Cell selective killing device based on surface and interface pH regulation and application thereof

Abstract

The invention relates to a selective cell killing device based on surface and interface pH regulation and application thereof. The method comprises the following steps: depositing a noble metal material capable of regulating and controlling extracellular proton concentration on one side of a substrate to form a regulation and control layer, and connecting the regulation and control layer to an external preset dynamic voltage control unit through a wire; the regulation and control layer is attached to the surface of the tissue; a dynamic voltage is periodically applied to the regulation and control layer through an external preset dynamic voltage control unit, so that the pH value of the interface microenvironment of the regulation and control layer and tissue cells is alternately changed between 5 and 8, and accurate selective killing of the cells is realized. A preset dynamic voltage control scheme based on a cancer cell glycolysis oscillation period is adopted, so that the pH value of a local surface interface can be regulated and controlled according to a set period, the excretion of lactic acid and protons of cancer cells is continuously and effectively interfered, and the cancer cells are prevented from generating adaptive reaction.

Description

Technical Field

[0001] The present invention relates to the technical field of precision cancer treatment, and particularly to a cell selective killing device based on surface and interface pH regulation and its application. Background Art

[0002] Traditional cancer treatment methods mainly include surgical treatment, radiotherapy, and chemotherapy with chemical drugs, etc. However, these methods usually lack selectivity. While killing cancer cells, they will also cause serious damage to normal cells, resulting in serious side effects and seriously affecting the quality of life of patients. Summary of the Invention

[0003] In view of the above problems, a cell selective killing device based on surface and interface pH regulation and its application are provided.

[0004] The specific technical solutions are as follows: The first aspect of the present invention is to provide a cell selective killing device based on surface and interface pH regulation, including a dynamic voltage control unit, a base layer, and a regulation layer deposited on the base layer. The regulation layer is connected to the dynamic voltage control unit through a wire.

[0005] Further, the base layer is made of one of anodic aluminum oxide nanopores, borate glass nanotubes, polyurethane fiber films, or indium tin oxide conductive glass.

[0006] Further, the regulation layer is formed by noble metal deposition.

[0007] Further, the noble metal is one of palladium or platinum.

[0008] Further, the dynamic voltage control unit is a DC regulated power supply or a controllable potentiostat.

[0009] The second aspect of the present invention is to provide the above cell selective killing device for selectively killing cancer cells.

[0010] The third aspect of the present invention is to provide a cell selective killing method based on surface and interface pH regulation, including: Depositing a noble metal material capable of regulating extracellular proton concentration on one side of the substrate to form a regulation layer, and connecting the regulation layer to an externally preset dynamic voltage control unit through a wire; Attaching the regulation layer to the tissue surface; Periodically applying a dynamic voltage to the regulation layer through the externally preset dynamic voltage control unit, so that the pH value of the interface microenvironment between the regulation layer and the tissue cells alternates between below 5 and above 8, thereby achieving precise selective killing of cells.

[0011] Further, the method of applying the dynamic voltage is: (i) Positive voltage stage: Apply a voltage of +0.5 V - +0.8 V for 15 - 30 seconds, causing the pH value of the interfacial microenvironment to rapidly drop below 5; (ii) Negative voltage stage: Apply a voltage of -0.3 V - -0.5 V for 15 - 30 seconds, causing the pH value of the interfacial microenvironment to rapidly rise above 8.

[0012] Studies have found that there are significant differences between cancer cells and normal cells in terms of physiological and metabolic characteristics. Under normal circumstances, cancer cells rely on aerobic glycolysis as the main energy source and will transiently ingest glucose and produce glycolytic oscillations under certain extreme culture conditions (such as starvation and hypoxia), that is, the lactate release of cancer cells shows periodic oscillation characteristics, where the rising and falling stages each last about 15 seconds, constituting an oscillation waveform with a total period of 30 seconds. In contrast, normal cells tend to mitochondrial oxidative phosphorylation, neither producing glycolytic oscillations nor having a large amount of lactate efflux. At the same time, cancer cells also generally highly express monocarboxylate transporter 4 (MCT4), which maintains intracellular acid-base balance by rapidly effluxing lactate and protons, promoting their proliferation and survival; while the expression level of MCT4 in normal cells is relatively low.

[0013] Based on the above strategy, if the pH can be forcibly changed to extremely acidic (pH < 5) or extremely alkaline (pH > 8) in a local area, it can significantly interfere with its efflux function, thereby inducing intracellular acidification in cancer cells and ultimately causing cell death, while normal cells are less affected due to the lack of significant oscillation characteristics. The above differences provide a potential targeted strategy for the selective killing of cancer.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: 1) By adopting a preset dynamic voltage control scheme based on the glycolytic oscillation period of cancer cells, it is possible to regulate the local surface pH value according to the set period, thereby continuously and effectively interfering with the lactate and proton efflux of cancer cells and preventing cancer cells from developing adaptive responses; 2) Provide a general interfacial regulation strategy. By selecting appropriate noble metal electrodes and electrode template materials, it can flexibly adapt to the metabolic characteristics of different cancer cells and achieve precise regulation of various target products and scenarios; 3) Utilize the significant differences between cancer cells and normal cells in terms of metabolic patterns and glycolytic oscillations to achieve precise selective killing of cancer cells, greatly reducing the side effects on normal cells, and significantly improving the precision, safety, and effectiveness of cancer treatment. Brief Description of the Drawings

[0015] Figure 1 It is a schematic diagram of the device of the present invention that can be prepared on different templates; Figure 2 It is a schematic diagram of the diverse usage scenarios of the device of the present invention; Figure 3 Schematic diagram of the working process of the device of the present invention; Figure 4 Verification experimental result diagram of the pH regulation effect after the positive voltage stage and the negative voltage stage in the present invention; Figure 5 Fluorescent staining diagram of the live and dead cell states after stimulation of the H8 cell line in the present invention; Figure 6 Fluorescent staining diagram of the live and dead cell states after stimulation of the HeLa cell line in the present invention. Detailed implementation manners

[0016] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0018] Next, the present invention will be further described in conjunction with the accompanying drawings and specific embodiments, but it is not a limitation of the present invention.

[0019] In an embodiment of the present invention, a cell selective killing device based on surface and interface pH regulation is provided, which includes a dynamic voltage control unit, a base layer, and a regulation layer deposited on the base layer. The regulation layer is connected to the dynamic voltage control unit through a wire. The preparation method of the above device is as follows: 1) Using the magnetron sputtering method, deposit a noble metal target, such as a palladium metal target, on the upper part of the ITO electrode surface for 100 s to form a surface regulation layer; 2) Incubate the prepared regulation layer (such as incubating with type I collagen of rat tail for 1 h) to ensure its stable attachment in the cell culture environment and excellent electrochemical regulation performance; 3) Connect the obtained regulation layer to an external DC regulated power supply with a preset dynamic voltage through a wire.

[0020] As Figure 1 shown, the base layer in the present invention is made of one of anodic aluminum oxide nanopores (one-dimensional porous channels), borate glass nanotubes (one-dimensional single pores), polyurethane fiber films (flexible fiber films), or indium tin oxide conductive glass (hard planar).

[0021] As Figure 2As shown, in the present invention, cancer cells can be directly cultured on the surface of the regulation layer in the above-mentioned device, or the regulation layer can be attached to the surface of cancer cells to facilitate the detection and dynamic regulation of the local cell microenvironment, such as pH value or other cancer cell characteristic products (such as electroactive molecules, biological small molecules, etc.), and thus the precise intervention and selective elimination of the metabolic processes of different types of cancer cells can be completed.

[0022] As Figure 3 As shown, in the present invention, the change of the interfacial pH value can be regulated by presetting a dynamic voltage, so as to achieve the selective killing of cancer cells. The principle is as follows: in the positive voltage stage (+0.5 - +0.8 V) for 15 seconds, noble metals in the regulation layer, such as palladium electrodes, rapidly release hydrogen ions in the lattice, that is, the stored hydrogen is released from the lattice by voltage drive, resulting in a rapid decrease of the local surface interfacial pH value to below 5, thereby inhibiting the excretion of lactic acid and protons through the MCT4 channel on the cancer cell membrane (MCT4 is a proton co-transport carrier protein, and its ability to transport lactic acid depends on the transmembrane proton concentration gradient. When the extracellular proton concentration is higher than the intracellular (i.e., the extracellular environment is acidic relative to the intracellular), the transmembrane proton gradient decreases or even reverses, and the MCT4-mediated excretion of lactic acid and protons is inhibited, resulting in the accumulation of intracellular lactic acid and H⁺, and then inducing cell acidification; on the contrary, when the extracellular proton concentration is lower than the intracellular (i.e., the extracellular environment is alkaline relative to the intracellular), the transmembrane proton gradient is enhanced, which will accelerate the ability of MCT4 to transport lactic acid and protons to the extracellular, disturbing the normal metabolic process in the cell), promoting intracellular acidification; then switch to the negative voltage stage (-0.3 to -0.5 V) for 15 seconds, so that the palladium electrode enters the hydrogen storage process, that is, the hydrogen generated on the electrode surface is stored in the palladium lattice, resulting in a rapid increase of the local surface interfacial pH value to above 8, thereby further disturbing the cancer cell metabolic rhythm, and finally leading to intracellular acidification and triggering apoptosis or disintegration and death. In the present invention, it is possible to first apply a negative voltage and then a positive voltage to achieve an effective cycle of hydrogen storage and hydrogen release.

[0023] As Figure 4 As shown, to verify that this device can change the local surface interfacial pH value by presetting a dynamic voltage, in the present invention, a phenol red solution is dropped on the surface of the regulation layer as a pH indicator. The results show that after stimulation in the negative voltage stage (-0.3 to -0.5 V, for 15 seconds), the phenol red solution changes from the original state to purple-red after 15 seconds, indicating that the local pH value rapidly rises to about 8 or above; then, after applying the positive voltage stage (+0.5 to +0.8 V, for 15 seconds), the color of the phenol red solution changes to yellow, indicating that the local pH value rapidly drops to about 5 or above. The experimental results fully prove that this device can achieve an effective dynamic conversion of the surface interfacial pH under periodic voltage regulation, laying an experimental basis for further intervening in cancer cell metabolism through pH regulation.

[0024] As shown above, the selective killing device constructed by the present invention has a simple structure and convenient operation, and can achieve precise selective killing of cancer cells by utilizing the significant difference in glycolytic oscillation between cancer cells and normal cells, demonstrating its potential application value in precision cancer treatment.

[0025] Furthermore, in the present invention, human normal cervical epithelial cells (H8) are used to evaluate the actual performance of this device in normal cell lines, as follows: H8 cells are inoculated on the surface of the regulation layer in the selective killing device, and cultured in DMEM high-glucose medium for 12 h. After 12 h of culture, the medium is replaced with DMEM glucose-free medium and cultured for 6 h to fully starve the cells. After connecting the device to the preset circuit and ensuring that all connections are normal, first apply 25 mM glucose stimulation to the starved normal cells (H8 cells) to activate the cell metabolic reaction. After glucose stimulation, wait for about 50 s to stabilize the cell state. Subsequently, start the dynamic voltage regulation scheme: continuously apply positive and negative voltages alternately according to a preset cycle. Each cycle includes 15 s of positive voltage (+0.5 V) and 15 s of negative voltage (-0.3 V), and the whole process lasts for 10 min, so as to form a periodically changing pH environment at the interface to interfere with the cell metabolic pathway; H8 cells are stained by live cell fluorescence staining (calcein-AM / propidium iodide).

[0026] As Figure 5 shown, since there is no lactate oscillation phenomenon in H8 cells, it indicates that no normal cell death occurs under the above periodic stimulation.

[0027] Furthermore, in the present invention, human cervical cancer epithelial cells (HeLa) are used to evaluate the actual performance of this device in cancer cell lines, as follows: HeLa cells are inoculated on the surface of the regulation layer in the selective killing device, and cultured in DMEM high-glucose medium for 12 h. After 12 h of culture, the medium is replaced with DMEM glucose-free medium and cultured for 6 h to fully starve the cells. After connecting the device to the preset circuit and ensuring that all connections are normal, first apply 25 mM glucose stimulation to the starved cancer cells (HeLa cells) to activate the cell metabolic reaction. After glucose stimulation, wait for about 50 s to stabilize the cell state. Subsequently, start the dynamic voltage regulation scheme: continuously apply positive and negative voltages alternately according to a preset cycle. Each cycle includes 15 s of positive voltage (+0.5 V) and 15 s of negative voltage (-0.3 V), and the whole process lasts for 10 min, so as to form a periodically changing pH environment at the interface to interfere with the cell metabolic pathway; HeLa cells were stained by live cell fluorescence staining (calcein-AM / propidium iodide).

[0028] As Figure 6 shown, due to the continuous and precise interference in the lactic acid oscillation process, all HeLa cells died. The above test results are consistent with the expectations, indicating that the cell selective killing device with surface interface pH regulation provided by the present invention can effectively achieve precise intervention in lactic acid oscillation and achieve the purpose of killing cancer cells.

[0029] It can be understood that in the present invention, materials that can induce corresponding microenvironment changes (such as platinum, palladium or other functionalized electrodes) can also be selected at the interface according to the physical and chemical properties of the target product or the protein transport mechanism, so as to complete precise intervention and selective elimination of the metabolic processes of different types of cancer cells.

[0030] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all the equivalent replacements and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A cell selective killing device based on surface and interface pH regulation, characterized in that, It includes a dynamic voltage control unit, a base layer, and a regulation layer deposited on the base layer, and the regulation layer is connected to the dynamic voltage control unit through a wire.

2. The cell selective killing device based on surface and interface pH regulation according to claim 1, wherein The base layer is made of one of anodic aluminum oxide nanopores, borate glass nanotubes, polyurethane fiber films, or indium tin oxide conductive glass.

3. The cell selective killing device based on surface and interface pH regulation according to claim 1, wherein The regulation layer is formed by depositing noble metals.

4. The cell-selective killing device based on surface and interface pH regulation according to claim 3, characterized in that, The noble metal is one of palladium or platinum.

5. The cell selective killing device based on surface and interface pH regulation according to claim 1, wherein The dynamic voltage control unit is a DC regulated power supply or a controllable potentiostat.

6. Use of a cell selective killing device based on surface and interface pH regulation in the treatment of cancer, characterized in that, Use the cell selective killing device according to any one of claims 1-5 to selectively kill cancer cells.

7. A method for selectively killing cells based on regulating the pH of the surface and interface, characterized in that, Use the cell selective killing device based on surface interface pH regulation according to any one of claims 1-5, including: Deposit a noble metal material capable of regulating the extracellular proton concentration on one side of the substrate to form a regulation layer, and connect the regulation layer to an externally preset dynamic voltage control unit through a wire; Attach the regulation layer to the tissue surface; Periodically apply a dynamic voltage to the regulation layer through an externally preset dynamic voltage control unit, so that the pH value of the interface microenvironment between the regulation layer and the tissue cells alternately changes below 5 and above 8, thereby achieving precise selective killing of cells.

8. The method for selectively killing cancer cells based on regulating pH at the surface of the table according to claim 7, characterized in that, The method for applying the dynamic voltage is: (i) Positive voltage stage: Apply a voltage of +0.5 V - +0.8 V to quickly reduce the pH value of the interface microenvironment below 5; (ii) Negative voltage stage: Apply a voltage of -0.3 V - -0.5 V to quickly increase the pH value of the interface microenvironment above 8.

9. The method for selectively killing cancer cells based on regulating the pH of the surface of a table according to claim 8, wherein, The duration of the positive voltage stage is between 15 seconds and 30 seconds.

10. The method for selectively killing cancer cells based on regulating the pH of the surface of the table according to claim 8, characterized in that, The duration of the negative voltage stage is between 15 seconds and 30 seconds.

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