompatibility, generating electrical signals without electrodes or external power, making it an ideal material for constructing electroresponsive systems. However, each single material has its limitations: magnetically responsive materials (such as Fe3O4), while capable of magneto-force conversion, lack electrical output, limiting their cellular stimulation; pure piezoelectric materials (such as BaTiO3), while electroactive, lack a stable and controllable source of mechanical force. Therefore, composite magneto-electric nanomaterials integrating magnetic response and piezoelectric properties can theoretically achieve a coupled amplification effect of magnetically controlled mechanical stimulation and electrical stimulation, further enhancing cell regulation capabilities.

[0006] A study reports that a core / shell structured magnetoelectric nanoparticle, Fe3O4@BaTiO3 (FO@BTO NPs), effectively improves magnetoelectric coupling efficiency and electrical signal output by increasing the contact area between its piezoelectric shell phase and its magnetic core phase. (Y. Zhang) et al. , 'Magnetoelectric Nanoparticles Incorporated BiomimeticMatrix for Wireless Electrical Stimulation and Nerve Regeneration', Adv. Healthc. Mater.(, vol. 10, no. 16, p. 2100695, Aug. 2021, doi: 10.1002 / adhm.202100695.). This undoubtedly enhances the magnetoelectric stimulation effect on cells and promotes the functional regulation of neurons and other electrically sensitive cells. However, existing magnetoelectric nanomaterials still have several problems in biological applications. First, the spatial range of magnetoelectric stimulation is wide and lacks specificity, making it difficult to achieve precise regulation of specific target cells (such as mast cells). Second, naked or unmodified magnetoelectric nanoparticles are prone to aggregation, sedimentation, or non-specific uptake, leading to reduced stimulation efficiency and uneven local responses. Third, magnetoelectric particles have limited stability in body fluid environments, and their surfaces are easily adsorbed or oxidized by proteins, thus affecting magnetoelectric coupling performance and biocompatibility.

[0007] On the other hand, the unstable binding of magnetoelectric nanoparticles to the tissue interface also limits their sustained effects in vivo. How to effectively immobilize them in a controllable local delivery carrier to achieve stable and sustainable magnetoelectric response stimulation, and ensure long-term and reproducible cell regulation without external power supply, has become another key technical problem that urgently needs to be solved in this field.

[0008] Therefore, how to provide an acupuncture material that can maintain the efficient magneto-electric coupling performance of magnetoelectric materials while endowing them with good biological targeting and stability, so as to achieve precise stimulation of specific cells and stable binding with the tissue interface, has become the technical problem that this invention urgently needs to solve. Summary of the Invention

[0009] The purpose of this invention is to solve the aforementioned technical problems, thereby providing a mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation, its preparation method, and its application. The technical objective of this invention is twofold: firstly, to provide a composite magnetoelectric nanoparticle that targets mast cells and possesses both magnetic responsiveness and piezoelectric properties, enabling controllable mechanical-electric signal coupling output under an external magnetic field, thus allowing for active localization and efficient activation of mast cells at acupoints without an external power source; secondly, to provide a magnetoelectric functionalized hydrogel acupuncture system capable of loading the aforementioned magnetoelectric nanoparticles and possessing deformability and tissue adhesion. This targeted magnetoelectric composite hydrogel can be loaded onto a threaded needle and delivered to the target acupoint during acupuncture, gradually releasing the magnetoelectric nanoparticles in vivo to generate continuous and controllable local magnetoelectric stimulation to activate mast cells, thereby regulating the immune-neuro-endocrine circuit and further achieving precise and lasting analgesic effects.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: One objective of this invention is to provide a method for preparing a mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation. The method involves functionalizing and coupling Fe3O4@BaTiO3 piezoelectric core-shell nanoparticles with magnetic responsiveness to enable them to target mast cells. Subsequently, the nanoparticles are dispersed in a hyaluronic acid bicomponent hydrogel and loaded into grooves on the surface of an acupuncture needle with a threaded structure. This assembly yields a magnetoelectric hydrogel acupuncture system that can be implanted subcutaneously and achieve targeted activation of mast cells under the action of an external magnetic field. The functionalized coupling involves chemically coupling Fe3O4@BaTiO3 nanoparticles with an anti-FcεRIA antibody to obtain FBTFA nanoparticles.

[0011] In the research of acupuncture analgesia techniques, achieving precise control of stimulation intensity and sustained analgesic effect has always been a technical challenge in clinical and materials science. Traditional acupuncture efficacy relies on the practitioner's experience, while electroacupuncture, although capable of quantifying stimulation intensity, requires an external power source and has a wide stimulation range, leading to issues such as non-target tissue activation and poor patient compliance. Therefore, designing a novel acupuncture system that can achieve localized, controllable, and sustained analgesia without an external power source has become a key scientific and technological problem urgently needing a breakthrough. Through extensive reading and research, the inventors have successfully discovered that by surface functionalizing Fe3O4@BaTiO3 piezoelectric core-shell nanoparticles (FO@BTO NPs) with magnetic responsiveness and chemically coupling them with an antibody targeting the mast cell membrane's highly expressed receptor FcεRIA, FBTFA nanoparticles can be obtained, possessing both magnetoelectric conversion capabilities and mast cell targeting recognition capabilities. When these nanoparticles are subjected to magnetic force under an external alternating magnetic field, they can effectively convert into locally controllable electrical stimulation signals, achieving precise activation of targeted mast cells.

[0012] Furthermore, the inventors dispersed the aforementioned FBTFA nanoparticles in a composite hydrogel consisting of methacryloyl hyaluronic acid (HAMA) and dopamine hydrochloride-modified hyaluronic acid (HADA) as two components, constructing a magnetoelectric functionalized hydrogel system with good biocompatibility and tissue adhesion. This system can be loaded into the grooves on the surface of an acupuncture needle with a threaded structure and precisely delivered to the subcutaneous target site during acupuncture, achieving a comprehensive function of implantability, controlled release, and continuous magnetoelectric stimulation.

[0013] The mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation provided by this invention not only overcomes the limitations of traditional acupuncture and electroacupuncture in terms of stimulation intensity, controllability of action, and duration, but also achieves the unity of targeted delivery, controllable magnetoelectric response, and sustainable analgesic effect.

[0014] The mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation obtained in this invention has been demonstrated in vitro to possess excellent superparamagnetism, piezoelectric response, biocompatibility, and targeting properties. It achieves microscale "mechanical-electric" coupling stimulation of subcutaneous mast cell membranes at acupoints under the drive of an external magnetic field, effectively inducing calcium influx and degranulation, and promoting the rapid release of analgesic mediators such as adenosine. Simultaneously, it activates calcium signaling and immune-related pathways, achieving a rapid functional response in mast cells. In vivo experiments further demonstrate that this system significantly enhances mast cell degranulation rate at the ST-36 acupoint in AA rats, inhibits TRPV1 expression in the nociceptive pathway, reduces peripheral inflammatory factor levels, and inhibits DRG neuron excitation through the extracellular ATP-adenosine-A1R pathway, while simultaneously increasing the release of endogenous analgesic factors from cerebrospinal fluid, forming a synergistic immune-neuro-endocrine regulatory loop, thereby achieving efficient and controllable local analgesia.

[0015] Furthermore, the functionalization coupling operation steps are as follows: first, Fe3O4@BaTiO3 nanoparticles are silanized to fill their surface with amino groups, and then chemically coupled with anti-FcεRIA antibody through EDC and NHS activation reaction.

[0016] Furthermore, the hyaluronic acid two-component composite hydrogel is prepared by adding a photoinitiator to a composite hydrogel precursor solution composed of methacrylamide hyaluronic acid (HAMA) and dopamine hydrochloride modified hyaluronic acid (HADA) and then performing a photocrosslinking reaction.

[0017] Furthermore, the hyaluronic acid is sodium hyaluronate with a molecular weight of 50 kDa and hyaluronic acid with a molecular weight of 800 kDa.

[0018] Furthermore, the weight ratio of methacryloyl hyaluronic acid (HAMA) to dopamine hydrochloride modified hyaluronic acid (HADA) is 2:1.

[0019] Furthermore, the mass percentage of the composite hydrogel precursor solution is 6% (w / v).

[0020] Furthermore, the photoinitiator is LAP (lithium phenyl-2,4,6-trimethylbenzoyl phosphate), and its addition concentration is 0.1% (w / v) of the total hydrogel precursor solution, and the crosslinking time is 30 s.

[0021] Furthermore, the preparation method of the Fe3O4@BaTiO3 piezoelectric core-shell nanoparticles includes the following steps: 1) Ferric chloride, sodium acetate, and PSSMA were dissolved in ultrapure water, and NaOH was added after thorough stirring. The mixture was reacted at 190 °C for 9 h. The product was magnetically separated and washed with ethanol and deionized water to obtain Fe3O4 nanoparticles. (2) Disperse the suspension of Fe3O4 nanoparticles, then add ammonia and TBOT in sequence, stir at room temperature for 1.5 h to obtain Fe3O4@TiO2 nanoparticles; (3) The suspension of Fe3O4@TiO2 nanoparticles was mixed with Ba(OH)2·8H2O solution, ammonia was added, and the mixture was subjected to hydrothermal reaction at 200 °C for 8 h to obtain Fe3O4@BaTiO3 piezoelectric nanoparticles with core-shell structure.

[0022] For example, the preparation and composition method of the mast cell-targeting magnetoelectric hydrogel acupuncture system provided by the present invention is as follows: Ferric chloride (0.26 g), sodium acetate (1.2 g), PSSMA (0.4 g), and a small amount of ultrapure water (20 μL) were added to 16 mL of ethylene glycol. After thorough stirring, NaOH (0.24 g) was added, and the mixture was transferred to a stainless steel high-pressure reactor and reacted at 190 °C for 9 h. The product was magnetically separated and washed with ethanol and deionized water to obtain Fe3O4 NPs.

[0023] Fe3O4 suspension (5 mL) was dispersed in a mixed solution of ethanol (85 mL) and acetonitrile (30 mL), followed by the addition of ammonia (0.5 mL) and TBOT (1 mL). After stirring at room temperature for 1.5 h, the mixture was washed to obtain Fe3O4@TiO2 (FO@TO) particles. Further, FO@TO suspension (20 mL) was mixed with Ba(OH)2·8H2O solution (0.315 g), and ammonia (2 mL) was added. The mixture was transferred to a high-pressure reactor and hydrothermally reacted at 200 °C for 8 h. The resulting product was washed with formic acid and ethanol and then lyophilized to obtain FO@BTO piezoelectric nanoparticles with a core-shell structure.

[0024] The obtained FO@BTO powder (400 mg) was placed in a polytetrafluoroethylene Erlenmeyer flask, and 30% H2O2 solution (25 mL) was added. After ultrasonic dispersion, the mixture was refluxed at 110 °C for 4 h and washed until no bubbles were generated to obtain FO@BTO-OH particles. These particles were then dispersed in anhydrous ethanol (25 mL), and APTES (400 µL) was added dropwise under stirring at 80 °C, followed by reflux for 24 h. After washing, the particles were lyophilized to obtain FO@BTO-NH2 particles. Subsequently, 4 µL of FcεRIA polyclonal rabbit anti-antibody was dissolved in 4 mL of PBS, and EDC (1 mg) and NHS (1 mg) were added sequentially, reacting for 30 min each time. 10 mg of FO@BTO-NH2 was resuspended in 1 mL of PBS by ultrasonication, and the activated antibody solution was added. The mixture was stirred at room temperature for 4 h, and after washing, the targeted nanoparticles FO@BTO-FcεRIA (FBTFA) were obtained.

[0025] Sodium hyaluronate (10 g) with a molecular weight of 50 kDa was dissolved in 500 mL of pre-cooled PBS. Methacrylic anhydride (10 mL) was slowly added dropwise under ice bath conditions, and the pH was maintained between 8 and 9 with 5 mol / L NaOH solution. After stirring in the dark for 4 h, the product was transferred to a dialysis bag for dialyzing and then lyophilized. The resulting HAMA was stored at −20 °C for later use. Hyaluronic acid (1 g) with a molecular weight of 800 kDa was dissolved in 100 mL of deionized water. Under nitrogen protection, EDC (574 mg) and NHS (344 mg) were added for activation for 20 min. Then, 404 mg of dopamine hydrochloride (Macklin, China) was added, and the pH was adjusted to 4.5–5 with hydrochloric acid solution. After stirring in the dark overnight, the mixture was dialyzed in acidic aqueous solution, lyophilized, and stored at −20 °C.

[0026] HAMA and HADA were dissolved in PBS at a mass ratio of 2:1, and FBTFA nanoparticles with a final concentration of 100 μg / mL and 0.1% (w / v) photoinitiator LAP were added. After mixing evenly, the composite hydrogel was loaded into the groove on the surface of the threaded acupuncture needle and crosslinked by UV irradiation for 30 s.

[0027] The second objective of this invention is to provide a mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation, prepared by the method described above.

[0028] A third objective of this invention is to provide an application of the mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation as described above, wherein the acupuncture system is used to deliver implantable materials such as magnetoelectric hydrogel that promote analgesia.

[0029] The beneficial effects of this invention are as follows: This invention constructs a mast cell-targeting magnetoelectric hydrogel acupuncture system with magnetoelectric coupling properties for acupoint stimulation. This system achieves a synergistic effect of magnetic response and electrical output, as well as specific recognition of mast cells, by functionally coupling Fe3O4@BaTiO3 magnetoelectric core-shell nanoparticles with an anti-FcεRIA antibody. Compared with existing single magnetic or piezoelectric materials, the magnetoelectric composite nanoparticles of this invention can generate more efficient and stable local electrical signals under an external magnetic field, thereby achieving precise activation of mast cells.

[0030] The magnetoelectric functionalized hydrogel provided by this invention employs a HAMA / HADA two-component system, exhibiting both excellent structural stability and tissue adhesion. This hydrogel can load FBTFA nanoparticles and embed them in grooves on the surface of a threaded needle, delivering them to the subcutaneous target site during acupuncture. Under the influence of an external magnetic field, the system can achieve controllable magnetoelectric stimulation and sustained-release effects, overcoming the shortcomings of traditional acupuncture and electroacupuncture, such as difficulty in controlling stimulation intensity, insufficient duration of action, and the need for an external power source.

[0031] (3) The magnetic-electric hydrogel acupuncture system of the present invention can achieve targeted activation and degranulation induction of mast cells. In vivo experiments have shown that the system can achieve local high-efficiency stimulation without external power source. By regulating the TRPV1 channel and A1R signaling pathway, it promotes the synergistic regulation of the immune-neural-endocrine circuit, thereby achieving efficient and controllable local analgesia. Attached Figure Description

[0032] Figure 1 A schematic diagram of the composition and analgesic mechanism of a mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation; A) Schematic diagram of the synthesis of magnetoresponsive piezoelectric nanoparticles (FBTFA) targeting mast cells; B) Composition of magnetoresponsive piezoelectric hydrogel (FBTFA@HAMA / HADA); C) Schematic diagram of the application of the mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation, demonstrating how external magnetic field stimulation induces local analgesia in arthritic rats through immune-neural interactions activated by mast cells at acupoints.

[0033] Figure 2 Characterization of the magnetically responsive piezoelectric nanoparticles: A) Scanning electron microscopy (SEM) images showing the morphology of FO, FO@TO, FO@BTO, and FO@BTO-NH2; B) Energy dispersive spectroscopy (EDS) elemental mapping of FO@BTO-NH2; C) Transmission electron microscopy (TEM) images of FO@TO, FO@BTO, and FO@BTO-NH2; D) Elemental composition of FO@BTO determined by ICP-OES analysis; EF) Particle size distribution and zeta potential measurement results of the above nanoparticles.

[0034] Figure 3Characterization of the magnetic and piezoelectric properties of magnetically responsive piezoelectric nanoparticles: A) VSM magnetization hysteresis loops of FO, FO@TO, FO@BTO, and FO@BTO-NH2; B) XRD analysis of FO, FO@BTO, and BaTiO3; C) TGA curves of FO@BTO and FBTFA; D) Atomic force microscopy morphology of FO@BTO-NH2; E) Amplitude and phase diagrams of FO@BTO-NH2 obtained by magnetic force microscopy; F–H) Magnetic force microscopy test results of FO@BTO-NH2, including phase switching loop, amplitude butterfly curve, and d... 33 Imaging.

[0035] Figure 4 The following images illustrate the synthesis and characterization of HAMA / HADA hydrogels, as well as the morphology and transport characteristics of ST-N: A) Schematic diagram of the synthesis of HAMA, HADA, and HAMA / HADA hydrogels; B) Photograph of HAMA and HAMA / HADA solidified into block hydrogels; C) Photograph showing the dynamic adaptability and tissue adhesion of HAMA / HADA hydrogels; D) SEM images of lyophilized HAMA and HAMA / HADA hydrogels (red arrows indicate FBTFA nanoparticles); E) [Images of HAMA, HAMA, HADA, and HAMA / HADA hydrogels are included.] 1 F) 1H NMR spectrum; G) Rheological properties of HAMA / HADA hydrogels at different concentrations; H) Macroscopic images of CA-N and ST-N; I) Schematic diagram of HAMA / HADA hydrogel filling ST-N; J) Microscopic images of the tips of CA-N and ST-N; JK) Photographs of CA-N and ST-N implanted tissues and schematic diagram of needles being pulled out against the reverse thread; LM) Hematoxylin-eosin (H&E) stained images of skin tissue after CA-N and ST-N were inserted into acupoints in rats and then pulled out (arrows indicate parts of the hydrogel).

[0036] Figure 5 To assess the biocompatibility of nanoparticles and hydrogels and the cell-targeting properties of FBTFA nanoparticles; A) Cell viability of RBL-2H3 cells after incubation with specified treatments for 24 and 48 hours, determined by the MTT assay (n=4); B) Live / dead cell staining analysis of RBL-2H3 cells after incubation with FBTFA nanoparticles (NPs), HAMA / HADA hydrogel, or a combination thereof for 48 hours; C) Time-series flow cytometry analysis of the targeting binding process of RBL-2H3 cells to FITC-labeled FBTFA nanoparticles; E) Confocal fluorescence microscopy images showing the targeting localization of FITC-labeled FBTFA nanoparticles in RBL-2H3 cells (red: cytoskeleton; green: FITC-labeled FBTFA nanoparticles).

[0037] Figure 6Magnetically induced calcium ion influx and degranulation in RBL-2H3 mast cells mediated by FBTFA nanoparticles; AB) Confocal fluorescence microscopy images and corresponding quantitative analysis of intracellular calcium ion influx in RBL-2H3 cells under magnetic field stimulation (green: Fluo-4AM-based calcium ion fluorescence; blue: cell nucleus, n=3); C–D) Flow cytometry analysis and quantification of intracellular calcium ion fluorescence in RBL-2H3 cells under magnetic field stimulation (n=3); E) Relative degranulation rate of RBL-2H3 cells treated with FBTFA nanoparticles under magnetic field stimulation (n=4); F–H) Histamine, 5-HT, and ATP levels released by degranulated RBL-2H3 cells under magnetic field stimulation (histamine n=4; 5-HT, ATP n=4); (All analyses: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Figure 7 The molecular pathway changes in RBL-2H3 cell activation and degranulation induced by FBTFA under external magnetic field regulation are shown below: A) Principal component analysis (PCA) plot of transcriptome data from the two groups of cells; B) Bar chart of differentially expressed genes; C) Volcano plot of differentially expressed genes; D) Bubble chart of GO and KEGG enrichment analysis of upregulated genes; E) GSEA analysis of the immune response and cytokine-cytokine receptor interaction pathway.

[0038] Figure 8 To investigate the effects of an external magnetic field on mast cell degranulation in acupoint stimulation using a mast cell-targeting magnetoelectric hydrogel acupuncture system, which significantly alleviates pain, the following data was analyzed: A) Schematic diagram of the animal experiment process; B) Von Frey mechanical pain threshold in rats of different treatment groups (untreated control group, CFA-induced model group, FBTFA non-magnetic field group, CA-N group, non-piezoelectric FOFA group, and FBTFA magnetic stimulation group); C) Toluidine blue staining images of skin tissue at ST-36 acupoints and statistical analysis of degranulation rates in each treatment group (green arrows indicate mast cells with intact membrane structures, red arrows indicate mast cells in different degranulation states, n=4); D) Immunofluorescence images of TRPV1 and MCP7 in skin tissue at ST-36 acupoints and fluorescence intensity analysis of each channel (red: TRPV1, green: MCP7, yellow arrows indicate mast cell degranulation state, n=3); E) ELISA was used to determine the serum levels of TNF-α and IL-6 in rats of each treatment group (n=4); (All results: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) Figure 9To reduce the excitability of dorsal root ganglion (DRG) neurons and induce endogenous analgesia through mast cell degranulation, thereby achieving synergistic analgesia from local to systemic levels; A–C) Immunofluorescence images of the dorsal root ganglion (DRG) of rats in different treatment groups and fluorescence intensity analysis of each channel (red: adenosine A1 receptor; green: c-Fos; n=3); D) Analysis of β-EP release levels in cerebrospinal fluid of rats in different treatment groups (n=4); (All analyses: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) Figure 10 XPS analysis images of FO@BTO and FBTFA nanoparticles.

[0039] Figure 11 EDS image of FBTFA@HAMA / HADA magnetoelectric hydrogel.

[0040] Figure 12 XRD image of FBTFA@HAMA / HADA magnetoelectric hydrogel.

[0041] Figure 13 For the adhesion properties and other physical characterization of HAMA / HADA hydrogel; A) Image of HAMA / HADA hydrogel at a mass ratio of 2:1 attached to ST-N substrate, with the overall structure being lifted when the needle is pulled upward; B) Image of the weight of ST-N loaded with FBTFA@HAMA / HADA and ST-N without hydrogel (n=3); C) Image of the expansion and degradation of HAMA / HADA and HAMA hydrogel (n=3); E) In vitro release kinetics analysis of FBTFA in HAMA / HADA hydrogel (n=3).

[0042] Figure 14 Macroscopic images of ST-N loaded with HAMA / HADA hydrogels at different mass ratios.

[0043] Figure 15 In vivo delivery depth image of ST-N-loaded HAMA / HADA hydrogel.

[0044] Figure 16 Enlarged images of H&E-stained skin tissue at the acupuncture sites in rats (A and D show the acupuncture sites in the ST-N group and CA-N group, respectively; B and C are enlarged sections of image A). Figure 17For A) MFI analysis of live / dead staining images of RBL-2H3 cells after treatment with FBTFA nanoparticles, HAMA / HADA, or a combination of both for 48 hours (n=3, ns indicates no significant difference); B) Bright-field images of cell localization under confocal fluorescence microscopy (used to confirm nanoparticle binding); C) Schematic diagram of cell magnetic field stimulation and dynamic magnetic field measurement images generated by the dynamic magnetic field device at 200 rpm and 500 rpm (n=3).

[0045] Figure 18 A) Representative images of SD rats before and after CFA injection into the left ankle joint to induce a walking model; B) The device used to allow the rats to move freely in an external magnetic field; permanent magnets are symmetrically embedded in the bottom, and the magnetic field strength (0-60 mT) at different locations is measured using a gaussmeter; C) A schematic diagram of the top of the stimulation chamber, showing eight symmetrically arranged permanent magnets (blue circles) and a food-guided walking path (orange rectangle). The animals were guided to walk sequentially from P1 to P4, forming a closed loop; D) Estimated dynamic magnetic flux density at the Zusanli acupoint during the complete walking cycle; the blue shaded area, based on gaussmeter measurements at the corresponding spatial locations, represents the potential exposure range considering changes in acupoint height with posture (z≈2–6 cm); the red line indicates the typical magnetic field trajectory measured at z≈4 cm; the horizontal axis represents the normalized time along the feeding-guided path, independent of absolute walking speed. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0047] Example 1

[0048] I. Preparation of Experimental Materials (I) Preparation of a mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation: The preparation and assembly process of a two-component hyaluronic acid hydrogel (denoted as FBTFA@HAMA / HADA) containing mast cell-targeting magnetically responsive piezoelectric nanoparticles that can be mounted in the grooves of a threaded needle is as follows: Figure 1 As shown, the process includes the preparation of core-shell magnetoelectric nanoparticles FO@BTO, mast cell-targeted functionalization of FO@BTO, synthesis of a two-component hydrogel HAMA / HADA with a balance of stability and adhesion, loading FBTFA@HAMA / HADA into the groove of a threaded needle and photocrosslinking molding, etc.

[0049] The specific preparation method of the FBTFA@HAMA / HADA magnetoelectric hydrogel is as follows: (1) Preparation of core-shell magnetoelectric nanoparticles FO@BTO: First, FeCl3 (0.26 g), sodium acetate (1.2 g), PSSMA (0.4 g), and a small amount of ultrapure water were dissolved in ethylene glycol (16 mL). After thorough stirring, NaOH (0.24 g) was added, and the mixture was transferred to a stainless steel high-pressure reactor and reacted at 190 °C for 9 h. The product was magnetically separated and washed with ethanol and deionized water to obtain Fe3O4 NPs. Subsequently, the Fe3O4 suspension (5 mL) was dispersed in a mixed solution of ethanol (85 mL) and acetonitrile (30 mL), and ammonia (0.5 mL) and TBOT (1 mL) were added sequentially. After stirring at room temperature for 1.5 h, the mixture was washed to obtain Fe3O4@TiO2 (FO@TO) particles. Further, the FO@TO suspension (20 mL) was mixed with Ba(OH)2·8H2O solution (0.315 g), and ammonia (2 mL) was added. The mixture was transferred to a high-pressure reactor and hydrothermally reacted at 200 °C for 8 h. The obtained product was washed with formic acid and ethanol and then freeze-dried to finally obtain FO@BTO piezoelectric nanoparticles with a core-shell structure. (2) FO@BTO mast cell-targeted functionalization: The prepared FO@BTO powder (400 mg) was placed in a polytetrafluoroethylene Erlenmeyer flask, and 30% H2O2 (25 mL) was added for ultrasonic dispersion. The mixture was then refluxed at 110 °C for 4 h and washed until no bubbles were generated, yielding OH particles. These particles were then dispersed in anhydrous ethanol (25 mL), and APTES (400 µL) was added dropwise under stirring at 80 °C, followed by reflux for 24 h. After washing, the particles were lyophilized to obtain FO@BTO-NH2 particles. Subsequently, FcεRIA antibody was modified onto the particle surface using an EDC / NHS conjugation method: 4 µL of FcεRIA polyclonal rabbit antibody was dissolved in 4 mL of PBS, and EDC (1 mg) and NHS (1 mg) were added sequentially, reacting for 30 min each time. 10 mg of FO@BTO-NH2 was resuspended in 1 mL of PBS by ultrasonication, and the antibody solution was added and stirred at room temperature for 4 h. After washing, the targeted nanoparticles FO@BTO-FcεRIA (FBTFA) were obtained. (3) Synthesis of a two-component hydrogel HAMA / HADA with both stability and adhesion ratio First, 10 g of sodium hyaluronate (50 kDa) was dissolved in 500 mL of pre-cooled PBS. 10 mL of methacrylic anhydride (MA) was slowly added dropwise under ice bath conditions, and the pH was maintained at 8–9 with 5 mol / L NaOH. After stirring in the dark for 4 h, the product was transferred to a dialysis bag and dialyzed for 3 days, followed by lyophilization. The resulting HAMA was stored at −20 °C for later use. Next, 1 g of hyaluronic acid (800 kDa) was dissolved in 100 mL of deionized water. Under nitrogen protection, EDC (574 mg) and NHS (344 mg) were added for activation for 20 min. Dopamine hydrochloride (404 mg) was then added, and the pH was adjusted to 4.5–5 with hydrochloric acid solution. After stirring in the dark overnight, the product was dialyzed in acidic aqueous solution for 2 days, then dialyzed in deionized water for 1 day, and finally lyophilized and stored at −20 °C. The synthesized HAMA and HADA were dissolved in PBS at a mass ratio of 2:1, and 0.1% (w / v) of photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) was added to form a prepolymer solution.

[0050] (4) Assembly of the magnetoelectric hydrogel acupuncture needle system FBTFA nanoparticles (100 μg / mL) were added to the prepolymer solution and dispersed evenly. The liquid was then filled into the groove of the threaded needle using a syringe and crosslinked with ultraviolet light to obtain the final magnetoelectric hydrogel acupuncture system.

[0051] (II) Local analgesic effect of mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation: A mast cell-targeted magnetoelectric hydrogel acupuncture system was inserted into the Zusanli (ST36) acupoint in a rat model of acute adjuvant arthritis pain. The needle was withdrawn counter-rotatingly to allow the magnetoelectric hydrogel to remain in the target tissue. Twenty-four hours later, the rat was placed in a self-made magnetic field device and moved along a designated route for 10 minutes to simulate dynamic magnetic field stimulation of the acupoint. The device, measuring 34 × 25 cm, had eight symmetrically arranged cylindrical permanent magnets (30 mm in diameter and 5 mm in thickness) fixed at its base, generating a magnetic field strength covering the range of 0–60 mT. Under free movement, the magnetic field strength in the Zusanli area of ​​the rat's leg exhibited continuous spatial variation, forming a dynamic magnetic field exposure within the range of 0–6 mT driven by movement. Finally, the paw withdrawal threshold (PWT) induced by abnormal mechanical pain was used as a quantitative indicator to assess the degree of pain in the rats.

[0052] II. Characterization of Experimental Materials 1. Characterization of a mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation (1) The morphology of the synthesized nanoparticles (Fe3O4, FO@TO, FO@BTO, FO@BTO-NH2) at each stage was observed by field emission scanning electron microscopy and transmission electron microscopy. The core-shell structure and surface modification uniformity of FO@BTO-NH2 were further analyzed by energy dispersive spectroscopy (EDS mapping). The elemental composition (Fe, Ba, Ti) of FO@BTO was quantitatively determined by inductively coupled plasma atomic emission spectrometry. The particle size and surface potential of the nanoparticles were characterized by dynamic light scattering to evaluate their dispersibility and surface stability. The magnetic properties were measured using a vibrating sample magnetometer in the range of 0–20 kOe to confirm their superparamagnetic properties. The crystal structure of the nanoparticles was analyzed by X-ray diffraction to verify the successful synthesis of FO@BTO. The successful coupling of the mast cell-targeting antibody FcεRIA was verified by thermogravimetric analysis of the mass change curves of FBTFA and FO@BTO under a nitrogen atmosphere (28–800 °C). The piezoelectric properties of FO@BTO were characterized using a piezoelectric response force microscopy (PFM) module equipped with an atomic force microscope. Typical butterfly-shaped amplitude-voltage curves were recorded under ±10 V AC bias, and the effective piezoelectric coefficient (d) was calculated based on the displacement-voltage relationship. 33 ).

[0053] (2) To obtain hydrogels with both stability and adhesion, HAMA and HADA were dissolved in PBS at mass ratios of 1:1, 2:1, and 3:1. 0.1% (w / v) of the photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate, MCE, China) was added, and after thorough mixing, ST-N was inserted into the solution and crosslinked under UV irradiation for 30 s. The adhesion effect between the gel and the needle was observed by lifting the needle handle. A 2:1 mass ratio was ultimately selected for subsequent experiments. To verify the hydrogel structure, HAMA, HADA, and HAMA / HADA hydrogels were dissolved in heavy water and analyzed using nuclear magnetic resonance spectroscopy (¹H NMR). The rheological properties of the hydrogels at different concentrations (3%, 4.5%, 6%, 9%, 18%) were measured using a rotational rheometer with a frequency range of 1–100 rad / s. The sample height was 2 mm and the diameter was 12 mm. A 6% concentration was ultimately selected for subsequent experiments. FBTFA nanoparticles (100 μg / mL) were added to the prepolymer solution and photocrosslinked to obtain FBTFA@HAMA / HADA hydrogel. The prepared sheet-like hydrogel (2 cm × 1 cm × 4 mm) was attached to the surface of the index finger joint, and its extensibility and adhesion were tested by bending. The lyophilized hydrogel sample was cut, and its internal porous structure and nanoparticle distribution were observed using scanning electron microscopy. Subsequently, the water absorption and degradation properties of the HAMA / HADA hydrogel were tested using the following method: First, the weight of the dried support (W0) was measured. Then, the dried hydrogel was placed in PBS at 37 °C and soaked for 0, 0.5, 1, 2, 4, 6, 12, and 24 hours. After timed removal, the surface moisture was wiped off with absorbent paper, and the wet weight (Wt) was measured. Simultaneously, under the same conditions, the sample was placed in a shaker and continuously shaken for 0, 1, 2, 4, 7, and 15 days, after which it was removed, dried, and the remaining dry weight (Wt') was measured.

[0054] The swelling ratio and degradation rate are calculated using the following formulas: Swelling ratio = (Wt – W0) / W0 Degradation rate (%) = Wt' / W0 × 100% FITC-labeled FBTFA nanoparticles (5 mg) were uniformly encapsulated in a HAMA / HADA hydrogel and incubated with gentle shaking in PBS solution at 37°C. The supernatant was collected at predetermined time points and replaced with an equal volume of fresh PBS solution. Fluorescence intensity was then measured using a microplate reader, and a cumulative release curve was plotted.

[0055] (3) Multi-angle photography using a macro lens was used to observe the morphological characteristics of CA-N and ST-N. To evaluate the hydrogel delivery capability of ST-N, muscle tissue (20 × 20 × 10 mm) was obtained from the hind limbs of domestic pigs. FBTFA@HAMA / HADA composite hydrogel stained with blue aqueous dye was loaded onto the surface of CA-N and ST-N. The needles were inserted 1 cm into different parts of the tissue. After ST-N was inserted, it was pulled out along the counter-thread direction. The needle path on the surface and inside of the domestic pig tissue was observed. Further simulation was carried out using rats. CA needles and ST needles loaded with magnetoelectric hydrogel were inserted into acupoints on the hind limbs of rats and then pulled out along the counter-thread direction. The rats were immediately euthanized by cervical dislocation. Skin tissue from the acupuncture site was taken and fixed in 4% paraformaldehyde. The fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to visually evaluate the delivery and distribution of hydrogel in subcutaneous tissue using histological methods.

[0056] 2. Biocompatibility testing To verify the analgesic performance of the mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation, its in vitro cytotoxicity was first evaluated. Basophilic leukemia granulocytes (RBL-2H3) are a widely used in vitro mast cell model, and this cell type was selected for validation.

[0057] RBL-2H3 cells were cultured in MEM medium containing non-essential amino acids, supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin, and placed at 37°C, 5% CO2, and saturated humidity. When cell confluence reached 80–90%, cells were passaged or plated every 2–3 days. The cytotoxicity of FO@BTO NPs and HAMA / HADA composite hydrogel was assessed using the MTT assay. After solidification with 6% (w / v) HAMA / HADA hydrogel, cells were immersed in serum-free medium for 48 h. The resulting supernatant was centrifuged and filtered through a 0.22 μm filter to prepare complete culture medium, which was stored at 4°C for later use. Cells were seeded at a density of 2000 cells / well in 96-well plates, with two experimental groups: ① FO@BTO NPs co-culture group; ② FO@BTO NPs co-treated group with hydrogel extract, with consistent nanoparticle concentrations (100, 500, 1000 μg / mL). After culturing for 24 or 48 h, cells were washed with PBS, incubated with 10 μL of MTT working solution for 4 h, and then 100 μL of Formazan dissolving solution was added until the purple crystals were completely dissolved. Absorbance was measured at 570 nm using a microplate reader to assess cell viability. Furthermore, biocompatibility was further assessed using the Calcein-AM / PI double staining method. After co-culturing with 100 μg / mL FO@BTO for 48 h, cells were stained with working solution containing Calcein-AM and PI, and the intensity of green fluorescence (live cells) and red fluorescence (dead cells) was observed under an inverted fluorescence microscope.

[0058] 3. Evaluation of FBTFA nanoparticle mast cell targeting First, FBTFA NPs were fluorescently labeled. 10 mg of FO@BTO-NH2 was dispersed in 5 mL of dichloromethane, and 1 mg of FITC-NHS was added. The mixture was stirred overnight in the dark. The product was washed multiple times with ethanol and PBS, and then resuspended in PBS to obtain FITC-labeled FO@BTO (FO@BTO-FITC). Subsequently, anti-FcεRIA antibody was conjugated to its surface using the aforementioned method to prepare FBTFA-FITC NPs, which were stored at -20 °C in the dark for later use. To visualize the targeting performance, RBL-2H3 cells were used at a concentration of 2 × 10⁻⁶ cells / mL. 4 RBL-2H3 cells were seeded at 5 × 10⁵ cells / well in 24-well plates and cultured for 24 h. Then, 100 μg / mL FO@BTO or FBTFA-FITC was added, and the cells were co-cultured for 2 h. After washing three times with PBS, the cells were fixed with 4% PFA for 30 min and then permeabilized with 0.5% Triton X-100. The cytoskeleton was stained with Actin-Tracker Red-555, and the cell nuclei were stained with DAPI. The cells were mounted, air-dried in the dark, and then observed under a confocal laser scanning microscope. Simultaneously, the binding kinetics of FBTFA-FITC to mast cells were analyzed by flow cytometry. RBL-2H3 cells were seeded at 5 × 10⁵ cells / well in 24-well plates, and 100 μg / mL FBTFA-FITC NPs were added. Samples were collected at 0, 0.5, 1, 2, 3, and 6 h, and the mean fluorescence intensity (MFI) at each time point was measured to assess the cell binding efficiency and time dependence of the nanoparticles. Furthermore, SEM and phalloidin staining were used to verify cell growth and permeation within the scaffold.

[0059] 4. In vitro detection of calcium influx in mast cells To evaluate the effect of FBTFA nanoparticle-mediated magnetoelectric stimulation on calcium ion influx in mast cells, calcium imaging experiments were performed using the Fluo-4 AM fluorescent probe (Beyotime, China). First, a dynamic magnetic field was generated by mechanically rotating a permanent magnet system, forming a continuous low-frequency rotating alternating magnetic field. A 35 mm diameter cell culture dish was placed at the center of the magnetic field, and the magnetic field strength B0 at this location was measured to be 0.92 mtesas. Unless otherwise specified, the rotating magnetic field was applied at a constant speed of 200 rpm for 10 minutes. To facilitate the quantification of the time-varying characteristics of this dynamic magnetic field, we approximate the axial magnetic field component as:

[0060] Given the rotation frequency is ≈3.33 Hz, therefore, its maximum rate of change is:

[0061] Substituting the above data, the rate of change of the dynamic magnetic field is approximately 19.26 mT / s. These parameters collectively define the physical conditions for in vitro magnetic stimulation in this study, thus ensuring the quantitative nature and reproducibility of the experimental stimulation protocol. The subsequent experimental setup was as follows: RBL-2H3 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μg / mL on 24-well glass coverslips and cultured overnight to allow adherence. They were then co-cultured at 37°C for 2 hours with specified nanomaterials (FOFA or FBTFA nanoparticles, 100 µg / mL). After incubation, Fluo-4 AM was loaded into cells in all groups according to the manufacturer's instructions. Subsequently, the FOFA and +MS groups underwent magnetic stimulation. Magnetic stimulation was performed using a dynamic rotating magnetic field with an intensity of approximately 0.92 mT, a rotation speed of 200 rpm, and a duration of 10 minutes. Cells were then gently washed with PBS buffer and fixed / mounted using anti-fading mounting medium (containing DAPI). Fluorescence images were acquired using a confocal laser scanning microscope under the same acquisition parameters. The mean fluorescence intensity of single cells was quantified using ImageJ software to assess intracellular Ca²⁺ levels. To further validate the results, flow cytometry analysis was performed under the same treatment and stimulation conditions. Cells were collected immediately after magnetic stimulation was terminated, and population-level Ca²⁺ influx data were obtained by quantifying the Fluo-4AM fluorescence intensity.

[0062] 5. In vitro mast cell degranulation and release experiment To evaluate the regulatory effect of FBTFA nanoparticles on mast cell degranulation, the release of β-hexosaminease (β-Hex) was quantitatively determined using a colorimetric substrate method. RBL-2H3 cells were inoculated at 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 100 µL per well in 96-well plates and cultured for 24 hours. Cells were divided into the following groups: Untreated control, positive control (C48 / 80), FBTFA piezoelectric nonmagnetic group (−MS), and FBTFA piezoelectric electromagnetic stimulation group (+MS). After 24 hours, the culture medium was removed, and cells were incubated for 2 hours in a benchtop medium (Yuan Ye, China) containing 100 µg mL⁻¹ FBTFA nanoparticles or 50 µg mL⁻¹ C48 / 80 (Sigma, USA). The +MS group was then placed in a rotating magnetic field (200 rpm, 0.92 mT) for 10 minutes, while the other groups received no magnetic stimulation under the same conditions. After stimulation, 50 µL of supernatant (ODa) was collected from each well and transferred to a new 96-well plate. The remaining supernatant was used to lyse adherent cells with 200 µL of 0.1% Triton X-100 solution, and 50 µL of cell lysis buffer (ODb) was used to determine the total β-hexosidase content. Tyrode buffer was used as a blank control (ODc). Subsequently, 50 µL of substrate solution (2 mmol L⁻¹ 4-nitrophenyl-N-acetyl-β-D-glucosamine peptide, MCE, China) was added to each well, and the cells were incubated at 37°C for 1 hour. The reaction was terminated with 200 µL of carbonate buffer (Yuan Ye, R20887). The absorbance was measured at 405 nm using a microplate reader. The relative degranulation rate was calculated using the following formula after normalization by dividing the released β-hexosidase content by the total cellular β-hexosidase content (considering the corresponding volume ratio):

[0063] 6. Detection of histamine, 5-HT, and ATP release in degranulation media In the mediator release experiment, the C48 / 80 group was omitted, and a new FOFA group was added (magnetic stimulation only, without piezoelectric stimulation). Cells were introduced at a rate of 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 µL in 96-well plates and cultured for 24 hours. Subsequently, 100 µg mL⁻¹ FOFA and FBTFA nanoparticles were co-incubated with the cells. After treatment with the same magnetic field (200 rpm, 0.92 mT, 10 min), the supernatant was collected and centrifuged at 4°C, 2500g for 10 min. Histamine and 5-HT concentrations were determined using ELISA kits (U-Bio and Ximei Bio, China) according to the manufacturer's instructions. Extracellular ATP (eATP) release was determined using a bioluminescent luciferase assay. Cells (1×10⁶ cells / well) were seeded at a density of 100 µL in 96-well plates and cultured for 24 hours. 6Cells / mL) were seeded in 96-well plates and cultured adherently for 3 hours. Subsequently, 100 µg mL⁻¹ FOFA or FBTFA nanoparticles were added, incubated for 2 hours, and then the same magnetic stimulation protocol was performed. The eATP level in the supernatant was quantitatively determined using a luciferase-based ATP detection kit (MCE, China).

[0064] 7. RNA-seq RNA was extracted from untreated and +MS-treated RBL-2H3 cells using TRIzol reagent (Saiweier, China). After constructing the library, its quality was verified, the libraries were merged and sequenced on the Illumina NovaSeq PE150 platform. The obtained clean read sequences were used for subsequent bioinformatics analysis.

[0065] 8. Animal experiment model establishment and intervention In this study, an acute adjuvant arthritis (AA) model was used as a pain model. Clean-grade male SD rats, 6 weeks old and weighing 190–220 g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK (Zhe) 2024-0001). All experiments were approved by the Animal Ethics Committee of Wenzhou Institute, Chinese Academy of Sciences (Approval No.: WIUCAS25010703) and strictly followed the relevant operating specifications and national standards (GB 14925-2001). The rats were randomly divided into 6 groups (n = 6): ① blank control group (Untreated); ② AA model group (CFA); ③ traditional acupuncture group (CA-N); ④ FOFA delivery combined with magnetic stimulation group (FOFA); ⑤ FBTFA delivery without magnetic field group (–MS); ⑥ FBTFA delivery combined with magnetic stimulation group (+MS). The method for establishing the AA model was as follows: Under 2% isoflurane inhalation anesthesia, 50 μL of CFA (MCE, China) was injected into the ankle joint cavity of the left hind limb of the rats. Approximately 48 hours after the model injection, the rats showed obvious local pain responses, manifested as obvious swelling and redness of the ankle, accompanied by behavioral characteristics such as movement disorders or lifting the leg to avoid weight-bearing.

[0066] Under 2.5% isoflurane gas anesthesia, in the hydrogel intervention group, the HAMA / HADA hydrogel containing 100 μg / mL FBTFA or FOFA nanoparticles was delivered to the subcutaneous area about 1 cm below the ST-36 acupoint (about 5 mm below the head of the fibula) of the rats through ST-N, and then the needle was withdrawn in a reverse spiral to make the hydrogel stay. After 24 hours, the rats in the +MS group and the FOFA group were placed in a self-made magnetic field device containing symmetrically arranged permanent magnets (diameter 30 mm, thickness 5 mm) and allowed to move freely for 10 minutes to simulate the action of a dynamic magnetic field. The control group CA-N received traditional acupuncture intervention (inserting the needle 1 cm and lifting and thrusting 100 times per minute) at the same acupoint.

[0067] 9. Behavioral pain threshold testing To assess pain intensity, we used the paw withdrawal threshold (PWT) induced by abnormal mechanical pain as a quantitative indicator. Measurements were taken before modeling (Day 0), on day 2 after modeling (Day 2), and one hour after each intervention (Days 3–7). All tests were blinded, and operators were unaware of group information. Thirty minutes before each measurement, rats were placed in a transparent observation box with a metal mesh bottom to acclimatize. After they settled, the center of the left paw sole was stimulated with Von Frey filaments in ascending order of pressure for 2–3 seconds until escape responses such as paw withdrawal and licking occurred. The stress value at this point was recorded. Each animal was tested five times consecutively, with intervals of at least two minutes between each test. The average of the three results with the smallest differences was taken as the final PWT value. A lower PWT value indicates higher mechanical pain sensitivity in the rat.

[0068] 10. Surgery and Sampling After all interventions were completed, samples were collected uniformly on Day 8. First, rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg). Once fully anesthetized, the rats' heads were curled inwards at approximately 45° to expose the foramen magnum. Cerebrospinal fluid (CSF) was collected from the subdural space using a 25G disposable intravenous infusion needle and immediately stored at -80°C. Subsequently, the heart was exposed for blood collection. After completion, the syringe needle was inserted from the apex of the heart through the left ventricle into the aortic arch and fixed. The right atrial appendage was cut open, and physiological saline was injected using a microinfusion pump until the rat's liver turned grayish-white. The spine was then exposed, and the DRG of the L4–L6 segments on the modeling side was carefully dissected. Skin tissue from the ST-36 acupoint area on the modeling side was excised and fixed in 4% PFA for preservation.

[0069] 11. Histological analysis Skin from the ST-36 acupoint area was fixed with 4% PFA for 24 h, dehydrated with 30% sucrose, and embedded in OCT to prepare approximately 10 μm frozen sections. Three sections were randomly selected from each group, stained with 0.15% toluidine blue, dehydrated, mounted, and observed using a digital pathology scanner. After staining, mast cells appeared purplish-red, and the nuclei appeared blue. Four fields of view were randomly selected from each section for counting, and the degranulation rate was calculated as the percentage of degranulated mast cells to the total number of mast cells. Immunofluorescence was used to analyze mast cell degranulation and TRPV1 expression in the acupoint area. After blocking with 10% goat serum (containing 0.1% Triton X-100), the sections were incubated at 4°C with primary antibodies, including anti-TRPV1 antibody (Affinity, China, 1:300) and anti-mast cell trypsin antibody (MCP7, Servicebio, China, 1:300). The next day, fluorescent secondary antibody was added, and the sections were incubated in the dark for 1 hour, stained with DAPI, and mounted. L4–L6 DRG sections were processed using the same method and incubated with anti-A1R (Abcam, USA, 1:500) and anti-c-Fos (Affinity, China, 1:300), respectively. All samples were confocally imaged, and fluorescence intensity was analyzed using ImageJ. Finally, serum TNF-α and IL-6 levels were measured using an ELISA kit (SIMIBIO, China) to assess inflammatory status; simultaneously, the content of opioid analgesics (β-endorphin, SIMIBIO, China) in CSF was detected to reflect the activation of central analgesic mechanisms.

[0070] III. Experimental Results and Analysis 1. Characterization and performance of the magnetoelectric hydrogel acupuncture system The FO, FO@TO, FO@BTO and FO@BTO-NH2 nanoparticles prepared stepwise in Example 1 were analyzed by field emission scanning electron microscopy (FESEM). Figure 2 Microscopic morphology observation was performed (see Figure A). FO nanoparticles are uniform spheres with a diameter of approximately 100 nm. After coating with a TiO2 layer, the size of FO@TO particles increased significantly, and the surface became rough with visible deposits. Further hydrothermal reaction resulted in the formation of a granular BTO shell on the surface of FO@BTO, significantly altering its morphology. After silanization modification, FO@BTO-NH2 still retained the BaTiO3 particle shell, indicating that the H2O2 treatment did not affect the BTO shell responsible for generating piezoelectric properties.

[0071] Next, further observation was conducted using transmission electron microscopy (TEM). For example... Figure 2As shown in the C-section diagram, both FO@BTO and FO@BTO-NH2 are coated with a dense, irregular shell of approximately 10 nm, confirming successful BTO coating. In FO@BTO-NH2, although slight corrosion marks caused by H2O2 are visible, the shell thickness remains essentially unchanged.

[0072] In addition, energy-dispersive X-ray spectroscopy (EDS Mapping) is also used. Figure 2 (Figure B) Fe, O, Ba, Ti, and N elements were detected, with Ba and Ti signals overriding the Fe signal, further confirming that the nanoparticles have a typical core-shell structure consisting of a ferrite core and a BaTiO3 shell. Simultaneously, the enrichment of nitrogen on the particle surface indicates that the silane coupling reaction successfully achieved functionalization modification. Figure 2 As shown in Figure D, the elemental mass percentage of FO@BTO nanoparticles was further determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Based on the calculations using Fe and Ba elements, the mass ratio of FO to BTO was approximately 1.109:1. Nanoparticle size analyzer results showed ( Figure 2 In the midpoint diagram (E), the average hydrated particle sizes of FO@TO, FO@BTO, and FO@BTO-NH2 are similar (approximately 300 nm), and all are significantly larger than FO (approximately 120 nm), indicating successful deposition of the outer shell layer. The corresponding Zeta potential ( Figure 2 The values ​​in the middle part (F) are -23.5 mV, -38.9 mV, -24.1 mV and -21.7 mV, respectively. It is worth noting that although the potential of FO@BTO-NH2 is still negative, the absolute value has decreased, which may be due to the introduction of amino groups neutralizing some of the surface negative charge.

[0073] Magnetic properties are one of the important indicators for evaluating the responsiveness of magnetoelectric composite nanomaterials, directly determining whether the material can achieve effective magnetoelectric coupling and bio-regulation functions under external magnetic field stimulation. Therefore, within a magnetic field range of ±2T, the hysteresis loop of Fe3O4 and its multilayer coated structure was measured using a vibrating sample magnetometer (VSM). Figure 3 (See Figure A). The results show that all samples exhibit typical S-shaped hysteresis loops. The saturation magnetization gradually decreases with increasing coating layer, but the composite nanoparticles still maintain good magnetic response and soft magnetism, proving their ability to be modulated by an external magnetic field, providing a reliable basis for subsequent magnetoelectric stimulation. Simultaneously, to further verify the crystal phase composition of the composite structure, X-ray diffraction (XRD) was performed using Jade 6.0 software and compared with standard PDF cards. Figure 3(See Figure B). The diffraction peaks of FO nanoparticles are consistent with those of Fe3O4 (PDF#19-0629), and the diffraction peaks of BaTiO3 are consistent with those of PDF#05-0626. The synthesized FO@BTO nanoparticles also exhibit characteristic peaks of BaTiO3, further confirming that their surface is coated with a perovskite-structured BTO shell. These results corroborate the TEM characterization results, further demonstrating that the constructed magnetoelectric composite nanoparticles possess a complete core-shell structure and good crystal characteristics.

[0074] Mast cells highly express the IgE receptor α subunit (FcεRIA) on their surface, exhibiting good membrane localization and antibody accessibility. Therefore, this study selected it as a targeting marker and used a low-density FcεRIA monoclonal antibody to modify the surface of FO@BTO-NH2 NPs.

[0075] To verify the success of peptide conjugation, thermogravimetric analysis (TGA) was used to detect FO@BTO and its FcεRIA antibody-modified composite nanoparticles (FBTFA). Figure 3 As shown in the C-plot, FO@BTO began to lose weight slowly after 229.0°C, with a residual mass of 92.7% at 800°C, demonstrating good thermal stability. The FBTFA group, on the other hand, showed significant weight loss after 248.3°C, with two peaks in the DTG curve at 280.8°C and 320.6°C, corresponding to the decomposition of the antibody protein backbone and branches. The final residual mass was 91.23%, a decrease of approximately 1.47% compared to the unmodified group, consistent with the mass changes caused by antibody modification. Furthermore, to investigate the surface chemical changes induced by antibody conjugation, high-resolution N 1s and C 1s X-ray photoelectron spectroscopy (XPS) analyses were performed on FO@BTO and FBTFA. Figure 10 As shown in Figure A, the N 1s spectrum of FO@BTO exhibits a single peak (~398.3 eV), attributed to the amine nitrogen species introduced by surface amination modification. In contrast, the antibody-conjugated FBTFA nanoparticles show an additional N 1s component at ~399.8–400.0 eV. Figure 10 (See C). This characteristic peak corresponds to the amide nitrogen (–CONH–), indicating the formation of a new amide bond during covalent coupling. In the C 1s spectrum (… Figure 10 In the midpoint diagrams (B and D), both groups of samples exhibit a dominant C–C / C–H peak (~284.8 eV), accompanied by weak oxygen / nitrogen-carbon (~286 eV) and weak carbonyl-related components in the high binding energy region. This carbonyl component is more pronounced in the antibody-conjugated sample, corresponding to a C=O chemical environment at approximately 287.7 eV, indicating the introduction of additional protein-related functional groups to the surface. These results demonstrate that the FcεRIA antibody has been successfully conjugated to the surface of FO@BTO nanoparticles, thus establishing preliminary single-cell targeting capability.

[0076] Piezoelectric properties directly affect the electrical response of magnetoelectric materials under an applied magnetic field. Therefore, piezoelectric power microscopy (PFM) was used to determine the morphology and piezoelectric properties of individual FO@BTO-NH2 nanoparticles. Figure 3 Sub-maps D–H). Result indication: 3D topographic map ( Figure 3 The middle (D) shows that the particle surface is rough and the average particle size is about 300 nm; the amplitude response diagram ( Figure 3 E(i) shows a distinct local high-response region, with a maximum amplitude of approximately 141 pm; the phase diagram ( Figure 3 The phenomenon of brightness reversal in E(ii) indicates the presence of a switchable polarization direction within it, exhibiting typical piezoelectric response characteristics. Furthermore, the phase loop under local voltage excitation ( Figure 3 F) shows a phase reversal of approximately 180°, and the amplitude curve ( Figure 3 The FO@BTO-NH2 nanoparticles exhibit an asymmetrical butterfly shape with slight horizontal displacement. Based on the slope of the amplitude curve, the effective piezoelectric coefficient d0 of the FO@BTO-NH2 nanoparticles is calculated. 33 Approximately 16.4 pm / V ( Figure 3 (H). The above results demonstrate that the prepared FO@BTO-NH2 nanoparticles possess excellent reversible polarization characteristics and stable electromechanical coupling performance, which can well meet the requirements of magnetoelectric coupling and subsequent biostimulation applications. Based on its d 33 Furthermore, we conservatively estimated the potential electrical stimulation intensity of this material under mechanical disturbance conditions. For piezoelectric materials, under the open-circuit approximation condition, the equivalent electric field inside the shell can be approximately expressed as , where the stress can be expressed as . Combining the typical dielectric constant range (εr ≈ 200–1000) of the BaTiO3 nanoshell-like localized structure with the characteristic Young's modulus of BaTiO3 (Y ≈ 100 GPa), and using the conservative strain range corresponding to the ppm-level ferrite magnetostriction value reported in the literature (ε ≈ 10⁻ 5 –10⁻ 4 The estimated local equivalent electric field is approximately 10³–10. 5 V / m. This corresponds to an equivalent interface potential of approximately 0.02–0.9 mV at a 10 nm shell scale. This estimate aims to quantify the magnitude of electrical stimulation that the piezoelectric response may generate, contributing to an understanding of the bioelectric modulation potential of this invention under external field driving.

[0077] To further leverage its magnetoelectric modulation effect in biological systems, this invention constructs a HAMA / HADA composite hydrogel system with loading and adhesion functions, used for the precise delivery and fixation of nanoparticles at specific acupoints. For example... Figure 4As shown in Figure A, HAMA is obtained by reacting hyaluronic acid (HA) with methacrylic anhydride (MA), and the introduction of the methacryloyl group endows it with polymerizability; HADA, on the other hand, forms an amide bond by coupling the carboxyl group of EDC / NHS-activated HA with the amino group of dopamine (DA), thereby increasing the adhesive properties of DA while maintaining the hydrophilicity and modifiability of HA, allowing it to bond with various inorganic / organic surfaces. Furthermore, as... Figure 4 As shown in Figure B, HAMA and HAMA / HADA were dissolved in PBS to form a pregel solution. Subsequently, the photoinitiator LAP was added and photocrosslinking was achieved by irradiation with ultraviolet (UV) light. Figure 4 Figure C shows a HAMA / HADA hydrogel measuring 2 cm × 1 cm × 0.3 cm applied to the skin of a human finger joint. Under natural extension (180°), slight bending (120°), over-bending stretching (90°), and flipping (360°) movements, the hydrogel can conform to the skin folds and adhere well to the skin surface without obvious breakage, peeling, or curling, demonstrating excellent flexibility and dynamic adaptability.

[0078] In addition, after FBTFA NPs were incorporated into the HAMA / HADA pregel solution, they were photocrosslinked and lyophilized, and then the cross-sections of the hydrogels without nanoparticles were observed by SEM. Figure 4 (See Figure D). The results show that both pure HAMA / HADA and FBTFA@HAMA / HADA hydrogels exhibit porous structures. Compared to the former, the FBTFA@HAMA / HADA surface displays a granular, rough morphology, indicating successful loading of nanoparticles. Further analysis using EDS energy dispersive spectroscopy and elemental surface distribution results... Figure 11 The XRD analysis clearly detected characteristic element signals such as Fe, Ba, and Ti, and their spatial distribution closely matched the hydrogel matrix region, indicating that the FBTFA-related inorganic components are not limited to local aggregation or surface adsorption, but are dispersed in a stable form within the HAMA / HADA polymer network. Simultaneously, XRD analysis ( Figure 12 The results show that the characteristic diffraction peaks of the magnetoelectric nanoparticles in the FBTFA@HAMA / HADA composite material are significantly weakened and broadened compared to the pure powder, reflecting that its crystal structure is restricted by the polymer network. These morphological, elemental composition, and crystal structure results corroborate each other, confirming from multiple levels the successful introduction and relatively stable encapsulation of FBTFA nanoparticles in the HAMA / HADA hydrogel, providing a structural basis for subsequent functional applications.

[0079] The successful synthesis of HAMA and HADA was demonstrated by 1H NMR spectroscopy ( Figure 4Structural testing was performed on the midpoint of Figure E: HAMA showed characteristic peaks of methacryloyl group at δ = 6.16, 5.73, and 1.85 ppm, while HADA exhibited a typical triplet peak of dopamine-catechol structure at δ = 6.7–6.9 ppm. The simultaneous display of these characteristic peaks by HAMA / HADA indicates accurate structural fusion. To optimize adhesion performance, HAMA and HADA were mixed at mass ratios of 1:1, 2:1, and 3:1, and then crosslinked using blue light after insertion into ST-N needles. The results showed ( Figure 13 As shown in Figure A, only the 2:1 ratio hydrogel could adhere firmly to the needle, indicating that this ratio has the best adhesion performance. Rheological tests were conducted using hydrogels of different concentrations (3–18 wt%) prepared with this 2:1 ratio. Figure 4 As shown in Figure F, the storage modulus G′ is greater than the loss modulus G″ in all concentration groups, indicating that the system is an elastic-dominated three-dimensional network structure. With increasing concentration, G′ increases significantly (from approximately 10¹ Pa to 10³–10⁴ Pa), indicating enhanced crosslinking density and network rigidity; the tanδ value (G″ / G′) decreases, further highlighting the elastic advantage. In the low-frequency region (0.1–1 rad / s), the G′ of the low-concentration groups (3%, 4.5%) fluctuates slightly and is close to G″, suggesting that the network structure is not yet fully stable and may exhibit some fluidity; the G′ curve of the high-concentration groups (9%, 18%) is stable and almost insensitive to frequency, indicating stronger structural stability and resistance to deformation, but potentially reduced operability and injectability. Therefore, a concentration range of 6 wt% can balance good elasticity and moderate flexibility, making it suitable as an ideal material for bioadhesion.

[0080] The swelling properties of hydrogels in body fluids directly regulate their interfacial adhesion to surrounding tissues. The swelling properties of hydrogels in body fluids directly affect their interfacial adhesion to surrounding tissues. Swelling and degradation experiments in PBS at 37℃ ( Figure 13The median (CD) shows that the 6 wt% HAMA hydrogel rapidly reached swelling equilibrium; in contrast, the swelling process of the HAMA / HADA composite hydrogel at the same concentration lasted approximately 6 hours, with a final swelling rate exceeding 53%, demonstrating superior water absorption and structural stability. Degradation curves indicate that both hydrogels gradually degraded over time in PBS, but the HAMA / HADA composite hydrogel exhibited a relatively mild and controllable mass loss process: retaining approximately 55-60% of its initial mass after 15 days, with a significantly lower degradation rate than the monomeric HAMA hydrogel. These results suggest that the composite network structure formed by introducing HADA can, to some extent, delay hydrogel degradation, implying that it can be maintained in target tissues in vivo for a longer period. Given that both hyaluronic acid-based hydrogels and Fe3O4-based or related magnetic nanoparticles possess acceptable biocompatibility in biological systems, their synergistic construction provides a relatively stable microenvironment for the role of magnetic nanoparticles in local tissues. Based on this, we further evaluated the in vitro release behavior of FBTFA nanoparticles in the HAMA / HADA composite hydrogel. Cumulative release curves (…) Figure 13 The results (E) indicate that the nanoparticles exhibit stable, continuous, and slow release characteristics in this hydrogel system. By day 20, the cumulative release reached 78% of the initial content. This suggests that the network structure formed by the HAMA / HADA composite hydrogel not only slows down the degradation process of the material itself but also effectively regulates the diffusion behavior of the nanoparticles, providing favorable conditions for achieving sustained action within local tissues.

[0081] Figure 4 As shown in diagram G, the commercially available standard needle (CA-N) was purchased from Zhongyan Taihe Company, made of 06Cr19Ni10 stainless steel, 3.4 cm long, and with a needle body diameter of 0.25 mm. Our designed acupuncture needle (ST-N) with a spiral grooved body is made of 314 medical-grade stainless steel, 3.2 cm long, with a needle body diameter of 0.25 mm, a thread length of 1.7 cm, a pitch of 1.3 mm, and a groove depth of 0.05 mm. Figure 4 As shown in the midpoint diagram H, HAMA / HADA hydrogel stained with blue aqueous dye was drop-coated into the groove of the needle using a 1 mL syringe and then cured by light for 30 seconds. (Needle tip macro image) Figure 4 As shown in Figure I), the hydrogel aggregates in droplets due to the smooth surface of CA-N, while the spiral grooves of ST-N can effectively retain the hydrogel. Figure 4 Midpoint J shows that in a 2 × 2 × 2 cm sample of porcine abdominal skin and muscle tissue, both CA-N and ST-N could penetrate the subcutaneous tissue. However, the solidified hydrogel only entered the subcutaneous tissue with ST-N, while the hydrogel on CA-N remained on the skin surface, indicating that ST-N possesses significant hydrogel transport capabilities. Furthermore, in Figure 14The HAMA / HADA system with a mass ratio of 2:1 exhibited relatively stable needle adhesion and controllable delivery characteristics. For example... Figure 4 As shown in diagram K, after the needle is withdrawn counterclockwise, the hydrogel detaches from the needle body under tissue traction and remains at the acupoint. The pig tissue treated with the hydrogel was then dissected along the needle path. Figure 15 As shown in section A, the blue hydrogel is clearly identifiable in the local tissue, and its distribution exhibits a gradual decrease along the needle path, with the signal intensity gradually weakening with increasing tissue depth. Simultaneously, in subcutaneous muscle tissue sections corresponding to the Zusanli acupoint in rats (… Figure 15 In (B), a distinct green fluorescent signal was observed in the needle tract region, indicating that the HAMA / HADA hydrogel loaded with FBTFA-FITC nanoparticles can also achieve local tissue retention in vivo. Skin tissue samples were collected from the acupuncture sites of rats. H&E-stained skin tissue sections showed residual hydrogel fragments adhering to the epithelial layer of the target tissue. Figure 4 Mid-part image (L–M). Further zoom in on the image ( Figure 16 (Partial diagrams A–C) show that the residual hydrogel appears as irregular, weakly stained, amorphous fragments within the needle tract, exhibiting significantly weaker affinity for hematoxylin and eosin compared to surrounding cells and extracellular matrix components. Furthermore, these characteristics are limited to the puncture site of the magnetoelectric hydrogel acupuncture system. This phenomenon was not observed in adjacent intact tissues or at the puncture site of conventional acupuncture needle CA-N. Figure 16 (D). The above in vitro and in vivo observations corroborate each other, jointly confirming that the spiral grooved needle can achieve the delivery and retention of hydrogel along the needle track in local tissue. Through simple weighing tests, each ST-N fiber can carry an average of approximately 1.2 mg of hydrogel (D). Figure 13 (B) Given the inherent non-uniformity of the manual coating process and the losses during insertion, future studies can further characterize the actual delivery volume by integrating more precise in vivo quantitative methods. These results demonstrate that ST-N can successfully load and accurately deliver magnetoelectric hydrogels to acupoint areas, providing a reliable physical basis for subsequent magnetically controlled degranulation and local analgesia.

[0082] 2. Biocompatibility and mast cell targeting of magnetoelectric hydrogels RBL-2H3 cells are a widely used in vitro mast cell model. To ensure the safety of the magnetoelectric hydrogel acupuncture system under subcutaneous application, this invention evaluates the biocompatibility of FBTFA nanoparticles and their application in the HAMA / HADA hydrogel system. MTT assay results ( Figure 5As shown in Figure A, after treatment with 100–1000 μg / mL FBTFA for 24 or 48 h, cell viability remained above 80%, with no obvious dose-dependent toxicity observed. Further co-treatment of cells with the same concentration of FBTFA and HAMA / HADA hydrogel extract (HAM / D-FBTFA) also did not result in a decrease in cell viability, suggesting that the hydrogel extract has no significant effect on cell proliferation and metabolism. Cell Calcein-AM / PI live / dead staining (…) Figure 5 (Figure B) further confirmed that all groups of cells showed strong green fluorescence signals after 48 h of culture. Figure 17 As shown in Figure A, there was no significant difference in the number of live cells among the groups, and the number of PI-positive dead cells was relatively small, indicating that the material has further application value for the construction of in vivo delivery systems.

[0083] Based on this, to evaluate the targeting and binding ability of FBTFA nanoparticles to mast cells, fluorescein isothiocyanate (FITC) was grafted onto the surface of the particles, and RBL-2H3 cells were co-incubated for 0–6 h. Flow cytometry was used to analyze the FITC fluorescence intensity of individual cells. The results showed that the proportion of positive fluorescent cells gradually increased over time (from 0 to 78.1%). Figure 5 The fractional part (CD) was observed, and the binding efficiency exceeded 50% at 0.5 h, indicating a high efficiency of particle-cell binding that increased with incubation time. Subsequently, for further observation, this study used SEM to visually evaluate the targeting performance of the FBTFA nanoparticles. Figure 5 Midpoint E and Figure 17 As shown in Figure B, after 2 hours of co-culture, FBTFA NPs were enriched and adhered closely to the cytoskeleton at the periphery of RBL-2H3 cells, while the binding of unmodified FO@BTO NPs was limited. This indicates that the modification of FcεRIA significantly promoted the specific binding of nanoparticles and had good targeting recognition performance.

[0084] 3. Effects of FBTFA magnetoelectric nanoparticle-generated "mechanical-electric" stimulation on calcium influx and degranulation in RBL-2H3 cells Previous studies have shown that, under the control of an external magnetic field, magnetoelectric core-shell nanoparticles Fe3O4@BaTiO3 can achieve energy conversion from magnetic signals to electrical signals through a magneto-electric coupling effect (Y. Zhang et al., 'Wireless-Powering Deep Brain Stimulation Platform Based on 1D-Structured Magnetoelectric Nanochains Applied in Antiepilepsy Treatment', ACS Nano, vol. 17, no. 16, pp.15796–15809, Aug. 2023, doi: 10.1021 / acsnano.3c03661.). Specifically, the magnetic core Fe3O4 generates directional mechanical stress under the action of an alternating magnetic field, which in turn causes a periodic reversal of the lattice polarization of the piezoelectric shell BaTiO3, thereby forming a significant instantaneous potential difference on the nanoparticle surface. This magnetically induced polarization mechanism provides a new approach to achieving non-contact, controllable local electrical stimulation. Based on the above principles, the FBTFA nanoparticles designed by the inventors can convert magnetic signals into synchronous "mechanical-electrical" coupled stimulation under the action of an external magnetic field, thereby achieving precise activation of mast cells. To verify its biological effects, this invention systematically evaluated the FBTFA-mediated magnetoelectric coupling stimulation by detecting changes in intracellular calcium ion current and degranulation release levels in RBL-2H3 cells. Specifically, as... Figure 17 As shown in Figure C, FBTFA-treated RBL-2H3 cells were placed on a dynamic rotating magnetic field device (DLAB, MS-H280-Pro, China). A low-frequency rotating magnetic field was generated by a high-speed rotating multipole magnet, causing microscale mechanical perturbations on the cell surface of the nanoparticles. These perturbations were converted into local electrical signals by the BaTiO3 shell, achieving a passive "force-electric" coupled stimulation process. The magnetic field strength was measured at a distance of 5 mm from the device surface, and three repeated tests were performed at 200 and 500 rpm. The results showed that the magnetic field strength was stable and the stimulation conditions were controllable.

[0085] Calcium ions (Ca²⁺) play a crucial role in the regulation of mast cell function as an important intracellular second messenger. During mast cell activation, the increase in intracellular Ca²⁺ concentration triggers the fusion of granular vesicles with the cell membrane, thereby releasing various neuroactive and immunomodulatory mediators, such as histamine, serotonin (5-HT), adenosine triphosphate (ATP), and β-endorphin. These mediators regulate pain signal transmission and central sensitization processes by acting on peripheral nerve endings and immune cells, exerting analgesic and anti-inflammatory effects. Based on the core role of calcium ions in mast cell activation and analgesia, this invention further evaluates the effect of magnetoelectric stimulation on the degree of mast cell activation and functional regulation by detecting changes in intracellular Ca²⁺ influx. RBL-2H3 cells were labeled with a Fluo-4 AM fluorescent calcium ion probe, and the intracellular Ca²⁺ influx level under a 200 rpm rotating magnetic field was detected by flow cytometry and confocal laser microscopy. Confocal imaging results ( Figure 6 As shown in Figure A, the Fluo-4 AM signal primarily reflects changes in calcium signal at the single-cell level, while DAPI, as a nuclear reverse staining agent, is used for cell labeling and recognition. Based on this, calcium imaging signals were standardized by cell counting to achieve quantitative analysis. Figure 6 (Figure B). The results showed that the fluorescence intensity of the FBTFA magnetic field treatment group (+MS group) was significantly higher than that of other intervention groups. Flow cytometry results ( Figure 6 The midpoint diagram (CD) also shows that the fluorescence intensity in the +MS group was higher than that in the control groups. These consistent findings indicate that FBTFA significantly enhances intracellular Ca²⁺ influx under magnetic field stimulation, suggesting an increased level of cell activation.

[0086] To verify whether magnetoelectric coupling stimulation further drives the degranulation process, we subsequently examined the release levels of typical degranulation markers and various neuroactive mediators. The relative release rate of β-Hex enzyme obtained from the β-Hexosaminidase release assay is generally considered to represent the relative degranulation rate of mast cells. The results showed that ( Figure 6 (Figure 6, center part E) Under the influence of a magnetic field, FBTFA significantly promoted degranulation and release, with a relative release rate of β-Hex enzyme reaching 50.65%, far higher than the non-magnetic field group (-MS group) and the blank control (Untreated group). ELISA detection showed that the levels of histamine (HIS) and serotonin (5-HT) in the cell supernatant of the +MS group were significantly increased (Figure 6, center part FG), and eATP detection also showed a significant increase in extracellular ATP concentration (384.17 nM). Figure 6(Figure H). Overall, FBTFA-mediated mechano-electric stimulation significantly activated RBL-2H3 cells, enhanced Ca²⁺ influx and degranulation release, and induced the secretion of key analgesic mediators.

[0087] RNA-seq analysis further revealed the changes in molecular pathways induced by FBTFA under external magnetic field stimulation. PCA results showed that ( Figure 7 (See Figure A). The FBTFA group and the Untreated group were clearly separated along the PC1 and PC2 dimensions, indicating a significant change in the overall transcriptional profile. (Difference analysis) Figure 7 (Partial diagram B) identified a total of 381 upregulated genes and 78 downregulated genes. Among them, upregulated genes ( Figure 7 Figure C shows calcium signaling mediators (Cacna2d1, Itpr2, Ahnak), immune and degranulation markers (IL3, CD69, Cxcl2), and early response regulators (Fos, Dusp1, Ptgs2, Gpr35). Functionally, these differentially expressed genes and their enriched pathways exhibit a consistent biological orientation, all closely related to mast cell activation and degranulation. Given that calcium ion influx is a key early event triggering mast cell degranulation, the significant upregulation of calcium signaling-related molecules (such as Cacna2d1) suggests that FBTFA may enhance calcium-dependent sensing and responsiveness to stimuli under magnetic field influence. Consistent with this, GO and KEGG enrichment results indicate a significant enhancement of Ca²⁺ signaling, MAPK activation, and immune response pathways. Figure 7 The results (D) are consistent with those obtained from Fluo-4 AM assays of Ca²⁺ influx and release of various degranulation mediators. Further GSEA analysis showed that the upregulated gene was significantly enriched in the immune response (GO:0006955) and cytokine-receptor interaction (rno04060) pathways. Figure 7 (Figure E). The simultaneous activation of MAPK pathway-related genes and multiple immune response molecules, coupled with the significant upregulation of early response genes, provides a crucial molecular basis for the release of inflammatory mediators and signaling molecules after degranulation. This indicates that magnetic field-induced FBTFA stimulation can be rapidly sensed at the transcriptional level and translated into functional cellular responses. In summary, these transcriptomic findings not only molecularly confirm the calcium-dependent signaling activation and immune pathway enhancement driven by magnetoelectric stimulation, but also provide a reasonable mechanistic explanation for the subsequently observed enhanced mast cell degranulation and its in vivo pain regulation mechanism.

[0088] 4. In vivo analgesic effect of mast cell-targeted magnetoelectric hydrogel acupuncture system In vitro experiments demonstrated that the targeted magnetoelectric hydrogel acupuncture system possesses good biocompatibility and can generate coupled magnetoelectric dual stimulation under external magnetic field modulation to promote rapid activation and degranulation function of mast cells. To evaluate the in vivo local analgesic effect of this system, we first established an AA pain model by injecting CFA into the left ankle joint cavity of SD rats according to previous modeling methods, and then... Figure 8 The experimental operation was carried out according to the process of the mid-section diagram A.

[0089] Approximately 48 hours after modeling, obvious redness and swelling of the foot and ankle, as well as persistent and stable local pain, can be observed. Figure 18 (See Figure A). Subsequently, HAMA / HADA hydrogels loaded with different nanoparticles were delivered subcutaneously to the ST-36 acupoint on the left hind limb at a depth of approximately 1 cm, and six groups were established: Untreated control group, AA pain model group (CFA), FBTFA without magnetic field group (–MS), FBTFA combined with magnetic field group (+MS), FOFA combined with magnetic field group (FOFA), and conventional acupuncture control group (CA-N). Two magnetic field intervention groups of rats were placed in a device with a permanent magnet embedded in the bottom and allowed free movement. Based on actual measurements of the magnetic field distribution within the chamber, the magnetic field strength of the device covered the range of 0–60 mT (…). Figure 18 (B). However, when the animal moves along the pre-set walking path, the magnetic field strength in the Zusanli area exhibits continuous spatial changes, forming a dynamic magnetic field exposure driven by movement (B). Figure 18 The magnetic field strength at this acupoint may fluctuate with time and location during free movement (0–6 mT). It should be noted that the instantaneous magnetic field strength at this acupoint may vary with time and location. In contrast, rats in the CA-N group received only routine acupuncture treatment (insertion depth approximately 1 cm, needle insertion and withdrawal frequency approximately 100 times / minute) without additional magnetic field intervention. All groups underwent continuous intervention for 5 days (3–7 days), with each treatment lasting 10 minutes. Pain intensity was assessed 2 hours after each intervention using the classic Von Frey filament test for mechanical pain threshold (PWT). Results showed ( Figure 8 (See Figure B). Both the FOFA and CA-N groups showed an increase in PWT, indicating a certain analgesic effect. However, the +MS group showed a more significant increase in pain threshold from Day 5, suggesting a stronger analgesic effect in this group. In contrast, there was no significant difference in PWT between the -MS group and the CFA model group, indicating that FBTFA failed to produce local analgesia in the absence of external magnetic field activation.

[0090] To further elucidate its analgesic mechanism, we evaluated the effects of different interventions on mast cell degranulation. Toluidine blue staining can specifically label mast cells because its dye reacts metastainingly with chondroitin sulfate glycosaminoglycans within the granules, thus clearly showing the distribution of mast cells and the degranulation process. Toluidine blue staining results of skin sections from the ST-36 acupoint showed ( Figure 8 (Figure C) The number of mast cells in the +MS group was significantly increased, and most of them were in a degranulated state (granules spilling out and cell membrane boundaries blurred). Statistical analysis ( Figure 8 As shown in Figure D, the degranulation rate in the +MS group was as high as 67.5%, significantly better than that in the FOFA group (48.1%) and the CA-N group (49.2%), while the rates in the CFA group and the -MS group were significantly lower, suggesting that the "mechanical-electrical" stimulation driven by the external magnetic field significantly enhanced the activation level of mast cells. Further immunofluorescence staining ( Figure 8 The results (Figure E) showed that mast cell trypsin-like enzyme (MCP7, a marker of mast cell degranulation) released during degranulation in the CFA group was co-expressed with the mechanopathic pain pathway TRPV1, suggesting that the mechanopathic pain pathway of mast cells was activated and degranulated. The staining results of each group were quantitatively analyzed for fluorescence intensity. Figure 8 (See the midpoint diagram FG). It is evident that MCP7 expression was significantly upregulated in the +MS group, while TRPV1 expression was decreased, indicating that this system not only promotes mast cell activation and degranulation but also inhibits peripheral pain signal transduction. Studies have shown that inflammatory factors (such as TNF-α and IL-6) are important signaling molecules mediating pain responses and tissue inflammation. To assess whether the analgesic effect is accompanied by an anti-inflammatory response, we used ELISA to detect the levels of relevant pro-inflammatory factors in serum. Results ( Figure 8 The midpoint plot (HI) showed that, compared with the CFA model group, the levels of TNF-α and IL-6 in the +MS group decreased to 27.3% and 15.1% of those in the model group, respectively (P < 0.0001), suggesting that this intervention also plays an important role in the regulation of peripheral inflammation.

[0091] Previous studies have shown that extracellular ATP released during mast cell degranulation is rapidly hydrolyzed by extracellular nucleotidases to form adenosine—a key signaling molecule that has been repeatedly validated in acupuncture analgesia studies. After binding to sensory neurons, adenosine exerts an inhibitory neuromodulatory effect by activating the adenosine A1 receptor (ADORA1), thereby reducing neuronal excitability. Based on this, we examined the expression of A1R and the neuronal activation marker c-Fos in DRG (L4–L6) neurons corresponding to the pain site in each group of rats. Immunofluorescence results ( Figure 9The midpoint plot (AC) showed that c-Fos was significantly elevated and A1R was compensatorily upregulated in the CFA model group, indicating that neurons were in an overactivated state. In all intervention groups, c-Fos levels decreased, but A1R expression was significantly enhanced in the +MS group (P < 0.0001). This indicates that the excitability of pain-related peripheral sensory neurons was effectively inhibited under the mast cell degranulation-promoted by magnetoelectric stimulation. Therefore, the three parallel changes observed in this study—enhanced mast cell degranulation, upregulation of ADORA1, and decreased TRPV1 expression—may collectively constitute a regulatory axis for converting immune signals into neural signals, thereby effectively weakening the intensity of pain signal transmission in the peripheral stage. This inhibitory effect on sensory afferent neurons may regulate the central opioid system through ascending or feedback pathways, thereby enhancing the analgesic effect. We further examined the level of β-endorphin (β-EP) in cerebrospinal fluid (CSF). Results ( Figure 9 D) The results showed that, compared with the CFA model group, the cerebrospinal fluid β-EP level in the +MS group was significantly increased to 783.83 pg / mL, indicating that under this intervention condition, the central endogenous opioid system may be mobilized to participate in analgesic regulation. Combined with the above findings, it can be inferred that magnetoelectric stimulation may enhance local mast cell degranulation while inhibiting the excitability of peripheral sensory neurons. Simultaneously, by reducing the peripheral pain afferent load, it creates favorable conditions for activating the central analgesic system. In conclusion, these findings support the ability of the magnetoelectric hydrogel acupuncture system to achieve a progressive analgesic effect from the peripheral to the central nervous system through synergistic regulation of local immune-neural interactions and central endogenous analgesic mechanisms.

[0092] IV. Conclusion This invention presents a mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation. This system utilizes mast cell-targeted, magnetically responsive piezoelectric nanoparticles (FBTFA) to achieve microscale "mechanical-electric" coupling stimulation of subcutaneous mast cell membranes at acupoints under an external magnetic field drive in in vitro experiments. This effectively induces calcium influx and degranulation, promoting the rapid release of analgesic mediators such as adenosine. Simultaneously, it activates calcium signaling and immune-related pathways, achieving a rapid functional response in mast cells. In vivo experiments further demonstrate that the spiral needle delivers the adhesive hydrogel loaded with FBTFA nanoparticles to the painful acupoints, significantly enhancing mast cell degranulation rate at the ST-36 acupoint in AA rats, inhibiting TRPV1 expression in the nociceptive pathway, reducing peripheral inflammatory factor levels, and inhibiting DRG neuron excitation through the extracellular ATP-adenosine-A1R pathway while simultaneously increasing the release of endogenous analgesic factors from cerebrospinal fluid, forming a synergistic immune-neuro-endocrine regulatory loop, thereby achieving efficient and controllable local analgesia. It is evident that the mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation prepared in this invention can provide experimental evidence and theoretical support for the modernization of acupuncture in China and for local precision analgesia strategies.

Claims

1. A method for preparing a mast cell-targeted magnetoelectric hydrogel acupuncture system for acupoint stimulation, characterized in that, The method involves functionalizing and coupling Fe3O4@BaTiO3 piezoelectric core-shell nanoparticles with magnetic responsiveness to enable them to target mast cells; then dispersing the nanoparticles in a hyaluronic acid bicomponent hydrogel and loading them into the grooves on the surface of an acupuncture needle with a threaded structure; and finally curing the hydrogel coating with ultraviolet light to obtain the mast cell-targeting magnetoelectric hydrogel acupuncture system. The functionalized coupling involves chemically coupling Fe3O4@BaTiO3 nanoparticles with an anti-FcεRIA antibody to obtain FBTFA nanoparticles.

2. The preparation method according to claim 1, characterized in that, The functionalized coupling operation steps are as follows: first, Fe3O4@BaTiO3 nanoparticles are silanized to fill their surface with amino groups, and then chemically coupled with anti-FcεRIA antibody through EDC and NHS activation reaction.

3. The preparation method according to claim 1 or 2, characterized in that, The hyaluronic acid two-component hydrogel is prepared by photocrosslinking a photoinitiator with a composite hydrogel precursor composed of methacrylamide hyaluronic acid and dopamine hydrochloride modified hyaluronic acid.

4. The preparation method according to claim 3, characterized in that, The hyaluronic acid is sodium hyaluronate with a molecular weight of 50 kDa and hyaluronic acid with a molecular weight of 800 kDa.

5. The preparation method according to claim 3, characterized in that, The weight ratio of the methacrylamide hyaluronic acid to the dopamine hydrochloride modified hyaluronic acid is 2:1; preferably, the mass percentage of the composite hydrogel precursor solution is 6% (w / v).

6. The preparation method according to claim 3, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate, and its concentration is 0.1% (w / v) of the composite hydrogel precursor solution. The crosslinking time is 30 s.

7. The preparation method according to claim 1, characterized in that, The preparation method of the Fe3O4@BaTiO3 piezoelectric core-shell nanoparticles includes the following steps: 1) Ferric chloride, sodium acetate, and PSSMA were dissolved in ultrapure water, and NaOH was added after thorough stirring. The mixture was reacted at 190 °C for 9 h. The product was magnetically separated and washed with ethanol and deionized water to obtain Fe3O4 nanoparticles. (2) Disperse the suspension of Fe3O4 nanoparticles, then add ammonia and TBOT in sequence, stir at room temperature for 1.5 h to obtain Fe3O4@TiO2 nanoparticles; (3) The suspension of Fe3O4@TiO2 nanoparticles was mixed with Ba(OH)2·8H2O solution, ammonia was added, and the mixture was subjected to hydrothermal reaction at 200 °C for 8 h to obtain Fe3O4@BaTiO3 piezoelectric nanoparticles with core-shell structure.

8. The acupuncture needle with a threaded structure according to claim 1, characterized in that, The structural parameters of the acupuncture needle are as follows: needle diameter D = 0.25 mm, total spiral height h = 17 mm, spiral pitch p = 1.3 mm, number of spiral turns n = 10, and thread groove depth d = 0.05 mm.

9. A mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation prepared by the method according to any one of claims 1-8.

10. The use of the mast cell-targeting magnetoelectric hydrogel acupuncture system for acupoint stimulation as described in claim 9 in the preparation of magnetoelectric hydrogel implant materials for delivering acupoint analgesia.

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