An electroporation device and its application

Abstract

The present invention provides an electroporation device and its application, the electroporation device comprises a positive electrode unit and a push-pull slide; the positive electrode unit comprises a connecting handle, a receiving portion and a positive electrode body; a push-pull slide is installed on the connecting handle, and the push-pull slide can move along the length direction of the connecting handle; the inner hollow of the receiving portion forms a receiving cavity, and the first end of the receiving portion is connected to the connecting handle; the material of the positive electrode body is a shape memory alloy, the first end of the positive electrode body passes through the receiving cavity and is arranged in the connecting handle, and the first end of the positive electrode body is connected to the push-pull slide; the second end of the positive electrode body has a disc-shaped spiral structure. One technical effect of the present invention is that the design is reasonable, not only can the micro-electric field be introduced into the eye, and the precise electroporation of the retina is realized, but also the influence range of the electric field is reduced, the damage to other non-target tissues of the eyeball is reduced, and the surgical risk is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of electroporation equipment, and in particular relates to an electroporation device and application thereof. Background Art

[0002] Inherited retinal diseases (IRDs) are a class of irreversible blinding eye diseases. IRDs are often single-gene diseases with genetic heterogeneity, and more than 300 pathogenic genes have been discovered. Gene therapy is an important method for the treatment of IRDs. This method aims to achieve therapeutic effects by transfecting normal exogenous genes into target cells to correct or compensate for diseases caused by defective or abnormal genes. Transfection methods are mainly divided into two categories: viral and non-viral.

[0003] Among viral vectors, adeno-associated virus (AAV) is one of the most commonly used viral vectors and the only FDA-approved vector for gene therapy drugs due to its advantages such as good cell targeting, high transduction rate, low immunogenicity, wide range of host cells, and ability to maintain long-term continuous expression of therapeutic genes. However, the capacity of AAV vectors is relatively limited (<4.7kb), which limits its clinical application to a certain extent. To this end, some researchers have modified and transformed AAV vectors to regulate the specific expression of genes, while improving their loading capacity and cell targeting and reducing immunogenicity. However, the potential biological risks, immunogenicity, short loading gene fragments, and high costs of viral vectors have greatly limited their application in gene therapy of retinal diseases. Non-viral methods circumvent potential biological risks, immunogenicity, short loading gene fragments, and high costs, have good potential carrying capacity, and can support longer exogenous gene lengths without restrictions. Non-viral vectors mainly transduce therapeutic genetic materials through physical and chemical methods, including needles, gene guns, electroporation, sonoporation, photoimaging, magnetic field conversion, hydrogenation, calcium phosphate co-precipitation, liposomes, nanoparticle-mediated methods, etc.

[0004] Electroporation is a physical transfection method that uses an appropriate electric field to instantly increase the permeability of the cell membrane. It has low manufacturing costs, high efficiency, simple preparation, and is suitable for large-scale production. It has high transfection efficiency and low toxicity, and has no restrictions on the length of gene fragments. It has the potential to become an alternative to adeno-associated virus retinal gene delivery. At present, this technology is still in the laboratory research stage and has only been verified through in vitro / in vivo experiments on small animal models. There are mainly three problems in its clinical transformation:

[0005] 1. The electroporation device has low surgical compatibility. The device developed by the institute is not operated on an eyeball that is close to the size of the human eye, has low portability, and has a high risk of being affected by the designed surgical site.

[0006] 2. The experimental voltage of the electroporation device is high. The voltage used in the study is significantly higher than the safe voltage for the human body, and the implementation of this voltage cannot be supported in labor research.

[0007] 3. Unable to accurately focus on the retinal transfection area. The devices developed by the institute usually discharge at a remote distance. The entire eyeball is within the high electric field range and cannot accurately focus on the retinal layer, which can easily cause unnecessary point damage.

[0008] Therefore, electroporation technology is currently limited by problems such as high electroporation voltage, low electrode compatibility, and large damage to the electroporation area. It has not yet been approved for clinical use in retinal gene therapy. Retinal transgenic technology that is safe, non-immunogenic, and suitable for long gene fragment transfection needs to be developed urgently. Summary of the invention

[0009] The present invention aims to solve at least one of the technical problems existing in the prior art and provide a new technical solution for an electroporation device and its application.

[0010] According to a first aspect of the present invention, there is provided an electroporation device, comprising:

[0011] A positive electrode unit and a push-pull slide, wherein the positive electrode unit comprises a connecting handle, a receiving portion and a positive electrode body; the connecting handle is provided with a push-pull slide, and the push-pull slide can move along the length direction of the connecting handle; the receiving portion is hollow inside to form a receiving cavity, and the first end of the receiving portion is connected to the connecting handle; the positive electrode body is made of a shape memory alloy, the first end of the positive electrode body passes through the receiving cavity and is arranged in the connecting handle, and the first end of the positive electrode body is connected to the push-pull slide; the second end of the positive electrode body has a disc-shaped spiral structure;

[0012] In the first temperature range, the positive electrode body undergoes non-elastic deformation, and the push-pull slide plate can move relative to the connecting handle and drive the disc-shaped spiral structure to be accommodated in the accommodating cavity or to extend from the accommodating cavity;

[0013] In the second temperature range, the disc-shaped spiral structure extending from the accommodating cavity maintains a preset shape and can act on the retinal treatment area to discharge electricity to the retinal treatment area.

[0014] Optionally, the positive electrode body comprises a positive electrode functional area, a first electrode connection area and a first connection reinforcement area; one end of the first electrode connection area is connected to the positive electrode functional area, and the other end is connected to the first connection reinforcement area; the first connection reinforcement area is fixedly connected to the push-pull slide;

[0015] The disc-shaped spiral structure forms the positive electrode functional area;

[0016] The first electrode connection area and the first connection reinforcement area are both in the shape of long strips, and the diameter of the first electrode connection area is smaller than the diameter of the first connection reinforcement area.

[0017] Optionally, in the second temperature range, the disc-shaped spiral structure extending from the accommodating cavity forms a preset angle with the first electrode connection area.

[0018] Optionally, the preset angle is 20°-60°.

[0019] Optionally, surfaces of the first electrode connection region and the first connection reinforcement region are both coated with an insulating layer.

[0020] Optionally, the connecting handle comprises a first clamping portion and an electrode connecting portion located at the ends, and a second clamping portion located in the middle;

[0021] The first clamping portion is fixedly connected to the first end of the accommodating portion; the push-pull slide is fixedly connected to the first connection reinforcement area at the second clamping portion; and the positive electrode body is electrically connected to the pulse generator through the electrode connecting portion.

[0022] Optionally, the electroporation device further comprises a negative electrode unit, the negative electrode unit comprising a negative electrode functional area, a second electrode connection area and a second connection reinforcement area; the negative electrode functional area is connected to the second connection reinforcement area through the second electrode connection area, and the second connection reinforcement area is used to connect to a pulse generator;

[0023] The negative electrode functional area, the second electrode connection area and the second connection reinforcement area constitute a hook negative electrode.

[0024] Optionally, the first temperature range is 0°C~25°C, and the second temperature range is 30°C~37°C.

[0025] Optionally, the accommodating portion is a tubular structure, and the inner diameter of the accommodating portion is 0.35 mm to 0.45 mm, and the outer diameter of the accommodating portion is 0.5 mm to 0.6 mm.

[0026] According to a second aspect of the present invention, there is provided a use of an electroporation device, wherein the electroporation device as described in the first aspect is used to perform gene therapy on inherited retinal diseases.

[0027] A technical effect of the present invention is:

[0028] In the embodiment of the present application, the positive electrode unit can introduce a micro-electric field into the eye through a special ophthalmic sheath needle, thereby realizing precise electroporation of the retina. The second end of the positive electrode body has a disc-shaped spiral structure, and the material of the positive electrode body is a shape memory alloy. When the disc-shaped spiral structure extending from the accommodating cavity maintains a preset shape and acts on the retinal treatment area, a uniformly distributed electric field on the retina can be achieved, and precise focusing of the retinal treatment area can be achieved.

[0029] In addition, by using a positive electrode body made of shape memory alloy, the positive electrode body can move freely in and out of the eyeball and ensure an electric field action area within a certain action requirement range, which can accurately achieve directional electroporation of the target retinal tissue, thereby reducing the electric field influence range, reducing damage to other non-target tissues of the eyeball, reducing surgical risks, and having higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic structural diagram of a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0031] Figure 2 This is a schematic diagram of the exploded structure of a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0032] Figure 3 This is a schematic structural diagram of a positive electrode body of a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0033] Figure 4 This is a schematic structural diagram of a connection handle of a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0034] Figure 5 This is a schematic structural diagram of a positive electrode functional area of ​​a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0035] Figure 6 A schematic diagram of the connection between a positive electrode functional area and a first electrode connection area of ​​a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0036] Figure 7 A reference diagram of a first use state of a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0037] Figure 8 A reference diagram of a second use state of a positive electrode unit of an electroporation device according to an embodiment of the present invention;

[0038] Fig. 9 It is a schematic structural diagram of a negative electrode unit of an electroporation device according to an embodiment of the present invention;

[0039] Fig.10 It is a schematic diagram of the details of various parts of a negative electrode unit of an electroporation device according to an embodiment of the present invention;

[0040] Fig.11 This is a schematic diagram of the matching relationship between a push-pull slide and a connecting handle of an electroporation device according to an embodiment of the present invention;

[0041] Fig.12 A schematic diagram of a push-pull slide of an electroporation device according to an embodiment of the present invention;

[0042] Fig.13 A reference diagram of an electroporation device in use according to an embodiment of the present invention;

[0043] Fig.14 This is a slice diagram of 293 cells electroporated using an electroporation device according to an embodiment of the present invention;

[0044] Fig.15 This is a New Zealand rabbit retinal electroporation slice diagram using an electroporation device according to an embodiment of the present invention.

[0045] In the figure: 1, positive electrode unit; 101, connecting handle; 1011, first clamping part; 1012, electrode connecting part; 1013, second clamping part; 102, accommodating part; 103, positive electrode body; 1031, positive electrode functional area; 1032, first electrode connecting area; 1033, first connection reinforcement area;

[0046] 2. Push and pull the slide;

[0047] 3. Negative electrode unit; 301. Negative electrode functional area; 302. Second electrode connection area; 303. Second connection reinforcement area;

[0048] 41. Positive electrode interface of the pulse generator device; 42. Negative electrode interface of the pulse generator device; 5. Eyeball; 6. Retina; 7. Plasmid bubble; 8. Sheath needle. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present application.

[0050] The embodiments of the present application will be described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and should not be construed as limitations on the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present application.

[0051] The term "first" or "second" in the specification and claims of this application may include one or more of the features explicitly or implicitly. In the description of this application, unless otherwise specified, "plurality" means two or more. In addition, "and / or" in the specification and claims means at least one of the connected objects, and the character " / " generally means that the objects connected before and after are in an "or" relationship.

[0052] In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0053] In the description of this application, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0054] According to a first aspect of the present invention, see Figures 1 to 15 , provides an electroporation device for retinal gene delivery.

[0055] Specifically, the electroporation device comprises:

[0056] A positive electrode unit 1 and a push-pull slide 2, wherein the positive electrode unit 1 comprises a connecting handle 101, a receiving portion 102 and a positive electrode body 103; a push-pull slide 2 is mounted on the connecting handle 101, and the push-pull slide 2 can move along the length direction of the connecting handle 101; the interior of the receiving portion 102 is hollow to form a receiving cavity, and the first end of the receiving portion 102 is connected to the connecting handle 101; the material of the positive electrode body 103 is a shape memory alloy, the first end of the positive electrode body 103 passes through the receiving cavity and is arranged in the connecting handle 101, and the first end of the positive electrode body 103 is connected to the push-pull slide 2; the second end of the positive electrode body 103 has a disc-shaped spiral structure; wherein the receiving portion 102 can enter the eyeball 5 through the sheath needle 8;

[0057] In the first temperature range, the positive electrode body 103 undergoes non-elastic deformation, and the push-pull slide 2 can move relative to the connecting handle 101 and drive the disc-shaped spiral structure to be accommodated in the accommodation cavity or to extend from the accommodation cavity;

[0058] In the second temperature range, the disc-shaped spiral structure extending from the accommodation cavity maintains a preset shape and can act on the retinal treatment area to discharge to the retinal treatment area. The preset shape is a disc-shaped spiral or a mosquito coil shape.

[0059] In the embodiment of the present application, the positive electrode unit 1 can introduce a micro-electric field into the eye through an ophthalmic sheath needle 8, thereby achieving precise electroporation of the retina. The second end of the positive electrode body 103 has a disc-shaped spiral structure, and the material of the positive electrode body 103 is a shape memory alloy. When the disc-shaped spiral structure extending from the accommodating cavity maintains a preset shape and acts on the retinal treatment area, a uniformly distributed electric field on the retina can be achieved, and precise focusing of the retinal treatment area can be achieved.

[0060] In addition, by using the positive electrode body 103 made of shape memory alloy, the positive electrode body 103 can freely enter and exit the eyeball 5, and the electric field action area within a certain action requirement range is guaranteed, which can accurately achieve directional electroporation of the target retinal tissue, thereby reducing the electric field influence range, reducing damage to other non-target tissues of the eyeball 5, reducing surgical risks, and having higher safety.

[0061] It should be noted that the disc-shaped spiral structure based on the positive electrode unit 1 can be accommodated in the accommodating cavity or extended from the accommodating cavity, and maintains a preset shape within the second temperature range and can act on the retinal treatment area. The electroporation device of this embodiment explores its feasibility in applying to intraocular surgical pathways and forms a standardized process, so that the surgical operation process can be completed within 10 minutes, greatly improving the surgical efficiency.

[0062] In this embodiment, see Figure 7 and Figure 8 During the use of the positive electrode unit 1, the disk-shaped spiral structure can be freely moved in and out of the accommodating portion 102 by the push-pull slide 2. Before the electroporation device is used, the push-pull slide 2 is at the far right, and the disk-shaped spiral structure is accommodated in the accommodating portion 102; when the electroporation device is used, that is, after the positive electrode unit 1 enters the eyeball 5 through the sheath needle 8, the push-pull slide 2 is at the far left, and the disk-shaped spiral structure extends out of the accommodating cavity.

[0063] Exemplarily, the connection port at the rear end of the connection handle 101 is used to connect an electric pulse generator; the material of the connection port is a metal material, an alloy material, an inorganic non-metallic material or a polymer conductive material.

[0064] The process of using the electroporation device is as follows:

[0065] The positive electrode body 103 is received in the accommodating portion 102 and passes through the sheath needle 8 and enters the inside of the eyeball 5. The positive electrode body 103 extends from the accommodating portion 102 and after sensing the temperature change inside the eyeball 5, it recovers to form a mosquito coil disc (i.e., a disc-shaped spiral structure), which greatly increases the range of the action area of ​​the positive electrode unit 1. In addition, the mosquito coil disc-shaped electrode functional area and the first electrode connection area 1032 have a certain degree of curvature, which can facilitate the doctor to insert the positive electrode unit 1 from the sheath needle 8 into the eyeball 5 and closely contact the positive electrode functional area 1031 with the plasmid bubble, which is beneficial to the doctor's operation. Among them, the accommodating portion 102 plays a key role both before and after the operation. Before the operation, it mainly serves as an isolation layer between the positive electrode unit 1 and the sheath needle 8, which can reduce the friction between the sheath needle 8 and the inner wall of the eyeball 5 when the positive electrode unit 1 enters the eyeball 5 and is deformed. After the operation, it can cooperate with the push-pull slide 2 to retract the positive electrode body 103, thereby withdrawing the electroporation device in situ without the need for additional incisions to remove the electrode.

[0066] In one embodiment, the material of the positive electrode body 103 is a one-way memory alloy, such as a nickel-titanium memory alloy. The one-way memory alloy deforms between two shapes. The reason is that during the manufacturing process, the positive electrode functional area 1031 in the second form (i.e., a mosquito coil disc or a flat spiral) is cooled to a low temperature and then molded into the first form. After the above-mentioned advance treatment, when the positive electrode functional area 1031 is affected by the temperature increase, the positive electrode functional area 1031 will change from the first form to the second form. The deeper reason is that the memory alloy will undergo phase change at different temperatures. For example, at low temperatures, it is mainly martensite, and at high temperatures, it is transformed from martensite to austenite, with austenite being the main form.

[0067] Optionally, the positive electrode body 103 includes a positive electrode functional area 1031, a first electrode connection area 1032 and a first connection reinforcement area 1033; one end of the first electrode connection area 1032 is connected to the positive electrode functional area 1031, and the other end is connected to the first connection reinforcement area 1033; the first connection reinforcement area 1033 is fixedly connected to the push-pull slide 2;

[0068] The disc-shaped spiral structure forms the positive electrode functional area 1031;

[0069] The first electrode connection area 1032 and the first connection reinforcement area 1033 are both in the shape of long strips, and the diameter of the first electrode connection area 1032 is smaller than the diameter of the first connection reinforcement area 1033 .

[0070] In the above embodiment, the design of the positive electrode body 103 is relatively reasonable, which not only helps to ensure the stability of the connection between the first connection reinforcement area 1033 and the push-pull slide 2, but also helps to ensure that the positive electrode functional area 1031 and the first electrode connection area 1032 can move freely in the accommodating portion 102, so that the positive electrode functional area 1031 can be smoothly received in the accommodating cavity or extended from the accommodating cavity.

[0071] Optionally, in the second temperature range, the disc-shaped spiral structure extending from the accommodating cavity forms a preset angle with the first electrode connection area 1032. This helps to align the disc-shaped spiral structure with the retinal treatment area, facilitating the doctor's operation.

[0072] Optionally, the preset angle is 20°-60°, which helps to smoothly achieve accurate focusing of the disc-shaped spiral structure and the retinal treatment area.

[0073] Optionally, the surfaces of the first electrode connection region 1032 and the first connection reinforcement region 1033 are coated with an insulating layer. The positive electrode functional region 1031 is treated with acid to remove the oxide layer to enhance the conductivity of the electrode.

[0074] The above implementation manner generally helps to ensure the safety of the surgical procedure.

[0075] Optionally, the connecting handle 101 includes a first clamping portion 1011 and an electrode connecting portion 1012 located at the end, and a second clamping portion 1013 located in the middle;

[0076] The first clamping portion 1011 is fixedly connected to the first end of the accommodating portion 102 ; the push-pull slide 2 is fixedly connected to the first connection reinforcement area 1033 at the second clamping portion 1013 ; and the positive electrode body 103 is electrically connected to the pulse generator via the electrode connecting portion 1012 .

[0077] In the above embodiment, the design of the connecting handle 101 is relatively reasonable, which helps the doctor to quickly perform the surgical operation.

[0078] Optionally, the electroporation device further comprises a negative electrode unit 3, wherein the negative electrode unit 3 comprises a negative electrode functional area 301, a second electrode connection area 302 and a second connection reinforcement area 303; the negative electrode functional area 301 is connected to the second connection reinforcement area 303 via the second electrode connection area 302, and the second connection reinforcement area 303 is used to connect to a pulse generator;

[0079] The negative electrode functional area 301, the second electrode connection area 302 and the second connection reinforcement area 303 constitute a hook negative electrode.

[0080] In the above embodiment, through the cooperation of the positive electrode unit 1 and the negative electrode unit 3, directional electroporation of the target retinal tissue can be achieved, thereby reducing the influence range of the electric field, reducing the damage to other non-target tissues of the eyeball 5, reducing surgical risks, and improving safety.

[0081] Exemplarily, the tail of the hook negative electrode (i.e., the second connection reinforcement area 303) is electrically connected to the electric pulse generator, and the head area (i.e., the negative electrode functional area 301) is provided with a conductive layer (i.e., a directional electroporation structure) for concentrating discharge to the target tissue (i.e., the retinal treatment area) to reduce the range of the electric field.

[0082] Exemplarily, the electrode material of the negative electrode unit 3 is at least one of platinum, gold, silver, cobalt-based alloys, titanium-based alloys, stainless steel, shape memory alloys, precious metals, pure metal niobium, zirconium, titanium, and tantalum.

[0083] Furthermore, the surfaces of the second electrode connection area 302 and the second connection reinforcement area 303 are both covered with an insulating material, and the insulating material is polychlorofluoroethylene, Teflon, polyimide or parylene; the thickness of the insulating material is 0.001 mm to 0.1 mm.

[0084] In a specific embodiment, the receiving portion 102 can be directly made of a metal material, or the surface of the metal material can be covered with an insulating coating. The metal material is at least one of platinum, gold, silver, cobalt-based alloy, titanium-based alloy, stainless steel, shape memory alloy, precious metal, pure metal niobium, zirconium, titanium, and tantalum, and the insulating material is polychlorofluoroethylene, Teflon, polyimide, or parylene.

[0085] Optionally, the first temperature range is 0° C. to 25° C., and the second temperature range is 25° C. to 37° C. Both the first temperature range and the second temperature range are within the acceptable range for human body, and can realize the morphological change of the positive electrode functional area 1031 (shape memory alloy).

[0086] Optionally, the accommodating portion 102 is a tubular structure, and the inner diameter of the accommodating portion 102 is 0.35 mm to 0.45 mm, and the outer diameter is 0.5 mm to 0.6 mm. This helps to accommodate the disc-shaped spiral structure in the accommodating cavity or extend it from the accommodating cavity.

[0087] In a specific embodiment, the main functional area of ​​the electroporation device is the electrode pair, that is, the positive electrode unit 1 and the negative electrode unit 3 of this embodiment. The head (that is, the second end) of the positive electrode body 103 of the positive electrode unit 1 is a disc-shaped spiral structure, and the disc-shaped spiral structure is provided with a conductive area for concentrated discharge to the target tissue (that is, the retinal treatment area) to reduce the range of the electric field. Among them, the disc-shaped spiral structure is also a mosquito coil disc-shaped structure. The tail (that is, the end of the first end) of the positive electrode body 103 of the positive electrode unit 1 is electrically connected to the electric pulse generator.

[0088] Exemplarily, the material of the disc-shaped spiral structure is nickel-titanium memory alloy material, and the wire diameter is 0.05-0.3 mm. The surfaces of the first electrode connection area 1032 and the first connection reinforcement area 1033 are coated with insulating material, and the insulating material is polychlorofluoroethylene, Teflon, polyimide or parylene, and the thickness of the insulating material is 0.001 mm to 0.1 mm.

[0089] Since the positive electrode body 103 has shape memory characteristics, it can be deformed freely at the temperature outside the human eyeball 5 , and when it is at the temperature inside the human eyeball 5 , it can restore the deformation to a preset shape, such as a disc spiral.

[0090] According to a second aspect of the present invention, there is provided a use of an electroporation device, wherein the electroporation device as described in the first aspect is used to perform gene therapy on inherited retinal diseases.

[0091] In a specific implementation, see Fig.13 , the application of the electroporation device includes the following steps:

[0092] Step 1, preoperative treatment: general anesthesia (Sutane+Sumianxin) → mydriasis (compound tropicamide) → surface anesthesia (procaine) → skin disinfection (Aner iodine) → drape → local disinfection (povidone iodine) → retrobulbar anesthesia (lidocaine).

[0093] Step 2, open the surgical chamber: on the one hand, make a 5mm incision parallel to the limbus of the cornea above the nose, and bluntly separate the conjunctival sac to the back of the eyeball 5; on the other hand, make a 23G trocar (also known as sheath needle 8) 3.5mm above the nose and limbus.

[0094] Step 3, plasmid injection: 8-0 suture fixation loop → place 30° oblique mirror; inject 15ul plasmid suspension subretinaly under the trocar nose to form a plasmid bubble7.

[0095] Step 4, inserting the electroporation device: the negative electrode unit 3 enters through the incision above the nose and is placed below the plasmid bubble 7; the positive electrode unit 1 enters the eye through the trocar, pushes out the positive electrode functional area 1031 (i.e., the disc-shaped spiral structure), waits for it to complete deformation in the second temperature range, and is placed on the retina 6 on the surface of the plasmid bubble;

[0096] Step 5, electrotransfer: the positive electrode unit 1 is connected to the positive electrode interface 41 of the pulse generator device, and the negative electrode unit 3 is connected to the negative electrode interface 42 of the pulse generator device, and the electrotransfer parameters are set, and pulses are applied to complete the electrotransfer; then, the positive electrode unit 1 is withdrawn, and the negative electrode unit 3 is withdrawn.

[0097] Step 6, postoperative treatment: cut the fixing sutures, suture the scleral opening with 8-0 sutures, and apply Diabeto eye ointment to the eyes.

[0098] To verify the effectiveness of the electroporation device, the functionality of the electroporation device was verified by using EGFP plasmid and 293 cells as a cell experiment paradigm. Fig.14 Under a fluorescence microscope, within a certain range, as the voltage increases, the electroporation efficiency gradually increases, and the cell EGFP expression is enhanced, indicating that the positive electrode unit achieves efficient cell transfection under a certain electric field strength; the operability and effectiveness of the electroporation device were verified using New Zealand rabbits as an animal experimental paradigm. The operability of the intraoperative route via the sheath needle device was verified for the first time on a large living animal model using the positive electrode unit, and the clinical use of the surgical device process was designed based on animal living experiments, which can achieve efficient transfection efficiency at low voltages. See Fig.15 , under a confocal microscope, EGFP expression was observed in the nerve fiber layer and RPE cell layer of retinal frozen sections.

[0099] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. An electroporation device, characterized in that: include: A positive electrode unit and a push-pull slide, wherein the positive electrode unit comprises a connecting handle, a receiving portion and a positive electrode body; the connecting handle is provided with a push-pull slide, and the push-pull slide can move along the length direction of the connecting handle; the receiving portion is hollow inside to form a receiving cavity, and the first end of the receiving portion is connected to the connecting handle; the positive electrode body is made of a shape memory alloy, the first end of the positive electrode body passes through the receiving cavity and is arranged in the connecting handle, and the first end of the positive electrode body is connected to the push-pull slide; the second end of the positive electrode body has a disc-shaped spiral structure; The positive electrode body comprises a positive electrode functional area, a first electrode connection area and a first connection reinforcement area; one end of the first electrode connection area is connected to the positive electrode functional area, and the other end is connected to the first connection reinforcement area; the first connection reinforcement area is fixedly connected to the push-pull slide; The disc-shaped spiral structure forms the positive electrode functional area; The first electrode connection area and the first connection reinforcement area are both in the shape of long strips, and the diameter of the first electrode connection area is smaller than the diameter of the first connection reinforcement area; In a first temperature range, the positive electrode body undergoes inelastic deformation, the push-pull slide can move relative to the connecting handle and drive the disc-shaped spiral structure to be accommodated in the accommodating cavity or to extend from the accommodating cavity; the first temperature range is 0°C to 25°C; In the second temperature range, the disc-shaped spiral structure extending from the accommodating cavity maintains a preset shape and can act on the retinal treatment area to discharge to the retinal treatment area, and the second temperature range is 30°C~37°C.

2. The electroporation device according to claim 1, characterized in that In the second temperature range, the disc-shaped spiral structure extending from the accommodating cavity forms a preset angle with the first electrode connecting area.

3. The electroporation device according to claim 2, characterized in that: The preset angle is 20°-60°.

4. The electroporation device according to claim 3, characterized in that The surfaces of the first electrode connection region and the first connection reinforcement region are both coated with an insulating layer.

5. The electroporation device according to claim 4, characterized in that The connecting handle comprises a first clamping portion and an electrode connecting portion located at the end, and a second clamping portion located in the middle; The first clamping portion is fixedly connected to the first end of the accommodating portion; the push-pull slide is fixedly connected to the first connection reinforcement area at the second clamping portion; and the positive electrode body is electrically connected to the pulse generator through the electrode connecting portion.

6. The electroporation device according to claim 5, characterized in that It also includes a negative electrode unit, the negative electrode unit includes a negative electrode functional area, a second electrode connection area and a second connection reinforcement area; the negative electrode functional area is connected to the second connection reinforcement area through the second electrode connection area, and the second connection reinforcement area is used to connect the pulse generator; The negative electrode functional area, the second electrode connection area and the second connection reinforcement area constitute a hook negative electrode.

7. The electroporation device according to claim 1, characterized in that: The accommodating portion is a tubular structure, and the inner diameter of the accommodating portion is 0.35mm-0.45mm, and the outer diameter is 0.5mm-0.6mm.

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