Zinc-manganese self-powered battery activated by tumor microenvironment and preparation method of zinc-manganese self-powered battery

Abstract

The invention discloses a zinc-manganese self-powered battery activated by a tumor microenvironment and a preparation method thereof, and belongs to the field of biomedical engineering. According to the self-powered battery, zinc serves as an anode, alpha-MnO2 loaded carbon cloth serves as a cathode, body fluid of TME and endogenous GSH serve as electrolytes directly, and self-powered discharging can be achieved without an external power source. When the battery works, Zn < 2 + > and Mn < 2 + > are released through an oxidation reaction of zinc and a reduction reaction of manganese dioxide, localized release of metal ions is realized through a self-energized electrochemical reaction, a series of reactions are initiated, that is, mitochondrial damage and ROS outbreak are induced through accumulation of Zn < 2 + >, and meanwhile, the released Mn < 2 + > also regulates and controls a tumor microenvironment (TME) index through a Fenton-like reaction. Besides, the GSH-mediated MnO2 reduction reaction rate is self-matched with the TME oxidation-reduction state, and ion release can be dynamically regulated and controlled, so that the growth of tumor cells is inhibited, and an innovative solution is provided for overcoming the limitation of the prior art.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and in particular relates to a tumor microenvironment activated zinc-manganese (Zn / / MnO2) self-powered battery and a preparation method thereof. Background Art

[0002] The application of metal ion delivery systems (such as nanoparticles, liposomes and polymer carriers) in related fields has been widely studied. Such systems release metal ions (such as Mn 2+ 、Zn 2+ ) plays a role, but it has significant limitations. Free metal ions can easily diffuse to non-target tissues through the blood circulation, leading to systemic toxicity, and the tumor accumulation efficiency of nanocarriers is limited by vascular permeability and tumor microenvironment (TME) heterogeneity. The retention time is usually less than 48 hours, and frequent administration is required to maintain the effect. In addition, existing metal ion delivery systems rely on chemical or enzymatic mechanisms to release ions, which makes it difficult to achieve sustained and stable release in the TME, and local ion concentration fluctuations may weaken the regulatory effect on the tumor microenvironment.

[0003] In response to the above problems, a variety of improvement solutions have been proposed in recent years. For example, targeted Mn 2+ Lipid nanocomplexes utilize surface modification to enhance tumor accumulation, but their degradation rate is significantly affected by the redox state of the TME, and ion release stability is insufficient. Furthermore, while cGAMP-based sustained-release hydrogels have some efficacy, they lack synergy with other beneficial effects and are expensive to synthesize, making clinical translation difficult.

[0004] In summary, existing technologies still face the following core challenges: first, how to design a self-powered device to utilize endogenous components of the TME (such as body fluids and glutathione) to drive the localized and sustained release of metal ions; second, how to achieve stable oxidative stress (ROS burst) through electrochemical mechanisms; and third, how to optimize the device structure and reaction kinetics to ensure stable operation under the dynamic physical and chemical conditions of the TME (such as pH = 6.5-7.0, GSH concentration 0.5-10mM). Summary of the Invention

[0005] The present invention aims to provide a tumor microenvironment (TME)-activated Zn / / MnO2 self-powered battery and its preparation method. By utilizing endogenous components of the tumor microenvironment to drive the self-powered discharge of the zinc-manganese battery, the present invention achieves the sustained release of metal ions in the tumor microenvironment, thereby regulating the tumor microenvironment and inhibiting tumor cell growth.

[0006] The self-powered battery uses zinc as anode and α-MnO2 loaded carbon cloth as cathode, and directly uses the body fluid of TME and endogenous GSH as electrolyte, and can achieve self-powered discharge without external power supply. When the battery is working, the zinc is oxidized (Zn→Zn 2+ +2e - ) and the reduction reaction of manganese dioxide (MnO2+2GSH+2H + →GSSG+Mn 2+ +2H2O) releases Zn 2+ With Mn 2+ The present invention realizes the localized release of metal ions through self-powered electrochemical reaction, triggering a series of reactions, namely, through Zn 2+ The accumulation induces mitochondrial damage and ROS burst, and the released Mn 2+ It also regulates TME indicators through a Fenton-like reaction. Furthermore, the GSH-mediated MnO2 reduction reaction rate self-matches with the TME redox state, dynamically regulating ion release and thereby inhibiting tumor cell growth, providing an innovative solution to overcome the limitations of existing technologies.

[0007] The method for preparing a tumor microenvironment (TME) activated Zn / / MnO2 self-powered battery described in the present invention comprises the following steps:

[0008] (1) Preparation of zinc anode

[0009] A medical-grade zinc foil with a purity of ≥99.9% and a thickness of 50 to 100 μm is ultrasonically cleaned with a 3 to 8% by mass dilute hydrochloric acid solution for 3 to 8 minutes to remove the surface oxide layer and impurities, and then rinsed with deionized water 3 to 5 times, each time for 8 to 15 minutes, to ensure that there is no acid residue on the surface of the zinc foil; the rinsed zinc foil is then dried under vacuum at 35 to 50° C. for 10 to 15 hours to obtain a smooth, non-oxidized zinc anode;

[0010] (2) Synthesis of α-MnO2 cathode by hydrothermal method

[0011] 2-3 g of MnSO4·H2O and 2-3 mL of 0.5 M H2SO4 were dissolved in 60-90 mL of deionized water and magnetically stirred for 3-8 minutes until completely dissolved; 1.5-2.0 g of KMnO4 powder was then slowly added and stirred for 20-40 minutes to form a uniform mixed solution; the mixed solution was transferred to a 100 mL polytetrafluoroethylene-lined reactor, sealed, and placed in a forced air drying oven for a constant temperature reaction at 110-130°C for 20-30 hours; after the reaction was completed, the mixture was naturally cooled to room temperature, and the reaction product was collected by vacuum filtration. The reaction product was then washed with deionized water and anhydrous ethanol 3-5 times each to remove unreacted ions and by-products; and finally dried under vacuum at 70-90°C for 10-15 hours to obtain a highly crystalline black α-MnO2 powder.

[0012] α-MnO2 black powder active material, acetylene black conductive agent and polyvinylidene fluoride (PVDF) binder are added together in an agate mortar in a mass ratio of 7:2:1, and the pestle is fully ground for 10 to 15 minutes using a combination of circular motion and up and down squeezing to fully mix the three materials and form a fine black powder; the carbon cloth is pre-ultrasonic cleaned with acetone and ethanol for 10 to 20 minutes each, and vacuum dried at 50 to 70°C to obtain a pretreated carbon cloth; the fine black powder is evenly coated directly on the surface of the pretreated carbon cloth, and the coating thickness is controlled to be 100 to 150 μm, so that the active material loading is 2 to 4 mg / cm 2 The coated carbon cloth is then cured in a vacuum at 50-70°C for 10-15 hours, and then rolled at a pressure of 8-15 MPa to ensure that the active material is tightly bonded to the carbon cloth substrate, ultimately obtaining an α-MnO2 cathode.

[0013] (3) Assembly of self-powered batteries

[0014] The zinc anode and α-MnO2 cathode were cut into 1cm×1cm sheets respectively, and connected with a copper wire with a diameter of 0.1mm. Polydimethylsiloxane (PDMS) sealant was evenly coated on the interface between the copper wire and the electrode and cured at room temperature for 20 to 30 hours to ensure that the interface was effectively isolated from body fluid corrosion. Medical-grade epoxy resin was then used to encapsulate the entire battery. Finally, the self-powered battery was obtained after ethylene oxide sterilization and stored in sterile packaging for future use.

[0015] When the self-powered battery is implanted in the human body, it is necessary to place the self-powered battery around the subcutaneous tumor through minimally invasive surgery to ensure that the electrode is in direct and good contact with the tumor tissue (the encapsulated epoxy resin does not affect the direct and good contact between the electrode and the tumor tissue), and use TME body fluid infiltration to activate the self-powered battery discharge. During the discharge process, the electrochemical workstation monitoring found that the initial voltage was 1.45V, which gradually decayed to 0V within 11 days (corresponding toFigure 4 ), and inductively coupled plasma optical emission spectrometry (ICP-OES) was used to quantify the local Zn 2+ With Mn 2+ In the 4T1 breast cancer model experiment, after a single implantation of the device for 14 days, the tumor volume was significantly suppressed (corresponding to Figure 7 ), its mechanism of action is: Zn 2+ Accumulation in tumor cell mitochondria leads to membrane potential collapse and a 3.8-fold increase in the level of reactive oxygen species (ROS) (corresponding to Figure 6 ), causing oxidative damage to tumor cells. Mn 2+ By participating in the redox reaction in cells, it further regulates the level of oxidative stress in cells. 2+ The ROS burst triggered by these factors synergizes and enhances the damage to tumor cells.

[0016] The biosafety of the device was verified by pathological analysis: no obvious inflammatory reaction was observed at the implantation site, and H&E staining revealed no tissue necrosis or fibrosis; the accumulation of Zn and Mn ions in major organs such as the heart, liver, spleen, lungs, and kidneys was below the median lethal dose (LD50) limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Schematic diagram of the working mechanism of the tumor microenvironment-activated Zn / / MnO2 self-powered battery prepared by the present invention in the tumor microenvironment;

[0018] Figure 2 : Cyclic voltammetry curves of the Zn / / MnO2 self-powered battery prepared by the present invention at different scan rates in 2M ZnSO4 and 0.5M MnSO4 electrolytes;

[0019] Figure 3 : Comparison of cyclic voltammetry of the Zn / / MnO2 self-powered battery prepared in the present invention in PB (phosphate buffer) and in PB electrolyte containing GSH;

[0020] Figure 4 : Comparison of discharge curves of the Zn / / MnO2 self-powered battery prepared in the present invention in PB and in PB electrolyte containing GSH;

[0021] Figure 5 :The present invention uses different treatment methods to act on 4T1 cells and then stains them with JC-1 probe to obtain mitochondrial membrane potential fluorescence imaging images;

[0022] Figure 6 : The present invention uses different treatment methods to treat 4T1 cells and then stain them with DCFH-DA probe to generate confocal images of intracellular reactive oxygen species (ROS);

[0023] Figure 7 : Tumor growth trend diagram of different treatment groups in the mouse 4T1 breast cancer model of the present invention.

[0024] Figure 1 The working principle of the tumor microenvironment activated Zn / / MnO2 battery of the present invention is demonstrated. The figure clearly shows that the zinc anode undergoes oxidation reaction to release Zn 2+ α-MnO2 cathode is converted into Mn under the action of endogenous glutathione (GSH) in the tumor microenvironment. 2+ , while GSH and oxidized glutathione (GSSG) are cyclically converted. This provides an intuitive basis for understanding how batteries use endogenous substances in the tumor microenvironment to produce metal ions, demonstrating the basic principle of how batteries regulate the tumor microenvironment.

[0025] Figure 2 The cyclic voltammetry curves of Zn / / MnO2 battery at different scan rates in 2M ZnSO4 and 0.5M MnSO4 electrolytes are presented. From the curve changes at different scan rates (1-5mV s-1), it is analyzed that at 1.2V and 1.3V (vs. Zn 2 + Characteristic peaks corresponding to ion insertion / deinsertion appear at the Zn / Zn region, and the peak current increases with increasing scan rate. Simultaneously, the capacitance contribution increases from 20% to 38%, which is of great significance for studying the electrochemical performance and reaction kinetics of batteries.

[0026] Figure 3 Comparison of cyclic voltammetry of a Zn / / MnO2 battery in PB and GSH-containing PB electrolytes. This comparison clearly demonstrates the presence of a significant redox peak in the GSH-containing electrolyte, while this peak is subtle or absent in the standard PB electrolyte. This difference strongly demonstrates that GSH, as a redox mediator, significantly promotes the reduction reaction of MnO2 and the overall redox process in the battery, highlighting the key role of GSH in improving battery performance and providing an important reference for optimizing battery performance in the tumor microenvironment.

[0027] Figure 4 A comparison of the discharge curves of a Zn / / MnO2 battery in PB and PB electrolytes containing GSH is shown. The figure shows that the battery's discharge time is significantly prolonged and the discharge plateau voltage is higher in the PB electrolyte with GSH at pH 6.8. This clearly demonstrates that GSH can effectively improve the battery's discharge performance, ensuring the continuous and stable generation of metal ions during battery operation.

[0028] Figure 5The figure shows fluorescence imaging of mitochondrial membrane potential obtained by staining with a JC-1 probe after 4T1 cells were treated with different methods. The figure compares the fluorescence of 4T1 cells after treatment with a control group (Ctr), a single zinc electrode (Zn), a single MnO2 electrode (MnO2), an unconnected battery (UB), and a discharged battery (DB). Among them, the green fluorescence (representing JC-1 monomers of damaged mitochondria) in the DB-treated group was significantly enhanced, while the red fluorescence (representing JC-1 aggregates of normal mitochondria) was weakened, which intuitively shows that the discharged battery can depolarize the mitochondrial membrane potential of tumor cells, thereby affecting the normal function of the cells, providing direct cellular evidence for the battery's inhibition of tumor cell growth. An unconnected battery refers to a battery structure, but no pathway is formed between the positive and negative electrodes, and no current flows. The difference from the control group is that the control group does not involve battery-related effects, while the unconnected battery, although not discharged, is physically in contact with cells. The discharged battery is the battery described in the present invention, which, after being implanted into the tumor microenvironment, uses body fluids as electrolytes and endogenous glutathione (GSH) as a redox medium to produce a self-powered discharge reaction.

[0029] Figure 6 Confocal images of intracellular reactive oxygen species (ROS) in 4T1 cells treated with different treatments were generated using a DCFH-DA probe. Comparison of images from the different treatment groups revealed significantly higher fluorescence intensity in the DB-treated group compared to the other groups. Since the DCFH-DA probe reacts with intracellular ROS to produce fluorescence, this result demonstrates that the discharge battery can significantly increase ROS levels in tumor cells. High concentrations of ROS can damage tumor cells through oxidative stress, playing a key role in regulating the tumor microenvironment.

[0030] Figure 7 The study presents tumor growth trends for different treatment groups in a mouse 4T1 breast cancer model. The figure clearly depicts the changes in tumor volume over time in the control, zinc electrode, MnO2 electrode, UB, and DB treatment groups. Tumor growth in the DB treatment group was significantly inhibited, in stark contrast to the other groups. This intuitively demonstrates the anti-tumor effect of the self-powered battery in vivo and is a key experimental result in validating the effectiveness of battery therapy. DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with examples. However, the content of the present invention is not limited to the following examples and cannot be understood as limiting the scope of protection of the invention.

[0032] Example 1: Preparation of Zn / / MnO2 batteries activated by tumor microenvironment

[0033] (1) Preparation of zinc anode

[0034] Medical-grade zinc foil with a purity of ≥99.9% and a thickness of 80 μm was ultrasonically cleaned with a 5% by mass dilute hydrochloric acid solution for 5 minutes to remove the surface oxide layer and impurities. The foil was then rinsed with deionized water four times for 10 minutes each time to ensure that there was no acid residue on the surface of the zinc foil. The rinsed zinc foil was then dried under vacuum at 45°C for 12 hours to obtain a smooth, non-oxidized zinc anode.

[0035] (2) Preparation of α-MnO2 electrode

[0036] Accurately weigh 2.5g of MnSO₄·H₂O and 2mL of 0.5M H₂SO₄ into 80mL of deionized water. To ensure complete dissolution and uniform mixing, use a magnetic stirrer at 250 rpm for 5 minutes. During this process, the MnSO₄·H₂O gradually dissolves in the H₂SO₄ and deionized water mixture, forming a uniform and stable solution. The addition of H₂SO₄ not only helps to maintain the acidic environment of the solution but also promotes subsequent chemical reactions.

[0037] After the solution is thoroughly mixed, 1.6g of KMnO₄ powder is slowly added. During this addition, the solution is stirred continuously for 30 minutes to allow the KMnO₄ to dissolve quickly and thoroughly, allowing it to fully contact the other components in the solution. As a strong oxidant, KMnO₄ undergoes a complex redox reaction with MnSO₄ in an acidic environment. The mixed solution, after the KMnO₄ is completely dissolved, is transferred to a 100mL Teflon-lined stainless steel autoclave. This autoclave offers excellent high-temperature and corrosion resistance, providing a stable high-temperature and high-pressure environment for the reaction. After sealing the autoclave, it is placed in a thermostatic drying oven set to 120°C for 24 hours. During this 24-hour period, the chemical reaction in the solution continues, ultimately producing α-MnO₂ through a series of intermediate steps. These reaction conditions, optimized through extensive experimentation, ensure a high yield of α-MnO₂ and a well-defined crystal structure.

[0038] After the reaction is complete, allow the reactor to cool naturally to room temperature and then open the reactor. Collect the brown product generated by the reaction by vacuum filtration. Select a Büchner funnel of appropriate specifications and accompanying filter paper, install the vacuum filtration apparatus, and ensure a good seal. Turn on the vacuum pump to create negative pressure within the apparatus. Slowly pour the reaction solution into the Büchner funnel for filtration to effectively separate the solid product from the reaction solution and obtain a preliminary α-MnO2 product. The collected solid product is first rinsed with deionized water several times, using 20 mL of deionized water each time. Gently stir the solid to fully dissolve any impurities adhering to the surface, then filter again. Repeat this rinsing process three times until the filtrate is nearly colorless and transparent, indicating that most water-soluble impurities have been removed. Next, rinse the solid product with ethanol, which further removes any remaining water-soluble impurities and any organic matter. The ethanol rinsing process is similar to the deionized water rinsing process. After three repetitions, place the solid product on a watch glass and place it in a fume hood to allow the ethanol to evaporate naturally.

[0039] After cleaning and ethanol evaporation, the α-MnO2 solid product was transferred to a drying oven, set at 80°C, and dried for 12 hours to obtain a highly crystalline black α-MnO2 powder. The 80°C temperature ensures that the water in the product evaporates completely without damaging the α-MnO2 crystal structure and chemical properties. The dried α-MnO2 electrode material exhibits a uniform brown color, a loose texture, and a high specific surface area, providing a high-quality base material for the subsequent preparation of high-performance electrodes.

[0040] (3) Preparation of cathode

[0041] According to the weight ratio of 7:2:1, the prepared α-MnO2 cathode active material, polyvinylidene fluoride (PVDF, average Mw ~ 1000000) and acetylene black (C) were accurately weighed using an analytical balance with an accuracy of 0.0001g. The three weighed materials were placed in an agate mortar and thoroughly ground and mixed using a pestle. During the grinding process, a combination of circular motion and up and down extrusion was used for 15 minutes to fully mix the three materials and form a fine black powder. This evenly mixed black powder can ensure the uniformity and consistency of the subsequent electrode coating, thereby improving the electrochemical performance of the electrode.

[0042] Prepare a clean carbon cloth and ultrasonically clean it in acetone and ethanol for 15 minutes each to effectively remove oil, dust and other impurities on the surface of the carbon cloth. After cleaning, place the carbon cloth in a vacuum drying oven and dry it at 60°C to achieve a suitable dry state for subsequent coating operations.

[0043] Use a glass rod to apply the evenly mixed black powder directly to the surface of the activated carbon cloth. During the coating process, try to keep the coating thickness consistent. By controlling the coating amount and coating area, the surface loading density of the active material is controlled at about 3mg / cm 2 After coating, the carbon cloth was carefully transferred to a vacuum drying oven, set at 60°C and maintained at a vacuum of -0.08 MPa for 12 hours, and then rolled at a pressure of 10 MPa. Vacuum drying accelerates the removal of moisture and other volatile substances, while preventing impurities in the air from adhering to the coating surface during the drying process, thus ensuring the quality and performance of the electrode. After drying, a firm and uniform black coating formed on the surface of the carbon cloth, which was the prepared α-MnO2 cathode.

[0044] (4) Battery assembly

[0045] Carefully remove the prepared α-MnO2 cathode, use clean tweezers during operation, and be cautious to avoid damaging the coating. At the same time, cut the zinc anode obtained in step (1) into 1cm×1cm sheets, use a copper wire with a diameter of 0.1mm to connect the zinc anode and the α-MnO2 cathode, and evenly apply polydimethylsiloxane (PDMS) sealant at the interface between the copper wire and the electrode. Curing at room temperature for 24 hours effectively isolates the interface from body fluid corrosion. Afterwards, use medical grade epoxy resin for sealing. After sealing is completed, the self-powered battery of the present invention is obtained after ethylene oxide sterilization, and then stored in sterile packaging for standby use.

[0046] Example 2: In vitro performance testing of Zn / / MnO2 batteries activated by tumor microenvironment

[0047] A variety of cancer cell lines, including 4T1, B16-F10, HepG2, Hepa 1-6, and Hela, as well as 3T3 fibroblasts and HL-1 cardiomyocytes, were selected as control cell lines. These cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% streptomycin / penicillin (PS) (percentages are expressed as volume fractions, referring to the proportion of each component's volume to the total volume). The culture flasks were placed in a cell culture incubator at 37°C and 5% CO2, and cell growth was regularly observed. Subsequent experiments were performed when the cells reached the logarithmic growth phase. Cells in the logarithmic growth phase have good proliferative activity and metabolic status, which can more accurately reflect the effect of the battery on the cells.

[0048] The cultured cells were divided into different treatment groups, including a control group (treated with 1x PBS only; PBS is phosphate-buffered saline, used to culture cells), a single zinc electrode group, a single MnO2 electrode group, an unconnected battery group (UB), and a discharged battery group (DB). 5Cells were seeded at a density of 100 cells / well in 6-well plates and treated with the above-mentioned different treatment groups for 12 hours. During the treatment process, the experimental conditions, including cell culture environment and treatment time, were kept consistent across the treatment groups to ensure the accuracy and reliability of the experimental results.

[0049] After treatment, JC-1 fluorescent probe was used for staining, and mitochondrial membrane potential (ΔΨ m ) changes. JC-1 is a cationic dye that, in normal cells, accumulates in mitochondria to form red fluorescent JC-1 aggregates. However, when the mitochondrial membrane potential depolarizes, JC-1 remains in the cytoplasm as a monomer, emitting green fluorescence. By measuring the intensity ratio of green to red fluorescence, changes in mitochondrial membrane potential can be accurately assessed.

[0050] BCECF AM and Hoechst 33342 fluorescent probes were used to label intracellular pH and nuclei, respectively. BCECF AM is a pH-sensitive fluorescent probe whose fluorescence intensity changes with changes in intracellular pH. CLSM was used to observe changes in intracellular pH. Cell supernatants were collected at different time points and their pH values were measured using a pH meter to understand the effects of cell treatment on the intracellular and extracellular pH environments.

[0051] The treated cells were incubated with a DCFH-DA probe. While DCFH-DA itself is non-fluorescent, upon entry into the cells, it is hydrolyzed by intracellular esterases to DCFH, which is then oxidized by ROS to the highly fluorescent DCF. Fluorescence emission was observed using CLSM, and quantitative analysis was performed using FCM to measure intracellular reactive oxygen species (ROS) levels and assess the effects of battery treatment on cellular oxidative stress.

[0052] The treated cells were lysed through three freeze-thaw cycles, and the supernatant was collected by centrifugation. The Ellman assay was used to measure intracellular GSH content. Ellman's reagent reacts with GSH to form a yellow compound. By measuring the absorbance of this compound at 412 nm, the intracellular GSH content can be calculated, thus understanding the effect of cell treatment on the intracellular antioxidant system.

[0053] The cell viability was detected by MTT assay, and the cells were plated at 1×10 5Cells were seeded at a density of 10 cells / well in a 24-well plate and incubated with different treatment groups for 6 hours. MTT solution was then added for a further 4 hours. The supernatant was then removed, and DMSO was added to dissolve the formed formazan crystals. The absorbance at 570 nm was measured using a microplate reader to calculate relative cell viability. Cells were stained with Calcein-AM / PI and observed for viability using two-photon CLSM. Cell apoptosis was assessed using an Annexin V-FITC / PI kit and FCM to assess the effects of the battery on tumor cell growth and survival.

[0054] Example 3: Tumor Microenvironment Activated Zn / / MnO2 Battery Regulation Test on Tumor Microenvironment

[0055] Six-week-old female Balb / c mice were selected and 4T1 cells (1×10 6 100 μL of PBS) was accurately injected subcutaneously into the right back of the mouse, ensuring uniform cell distribution. After inoculation, the mice were housed in an environment with a temperature of 22-25°C, a humidity of 40-60%, and a 12-hour light / dark cycle. The long diameter (a) and short diameter (b) of the tumor were measured daily using a vernier caliper. The formula V = 1 / 2ab was used. 2 Calculate the tumor volume and closely observe the mouse status and tumor growth. When the tumor volume reaches 50-60 mm 3 The mice were randomly divided into groups for subsequent experiments.

[0056] Tumor-bearing mice were randomly divided into five groups (n=8): a control group (implanted with 1×PBS), a zinc electrode group, a MnO2 electrode group, an unconnected battery group (UB), and a discharged battery group (DB). Mice were anesthetized with isoflurane gas on a sterile operating table. The depth of anesthesia was determined to be such that the mice lost their pain reflexes and had stable breathing. After anesthesia, the corresponding battery or control was implanted in the subcutaneous tissue surrounding the tumor. The skin of the mouse was carefully incised using surgical instruments. The battery or control was accurately placed around the tumor, and the skin was sutured to ensure accurate battery placement, minimize damage to surrounding tissue, and ensure adequate contact between the battery and the tumor microenvironment.

[0057] Every day, a vernier caliper was used to measure the long diameter (a) and short diameter (b) of the mouse tumor to calculate the tumor volume. At the same time, a balance was used to weigh the mouse, and the changes in tumor volume and weight were recorded. By drawing the tumor growth curve and the weight change curve, the effect of the battery on tumor growth and its effect on the physical condition of the mouse were intuitively understood.

[0058] After 14 days of treatment, three mice in each group were euthanized and their hearts, livers, spleens, lungs, kidneys, tumors, lymph nodes, and epidermal tissues were collected. These tissues were immediately fixed overnight in 4% paraformaldehyde solution. After fixation, 5-μm-thick sections were prepared and stained with hematoxylin-eosin (H&E). Pathological changes in the tumor tissue, including cell morphology and tissue structure, were observed microscopically to assess the battery's effects on the tumor tissue.

Claims

1. A method for preparing a tumor microenvironment-activated Zn / / MnO2 self-powered battery, comprising the following steps: (1) Preparation of zinc anode Medical-grade zinc foil with a purity of ≥99.9% and a thickness of 50-100 μm is ultrasonically cleaned with a 3-8% by mass dilute hydrochloric acid solution for 3-8 minutes to remove the surface oxide layer and impurities, and then rinsed with deionized water to ensure that there is no acid residue on the surface of the zinc foil; the rinsed zinc foil is then dried under vacuum at 35-50° C. for 10-15 hours to obtain a smooth, non-oxidized zinc anode; (2) Synthesis of α-MnO2 cathode by hydrothermal method 2-3 g of MnSO4·H2O and 2-3 mL of 0.5 M H2SO4 were dissolved in 60-90 mL of deionized water and magnetically stirred for 3-8 minutes until completely dissolved; 1.5-2.0 g of KMnO4 powder was then slowly added and stirred for 20-40 minutes to form a uniform mixed solution; the mixed solution was transferred to a 100 mL polytetrafluoroethylene-lined reactor, sealed, and placed in a forced air drying oven for a constant temperature reaction at 110-130° C. for 20-30 hours; after the reaction was completed, the mixture was naturally cooled to room temperature, and the reaction product was collected by vacuum filtration. The reaction product was then washed with deionized water and anhydrous ethanol 3-5 times each to remove unreacted ions and by-products; and finally dried under vacuum at 70-90° C. for 10-15 hours to obtain a highly crystalline α-MnO2 black powder active material. The dried α-MnO2 black powder active material, acetylene black conductive agent and polyvinylidene fluoride binder are added together in an agate mortar at a mass ratio of 7:2:1, and fully ground for 10 to 15 minutes to fully mix the three materials to form a fine black powder; the fine black powder is evenly coated directly on the surface of the pretreated carbon cloth to control the coating thickness to 100 to 150 μm; The coated carbon cloth is then cured under vacuum at 50-70°C for 10-15 hours, and then rolled at a pressure of 8-15 MPa to ensure that the active material is tightly bonded to the carbon cloth substrate, ultimately obtaining an α-MnO2 cathode. (3) Assembly of self-powered batteries The zinc anode and the α-MnO2 cathode are cut into 1cm×1cm sheets respectively, and the zinc anode and the α-MnO2 cathode are connected by copper wire; polydimethylsiloxane sealant is evenly coated on the interface between the copper wire and the electrode, and cured at room temperature for 20 to 30 hours to ensure that the interface is effectively isolated from body fluid corrosion; then medical-grade epoxy resin is used to encapsulate the entire battery, and finally the self-powered battery is obtained after ethylene oxide sterilization.

2. The method for preparing a tumor microenvironment activated Zn / / MnO2 self-powered battery according to claim 1, characterized in that: In step (1), the sample is rinsed with deionized water for 3 to 5 times, each time for 8 to 15 minutes.

3. The method for preparing a tumor microenvironment activated Zn / / MnO2 self-powered battery according to claim 1, characterized in that: In step (2), the pestle is used to grind the mixture thoroughly by combining circular motion and up and down squeezing.

4. The method for preparing a tumor microenvironment activated Zn / / MnO2 self-powered battery according to claim 1, characterized in that: In step (2), the carbon cloth is ultrasonically cleaned with acetone and ethanol for 10 to 20 minutes each, and vacuum-dried at 50 to 70° C. to obtain a pretreated carbon cloth.

5. The method for preparing a tumor microenvironment activated Zn / / MnO2 self-powered battery according to claim 1, characterized in that: The active material loading in step (2) is 2-4 mg / cm 2 .

6. A tumor microenvironment-activated Zn / / MnO2 self-powered battery, characterized by: The method is prepared by any one of claims 1 to 5.

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