Flexible electrode and preparation method and application thereof

Abstract

The invention discloses a flexible electrode and a preparation method and application thereof, and relates to the technical field of medical instruments. The flexible electrode comprises a flexible substrate layer, a conductive electrode layer, a packaging layer and an electrode contact which are arranged in a stacked mode. The conductive electrode layer comprises a wire and a conductive contact which are communicated with each other; the packaging layer is provided with a through hole structure, and the through hole structure exposes the conductive contact; and the electrode contact is arranged on the conductive contact. The electrophysiology mapping and electroporation functions are integrated, switching and operation complexity among multiple devices is reduced, and organic combination of real-time monitoring and accurate treatment is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a flexible electrode and a preparation method and application thereof. Background Art

[0002] Electroporation is a technology that uses an electric field to transiently change the permeability of cell membranes to facilitate the entry of exogenous substances into cells. This technology is widely used in gene transfection, drug delivery, and cell engineering. The process of electroporation involves applying a short, high-intensity electric field to the cell membrane, which forms reversible holes in the membrane, allowing large molecules (such as DNA, RNA, or drug molecules) to enter the cell.

[0003] Traditional electroporation equipment mainly includes plate electrodes and needle electrodes. Plate electrodes are generally made of metal materials and are suitable for electroporation of large areas such as skin or tissue surfaces. However, they are insufficient in flexibility and adaptability and are difficult to fit tightly with complex curved tissues such as the heart. Needle electrodes are suitable for electroporation of smaller areas, but their operating accuracy and stability are limited.

[0004] In addition, when performing electroporation on complex curved tissues such as the heart, synchronous monitoring of electrical activity is usually required. In existing technologies, electrophysiological mapping and electroporation functions are usually performed by different devices. The separate operations of the two increase the complexity of experiments and treatments, and also limit the combination of real-time monitoring and precision treatment. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a flexible electrode that integrates electrophysiological mapping and electroporation functions, reduces the switching and operation complexity between multiple devices, and realizes the organic combination of real-time monitoring and precise treatment.

[0006] The present invention also provides a method for preparing the flexible electrode.

[0007] The invention also provides an electrotransfection method for non-disease diagnosis and treatment.

[0008] The present invention also provides the use of the above-mentioned flexible electrode in preparing products for repairing heart tissue damaged after myocardial infarction, gene editing products, electroporation systems or electrophysiological monitoring systems.

[0009] The present invention also provides a gene editing product.

[0010] The invention also provides an electroporation system.

[0011] The invention also provides an electrophysiological monitoring system.

[0012] According to the flexible electrode of the embodiment of the first aspect of the present invention, the flexible electrode includes a flexible substrate layer, a conductive electrode layer, a packaging layer and electrode contacts which are stacked together; the conductive electrode layer includes a wire and a conductive contact which are connected; the packaging layer is provided with a through hole structure, and the through hole structure exposes the conductive contact; the electrode contact is arranged on the conductive contact.

[0013] The flexible electrode according to the embodiment of the present invention has at least the following beneficial effects: The flexible electrode of the embodiment integrates the functions of electrophysiological mapping and electroporation (including reversible electroporation for gene delivery, cell transfection, etc. and irreversible electroporation for cell killing and tissue ablation), reduces the switching and operation complexity between multiple devices, realizes the organic combination of real-time monitoring and precision treatment, and improves the convenience and efficiency of overall treatment. It can perform large-area, multi-channel electrophysiological mapping of the epicardium of living animals, etc., can quickly and accurately identify areas with abnormal electrical activity, and has good stability and safety after implantation in the body. The flexible electrode of the embodiment has good application prospects in the treatment of diseases such as myocardial infarction.

[0014] When the flexible electrodes of the embodiments are used to deliver nucleic acid molecules, etc., they have higher efficiency than traditional viral vectors, and can avoid virus-related safety risks and possible immune responses, thereby improving the safety and reliability of the overall treatment.

[0015] According to some embodiments of the present invention, the flexible substrate layer includes at least one of thermoplastic polyurethane (TPU), polyester (PET), and silicone. The flexible substrate layer has good flexibility, stretchability, and biocompatibility (complying with the ISO 10993 biocompatibility standard. Thus, it is ensured that the flexible electrode will not cause adverse biological reactions such as cytotoxicity, sensitization, or induced inflammatory reactions when in contact with human tissues), and can not only form a good fit with tissues such as the heart, but also will not have a negative impact on the normal function of the heart, and can enhance the accuracy of signal acquisition; it can also withstand the dynamic movement of the heart tissue to avoid discomfort or damage to the subject caused by hardness.

[0016] According to some embodiments of the present invention, the Shore hardness of the flexible substrate layer is 70A-90A. For example, it can be 70A, 71A, 72A, 73A, 74A, 75A, 76A, 77A, 78A, 79A, 80A, 81A, 82A, 83A, 84A, 85A, 86A, 87A, 88A, 89A or 90A. Thus, it can be ensured that it has sufficient flexibility, elasticity and mechanical strength, and can fit closely to complex curved tissues such as the heart, without discomfort caused by being too hard or insufficient mechanical stability caused by being too soft.

[0017] According to some embodiments of the present invention, the tensile modulus of the flexible substrate layer is 5 MPa to 15 MPa. For example, it can be 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 11 MPa, 12 MPa, 13 MPa, 14 MPa or 15 MPa. It can be 15 MPa, thereby ensuring that it can maintain good structural integrity and fit under repeated stretching during heart beating.

[0018] According to some embodiments of the present invention, the elongation at break of the flexible substrate layer is 300% to 600%. For example, it may be 300%, 350%, 400%, 450%, 500%, 550% or 600%. Thus, it can be ensured that it can withstand large deformation without breaking during the stretching process, which is crucial for adapting to the deformation of dynamic tissues such as the heart.

[0019] According to some embodiments of the present invention, the moisture permeability coefficient of the flexible substrate layer is greater than 10 g / m² / day, and the air permeability coefficient is 0.5~5 cm³ / cm² / s. For example, the moisture permeability coefficient may be 10 g / m² / day, 12 g / m² / day, 14 g / m² / day, 16g / m² / day, 18 g / m² / day or 20 g / m² / day; the air permeability coefficient may be 0.5 cm³ / cm² / s, 1 cm³ / cm² / s, 2cm³ / cm² / s, 3 cm³ / cm² / s, 4 cm³ / cm² / s or 5 cm³ / cm² / s. Thus, it is possible to ensure that the flexible electrode has certain air permeability and moisture permeability to avoid water vapor accumulation when in contact with the skin or tissue for a long time, causing discomfort or material degradation; and it can still maintain good comfort and material performance during long-term use.

[0020] According to some embodiments of the present invention, the conductive electrode layer is at least one of liquid metal, silver paste, and carbon nanotubes. Compared with other conductive materials, liquid metal is liquid at room temperature, easy to apply, and can form a uniform conductive electrode layer, ensuring efficient current conduction of the electrode, providing a stable electric field, and more adaptable to the bending and stretching of the flexible substrate layer; and it exhibits higher biocompatibility and flexibility on the surface of the heart of large animals, and is not prone to causing inflammatory reactions.

[0021] The term "not less than" means higher than or equal to, and should be understood to include the number.

[0022] According to some embodiments of the present invention, the conductivity of the conductive electrode layer is not less than 3.4×10 6 S / m. Preferably 3.4×10 6 S / m to 6.0×10 6 S / m. For example: it can be 3.4×106 S / m, 3.6×10 6 S / m, 3.8×10 6 S / m, 4×10 6 S / m, 4.2×10 6 S / m, 4.4×10 6 S / m, 4.6×10 6 S / m, 4.8×10 6 S / m, 5×10 6 S / m, 5.2×10 6 S / m, 5.4×10 6 S / m, 5.6×10 6 S / m, 5.8×10 6 S / m or 6×10 6 S / m. Thus, it can meet the requirements of efficient current conduction while maintaining flexibility.

[0023] According to some embodiments of the present invention, the Young's modulus of the conductive electrode layer is 0.01 MPa to 10 MPa. For example, it may be 0.01 MPa, 0.05 MPa, 1 MPa, 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa or 10 MPa. Thus, it can be ensured that it has good flexibility and deformation ability during actual use, and can adapt to the surface of complex dynamic tissues such as the heart.

[0024] According to some embodiments of the present invention, the elongation at break of the conductive electrode layer is not less than 300%. For example, it may be 300%, 350%, 400%, 450% or 500%. Thus, it can be ensured that it is not easy to break under large deformation and can be stably attached to the heart surface for a long time.

[0025] According to some embodiments of the present invention, the conductive electrode layer can maintain its flexibility and conductivity within the operating temperature range of 0°C to 50°C to adapt to the temperature environment inside the human body; and comply with ISO 10993 or similar biocompatibility standards; and have good chemical stability, especially when exposed to a biological fluid environment for a long time, it is not easy to degrade or react, so as to ensure the long-term functionality of the material.

[0026] The conductive contacts are distributed in an array. Compared with conventional electroporation devices that usually support a single channel or a small number of channels, the flexible electrode of the embodiment is more suitable for application on large-area tissues by designing the size and arrangement of the conductive contacts, and has high efficiency and accuracy.

[0027] According to some embodiments of the present invention, the liquid alloy includes at least one of pure gallium, gallium alloy, pure bismuth, and bismuth alloy.

[0028] According to some embodiments of the present invention, the gallium alloy includes at least one of a gallium-indium alloy, a gallium-tin alloy, a gallium-zinc alloy and a gallium-indium-tin alloy.

[0029] According to some embodiments of the present invention, the bismuth alloy includes at least one of a bismuth indium tin alloy and a bismuth indium tin zinc alloy.

[0030] According to some embodiments of the present invention, the encapsulation layer includes at least one of TPU, polyvinylidene fluoride (PVDF), polyvinyl alcohol (PVA), and polytetrafluoroethylene (PTFE). The encapsulation layer is used to protect the flexible electrode from mechanical damage and erosion by biological fluids to ensure its long-term stability. Those skilled in the art may also select other polymer materials with high biocompatibility as needed. An encapsulation layer with suitable flexibility (Young's modulus, elongation at break), mechanical strength, and chemical stability is conducive to ensuring the contact between the flexible electrode and the surface of the tissue, etc., improving the service life, and facilitating the transmission and collection of signals.

[0031] According to some embodiments of the present invention, the Young's modulus of the encapsulation layer is 0.1 MPa to 10 MPa. For example, it may be 0.1 MPa, 1 MPa, 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa or 10 MPa.

[0032] According to some embodiments of the present invention, the elongation at break of the encapsulation layer is 50% to 500%, for example, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or 500%.

[0033] According to some embodiments of the present invention, the flexural strength of the encapsulation layer is 1 MPa to 100 MPa. For example, it may be 1 MPa, 5 MPa, 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa or 100 MPa.

[0034] According to some embodiments of the present invention, the tensile strength of the encapsulation layer is 5 MPa to 50 MPa. For example, it may be 5 MPa, 10 MPa, 15 MPa, 20 MPa, 25 MPa, 30 MPa, 35 MPa, 40 MPa, 45 MPa or 50 MPa.

[0035] According to some embodiments of the present invention, the tear strength of the encapsulation layer is 10 kN / m to 200 kN / m. For example, it can be 10 kN / m, 20 kN / m, 30 kN / m, 40 kN / m, 50 kN / m, 60 kN / m, 70 kN / m, 80 kN / m, 90 kN / m, 100 kN / m, 110 kN / m, 120 kN / m, 130 kN / m, 140 kN / m, 150 kN / m, 160 kN / m, 170 kN / m, 180 kN / m, 190 kN / m or 200 kN / m.

[0036] According to some embodiments of the present invention, the electrode contact modification layer includes at least one of gold and aluminum. The electrode contact modification layer is used to improve electrical conductivity and corrosion resistance.

[0037] According to some embodiments of the present invention, the thickness of the flexible substrate layer is 0.1 mm to 1 mm. For example, it can be 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm or 1 mm. This is conducive to achieving extremely high flexibility and fit. Those skilled in the art can select the thickness as needed, such as: 0.1 mm is the minimum thickness that can achieve extremely high flexibility and fit, which is suitable for application scenarios with extremely high flexibility requirements (such as cardiac electrophysiological mapping, which requires close contact with the surface of the heart); 1 mm is the upper limit thickness to ensure that the flexible substrate layer has sufficient mechanical strength and durability, and is suitable for applications that may face greater mechanical stress.

[0038] According to some embodiments of the present invention, the flexible substrate layer has a radius of curvature consistent with the surface of the target tissue (such as the surface of the heart) to adapt to the surface of the target tissue. The radius of curvature is 2 cm to 8 cm. For example, it can be 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm or 8 cm. Those skilled in the art can design the radius of curvature according to the measurement results of the radius of curvature of the target tissue surface to ensure the tight fit and precise function of the flexible electrode to the target tissue and avoid poor contact. For example, the radius of curvature designed for the apex of the heart or certain areas of the epicardium is 2 cm; the radius of curvature designed for the larger part of the heart surface or the side wall of the heart is 8 cm.

[0039] According to some embodiments of the present invention, the thickness of the encapsulation layer is 50 μm to 200 μm, for example, 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm or 200 μm.

[0040] According to some embodiments of the present invention, the thickness of the electrode contact modification layer is 20 nm to 50 nm, for example, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm or 50 nm.

[0041] By controlling the thickness of the flexible electrode, it can provide sufficient flexibility and stability to adapt to the dynamic movement of tissues such as the heart.

[0042] According to some embodiments of the present invention, the flexible electrode further comprises a conductive polymer layer, and the conductive polymer layer is coated outside a composite body formed by the flexible substrate layer, the conductive electrode layer, the packaging layer, and the electrode contacts.

[0043] According to some embodiments of the present invention, the thickness of the conductive polymer layer is 50 nm to 500 nm. For example, it can be 50 nm, 100 nm, 200 nm, 300 nm, 400 nm or 500 nm. This helps to ensure good conductive properties while providing a certain mechanical strength.

[0044] According to some embodiments of the present invention, the conductive polymer layer includes at least one of poly(3,4-ethylenedioxythiophene) (PEDOT), polyaniline (PANI), and polypyrrole (PPy). Compared with PANI and PPy, PEDOT has better conductivity, flexibility, and biocompatibility. Compared with PEDOT and PPy, PANI has higher mechanical strength.

[0045] According to some embodiments of the present invention, the conductivity of the conductive polymer layer is 1 S / cm to 1000 S / cm. For example, it may be 1 S / cm, 100 S / cm, 200 S / cm, 300 S / cm, 400 S / cm, 500 S / cm, 600 S / cm, 700 S / cm, 800 S / cm, 900 S / cm or 1000 S / cm.

[0046] According to some embodiments of the present invention, the tensile strength of the conductive polymer layer is 1 MPa to 10 MPa. For example, it may be 1 MPa, 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa or 10 MPa.

[0047] According to some embodiments of the present invention, the Shore hardness of the conductive polymer layer is 5A to 30A. For example, it may be 5A, 8A, 10A, 12A, 15A, 17A, 20A, 22A, 25A, 27A or 30A. If the Shore hardness is too high, the flexible electrode may not be able to fit the curved surface, while if it is too low, the conductivity may be affected.

[0048] According to some embodiments of the present invention, at 1 Hz to 1000 Hz (core frequency band of ECG signals), the impedance value of the flexible electrode is 1 kΩ to 100 kΩ.

[0049] According to some embodiments of the present invention, the diameter of the conductive contact is 1 mm to 2 mm, the wire width is 0.2 mm, and the side-to-side spacing (referring to the side-to-side spacing of two parallel sides corresponding to adjacent electrodes) is 1 to 4 mm.

[0050] According to some embodiments of the present invention, the conductive contact has a diameter of 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 1 mm. This makes it more suitable for high-density electrophysiological mapping (such as detailed electrical activity monitoring of the heart); close electrode contacts and smaller spacing can provide high-resolution data, which is suitable for detecting small changes in electrical activity.

[0051] According to some embodiments of the present invention, the conductive contact has a diameter of 2 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 2-5 mm. This is more suitable for large-area electrical activity detection (such as: extensive electrical activity detection on the surface of the heart); larger conductive contacts and wider spacing help reduce interference between electrodes while covering more detection areas.

[0052] According to some embodiments of the present invention, the electrode conductive contact has a diameter of 2 mm, a wire width of 0.2 mm, and an edge-to-edge spacing of 2 mm. This is more conducive to accurate electrical measurement of detailed areas (such as detailed analysis of specific cardiac areas); relatively large contacts and moderate spacing can provide better signal capture capabilities while maintaining sufficient resolution.

[0053] According to some embodiments of the present invention, the conductive contact has a diameter of 1 mm, the wire width is 0.3 mm to 0.6 mm, and the edge-to-edge spacing is 2 mm. This makes it more suitable for use in medium-density mapping areas; it balances the contact density and spacing, and is suitable for most standard electrophysiological mapping applications.

[0054] According to some embodiments of the present invention, the conductive contact has a diameter of 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 4 mm, which is more suitable for electrophysiological mapping in a wide area.

[0055] According to the second aspect of the present invention, the method for preparing the above-mentioned flexible electrode comprises the following steps: S1. providing a flexible base layer; S2, printing the conductive electrode layer on the flexible substrate layer; S3, manufacturing the encapsulation layer on the conductive electrode layer; S4. Making the electrode contacts on the conductive contacts.

[0056] According to some embodiments of the present invention, a method of printing the conductive electrode layer comprises a screen printing method.

[0057] According to some embodiments of the present invention, the method for manufacturing the encapsulation layer includes a hot pressing method, wherein the conditions of the hot pressing method include: a temperature of 150-200° C., a pressure of 1-5 MPa, and a time of 30-120 s.

[0058] According to some embodiments of the present invention, the method for making the electrode contact includes electron beam evaporation. The conditions of the electron beam evaporation method include: the evaporation temperature is 20° C. to 60° C., the evaporation pressure is 1×10 -6 Torr~1×10 -5 Torr, high vacuum environment is 1×10 -6 Torr~1×10 -5 Torr, and the evaporation rate is 0.1 nm / s~1 nm / s.

[0059] According to a third aspect of the present invention, a non-disease diagnosis and treatment electrotransfection method comprises the following steps: The target cells or target tissues are electrotransfected by the flexible electrodes to introduce exogenous substances into the target cells or target tissues.

[0060] According to some embodiments of the present invention, the parameters of the pulsed electric field set for electrofection include: electric field strength: 50 V / cm ~ 150 V / cm, pulse width: 1 millisecond ~ 20 milliseconds, pulse frequency: 0.5 Hz ~ 10 Hz, duration: 1 ~ 15 minutes.

[0061] According to the fourth aspect of the present invention, the above-mentioned flexible electrode is used in the preparation of products for repairing heart tissue damaged after myocardial infarction, gene editing products, electroporation systems or electrophysiological monitoring systems.

[0062] According to some embodiments of the present invention, the product for repairing damaged cardiac tissue after myocardial infarction comprises a miRNA combination; the miRNA combination comprises miR-1 (5'-UGGAAUGUAAAGAAGUAUGUAU-3'), miR-133 (5'-UUUGGUCCCCUUCAACCAGCUG-3'), miR-208 (5'-AUAAGACGAGCAAAAAGCUUGU-3') and miR-499 (5'-UUAAGACUUGCAGUGAUGUUU-3').

[0063] According to some embodiments of the present invention, the product for repairing cardiac tissue damaged after myocardial infarction further comprises an mRNA combination; the mRNA combination comprises Myocardin mRNA, Mef2c mRNA, TBX5 mRNA, and miR-133.

[0064] A gene editing product according to a fifth aspect of the present invention comprises the flexible electrode as described in the first aspect of the present invention.

[0065] According to some embodiments of the invention, the product is selected from a kit.

[0066] According to an embodiment of the sixth aspect of the present invention, an electroporation system comprises the flexible electrode as described in the embodiment of the first aspect above, and a power source connected to the flexible electrode. Since the electroporation system adopts all the technical solutions of the flexible electrode of the above embodiment, it at least has all the beneficial effects brought by the technical solutions of the above embodiment. The power source is used to provide a pulsed electric field.

[0067] According to some embodiments of the present invention, when the electroporation system is used for nucleic acid molecule delivery, the parameters of the pulsed electric field include: electric field strength: 50 V / cm ~ 150 V / cm, pulse width: 1 millisecond ~ 20 milliseconds, pulse frequency: 0.5 Hz ~ 10Hz, duration: 1 ~ 15 minutes. Thus, efficient delivery of nucleic acid molecules can be achieved without causing significant damage to tissues such as the heart. If the electric field strength is too low, the pulse width is too short, or the pulse frequency is too low, the cell membrane may not be effectively opened. If the electric field strength is too high, the pulse width is too long, or the pulse frequency is too high, cell or tissue damage may be caused. If the duration is too short, effective transfection may not be achieved. If the duration is too long, it may cause damage to cells or tissues.

[0068] According to an embodiment of the seventh aspect of the present invention, an electrophysiological monitoring system comprises a flexible electrode, an electrical signal acquisition interface and an electrical signal analysis device as described in the embodiment of the first aspect connected in sequence. Since the electrophysiological monitoring system adopts all the technical solutions of the flexible electrode of the above embodiment, it at least has all the beneficial effects brought by the technical solutions of the above embodiment. The electrical signal acquisition interface is used to realize real-time electrocardiogram signal acquisition and transmission.

[0069] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1is a schematic diagram of a pattern mask of a flexible electrode in Example 1; Figure 2 is a schematic diagram of the flexible electrode in Example 1; Figure 3 is a schematic diagram of a pattern mask for a flexible electrode in Example 2; Figure 4 is a schematic diagram of a pattern mask for a flexible electrode in Example 3; Figure 5 is a schematic diagram of a pattern mask for a flexible electrode in Example 4; Figure 6 is a schematic diagram of a pattern mask for a flexible electrode in Example 5; Figure 7 is a schematic diagram of a pattern mask for a flexible electrode in Example 6; Figure 8 is a schematic diagram of a pattern mask for a flexible electrode in Example 7; Fig. 9 The impedance test result of the flexible electrode of Example 1; Fig.10 These are the flexibility and stretchability test results of the flexible electrode of Example 1. DETAILED DESCRIPTION

[0071] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0072] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0073] In the description of the present invention, it should be noted that, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific content of the technical solution.

[0074] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0075] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0076] In the embodiments of the present application, the thickness of the TPU substrate is 100 μm, the Shore hardness is 85A, the tensile modulus is 10 MPa, the breaking strength is 50 MPa, the elongation at break is 500%, the moisture permeability is 15 g / m² / day, the air permeability is 3 cm³ / cm² / s, and it has been certified according to the ISO 10993 biocompatibility standard. The supplier is Lubrizol.

[0077] The main components of the gallium alloy are 75.5% gallium and 24.5% indium. The model is Gallium-Indium Alloy, and the conductivity is 3.4×10 6 S / m, supplier is Alfa Aesar. Gallium alloy has excellent conductivity and flexibility, and can maintain stable conductivity under large electric field strength.

[0078] PEDOT conductive polymer, model number 739316.

[0079] Example 1 The present application provides a flexible electrode, and the preparation method thereof is as follows: S1. Obtain imaging data through cardiac CT or MRI scanning to obtain the radius of curvature of the heart surface area, cut the TPU substrate according to the shape of the heart surface, and use ultrasonic cleaning (40 kHz, 5-10 minutes) to remove surface pollutants to ensure the cleanliness and good adhesion of the TPU substrate to obtain a flexible base layer.

[0080] S2, use CAD to design the pattern mask of the conductive layer (such as Figure 1As shown). Gallium alloy and n-decanol were mixed in a mass ratio of 1:4, and ultrasonically crushed (pulse time was 5 seconds, stop time was 5 seconds, ultrasonic energy was set to 22, and the total ultrasonic crushing time was 4 minutes) to obtain a coating ink. The coating ink was evenly coated on the flexible substrate layer by screen printing technology (mesh size was 300 mesh, printing pressure was 0.2 MPa, and printing speed was 5 cm / s), and dried at 50°C for 30 minutes to remove excess solvent, and cured to form a conductive electrode layer having a conductive wire and a conductive contact connected to one end of the conductive wire.

[0081] The conductive contacts have a diameter of 1 mm, a wire width of 0.2 mm, and an edge-to-edge spacing of 4 mm.

[0082] S3. Use a high-precision laser cutting machine to punch holes (aperture set to 500 μm) on the TPU substrate through laser technology to form through holes to expose the conductive contacts of the conductive electrode layer to ensure close contact with the heart tissue and transmit the electric field. Cover the TPU substrate on the conductive electrode layer and hot-press for 120 seconds at a temperature of 180°C and a pressure of 3 MPa to form an encapsulation layer to improve the mechanical strength and protection performance of the flexible electrode.

[0083] S4, through electron beam evaporation technology (evaporation temperature is 40 ° C, evaporation pressure is 5 × 10 -5 Torr, high vacuum environment is 5×10 -6 Torr, evaporation rate of 0.5 nm / s) was used to deposit a 30 nm thick gold layer (electrode contact) on the conductive contact to enhance its conductivity and corrosion resistance, thus obtaining a flexible electrode (such as Figure 2 as shown).

[0084] Example 2 The present application provides a flexible electrode, and its preparation method is basically the same as that of Example 1, with the only difference being that the parameter settings of the conductive electrode layer are different, specifically, the conductive contact diameter is 2 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 2 mm.

[0085] Pattern mask Figure 3 shown.

[0086] Example 3 The present application provides a flexible electrode, whose preparation method is basically the same as that of Example 1, with the only difference being that the parameter settings of the conductive electrode layer are different, specifically, the diameter of the first row of conductive contacts is 2 mm, the diameters of the remaining contacts are 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is arranged in the order of 2 mm, 5 mm, and 2 mm.

[0087] Pattern mask Figure 4 shown.

[0088] Example 4 The present application provides a flexible electrode, and its preparation method is basically the same as that of Example 1, with the only difference being that the parameter settings of the conductive electrode layer are different, specifically, the conductive contact diameter is 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 2 mm.

[0089] Pattern mask Figure 5 shown.

[0090] Example 5 The present application provides a flexible electrode, and its preparation method is basically the same as that of Example 1, with the only difference being that the parameter settings of the conductive electrode layer are different, specifically, the conductive contact diameter is 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 2 mm.

[0091] Pattern mask Figure 6 shown.

[0092] Example 6 The present application provides a flexible electrode, and its preparation method is basically the same as that of Example 1, with the only difference being that the parameter settings of the conductive electrode layer are different, specifically, the conductive contact diameter is 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is arranged in the order of 2 mm, 5 mm, and 2 mm.

[0093] Pattern mask Figure 7 shown.

[0094] Example 7 The present application provides a flexible electrode, and its preparation method is basically the same as that of Example 1, with the only difference being that the parameter settings of the conductive electrode layer are different, specifically, the conductive contact diameter is 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 4 mm.

[0095] Pattern mask Figure 8 shown.

[0096] Example 8 The present application provides a flexible electrode, the preparation method of which is basically the same as that of Example 1, except that: (1) The parameter settings of the conductive electrode layer are different, specifically: the conductive contact diameter is 1 mm, the wire width is 0.2 mm, and the edge-to-edge spacing is 0.2 mm; (2) also includes step S6: coating the outer surface of the prepared flexible electrode with a layer of PEDOT conductive polymer (with a thickness of 50 nm).

[0097] Test Example 1 This test example performs impedance measurement on the flexible electrode of the embodiment to verify its low impedance performance at the ECG sampling frequency.

[0098] The flexible electrode was connected to an impedance analyzer, and the impedance value of the flexible electrode at a frequency of 1 Hz to 1000 Hz was measured using an impedance analyzer (Keysight 4294A) at 25°C.

[0099] The measurement results show that the impedance values ​​of the flexible electrodes of Examples 1 to 7 are 1 kΩ to 100 kΩ in the 1 Hz to 1000 Hz (ECG signal core frequency band). The impedance value range of the ECG signal core frequency band of Example 1 is 1.5 k to 95 kΩ, which meets the expected performance indicators (such as Fig. 9 As shown in the figure, the black curve is the result measured in physiological saline to simulate the body fluid environment; the red curve is the result obtained by directly conformally attaching to the epicardium surface. The fluctuation is caused by the beating of the living heart at a heart rate of about 250 beats / minute. The two results are highly coincident, and the impedance value is within a good range, indicating that the impedance performance is excellent). This shows that the flexible electrode of the present invention has high-quality electrode-biological interface impedance and can effectively perform electrical signal acquisition and electric field application.

[0100] Test Example 2 This test example tests the flexibility and stretchability of the flexible electrode of the embodiment.

[0101] Take several flexible electrode samples, each with a size of 10 cm × 10 cm. Make sure there is no obvious damage on the sample surface.

[0102] (1) Flexibility test: Fix the sample on a bending test machine. Set the bending radius to 5 mm and test the bending performance of the sample at this radius. Record the changes in the sample's appearance (such as cracks, wrinkles, etc.) and electrical properties after each bending.

[0103] (2) Stretchability test: Fix the sample on a universal material testing machine, set the initial distance between the clamps to 20 mm, and stretch it at a speed of 0.5 mm / s until the sample breaks or stretches to 150% of its original length. Record the change in resistance value of the sample at 50% strain. Repeat the test 5 times, and record the deformation and electrical property changes of the sample after each stretching test.

[0104] The flexible electrode of Example 1 can maintain its structural integrity and conductivity in a bent state without cracks or breaks, showing excellent flexibility; it maintains good conductivity and structural stability under a strain of 50%, and the resistance value of the flexible electrode after stretching does not change by more than 5%, proving that it has good stretchability. The test results of the flexible electrodes of the other examples are similar to those of Example 1.

[0105] Test Example 3 This test example tests the in vitro myocardial reprogramming effect of the flexible electrode of the embodiment.

[0106] (1) Mix miR-1 (5'-UGGAAUGUAAAGAAGUAUGUAU-3'), miR-133 (5'-UUUGGUCCCCUUCAACCAGCUG-3'), miR-208 (5'-AUAAGACGAGCAAAAAGCUUGU-3'), and miR-499 (5'-UUAAGACUUGCAGUGAUGUUU-3') in a molar ratio of 1:1:1:1 and dissolve them in sterile phosphate buffered saline. The final concentration of each miRNA was 1 μM to obtain an electroporation solution.

[0107] (2) Cardiac fibroblasts (primary cells isolated from PDGFRa-DreER:rox-tdTomato transgenic mice) were cultured at a cell density of 1×10 4 cells / well were inoculated into 24-well plates and cultured until the confluence was about 80%. The cardiac fibroblasts were digested with trypsin (0.25% trypsin-EDTA) for 2-3 minutes, and an equal volume of complete culture medium (basal culture medium containing 10% fetal bovine serum) was added to terminate the digestion. The cells were centrifuged at 300 × g for 5 minutes and the supernatant was removed. The cells were washed twice with PBS to remove residual trypsin. The cells were resuspended with electroporation solution (cell density was 1 × 10 6 cells / mL), transferred to an electrotransfection device, the flexible electrode of Example 4 was placed in the cell suspension, the electrode contacts were connected to the positive and negative electrodes of the BTX ECM 830 electrotransfection instrument, and electrotransfection was performed using an electrotransfection instrument (BTX ECM 830). The electrotransfection parameters were: electric field strength of 300 V / cm, pulse width of 5 ms, pulse frequency of 1 Hz, and number of pulses of 4. After the electrotransfection was completed, the cells were immediately transferred to a centrifuge tube containing complete culture medium, centrifuged at 300×g for 5 minutes, and the electrotransfection solution was removed. The cells were resuspended in DMEM / F12 medium (containing 10% FBS) and inoculated into a 60 mm culture dish at a cell density of 1×10 5cells / dish, and cultured in an incubator at 38.5℃, 5% CO2. The morphology and growth of the cells were observed, and the culture medium was replaced regularly to maintain the good condition of the cells. MiR-1, miR-133, miR-208, and miR-499 were transfected into the cells by Lipofectamine 3000 as the liposome control group, and the operation was carried out according to the instructions. The difference between the negative control group and the experimental group was that miR-1, miR-133, miR-208, and miR-499 were replaced by miR-NC1 (5'-UAAUACUGCCUGCUAAUGUUGG-3') and miR-NC2 (5'-AGAGAGAGAGAGAGAGAGAGAG-3'). Cardiac fibroblasts without any treatment were used as the blank control group. The expression of myocardial-specific genes (α-MHC, cTnT, and GATA4) was analyzed by qPCR technology, and GAPDH was used as the internal reference gene to calculate the relative expression. The number of cTnT and tdTomato double-positive cells and single-positive cells (only tdTomato positive) were detected by flow cytometry, and the reprogramming efficiency was calculated (reprogramming efficiency was the percentage of double-positive cells to the total number of tdTomato fluorescent protein-labeled cells).

[0108] The results showed that compared with the blank control group, the expression of myocardial-specific genes in the experimental group of cardiac fibroblasts treated with the flexible electrode of Example 4 increased significantly, and the reprogramming efficiency was about 20%, indicating that the cardiac fibroblasts were effectively transformed into cardiomyocytes. The cell survival state of the liposome control group was poor, and the cell morphology was poor, which could not be used for flow cytometry detection. After transfection, the expression of myocardial-specific genes in the cardiac fibroblasts of the blank control group and the negative control group was significantly lower than that in the experimental group, and the reprogramming efficiency was about 0%.

[0109] Test Example 4 In this detection example, mice were used as experimental subjects to test the in situ myocardial reprogramming effect of the flexible electrode of the embodiment.

[0110] (1) Perform left coronary artery ligation on rox-tdTomato mice (20 g~25 g) to expose the anterior wall of the heart. Gently grasp the pericardial edge with blunt-edged curved forceps and gently cut to expose the anterior wall of the left ventricle. Determine the course of the left anterior descending coronary artery (LAD) and ligate it at the desired location. Use 7-0 absorbable sutures to suture 3~4 mm from the origin of the LAD. When the myocardium in the area supplied by the coronary artery becomes pale, the mouse model of myocardial infarction is successfully established.

[0111] (2) Mix miR-1 (5'-UGGAAUGUAAAGAAGUAUGUAU-3'), miR-133 (5'-UUUGGUCCCCUUCAACCAGCUG-3'), miR-208 (5'-AUAAGACGAGCAAAAAGCUUGU-3'), and miR-499 (5'-UUAAGACUUGCAGUGAUGUUU-3') in a molar ratio of 1:1:1:1 and dissolve them in sterile saline. The final concentration of each miRNA is 1 μM to obtain an electro-transfection solution.

[0112] (2) ① Experimental group (n=8): The flexible electrodes of Example 4 (with the encapsulation layer attached to the heart surface) were implanted into the heart surface of the myocardial infarction mouse model by thoracic surgery to ensure that the flexible electrodes were closely attached to the heart surface and stable on the heart surface to avoid mechanical damage to the heart tissue. 50 μL of the electrotransfection solution was evenly distributed on the surface of the encapsulation layer of the flexible electrode using a sterile syringe, and a pulsed electric field (electric field strength: 100 V / cm, pulse width: 10 milliseconds, pulse frequency: 1 Hz, duration: 1 minute) was applied to the flexible electrode.

[0113] ②Sham operation group (n=8): rox-tdTomato mice underwent thoracic surgery, but no electrodes were implanted.

[0114] ③ Modeling group (n=8): Mice with myocardial infarction underwent thoracic surgery, but no electrodes were implanted.

[0115] ④Negative control group (n=8): The treatment method was basically the same as that of the experimental group, except that miR-1, miR-133, miR-208, and miR-499 were replaced by miR-NC1 and miR-NC2.

[0116] ⑤Comparative Example 1 (n=8): BTX ECM 830 electroporation system and electrode (Tweezertrode, 3 mmDiameter (45-0486)) were used to connect the electrode to the BTX ECM 830 electroporation system, 50 μL of electrotransfection solution was evenly injected into the surface of the mouse heart, and then the heart was clamped with electrodes. The pulse parameters were set as follows: electric field strength of 100 V / cm, pulse width of 10 ms, pulse frequency of 1 Hz, and duration of 1 min.

[0117] ⑥Comparative Example 2 (n=8): The miRNA combination (such as miR-1, miR-133, miR-208, miR-499) was cloned into the AAV9 vector to ensure that the expression of each miRNA was controlled by the cardiac-specific promoter to obtain the AAV9-miRNA vector. The virus titer was set to 10^12 vg / mL, and 100 μL of AAV9-miRNA virus solution was directly injected into the heart tissue (LAD area) of the myocardial infarction mouse model, using ultrasound guidance to ensure the injection location and depth.

[0118] During the miRNA delivery process, the working status of the electrode system was monitored in real time to ensure that miRNA could be evenly distributed and enter the heart cells. The fitting effect of each electrode on the heart surface (whether there were bubbles, wrinkles or local non-fitting between the electrode and the heart surface) was observed and recorded by optical tomography (OCT), high-definition video microscopy, and magnetic resonance imaging (MRI), and the uniformity and area of ​​contact between the electrode and the heart surface were analyzed. After 1 week, 2 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, and 6 months after transfection, each group of mice was subjected to ultrasound cardiology examination to record and evaluate the recovery of cardiac function. After the experiment, the myocardial tissue near the electrode was removed and H&E staining was performed to observe the tissue morphology and evaluate whether there was mechanical damage, inflammatory response or fibrosis.

[0119] The results showed that in the experimental group, the flexible electrode showed excellent fitting effect on the surface of the heart. No obvious bubbles, wrinkles or uneven contact were found in visual observation and OCT scanning, and the electrode contact area was more than 98%. In the histological analysis, no obvious mechanical damage or fibrosis was found in the myocardial tissue near the electrode, and the inflammation score was low, indicating that the flexible electrode had less mechanical stimulation to the heart tissue. In the electrophysiological recording, the amplitude of the ECG signal captured by the flexible electrode was stable and clear, and the SNR reached more than 12 dB. The long-term stability test results showed that during the entire experimental period, the waveform of the electrophysiological signal changed by less than 5%, and the electrode showed good stability and reliability on the surface of the heart.

[0120] After the experiment, the EF of the mice in the experimental group increased from 40% before myocardial infarction to 60%, and the heart function recovered significantly. There was no obvious fibrosis or other side effects in the heart tissue. The control group 1 was prone to affect the heart pumping of mice during the operation, which was not conducive to the survival of mice. The EF of the surviving mice increased from 40% before myocardial infarction to only 45%~50%, and the heart function recovery was limited; compared with the experimental group, the myocardial tissue of the mice in the control group 1 was higher, and there were signs of tissue damage locally. The EF of the mice in the control group 2 increased from 40% before myocardial infarction to about 50%, the heart function recovery was not obvious, the reprogramming efficiency was low, and it was difficult to significantly improve the heart function; and the operation was difficult and the effect fluctuated greatly. If the flexible electrode of Example 4 is used to transfect a single miRNA (miR-1, miR-133, miR-208 or miR-499), the reprogramming efficiency is generally less than 10%, which is significantly lower than that of the experimental group. Some mice only show a small number of cells expressing myocardial specific markers, and cannot achieve effective myocardial reprogramming effects, lacking the synergistic effect of multiple signals. The increase in EF was also low, from 40% to only between 42% and 45%, and the degree of fibrosis in cardiac tissue was high, indicating that a single miRNA could not provide sufficient signals to drive effective myocardial regeneration and functional recovery.

[0121] After myocardial infarction, the ventricular internal diameter (especially left ventricular end-diastolic diameter, LVEDD) of the experimental group mice gradually decreased, indicating that the degree of ventricular remodeling was reduced. Compared with the model group and the sham operation group, the LVEDD of the experimental group decreased by an average of 15% after 1 month and by about 25% at 6 months.

[0122] Left ventricular wall thickness (particularly left ventricular posterior wall thickness, LVPW) increased after transfection, indicating myocardial remodeling and repair. The average LVPW of the experimental group increased by about 10% at 2 months and by about 18% at 6 months.

[0123] The reprogramming efficiency was evaluated by isolating the cardiomyocytes of each group of mice, and then quantitatively analyzing the proportion of cardiac cells expressing cardiomyocyte-specific markers using confocal microscopy and flow cytometry. The reprogramming efficiency of mice in the experimental group was higher than that in the control group. Observed by confocal microscopy, the proportion of cells expressing the cardiomyocyte-specific marker cTnT in the experimental group was significantly higher than that in the control group. At 6 months, the proportion of reprogrammed cells in the experimental group reached more than 40%, while the reprogramming efficiencies of comparison groups 1 and 2 were about 15% and 20%, respectively. Flow cytometric analysis showed that the proportion of cells positive for cardiomyocyte markers in the experimental group increased significantly 2 months after transfection, reaching 35%, and stabilized at about 40% at 6 months.

[0124] In summary, the flexible electrode combined with miRNA transfection of the present invention has a significant effect on myocardial remodeling and cardiac function recovery, and has high efficiency and long-term stability in myocardial reprogramming.

[0125] Test Example 5 In this test example, pigs were used as experimental subjects to test the in situ myocardial reprogramming effect of the flexible electrode of the embodiment.

[0126] Healthy pigs (60-80 kg) were fasted for 12 h and water deprived for 4 h before surgery. Anesthesia was induced by intramuscular injection of droperidol (0.5 mg / Kg), ketamine (12 mg / Kg), and diazepam (1 mg / Kg). The pigs were fixed on the operating table in a supine position, the ear vein was opened, and propofol (5 mg / Kg / h) was administered to maintain anesthesia. The pigs were intubated and connected to a ventilator to control ventilation. The tidal volume was controlled at 0.4-0.6 L / min, the respiratory rate was 18 times / min, the respiratory ratio was 1:2, and the oxygen concentration was 60%. After skin preparation, the ECG monitor was connected to observe vital signs and ECG changes. The skin was prepared in the inguinal area of ​​the experimental pigs, iodine disinfection and draping were performed, the right femoral artery was punctured, the radial artery sheath was inserted, and 10,000 U of heparin was injected for systemic heparinization, followed by an additional 2,000 U every half an hour. The guiding catheter was inserted through the arterial sheath to the left coronary artery ostium for angiography. The guidewire was sent to the distal end of the left anterior descending artery (LAD), and the balloon was sent along the guidewire to the junction of the middle and lower 1 / 3 of the LAD and 2 mm distal to the opening of the second diagonal branch. The balloon was expanded and completely blocked for 120 minutes. The electrocardiogram showed that the ST segments of multiple leads were clearly arched upward, indicating that the model was successfully constructed. At the same time, blood samples were drawn to check for myocardial infarction markers to verify the success of the model. A pig model of myocardial infarction was obtained.

[0127] The miRNA combination (miR-1, miR-133, miR-208, miR-409 were mixed at a molar ratio of 1:1:1:1, and the final concentration of each miRNA was 1 μM) and the mRNA combination (Myocardin mRNA (final concentration 2 μM), Mef2c mRNA (final concentration 2 μM), TBX5 mRNA (final concentration 2 μM), miR-133 (final concentration 1 μM)) were dissolved in sterile phosphate buffer to obtain electrotransfection solution. Twelve myocardial infarction pig models were randomly divided into three groups, with 4 pigs in each group.

[0128] (1) Experimental group (n=4): The flexible electrode of Example 2 (the electrode is designed as a 10×10 array) was implanted into the heart surface of a pig model with myocardial infarction through thoracic surgery (the encapsulation layer adheres to the heart surface), ensuring that the flexible electrode is closely attached to the heart surface. Use a sterile syringe to evenly distribute 500 μL of electrotransfection solution on the surface of the flexible electrode encapsulation layer, and apply a pulsed electric field to the flexible electrode (electric field strength: 100 V / cm, pulse width: 10 milliseconds, pulse frequency: 1 Hz, duration: 10 minutes).

[0129] (2) Control group (n=4): The flexible electrode of Example 2 (the electrode is designed as a 10×10 array) was implanted into the heart surface of a myocardial infarction pig model through thoracic surgery (the encapsulation layer fits the heart surface), ensuring that the flexible electrode fits the heart surface tightly. Use a sterile syringe to evenly distribute 500 μL of the electrotransfection solution without the miRNA combination on the surface of the flexible electrode encapsulation layer, and apply a pulsed electric field (electric field strength: 100 V / cm, pulse width: 10 milliseconds, pulse frequency: 1 Hz, duration: 10 minutes) to the flexible electrode.

[0130] (3) Sham operation group (n=4): The pig model of myocardial infarction underwent thoracic surgery without implantation of any electrodes.

[0131] Cardiac function was tested using echocardiography and magnetic resonance imaging at 1 week, 1 month, and 3 months after surgery; the conductivity of the electrodes, including conductivity and resistance, was measured regularly. Thirty days after surgery, cardiac tissue samples were taken from some pigs for histological analysis to detect the expression level of inflammatory marker TNF-α in cardiac tissue to evaluate the inflammatory response of cardiac tissue. The cardiac tissue was processed using tissue sectioning technology, and H&E staining was used to evaluate the infiltration of inflammatory cells, tissue structural damage, and cell apoptosis in the tissue sections; the expression levels of fibrosis-related genes (such as TGF-β1 and α-SMA) in cardiac tissue were detected, and Masson trichrome staining was used to evaluate the deposition of collagen fibers in cardiac tissue. After 3 months, myocardial fibroblasts in the heart tissue of each group were isolated, and the expression levels of Myocardin mRNA, Mef2c mRNA, TBX5 mRNA, and miR-133 were detected by qPCR; immunofluorescence staining was used to evaluate cell reprogramming and calculate the reprogramming efficiency (reprogrammed cells / total number of cells); Masson staining was used to evaluate the degree of fibrosis; and H&E staining was used to observe the pathological changes of heart tissue. The growth of tissue around the electrode was observed under a microscope to evaluate whether the electrode was firmly bonded to the heart tissue and whether tissue peeling or incomplete integration occurred. The degree of mechanical stimulation of the electrode on the heart tissue was detected to avoid causing adverse mechanical damage or myocardial degeneration.

[0132] The LVEF of the experimental group pigs gradually increased 1 week, 1 month, and 3 months after surgery, showing a high reprogramming efficiency (25%), and no significant fibrosis in the heart tissue. The LVEF of the representative pig No. 1 was 40%, 55%, and 65% at 1 week, 1 month, and 3 months after surgery, respectively, and the LVEF of the representative pig No. 2 was 32%, 40%, and 55% at 1 week, 1 month, and 3 months after surgery, respectively.

[0133] The LVEF of the control group pigs did not change significantly at 1 week, 1 month, and 3 months after surgery, and the reprogramming efficiency was 0%. The LVEF of the representative pig No. 3 was 30% at 1 week, 1 month, and 3 months after surgery, and the LVEF of the representative pig No. 4 was 30%, 32%, and 30% at 1 week, 1 month, and 3 months after surgery, respectively.

[0134] The resistance value of the flexible electrode of Example 2 was 0.5 Ω at the initial stage of implantation, and increased to 0.53 Ω (increased by about 5%) 30 days after implantation. The conductive performance was still stable 30 days after implantation, and no obvious decrease in conductive performance was observed.

[0135] Compared with the sham operation group, the expression of TNF-α, an inflammatory marker in the heart tissue of pigs in the experimental group was reduced by more than 50%. The experimental group showed less tissue inflammation and rejection reaction after surgery, indicating good tissue compatibility.

[0136] After the flexible electrode of Example 2 was coated with a layer of 50 nm thick PEDOT conductive polymer, its in situ myocardial reprogramming effect, performance stability, biosafety, etc. on pigs were similar to those of the flexible electrode of Example 2.

[0137] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A flexible electrode, It is characterized in that The flexible electrode comprises a flexible substrate layer, a conductive electrode layer, a packaging layer and electrode contacts which are stacked together; the conductive electrode layer comprises a wire and a conductive contact which are connected to each other; a through-hole structure is provided on the packaging layer, and the through-hole structure exposes the conductive contact; and the electrode contact is arranged on the conductive contact.

2. The flexible electrode according to claim 1, It is characterized in that The flexible base layer includes at least one of TPU, PET, and silicone; And / or, the thickness of the flexible substrate layer is 0.1 mm to 1 mm; And / or, the Shore hardness of the flexible base layer is 70A-90A; And / or, the tensile modulus of the flexible substrate layer is 5 MPa to 15 MPa; And / or, the elongation at break of the flexible substrate layer is 300% to 600%; and / or, the moisture permeability coefficient of the flexible substrate layer is greater than 10 g / m² / day; And / or, the air permeability coefficient of the flexible substrate layer is 0.5~5 cm³ / cm² / s.

3. The flexible electrode according to claim 1, It is characterized in that The conductive electrode layer is at least one of liquid metal, silver paste, and carbon nanotubes; And / or, the conductivity of the conductive electrode layer is not less than 3.4×10 6 S / m; And / or, the Young's modulus of the conductive electrode layer is 0.01 MPa to 10 MPa; And / or, the elongation at break of the conductive electrode layer is not less than 300%.

4. The flexible electrode according to claim 1, It is characterized in that The encapsulation layer includes at least one of TPU, PVDF, PVA, and PTFE; And / or, the thickness of the encapsulation layer is 50 μm to 200 μm And / or, the Young's modulus of the encapsulation layer is 0.1 MPa to 10 MPa; And / or, the elongation at break of the encapsulation layer is 50% to 500%; And / or, the flexural strength of the encapsulation layer is 1 MPa to 100 MPa; And / or, the tensile strength of the encapsulation layer is 5 MPa to 50 MPa; And / or, the tear strength of the encapsulation layer is 10 kN / m~200 kN / m.

5. The flexible electrode according to claim 1, It is characterized in that The flexible base layer has a radius of curvature that is consistent with the surface of the targeted tissue; And / or, the thickness of the electrode contact modification layer is 20 nm to 50 nm.

6. The flexible electrode according to claim 3, It is characterized in that The flexible electrode also includes a conductive polymer layer, which is coated on a composite body formed by the flexible substrate layer, the conductive electrode layer, the encapsulation layer, and the electrode contacts; preferably, the thickness of the conductive polymer layer is 50 nm ~ 500 nm; preferably, the conductive polymer layer includes at least one of PEDOT, PANI, and PPy; preferably, the tensile strength of the conductive polymer layer is 1 MPa ~ 10 MPa; preferably, the Shore hardness of the conductive polymer layer is 5A ~ 30A.

7. The method for preparing the flexible electrode according to any one of claims 1 to 7, It is characterized in that The following steps are involved: S1. providing a flexible base layer; S2, printing the conductive electrode layer on the flexible substrate layer; S3, manufacturing the encapsulation layer on the conductive electrode layer; S4. Making the electrode contacts on the conductive contacts.

8. An electrotransfection method for non-disease diagnosis and treatment, It is characterized in that The following steps are involved: The target cell or target tissue is electrotransfected by the flexible electrode according to any one of claims 1 to 7, so as to introduce exogenous substances into the target cell or target tissue.

9. Use of the flexible electrode according to any one of claims 1 to 7 in the preparation of a product for repairing cardiac tissue damaged after myocardial infarction, a gene editing product, an electroporation system or an electrophysiological monitoring system.

10. A gene editing kit, an electroporation system or an electrophysiological monitoring system, comprising the flexible electrode according to any one of claims 1 to 5.

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