Injectable soft bioelectrode hydrogel and its preparation method and application

Abstract

The present invention belongs to the field of bioelectrode technology, and specifically relates to an injectable soft bioelectrode hydrogel and its preparation method and application. The preparation method includes preparing an E-A complex and preparing a hydrogel. The hydrogel of the present invention is used to prepare an injectable soft bioelectrode, and the hydrogel is directly injected into the target tissue site through a needle to form a flexible and well-adhesive electrical stimulation interface, which significantly improves the accuracy and stability of electrical stimulation. The hydrogel composition principle and preparation method make it easier to regulate and micro-precisely inject according to different human body parts or clinical needs. Implanting the hydrogel into acupuncture points or other specific tissue sites and applying electrical stimulation can effectively avoid local tissue trauma and immune overreaction caused by traditional hard electrodes. For example, for pancreatic cancer, the electrode of the present invention can achieve more controllable and continuous electrical stimulation, and has the potential to regulate local immune response and inflammation levels, providing a safer and more cost-effective new treatment strategy for clinical diagnosis and treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of bioelectrodes, and in particular relates to an injectable soft bioelectrode hydrogel and a preparation method and application thereof. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignant tumor of the digestive system, and its treatment faces multiple challenges. The complex tumor microenvironment (TME) of PDAC contains multiple components, including inflammatory factors, immunosuppressive cells, and metabolic byproducts, which not only promote tumor progression but also seriously affect the efficacy of treatment.

[0003] In the exploration of new avenues beyond traditional treatments, electrical stimulation, as a non-drug therapy, has demonstrated significant advantages in immunomodulation and anti-inflammatory treatment. In particular, electrical stimulation of specific acupuncture points can activate the vagus-adrenal axis, prompting the adrenal glands to release catecholamines, thereby regulating immune responses and inflammation. Compared with traditional treatments, electrical stimulation has fewer side effects and better control of systemic immune responses, reducing the expression of inflammatory cytokines and modulating immune cell function through catecholamines.

[0004] Due to the rigidity and volume limitations of conventional electroacupuncture electrodes, electrical stimulation using rigid electrodes can cause local tissue damage or inflammation, leading to inconsistent treatment outcomes. Furthermore, repeated stimulation with rigid electrodes can cause discomfort, limiting their application in long-term treatment. Therefore, developing flexible, injectable soft bioelectrodes to replace traditional rigid electrodes is crucial for achieving more precise and sustained electrical stimulation. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art. In a first aspect, a method for preparing an injectable soft bioelectrode using a hydrogel is provided;

[0006] A second aspect of the present invention provides a hydrogel for a soft bioelectrode;

[0007] The third aspect of the present invention provides applications of the hydrogel.

[0008] The object of the present invention is achieved by the following technical solution: a method for preparing an injectable soft bioelectrode using hydrogel, which comprises the following steps:

[0009] S1. Preparation of EA complex: Epigallocatechin gallate and 3-acrylamidophenylboronic acid were dissolved in ethanol and allowed to stand to form an EA complex;

[0010] S2. Prepare hydrogel: Dissolve acrylamide in PEDOT:PSS, then add dodecylbenzenesulfonic acid, the EA complex prepared in step S1, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate in sequence, and mix well to form an injectable and highly tissue-adhesive hydrogel.

[0011] As a preferred technical solution, in step S1, the mass ratio of epigallocatechin gallate to 3-acrylamidophenylboronic acid is 0.8-2:1, and the mass volume ratio of 3-acrylamidophenylboronic acid to ethanol is 35-45 mg:1 ml.

[0012] As a preferred technical solution, the standing time in step S1 is 10 to 20 minutes.

[0013] As a preferred technical solution, in step S2, the mass volume ratio of acrylamide to PEDOT:PSS is 500-600 mg:1 ml, and the mass ratio of acrylamide to ammonium persulfate is 40-50:1.

[0014] As a preferred technical solution, the volume ratio of dodecylbenzenesulfonic acid, EA complex and N,N,N',N'-tetramethylethylenediamine in step S2 is 1-4:3-10:1.

[0015] The hydrogel prepared by the above method.

[0016] The hydrogel has the following properties:

[0017] By hydrogen bonding with amino groups in tissues, it has good tissue adhesion, ensuring a firm bond with the target tissue after injection;

[0018] It provides self-healing properties through coordination or other intermolecular forces and has the ability to degrade in vivo to achieve biocompatibility and long-term therapeutic effects.

[0019] The hydrogel is used in the preparation of injectable soft bioelectrodes. The hydrogel is injected into acupuncture points or specific targeted tissue sites, and through external electrical stimulation or a built-in micro-electrical stimulation system, it can achieve precise and continuous electrical stimulation of local tissues.

[0020] As a preferred technical solution, the soft bioelectrode regulates immune response and inflammation levels by electrically stimulating the injection site.

[0021] As a preferred technical solution, the hydrogel is used in the preparation of soft bioelectrodes for the prevention and treatment of pancreatic cancer. After injection, the hydrogel can continuously release electrical stimulation and regulate the local immune environment, thereby assisting in the prevention and treatment of pancreatic cancer.

[0022] As a novel flexible bioelectrode material, the hydrogels prepared in this invention exhibit excellent biocompatibility and flexibility, adapting to tissue deformation and movement and reducing mechanical damage to local tissues. Their electrical conductivity allows for precise electrical stimulation of specific acupuncture points, while their flexibility enables continuous, low-intensity electrical stimulation during treatment, effectively reducing local inflammatory responses and improving the safety and effectiveness of treatment. These hydrogels may play a significant role in synergistic therapies.

[0023] The EA complex component prepared in step S1 of the present invention can be inserted into the polymer chain system through free radical polymerization, provide tissue adhesion to the hydrogel by hydrogen bonding with amino groups in the tissue, and provide self-healing properties and in vivo degradation properties through coordination or other intermolecular forces.

[0024] Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) is a promising conductive polymer, and its hydrogel exhibits a conductivity of approximately 40 S cm -1 High electrical conductivity. However, the brittleness of pure PEDOT:PSS networks limits their applications. By combining a conductive polymer with another polymer network to form a second network within the existing PEDOT:PSS network to construct an interpenetrating polymer network (IPN), the stretchability of the hydrogel can be significantly enhanced.

[0025] The present invention has the following advantages:

[0026] (1) The present invention discloses an injectable soft bioelectrode hydrogel, which is a PEDOT / PAAM dual-network hydrogel with high conductivity, high injectability, and excellent tissue adhesion. The conductive gel provided by the present invention can be directly injected into the target tissue site through a needle, forming a flexible and well-adhesive electrical stimulation interface, significantly improving the accuracy and stability of electrical stimulation. The present invention overcomes the problem of local tissue damage or inflammation caused by the hardness and volume limitations of traditional electroacupuncture, and provides a more accurate and continuous electrical stimulation method.

[0027] (2) The present invention uses dodecylbenzenesulfonic acid (DBSA) as a secondary dopant and EA complex (a borate formed by the reaction of one EGCG molecule with two APBA molecules) as a crosslinker, and polymerizes with AM in the presence of N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS), successfully constructing a PEDOT / PAAM DN hydrogel. The construction principle and preparation method of this hydrogel make it easier to regulate and precisely inject micro-amounts according to different human body parts or clinical needs. This method improves the conductivity and mechanical properties of the hydrogel, enhancing its potential in biomedical applications.

[0028] (3) The hydrogel can regulate the tumor inflammatory microenvironment and enhance the anti-tumor immune response. It can be used to prepare injectable soft bioelectrodes. The hydrogel can activate the vagus-adrenal axis and promote the adrenal gland to release catecholamines, thereby regulating the immune response and inflammation level. It can not only reduce the expression of inflammatory cytokines (IFN-γ, TNF-α and IL-1β), but also regulate the function of immune cells through catecholamines. For example, adrenaline can mobilize NK cells into the bloodstream by activating the β-adrenergic receptors of NK cells and increase the anti-tumor CD8 + T cell infiltration.

[0029] (4) In the Panc02-H7 (mouse pancreatic cancer cell) tumor model, the conductive hydrogel demonstrated excellent therapeutic effects. Its dual effects synergistically and effectively solved the problem of immune escape. The injectable soft bioelectrode provided by the present invention can achieve more controllable and continuous electrical stimulation, providing a safer and more cost-effective treatment strategy for pancreatic cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of mechanical property evaluation of hydrogels are shown in Figure 1. In the figure, a is the stress-strain curve of hydrogels at different EA complex concentrations, and b is the cyclic loading-unloading test.

[0031] Figure 2 Pictures of hydrogels adhering to various organs of mice.

[0032] Figure 3 The experimental results of 9 mM EA composite hydrogel under 1% to 1000% strain and 3 cycles of G′ and G″.

[0033] Figure 4 Evaluation results of the electrical conductivity performance of the hydrogel, where a is the electrical conductivity of the hydrogel at different EA complex concentrations; b is the normalized change of the hydrogel resistance with strain; c is the normalized resistance change of the hydrogel when loaded to 100% strain after 1000 cycles.

[0034] Figure 5 Comparison results of hydrogel injectability. In the figure, a is a comparison of hydrogel injectability: i) hydrogel containing 11mm EA complex and ii) hydrogel containing 9mm EA complex, b is the "SCI" ​​injection mode showing the precise injectability of 9mm EA composite hydrogel, and c is a demonstration of hydrogel injection in chicken breast tissue.

[0035] Figure 6 This is a diagram of the in vitro degradation experimental results of the hydrogel. In the figure, a is a schematic diagram and physical diagram of the in vitro degradation of the hydrogel, and b is the dynamic changes of the mass retention and conductivity during the monitoring of the in vitro degradation process of the hydrogel.

[0036] Figure 7 The graph shows the cell viability results after incubation with hydrogel extracts at different concentrations for 24 hours.

[0037] Figure 8 The live-dead staining results of L929 cells co-cultured with hydrogels.

[0038] Figure 9 In the hemolysis test, the hemolytic ability of hydrogels at different concentrations was compared. In the figure, (a) shows the hemolysis physical diagram of different concentrations of hydrogel (1 mg / mL, 5 mg / mL, and 10 mg / mL) added to the red blood cell suspension, with normal saline and 1% Triton X-100 as negative and positive controls, respectively. (b) shows the absorbance of the solution measured using a microplate reader to quantify its hemolytic ability.

[0039] Figure 10 This is a graph showing the results of gel electrical stimulation of the mouse sciatic nerve. In the figure, a is the in vivo electrical stimulation of the sciatic nerve of a mouse coated with hydrogel; b is the relationship between the number of leg vibrations per second and the stimulation frequency; and c is the amplitude of the hind limb movement when the sciatic nerve is subjected to different electrical stimulation intensities.

[0040] Figure 11 The figures show the anti-tumor effects of different treatments on Pan02-H7 tumor-bearing mice. In the figure, a shows the weight changes of mice during treatment; b shows the survival curves of mice after different treatments; c shows the tumor growth curves of mice under different treatments; and d shows the comparison of tumor weights at the end of treatment.

[0041] Figure 12 This figure shows the biosafety evaluation of Pan02-H7 tumor-bearing mice after different treatments. (a) Representative H&E staining of major organs (heart, liver, spleen, lung, and kidney) after each treatment; (b) Analysis of serum biochemical indicators (ALT, AST, BUN) and liver and kidney function 15 days after each treatment.

[0042] Figure 13 To evaluate the apoptosis of tumors after different treatments, in the figure, a is TUNEL staining of tumors after different treatments; b is the Merge diagram of TUNEL and DAPI staining of tumors after different treatments; c is H&E staining of tumors after different treatments.

[0043] Figure 14 The experimental results of using the injectable soft bioelectrode prepared by the hydrogel of the present invention to improve the tumor immune microenvironment in vivo. In the figure, a is CD80 + and CD86 + DC cells in CD45 + b is the percentage of CD3 + CD8 +T cells in different 32 treatment groups CD45 + The percentage of cells, c is CD3 - NK1.1 + The percentage of NK cells in cells; d is the CD45 + Intracellular CD80 + CD86 + Quantitative analysis of cells; e is CD3 + CD8 + T cells in different treatment groups CD45 + Quantitative analysis of T cells; f is CD3 in different treatment groups - NK1.1 + Quantitative analysis of cells; g is the cytokine level of IFN-γ in the serum of each treatment group on the 15th day; h is the cytokine level of TNF-α in the serum of each treatment group on the 15th day; i is the cytokine level of IL-1β in the serum of each treatment group on the 15th day. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following: Example 1: A method for preparing a hydrogel for a soft bioelectrode, comprising the following steps:

[0045] S1. Preparation of EA complex: Epigallocatechin gallate and 3-acrylamidophenylboronic acid were dissolved in ethanol and allowed to stand for 10 min to form an EA complex, wherein the mass ratio of epigallocatechin gallate to 3-acrylamidophenylboronic acid was 0.8:1, and the mass volume ratio of 3-acrylamidophenylboronic acid to ethanol was 35 mg:1 ml;

[0046] S2. Preparation of a hydrogel: Dissolve acrylamide in PEDOT:PSS, then add dodecylbenzenesulfonic acid, the EA complex prepared in step S1, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate in sequence, and mix evenly to form an injectable and highly tissue-adhesive hydrogel; wherein the mass-to-volume ratio of acrylamide to PEDOT:PSS is 500 mg:1 ml, the mass ratio of acrylamide to ammonium persulfate is 40:1, and the volume ratio of dodecylbenzenesulfonic acid, EA complex, and N,N,N',N'-tetramethylethylenediamine is 1:3:1.

[0047] Example 2: A method for preparing a hydrogel for a soft bioelectrode, comprising the following steps:

[0048] S1. Preparation of EA complex: Epigallocatechin gallate and 3-acrylamidophenylboronic acid were dissolved in ethanol and allowed to stand for 20 min to form an EA complex, wherein the mass ratio of epigallocatechin gallate to 3-acrylamidophenylboronic acid was 2:1, and the mass volume ratio of 3-acrylamidophenylboronic acid to ethanol was 45 mg:1 ml;

[0049] S2. Preparation of a hydrogel: Dissolve acrylamide in PEDOT:PSS, then add dodecylbenzenesulfonic acid, the EA complex prepared in step S1, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate in that order, and mix well to form an injectable and highly tissue-adhesive hydrogel; wherein the mass-to-volume ratio of acrylamide to PEDOT:PSS is 600 mg:1 ml, the mass ratio of acrylamide to ammonium persulfate is 50:1, and the volume ratio of dodecylbenzenesulfonic acid, EA complex, and N,N,N',N'-tetramethylethylenediamine is 4:10:1.

[0050] Example 3: A method for preparing a hydrogel for a soft bioelectrode, comprising the following steps:

[0051] S1. Preparation of EA complex: Epigallocatechin gallate and 3-acrylamidophenylboronic acid were dissolved in ethanol and allowed to stand for 15 min to form an EA complex, wherein the mass ratio of epigallocatechin gallate to 3-acrylamidophenylboronic acid was 1.5:1, and the mass volume ratio of 3-acrylamidophenylboronic acid to ethanol was 38 mg:1 ml;

[0052] S2. Preparation of a hydrogel: Dissolve acrylamide in PEDOT:PSS, then add dodecylbenzenesulfonic acid, the EA complex prepared in step S1, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate in that order, and mix well to form an injectable and highly tissue-adhesive hydrogel; wherein the mass-to-volume ratio of acrylamide to PEDOT:PSS is 540 mg:1 ml, the mass ratio of acrylamide to ammonium persulfate is 47:1, and the volume ratio of dodecylbenzenesulfonic acid, EA complex, and N,N,N',N'-tetramethylethylenediamine is 2:7:1.

[0053] The beneficial effects of the present invention are described below by experiments:

[0054] 1. Preparation of the hydrogel of the present invention:

[0055] (1) Preparation of EA complex

[0056] 46 mg of epigallocatechin gallate (EGCG) and 38 mg of 3-acrylamidophenylboronic acid (APBA) were dissolved in 1 mL of anhydrous ethanol and allowed to stand for 15 minutes to form an EA complex.

[0057] (2) Preparation of PEDOT / PAAM DN hydrogel

[0058] 1. Prepare PEDOT / PAAM DN hydrogel according to the formula ratio in Table 1

[0059] Table 1: Amount of each raw material

[0060] hydrogel PEDOT:PSS AM DBSA EA TEMED APS PEDOT / PAAM DN hydrogel 1mL 560mg 30μL 50 μL 10 μL 12.5mg

[0061] (a) 560 mg of AM was accurately weighed and mixed using a vortex mixer at room temperature for 5 min to ensure complete dissolution in 1 mL of PEDOT:PSS PH1000 aqueous solution.

[0062] (b) 30 μL DBSA, 50 μL EA, 10 μL TEMED, and 12.5 mg APS were added to the above mixture in sequence, vortexing at room temperature to ensure that each component was evenly mixed.

[0063] (c) After the addition of APS, the gel preparation solution was quickly transferred into the prepared mold by vortexing for 10 seconds and allowed to gel at room temperature for 3 minutes.

[0064] 2. Characterization of PEDOT / PAAM DN Hydrogel

[0065] (1) Mechanical property evaluation of hydrogel: The hydrogel was solidified in a dog-bone-shaped mold (35 mm long, 4 mm wide, and 2 mm high). The mechanical properties were evaluated by tensile testing using a CMT5000 electronic universal material testing machine (Sasck, Zhuhai, China). The test was performed in tensile mode with a set tensile rate of 5 mm / min. The results are shown in Figure 2. Figure 1 As shown. Figure 1 It can be seen that the elongation at break of the hydrogel increased with increasing EA complex concentration from 3mM to 9mM, indicating a positive correlation between concentration and mechanical integrity. Based on the balance between mechanical strength and syringeability, 9mM EA complex concentration was selected as the optimal concentration for subsequent experiments. When the EA complex concentration was 9mM, the hydrogel had an elongation at break of 320% and an elastic modulus of 59.25kPa. The hydrogel showed minimal hysteresis during 50%, 100%, and 150% cyclic loading. This phenomenon is attributed to the stretching of the molecular chains in the conductive polymer network, rather than fracture, meeting the requirements of long-term use of implantable electrodes.

[0066] (2) Tissue adhesion of hydrogel: PEDOT:PSS / PEAM hydrogel was cut into rectangular block samples with a length of 30 mm, a width of 4 mm, and a height of 2 mm. The mice were dissected to obtain the organs, and the organs were washed with phosphate buffered saline (PBS) to remove residual blood, and then the surface of the organs was wiped dry. Use tweezers to clamp one corner of the hydrogel sample and the corresponding organ, keep contact for about 10 seconds, then release and lift the hydrogel sample, record and photograph the adhesion between the organ and the hydrogel, and the results are shown in Figure 2. Figure 2 As shown, the hydrogel was observed to adhere well to various organs and tissues of mice.

[0067] (3) Evaluation of hydrogel rheological properties: All hydrogel samples to be tested were uniformly cut into pieces with a diameter of 10 mm and a thickness of 3 mm to ensure consistency. Small amplitude shear oscillation tests were performed within the linear viscoelastic range of the hydrogel, applying a constant shear rate of 0.1 to 10 rad / s. HAAKE TM MARS TM Rheometer (Thermo Scientific TM , China) measured the storage modulus (G′) and loss modulus (G′) of the composite hydrogel, and the results are shown in Figure 3 As shown, strain amplitude sweep tests determined a cyclic test strain threshold of 1000%. At high strain, the storage modulus (G') dropped significantly below the loss modulus (G"), indicating structural damage. However, reducing the strain to 1% allowed the network to self-heal. Even after three cycles, no significant change in the modulus was observed, confirming the hydrogel's strong self-healing ability.

[0068] (4) Evaluation of hydrogel conductivity: The conductivity of PEDOT:PSS / PEAM hydrogel was measured using a standard four-probe method using an ST2242 four-probe resistivity tester (Suzhou Lattice Electronics Co., Ltd, China). The hydrogel was processed into a 1 cm × 1 cm circular sheet to fit the testing machine to ensure the accuracy of the measurement results. The conductivity under strain was measured using a CMT5000 electronic universal material testing machine with a 1 mm s -1 The resistance between the two terminals was measured by stretching the sample at a rate of 100 nm and recording it using a digital multimeter (Keithley DAQ6510). Figure 4As shown, at EA complex concentrations of 3mM, 6mM, and 9mM, the conductivity of the hydrogel stabilized at around 2.8S / m, which is higher than the conductivity of cerebrospinal fluid (CSF), the most conductive fluid in the human body (1.538S / m). In addition, the resistivity of the hydrogel slowly increased during stretching, which is closely related to the network structure of the hydrogel. At low strain levels, the molecular chains in the conductive network are stretched, with minimal damage to the PEDOT chains. Therefore, at 100% strain, the resistance change (ΔR / R0) of the PEDOT / PAAM DN20 hydrogel is only about 1.15, and its resistance remains unchanged even after 1000 repeated stretching cycles. This excellent conductivity and stability provide a solid foundation for the long-term application of hydrogel electrodes at specific acupuncture points.

[0069] (5) Hydrogel injectability evaluation: PEDOT:PSS / PAAM hydrogels containing different concentrations of EA complex (9mM and 11mM) were prepared and loaded into 1ml syringes to promote gel polymerization. During the test, the hydrogels were injected from the syringe into glass slides, culture dishes, and chicken breasts to simulate real clinical injection conditions. The morphology of the hydrogels after injection was carefully observed and recorded. The results are shown in Figure 2. Figure 5 As shown, hydrogels containing more than 9mM EA complexes could not be smoothly injected through the needle. In contrast, hydrogels containing 9mM EA complexes exhibited stable injectability, forming a solid structure shortly after extrusion (approximately 90 seconds). This precise injectability further demonstrated the ability of the hydrogel to be extruded into letters on a petri dish, highlighting its excellent performance. In addition, to evaluate the applicability of the hydrogel in biological tissues, this study carried out an experimental procedure to inject the hydrogel into chicken breast muscle, which not only demonstrated the good injectability of the hydrogel, but also highlighted its potential for application in biological tissues. The "SCI" ​​injection mode shows the precise injectability of the 9mM EA composite hydrogel. Demonstration of hydrogel injection in chicken breast tissue.

[0070] 3. In vitro degradation of PEDOT / PAAM DN hydrogel

[0071] To evaluate the in vitro degradation performance of PEDOT:PSS / PAAM DN hydrogels, 3.0 mL of phosphate buffered saline (PBS) was added to a six-well culture plate and the hydrogel sample was placed on a cell culture insert. The culture plate was then placed in an incubator at 37°C and shaken at 70 rpm. Every 48 hours, the PBS was removed, the remaining hydrogel sample on the cell culture insert was weighed, and its mass change was recorded. These data were used to draw the in vitro degradation curve of the hydrogel, and the results are shown in Figure 2. Figure 6As shown, the hydrogel initially swelled over the first 7 days and then gradually degraded over 15 days while maintaining its overall morphology and conductivity. This sustained performance may be attributed to the interpenetrating polymer network structure, which provides enhanced cross-linking density and hydrophilicity.

[0072] 4. Biosafety Experiment

[0073] (1) Cell culture

[0074] L929 cells were cultured in DMEM supplemented with 10% FBS and 1% PS in an incubator at 37°C and 5% CO2.

[0075] (2) The biosafety of the gel can be verified in in vitro experiments

[0076] (a) The effect of hydrogel on L929 cell viability was determined by CCK8 method. 8000 L929 cells were seeded in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. Subsequently, hydrogel extracts of different concentrations (0.1, 1, 2, 5 and 10 mg / mL) were co-cultured with L929 cells for 24 hours. After the co-culture, complete culture medium containing 10% CCK-8 reagent was added to the cells and cultured at 37°C for another hour. The absorbance was measured at a wavelength of 450 nm using a microplate reader (MOLECULAR DEVICES, America) to evaluate cell viability. The experimental results are shown in Figure 2. Figure 7 As shown, after incubation with L929 cells for 24 h, PEDOT:PSS / PAAM DN showed no obvious cytotoxicity compared with the control group even when the hydrogel suspension concentration was increased from 1 mg / mL to 10 mg / mL.

[0077] (b) The effect of hydrogel on L929 cell viability was determined by live-dead staining. L929 cells were also seeded into confocal culture dishes and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, the cells were treated with hydrogel extract (10 mg / mL) for 24 hours. Afterwards, the culture medium was replaced with a PBS solution containing 2mM Calcein-AM and 4.5mM propidium iodide (PI). Cell viability was assessed using FV3000 (Olympus, Tokyo, Japan), where live cells emitted green fluorescence (Ex: 490nm, Em: 515nm) due to Calcein-AM staining, and dead cells emitted red fluorescence (Ex: 535nm, Em: 617nm) due to PI staining. The experimental results are shown in Figure 2. Figure 8 As shown, the confocal images showed strong green fluorescence signals from the cells, indicating that the cytotoxicity of the hydrogel was negligible.

[0078] (c) Hemocompatibility of the hydrogel was determined by its hemolytic activity: Mouse blood was collected and centrifuged at 1500 rpm for 5 minutes at 4°C. The serum was removed and an equal volume of freshly prepared 0.9% saline solution was added. This process was repeated three times to completely remove residual serum. After the final rinse, the saline was discarded and the red blood cells (RBCs) were resuspended in 0.1 M PBS and diluted 25-fold to a concentration of 1.11 × 10 8 mL -1 Subsequently, hydrogels at concentrations of 1 mg / mL, 5 mg / mL, and 10 mg / mL were added to the red blood cell suspension. The negative control was normal saline, and the positive control was 1% TritonX-100. After incubation at 37°C for 60 minutes, the suspension was collected and centrifuged at 1500 rpm for 5 minutes. The supernatant was transferred to a 96-well plate, and the absorbance of the solution was measured using a microplate reader (MOLECULAR DEVICES, America). The experimental results are shown in Figure 2. Figure 9 As shown, compared with triton X-100, the hydrogel had no obvious hemolytic effect even at a concentration of 10 mg / mL, highlighting its good biocompatibility

[0079] 5. Verification of the Gel's Electrical Stimulation Ability in Mouse Sciatic Nerve

[0080] In the experiment, an incision of approximately 1 cm was made in the right sciatic nerve region of the mouse to expose the sciatic nerve, and the sciatic nerve coated with hydrogel was injected. Using the SDZ-II six-channel electronic acupuncture device, the hydrogel on the sciatic nerve was electrically stimulated at different frequencies (5, 10, 20, and 50 Hz) and amplitudes (0.1-0.9 V). The contraction response of the hind limb muscles was observed and recorded. The experimental results are shown in Figure 2. Figure 10 As shown in the figure, the number of vibrations per second (NO.of vibrations per second) did not increase with the increase of stimulation frequency, which may indicate that within a certain frequency range, the muscle response to electrical stimulation is not linear. This nonlinear response may be related to the physiological properties of the muscle and the conductivity of the hydrogel. Under other stimulation parameters, such as amplitude (0.1-0.9V), we observed that the change in muscle contraction angle increased with the increase of stimulation voltage, further confirming that hydrogels can effectively transmit nerve signals at different intensities, thereby promoting muscle contraction. These findings collectively emphasize the potential of hydrogels as an effective medium for neuromuscular signal transmission under in vitro conditions.

[0081] 6. Anti-tumor ability of hydrogel

[0082] (1) Cell culture

[0083] Panc02-H7 cells were cultured in DMEM supplemented with 10% FBS and 1% PS in an incubator at 37°C and 5% CO2.

[0084] (2) ST36 accuracy verification

[0085] (a) 6- to 8-week-old C57 / 6J female mice were purchased from Guizhou Bio-Han Biotechnology Co., Ltd. Mouse feeding and in vivo experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Ethical Guide for Animal Experimentation.

[0086] (b) For the construction of mouse tumor model, 1×10 7 Panc02-H7 cells (100 μL) were injected subcutaneously into the right hind thigh of each mouse. Tumor volume was calculated by length (mm) × width (mm) × width (mm) / 2. Tumors grew to 20 mm. 3 Get treatment.

[0087] (c) Mice were randomly divided into five groups: control group, non-acupoint injection group, non-acupoint electrical stimulation group (ENA), ST36 acupoint injection group, and ST36 acupoint electrical stimulation group (EA). The EA group received 10 Hz, 1 mA EA treatment every other day for a total of seven times.

[0088] (d) Immediately after the last EA treatment, blood samples were collected from mice, and the levels of inflammation-related cytokines in the serum were analyzed using enzyme-linked immunosorbent assay (ELISA) kits.

[0089] (3) In vivo anti-tumor experiments

[0090] (a) 6- to 8-week-old C57 / 6J female mice were purchased from Guizhou Bio-Han Biotechnology Co., Ltd. Mouse feeding and in vivo experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Ethical Guide for Animal Experimentation.

[0091] (b) For the construction of mouse tumor model, 1×10 7 Panc02-H7 cells (100 μL) were injected subcutaneously into the right hind thigh of each mouse. Tumor volume was calculated by length (mm) × width (mm) × width (mm) / 2. Tumors grew to 20 mm. 3 Get treatment.

[0092] (c) Mice were randomly divided into six groups (n = 5 per group): PBS, free MMAE, electroacupuncture (EA), PLGA, MMAE@PLGA, and EA+MMAE@PLGA. PBS, free MMAE, PLGA, and MMAE@PLGA (MMAE at 4 mg / kg each) were injected via the tail vein, and 50 μL of PEDOT:PSS / PAAM DN hydrogel was injected into the ST36 acupoint. The EA group received 10 Hz, 1 mA EA every other day for a total of seven sessions.

[0093] (d) The body weight and tumor volume of mice were measured every day, and the tumor volume was calculated according to the formula: 0.5×L×W2. After 15 days of treatment, blood, tumor tissues and major organs (heart, liver, spleen, lung, and kidney) were collected from each group. Hematoxylin and eosin (H&E) staining was used to evaluate the morphological abnormalities of the organs. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) levels were measured to evaluate liver and kidney function. The experimental results are shown in Figure 2. Figure 11 and Figure 12 As shown in the figure, during the treatment period, there was no significant change in body weight in the different treatment groups except the free MMAE treatment group. Due to the high systemic toxicity of MMAE, Pan02-H7 tumor-bearing mice died on the 5th day, while the survival rate of mice in the other treatment groups was 100%. Compared with the PBS group, the EA treatment group slightly inhibited tumor growth, while the tumor volume in the MMAE@PLGA and EA+MMAE@PLGA treatment groups was the smallest (26.37±10.67mm 3 ), compared with the EA group (96.63±49.34mm 3 ) and MMAE@PLGA group (52.00±13.26mm 3 ), the inhibition rates were 72.7% and 49.3%, respectively. The results showed that the combined strategy of MMAE@PLGA and EA significantly inhibited tumor growth, showing a synergistic effect. No obvious damage or tissue degeneration occurred in all treatment groups, further demonstrating the excellent stability of MMAE@PLGA and the safety of the conductive hydrogel EA. In addition, ALT, AST, and BUN were all within the normal range, confirming that EA+MMAE@PLGA treatment did not cause obvious liver and kidney dysfunction. In summary, these findings indicate that PEDOT:PSS / PAAM DN hydrogels are biocompatible for anti-tumor applications.

[0094] (4) Causes cell apoptosis in tumor tissues in vivo

[0095] The tumor tissue was fixed by immersion in 4% paraformaldehyde solution at 4°C for 12 hours and then dehydrated with 30% sucrose solution. The dehydrated tumor tissue was embedded in OCT compound (Sakura, Torrance, CA, USA) and sectioned using a freezing microtome (Leica, Germany). The sectioned tumor tissue samples were then stained with 4',6-diamino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) dye to mark the cell nucleus and detect apoptotic cells. Finally, a confocal fluorescence microscope (Olympus FV3000, Tokyo, Japan) was used to collect fluorescence image data of the stained samples. The collected image data were analyzed by ImageJ software to quantitatively evaluate the apoptosis of tumor cells. The experimental results are shown in Figure 2. Figure 13 As shown, stronger fluorescence signals were observed in the MMAE@PLGA and EA+MMAE@PLGA groups compared to the PBS group, indicating increased tumor cell apoptosis. In contrast, only a small number of apoptotic cells were detected in the PLGA and EA-treated groups. These findings confirm the cytotoxic effects of chemotherapy combined with EA treatment on tumor cells and provide a basis for future mechanistic studies.

[0096] (5) Improving the tumor immune microenvironment in vivo

[0097] Local tumor tissue obtained from Pan02-H7 tumor-bearing mice was processed and prepared into a single-cell suspension. The cell suspension was washed with sterile phosphate-buffered saline (PBS) to remove residual tissue debris and unbound antibodies. The cell suspension was incubated for 30 minutes at room temperature with the following fluorescein-conjugated specific antibodies: anti-CD45 antibody conjugated to PE (R-phycoerythrin), anti-CD3 antibody conjugated to APC / Cy7 (Allophycocyanin / Cyanine7), anti-CD8a antibody conjugated to AF647 (Alexa Fluor 647), anti-CD11c antibody conjugated to BV421 (Brilliant Violet 421), anti-CD80 antibody conjugated to PE, anti-CD86 antibody conjugated to AF488 (Alexa Fluor 488), and anti-NK1.1 antibody conjugated to PE. All antibodies were purchased from BioLegend. After incubation, the cell suspension was filtered through a nylon cell strainer with a pore size of 70 μm to ensure uniform distribution of cells and analyzed using a flow cytometer (Beckman Coulter, Cytoflex / Dxflex). The collected data were then further analyzed and processed using FlowJo software. Figure 14As shown in Figure 3, the percentage of mDCs in the MMAE@PLGA and EA+MMAE@PLGA treatment groups increased significantly, reaching 34.97% and 45.09%, respectively, which were 2.6-fold and 3.3-fold higher than those in the PBS control group. In contrast, the EA group only increased slightly to 20.86%. Similarly, the CD8 + T cell infiltration increased to 5.37%, 7.02% and 11.28%, respectively, which were 6.0 times, 4.6 times and 9.6 times higher than those in the PBS group. For NK cells, the EA and EA+MMAE@PLGA groups increased significantly to 8.08% and 7.85%, while the MMAE@PLGA group (3.1%) showed no significant change compared with the PBS control group (1.52%). In addition, mice treated with EA+MMAE@PLGA showed significantly reduced levels of proinflammatory cytokines, including IFN-γ, TNF-α and IL-1β, indicating that the inflammatory response mediated by EA intervention was inhibited at ST36. The results showed that MMAE@PLGA nanoparticles effectively induced tumor cell apoptosis by continuously releasing MMAE, while promoting DC maturation and T cell infiltration, activating anti-tumor immunity. At the same time, EA at ST36 reduced the inflammatory infiltration of tumor tissue, downregulated proinflammatory factors at local and systemic levels, and enhanced CD8 + The infiltration and anti-tumor activity of T cells and NK cells were detected, although the effect on DC maturation was not obvious.

[0098] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. A method for preparing an injectable soft bioelectrode using hydrogel, characterized in that: It includes the following steps: S1. Preparation of EA complex: Epigallocatechin gallate and 3-acrylamidophenylboronic acid were dissolved in ethanol and allowed to stand to form an EA complex; S2. Preparation of hydrogel: Dissolve acrylamide in PEDOT:PSS, then add dodecylbenzenesulfonic acid, the EA complex prepared in step S1, N,N,N',N'-tetramethylethylenediamine and ammonium persulfate in sequence, mix well to form a hydrogel; the concentration of the EA complex in the hydrogel is 3-9mM.

2. The method for preparing an injectable soft bioelectrode hydrogel according to claim 1, characterized in that: In step S1, the mass ratio of epigallocatechin gallate to 3-acrylamidophenylboronic acid is 0.8-2:1, and the mass volume ratio of 3-acrylamidophenylboronic acid to ethanol is 35-45 mg:1 ml.

3. The method for preparing an injectable soft bioelectrode hydrogel according to claim 1, characterized in that: The standing time in step S1 is 10 to 20 minutes.

4. The method for preparing an injectable soft bioelectrode hydrogel according to claim 1, characterized in that: In step S2, the mass volume ratio of acrylamide to PEDOT:PSS is 500-600 mg:1 ml, and the mass ratio of acrylamide to ammonium persulfate is 40-50:

1.

5. The method for preparing an injectable soft bioelectrode hydrogel according to claim 1, characterized in that: The volume ratio of dodecylbenzenesulfonic acid, EA complex and N,N,N',N'-tetramethylethylenediamine in step S2 is 1-4:3-10:

1.

6. A hydrogel prepared by the method according to any one of claims 1 to 5.

7. Use of the hydrogel according to claim 6 in preparing an injectable soft bioelectrode.

8. The use according to claim 7, characterized in that The soft bioelectrode regulates immune response and inflammation levels by electrically stimulating the injection site.

9. Use of the hydrogel according to claim 6 in preparing a soft bioelectrode for preventing and treating pancreatic cancer.

Images