Retinal or choroidal electrical transgenic stimulator containing flexible electrodes and its application

Abstract

The present invention provides a retinal or choroidal electrical transgenic stimulator containing a flexible electrode and its application, wherein the flexible electrode comprises a flexible substrate, the flexible substrate has a concave portion, and a metal electrode layer is formed on at least the inner wall of the concave portion of the flexible substrate. The flexible electrode of the present invention can be used to simply and efficiently introduce genes into specific parts of the retina or choroid, can achieve accurate positioning and quantitative transgenic, will not cause damage to extraocular tissues, and has the advantages of low invasiveness, convenient and safe operation, low manufacturing cost, and easy large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of medical devices, and in particular to a retinal or choroidal electrical transgenic stimulator containing a flexible electrode and an application thereof. Background Art

[0002] Retinal and choroidal degeneration diseases are a type of hereditary eye disease caused by genetic abnormalities, resulting in degeneration and apoptosis of retinal or choroidal cells. The disease is mainly manifested by progressive visual acuity loss, narrowing of the visual field, night blindness, and abnormal electroretinogram (ERG), which seriously affect the patient's vision and life. Common diseases include retinitis pigmentosa, Leber congenital amaurosis (LCA), macular dystrophy, choroidal atrophy, etc. These hereditary diseases are often single-gene diseases with high genetic heterogeneity. There is no effective treatment yet, and the most promising treatment method is gene therapy.

[0003] Gene therapy is the process of introducing exogenous normal genes into corresponding target cells through gene transfer technology, correcting diseases caused by gene defects, and thus treating lesions. Currently, commonly used gene vectors include viral vectors and non-viral vectors. Viral vectors mainly include adeno-associated virus vectors, lentiviral vectors, adenoviral vectors, etc. Adeno-associated virus vectors (AAV) are currently more commonly used in ophthalmology.

[0004] Since 2007, there have been more than 110 clinical trials of gene therapy for retinal diseases, including about 60 clinical trials of AAV-mediated retinal gene therapy, which have achieved certain therapeutic effects. For example, AAV vectors are used to deliver gene editing tools to photoreceptor cells to eliminate or reverse the pathogenic IVS26 mutation on the CEP290 gene to achieve the purpose of treating LCA10 patients. However, previous studies have shown that AAV has the following defects: (1) It is potentially carcinogenic; (2) High-dose gene therapy can cause liver and nervous system damage; (3) After intraocular administration, local or systemic immune responses and intraocular inflammation will occur; (4) AAV-mediated retinal gene therapy loses its therapeutic effect after a period of time; (5) The biggest defect of AAV vectors is that their vector capacity is limited, and its maximum vector capacity does not exceed 5kb; (6) Gene therapy with AAV vectors is generally administered through vitreous injection or subretinal injection. The former has low transgenic efficiency, and the latter has the possibility of reflux and overflow of subretinal fluid; (7) Gene therapy drugs are very expensive. Taking Luxturna as an example, the treatment cost per eye is US$425,000. All these have greatly limited the clinical application of AAV vectors in gene therapy of retinal diseases.

[0005] In the 1980s, electroporation, a technique that uses electric pulses to instantly penetrate the cell membrane, emerged as a powerful intracellular delivery method that is applicable to a variety of cell types, from animal cells to plants and lower organisms. In retinal gene therapy, electroporation has the following advantages over AAV vector transfection: (1) The gene delivery capacity of electroporation is much larger than that of AAV vectors; (2) Multiple plasmids can be mixed together and electroporated and delivered into the same cell at the same time, and the efficiency of transferring several plasmids into the retina at the same time is not reduced.

[0006] Retinal electrotransfer therapy is still in the animal research stage. The commonly used technology is the invention of Matsuda and Cepko in 2003. They first injected the DNA solution into the subretinal space, and then clamped the tweezer electrodes of the electroporator on both sides of the eyes of the young mouse to perform DNA transfection. This method has the following disadvantages: (1) The application electrode is located outside the eye, and it is impossible to accurately locate the retinal electrotransfer; (2) Using the subretinal space injection technology, the DNA solution is easy to flow out and cannot be accurately quantified; (3) The voltage required for electrotransfer is high, which can easily cause damage to the intraocular and extraocular tissue cells; (4) When performing cell electroporation, the cell mortality rate is high due to uncontrollable membrane rupture, resulting in low efficiency of intracellular delivery.

[0007] In summary, transgenic technology is an effective method for treating inherited retinal diseases, but there is currently a lack of ideal technology for retinal transgenic technology. Therefore, safe, efficient, low-damage, and low-side effect retinal transgenic technology is urgently needed to be developed. Summary of the invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention proposes a flexible electrode and a preparation method and application thereof, a retinal or choroidal electrical transgenic stimulator and a retinal or choroidal electrical transgenic system, an application of a flexible electrode, a choroidal electrical transgenic stimulator or a retinal or choroidal electrical transgenic system in retinal or choroidal electrical transgenic, and a method for introducing genes into the retina and / or choroid. The flexible electrode of the present invention can be used to simply and efficiently introduce genes into specific parts of the retina or choroid, and can achieve precise positioning and quantitative transgenic without causing damage to extraocular tissues. It has the advantages of low invasiveness, easy operation, safety, low manufacturing cost, and easy large-scale production.

[0009] In one aspect of the present invention, the present invention provides a flexible electrode. According to an embodiment of the present invention, the flexible electrode comprises a flexible substrate, the flexible substrate has a concave portion, and a metal electrode layer is formed on at least an inner wall of the concave portion of the flexible substrate.

[0010] In the flexible electrode according to the embodiment of the present invention, the flexible electrode made of a flexible substrate has flexibility and compliance, and the concave structure is designed on the flexible substrate to adapt to the shape of the eyeball wall. According to different situations, the flexible electrode is placed on the retinal surface, the subretinal cavity or the suprachoroidal cavity to achieve electrical transgenesis at different positions. At the same time, the concave portion can also be used to place the solution to be transduced, and after applying voltage to the metal electrode layer, electrical transgenesis can be achieved simply and efficiently.

[0011] In another aspect of the present invention, a method for preparing the flexible electrode described above is provided. According to an embodiment of the present invention, the method comprises: (1) forming a metal electrode layer on a flexible substrate; (2) performing a molding process on the flexible substrate having the metal electrode layer formed thereon, so as to form a recess on the flexible substrate, and the metal electrode layer is formed on at least the inner wall of the recess of the flexible substrate.

[0012] In another aspect of the present invention, the present invention provides a retinal or choroidal electrical transgenic stimulator. According to an embodiment of the present invention, the retinal or choroidal electrical transgenic stimulator comprises: the flexible electrode described above.

[0013] In another aspect of the present invention, the present invention provides a retinal or choroidal electrical transgenic system. According to an embodiment of the present invention, the retinal or choroidal electrical transgenic system comprises: the retinal or choroidal electrical transgenic stimulator described above; and a power supply device, wherein the power supply device is connected to the metal electrode layer.

[0014] In another aspect of the present invention, the present invention proposes the use of the above-mentioned flexible electrode in the preparation of a medical device. According to an embodiment of the present invention, the medical device is used to treat eye diseases.

[0015] In another aspect of the present invention, the present invention proposes the use of the aforementioned flexible electrode, the aforementioned retinal or choroidal electrical transgenesis stimulator, or the aforementioned retinal or choroidal electrical transgenesis system in electrical transgenesis to the retina or choroid.

[0016] In another aspect of the present invention, a method for introducing genes into the retina and / or choroid is provided. According to an embodiment of the present invention, the method comprises: providing the retinal or choroidal electrical transgenic system described above; contacting the concave portion of the flexible electrode, at least one of the retina and choroid, and the solution containing the transgenic material; and turning on the power supply device.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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:

[0019] Figure 1 and Figure 2 A schematic diagram of a flexible electrode structure according to an embodiment of the present invention is shown respectively;

[0020] Figure 3 A schematic diagram of a process for preparing a flexible electrode according to an embodiment of the present invention is shown;

[0021] Figure 4 A MEMS processing effect diagram according to an embodiment of the present invention is shown, wherein A is a mask effect diagram, B is a flexible microelectrode array on a 4-inch silicon wafer, and C is a single microelectrode array cut out;

[0022] Figure 5 shows a schematic diagram of a flexible electrode structure according to another embodiment of the present invention;

[0023] Figure 6 The electric field simulation diagram of the electroporation device under different voltages according to one embodiment of the present invention is shown, wherein A is a simulation diagram of electric field distribution, and B is a curve showing the change of electric field intensity with vertical depth;

[0024] Figure 7 A diagram showing the in vitro culture of neonatal rat retina according to one embodiment of the present invention is shown;

[0025] Figure 8 The figure shows frozen sections and confocal microscope images of retina flat mounts after in vitro retinal electrogenic transformation of a flexible electrode chip according to an embodiment of the present invention, and a schematic diagram of one-way analysis of variance of the mean fluorescence intensity (MFIs) of retina flat mounts, wherein A is an electron microscope image of the flexible electrode electrotransfected with EGFP plasmid into retinal tissue under voltage conditions of 5V, 10V, and 15V, with a scale bar of 50μm; B is a laser confocal microscope image of the full field of view (upper figure) and retinal flat mount (lower figure), with a scale bar of 500μm; C is a statistical analysis graph of MFIs of retinal flat mounts under different electrotransfection voltages, *∶0.0001 <P<0.05,****∶P<0.0001,ns∶P> 0.9, where P < 0.05 indicates that there is a significant difference between the two groups of data, and ns indicates that there is no difference between the two groups of data;

[0026] Fig. 9 The HE staining results after retinal electroporation according to one embodiment of the present invention are shown, wherein A is a normal retinal HE staining image; BD are retinal staining images after retinal tissue is electroporated at 5V, 10V, and 15V voltages of the flexible electrode; E is a staining image of retinal tissue after electroporation at 40V voltage of the traditional electrode cup, and the scale bar is 50μm;

[0027] Fig.10 The diagram shows the evaluation of retinal damage caused by electroporation with a flexible electrode and an electrode cup according to an embodiment of the present invention, wherein A is a normal retina; BD are cell staining images of retinal tissue after electroporation with the flexible electrode at 5V, 10V, and 15V voltages, with the number of apoptotic cells less than 1%; E is a cell staining image after electroporation with a traditional electrode cup at 40V voltage, with the number of apoptotic cells greater than 25%, and the scale bar is 100μm. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0029] The present invention provides a flexible electrode and a preparation method and application thereof, a retinal or choroidal electrical transgenic stimulator and a retinal or choroidal electrical transgenic system, an application of the flexible electrode, the choroidal electrical transgenic stimulator or the retinal or choroidal electrical transgenic system in electrical transgenic to the retina or choroid, and a method for introducing genes into the retina and / or choroid. Each of these will be described in detail below.

[0030] Flexible Electrode

[0031] In a flexible electrode of the present invention. According to an embodiment of the present invention, see Figure 1 and Figure 2 The flexible electrode comprises a flexible substrate 100 , wherein the flexible substrate has a concave portion 110 , and a metal electrode layer 200 is formed on at least an inner wall of the concave portion 110 of the flexible substrate 100 .

[0032] In the flexible electrode according to the embodiment of the present invention, the flexible electrode made of a flexible substrate has flexibility and compliance, and the concave structure is designed on the flexible substrate to adapt to the shape of the eyeball wall, so that the retina or choroid fits the concave. Therefore, according to different situations, the flexible electrode can be placed on the retinal surface, the subretinal cavity or the suprachoroidal cavity to achieve electrical transgenesis at different positions. At the same time, the concave can also be used to place the solution to be transduced, and after applying voltage to the metal electrode layer, electrical transgenesis can be achieved simply and efficiently.

[0033] It should be noted that the term "flexible substrate" used in the present invention refers to a substrate made of flexible materials, and the flexible material can be PI, PET, etc. The flexible substrate is used to improve the compliance of the flexible electrode, which can be stretched and bent, can fit the human tissue, and better adapt to the shape of the eyeball wall. According to different situations, the electrode is placed on the retinal surface, the subretinal cavity or the suprachoroidal cavity to achieve electrical transgenesis at different positions to meet different needs. In some embodiments, the flexible material is preferably polyethylene terephthalate (PET), which has the following advantages: (1) The PET substrate has good dimensional stability, excellent toughness, fatigue resistance, tensile resistance, and high impact strength; (2) PET has good electrical insulation and is almost unaffected by temperature; (3) PET is non-toxic, has good weather resistance, good chemical resistance, low water absorption, and is resistant to weak acids and organic solvents; (4) The PET substrate has poor temperature resistance. When the process temperature exceeds 100°C, PET will undergo permanent deformation; (5) PET is cheap, and the film substrate is easy to make. PET films of different thicknesses can be purchased directly from the market.

[0034] According to an embodiment of the present invention, the metal electrode layer includes: a gold layer and a chromium layer, and the chromium layer is formed between the gold layer and the flexible substrate. Since the resistivity of gold is only 2.4×10-8Ω·m, it has good conductivity. In addition, gold has good biocompatibility and good resistance to oxidation and corrosion, thereby improving the overall performance of the flexible electrode.

[0035] When the sputtering technology is used to form a metal electrode layer on a flexible substrate, due to the limitations of the current sputtering technology, the sputtering thickness of the gold electrode can only be less than 0.4μm, which may result in it being unable to withstand a large current. When a high voltage is applied, the gold electrode may break. In order to increase the voltage strength that can be withstood, a chromium layer is first sputtered on the substrate as a seed layer, and then a gold layer is sputtered on the chromium layer. The chromium used as the seed layer allows the gold to form a continuous ultra-thin layer with good conductive properties on the top. The role of the chromium seed layer is to evenly disperse the gold into a continuous film.

[0036] According to an embodiment of the present invention, the metal electrode layer 200 has a hollow portion. Specifically, the metal electrode layer 200 contains interdigitated electrodes. The advantages of interdigitated electrodes are mainly as follows: (1) The flexible substrate enables it to match the different surface contours of the target tissue; (2) It can provide a uniform electric field. The rectangular interdigitated electrodes provide a uniformly distributed electric field to maintain a consistent electroporation efficiency throughout the electrode working area; (3) Using this electrode, only non-invasive operation and low electroporation voltage are required, which significantly reduces tissue damage; (4) The size of the electroporation area can be easily adjusted by controlling the voltage size or the spacing between the rectangular microelectrodes. By properly arranging the microelectrodes and tightly covering the tissue surface, the electrode focuses the electric field on the target tissue to avoid unnecessary effects or safety risks on irrelevant tissues.

[0037] According to an embodiment of the present invention, the metal electrode layer 200 comprises a microelectrode array formed by rectangular interdigitated microelectrodes. Figure 2 The ratio of the thickness a of the rectangular interdigital microelectrode, the width b of each rectangular interdigital and the distance c between the rectangular interdigital of the adjacent positive and negative electrodes is (0.05-0.10): (50-100): (150-250), and the ratio of the thickness d, length e and width f of the flexible substrate is (40-60): (1×10 4 ~2×10 4 ):(5×10 3 ~10×10 3 According to a specific embodiment of the present invention, the thickness a of the rectangular interdigital microelectrode is 0.05-0.10 μm, the width b of each rectangular interdigital is 50-100 μm, the distance c between the rectangular interdigital of adjacent positive and negative electrodes is 150-250 μm, the thickness d of the flexible substrate is 40-60 μm, and the length e is 1-2×10 4 μm, width f is 5~10×10 3μm. The inventors obtained the above-mentioned preferred dimensions through a large number of experiments, optimization and screening. Specifically, (1) the electrode thickness a should not be too high. On the one hand, it is due to the limitations of cost and sputtering process. Since it is sputtered gold, it is made on a 4-inch silicon wafer and many small electrodes are made at one time, so increasing the thickness of the electrode will obviously increase the cost a lot. On the other hand, the sputtering process makes the gold thickness cannot exceed 0.4μm. The electrode thickness a should not be too low because it will not withstand higher voltages. If the voltage is slightly higher, it may cause the microelectrode to break. The inventors expect that electroporation will be achieved at about 10V, so this thickness value is selected for the electrode. (2) The width b of a single rectangular microelectrode is obtained by balancing the cost and the maximum withstand voltage without breaking. (3) In order for the electrode to achieve the expected effect, the main consideration is the spacing c of the interdigitated electrodes, because the electrode spacing will affect the electric field strength that can be generated at the same position under the same voltage. The lower the spacing c of the interdigitated electrodes, the higher the electric field strength at the same position under the same voltage. The inventor first calculated the electric field strength value for effective electrotransfection based on the traditional parallel plate electrode, which is the target field strength to be achieved by the device. By simulating the electric field distribution, it is calculated that when the interdigital electrode spacing is c, the target field strength can be achieved at the expected voltage (such as 5 to 30V), thereby achieving the electric field strength for effective electrotransfection. (4) The substrate has a good flexibility when the thickness is d; (5) The length e and width f are mainly to ensure that the size of the retina / choroid can be matched.

[0038] The present invention does not impose strict restrictions on the design position of the metal electrode layer, as long as the metal electrode layer is formed on the inner wall of the concave portion of the flexible substrate, and whether to design the metal electrode layer at other positions on the flexible substrate other than the inner wall of the concave portion can be flexibly selected according to actual conditions. Specifically, the metal electrode layer can be formed on the flexible substrate other than the inner wall of the concave portion and on the same side as the inner wall of the concave portion, and connected to the metal electrode layer on the inner wall of the concave portion to form an integrated microelectrode array.

[0039] According to an embodiment of the present invention, the concave portion 110 is hemispherical, so that the flexible electrode can better fit the retina or choroid, and at the same time, it is also convenient to place the transgenic material to be electrified in the hemispherical concave portion.

[0040] According to an embodiment of the present invention, the radius of the hemisphere is 1 to 1.5 times the radius of the eyeball axis. Thus, the flexible electrode can be placed on the retinal surface, the subretinal cavity, or the suprachoroidal cavity to better fit the retina or choroid. Specifically, the radius of the hemisphere can be determined based on the actual situation and the length of the eyeball axis, in view of the differences in the size of the eyeball.

[0041] The flexible electrode of the present invention is applied to retinal or choroidal electrotransfer, and has the following advantages: (1) the flexible electrode is located in the eye, and gene delivery can be achieved in the eye by applying a low voltage; (2) it will not cause damage to extraocular tissues; (3) the electrode is flexible and compliant, and can adapt to the shape of the eyeball wall. According to different situations, the electrode is placed on the retinal surface, the subretinal cavity or the suprachoroidal cavity for electrotransfer; (4) the gene can be introduced into a specific part of the retina / choroid by adjusting the position of the electrode; (5) the gene can be loaded on the surface of the flexible electrode, and gene electrotransfer can be achieved in one operation; (6) the target retinal cell layer can be accurately positioned and quantitatively transfected by voltage regulation, and the gene can be introduced into a specific cell layer by changing the current and voltage; (7) electrotransfer can transfer longer gene fragments into cells, which is beneficial to gene editing; (8) it has the advantages of low invasiveness, convenient and safe operation, low manufacturing cost, and easy large-scale production.

[0042] Method for preparing flexible electrodes

[0043] In another aspect of the present invention, a method for preparing the flexible electrode described above is provided. According to an embodiment of the present invention, the method comprises: (1) forming a metal electrode layer on a flexible substrate; (2) performing a molding process on the flexible substrate having the metal electrode layer formed thereon, so as to form a recess on the flexible substrate, and the metal electrode layer is formed on at least the inner wall of the recess of the flexible substrate.

[0044] According to an embodiment of the present invention, see Figure 3 , step (1) includes: (1-1) coating a flexible substrate material on a base material to form a flexible substrate on the base material; (1-2) first sputtering metal chromium on the flexible substrate to form a chromium layer, and then sputtering metal gold on the chromium layer to form a gold layer; (1-3) performing mask photolithography on the gold layer, and then etching and degumming to form a metal electrode layer on the flexible substrate, and finally peeling the flexible substrate from the base material.

[0045] It should be noted that the present invention does not impose strict restrictions on the specific type of substrate material, and it can be flexibly selected according to actual conditions, for example, it can be a polished silicon wafer.

[0046] In step (1-2), due to the limitation of sputtering technology, the sputtering thickness of the gold electrode can only be less than 0.4 μm, which may result in it being unable to withstand a large current. When a high voltage is applied, the gold electrode may break. In order to increase the voltage intensity that can be withstood, a chromium layer is first sputtered on the substrate as a seed layer, and then a gold layer is sputtered on the chromium layer.

[0047] Photolithography is an important step in the MEMS process. It is a method of accurately copying the pattern on the film mask onto a photoresist layer by photolithography. At present, photoresists are mainly divided into positive photoresists and negative photoresists. The light-receiving part of the positive photoresist will undergo a degradation reaction and can be dissolved by the developer. After development, the non-exposed part, that is, the pattern of the mask, is left. The negative photoresist is the opposite. The non-exposed part can be dissolved by the developer. The resulting pattern is complementary to the pattern of the mask. According to an embodiment of the present invention, the photoresist used is RZJ304 photoresist. The characteristics of RZJ304 photoresist are as follows: (1) Positive photoresist, the illuminated area is developed away, leaving the photoresist in other areas that are not illuminated. Since most areas of the mask are opaque, the defect rate of the exposed pattern is reduced; (2) High resolution, high contrast, high sensitivity, and high adhesion; (3) Easy to remove the resist.

[0048] According to an embodiment of the present invention, the molding process is performed by thermoplastic molding, so as to form the concave structure quickly and accurately.

[0049] It should be noted that the features and advantages described above for the flexible electrode are also applicable to the method for preparing the flexible electrode, and will not be described in detail here.

[0050] Retinal or choroidal electrical transgenic stimulator and system

[0051] In another aspect of the present invention, the present invention provides a retinal or choroidal electrical transgenic stimulator. According to an embodiment of the present invention, the retinal or choroidal electrical transgenic stimulator comprises the flexible electrode described above.

[0052] In another aspect of the present invention, the present invention provides a retinal or choroidal electrical transgenic system. According to an embodiment of the present invention, the retinal or choroidal electrical transgenic system comprises: the retinal or choroidal electrical transgenic stimulator described above; and a power supply device, wherein the power supply device is connected to the metal electrode layer.

[0053] Therefore, the stimulator or system of the present invention can be used to simply and efficiently introduce genes into specific parts of the retina or choroid, and can achieve precise positioning and quantitative transgenesis without causing damage to extraocular tissues. It has the advantages of low invasiveness, easy and safe operation, low manufacturing cost, and easy large-scale production.

[0054] It should be noted that the present invention does not strictly limit the type of power supply device, as long as it can apply voltage to the flexible electrode to achieve electroporation. In some embodiments, the power supply device is selected from an electroporator, which uses high-voltage electric pulses to cause instantaneous reversible perforation of the cell membrane in order to introduce genes into the cell. Specifically, the voltage applied by the power supply device is 5-30V, so that gene delivery can be achieved in the eye by applying a low voltage.

[0055] It should also be noted that the features and advantages described above for the flexible electrode are also applicable to the retinal or choroidal electrical transgenic stimulator and the retinal or choroidal electrical transgenic system, and will not be repeated here.

[0056] application

[0057] In another aspect of the present invention, the present invention proposes the use of the flexible electrode described above in the preparation of a medical device. According to an embodiment of the present invention, the medical device is used to treat eye diseases. The use of the flexible electrode described above can achieve electrical transgenesis, thereby helping to treat eye diseases, and has good application prospects.

[0058] According to an embodiment of the present invention, the eye disease is selected from retina or choroid related diseases; the medical device is selected from the aforementioned retinal or choroid electrical transgenic stimulator or retinal or choroid electrical transgenic system.

[0059] In another aspect of the present invention, the present invention proposes the use of the flexible electrode, retinal or choroidal electrical transgenic stimulator, and retinal or choroidal electrical transgenic system described above in electrical transgenic of the retina or choroid. The use of the flexible electrode described above can realize electrical transgenic, thereby facilitating the study of the retina or choroid, which is of great significance for the scientific research and clinical diagnosis and treatment of the retina and choroid.

[0060] It should be noted that the features and advantages described above for the flexible electrode, retinal or choroidal electrical transgenic stimulator, and retinal or choroidal electrical transgenic system are also applicable to this application and will not be repeated here.

[0061] Method for introducing genes into retina and / or choroid

[0062] In another aspect of the present invention, the present invention proposes a method for introducing genes into the retina and / or choroid. According to an embodiment of the present invention, the method includes: providing the retinal or choroidal electrical transgenesis system described above; contacting the concave portion of the flexible electrode, at least one of the retina and the choroid, and the solution containing the genetically modified organism; and turning on the power supply device. By turning on the power supply device, it is convenient to apply a voltage to the metal electrode layer of the flexible electrode, thereby achieving electrical transgenesis to the retina and / or choroid, which is of great significance for the scientific research and clinical diagnosis and treatment of the retina and choroid.

[0063] It should be noted that the present invention does not strictly limit the contact method between the retina and / or choroid, the concave part of the flexible electrode and the solution containing the genetically modified organism. The flexible electrode can be placed on the retinal surface, the subretinal cavity, or the suprachoroidal cavity, and the solution containing the genetically modified organism can be placed in the concave part of the flexible electrode, or injected into the vitreous cavity, the subretinal cavity, or the suprachoroidal cavity. The flexible electrode and the solution containing the genetically modified organism can also be placed in a solution containing the retina and / or choroid. The specific method can be flexibly selected according to the actual situation.

[0064] According to an embodiment of the present invention, the voltage applied by the power supply device is 5-30 V. Thus, gene delivery can be achieved in the eye by applying a low voltage.

[0065] It should be noted that the features and advantages described above for the retinal or choroidal electrical transgenic system are also applicable to this method and will not be repeated here.

[0066] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0067] Example 1

[0068] In this embodiment, the flexible electrode is prepared according to the following method. Figure 3 .

[0069] (1) Coating a flexible substrate layer: Prepare a substrate film with a thickness of 50 μm and coat it on a 4-inch polished silicon wafer as a substrate;

[0070] (2) Sputtered metal layer: Due to the limitation of sputtering technology, the sputtered thickness of the gold electrode can only be less than 0.4 μm, which may result in it being unable to withstand large currents. When a high voltage is applied, the gold electrode may break. In order to increase the voltage strength that can be tolerated, a 30 nm chromium layer is first sputtered on the substrate as a seed layer, and then a 50 nm gold layer is sputtered on the chromium layer;

[0071] (3) Mask photolithography: Using ultraviolet photolithography technology, first spin-coat 2μm thick RZJ304 photoresist on the gold layer, let it stand for ten minutes after spin-coating, then place the silicon wafer on a heating plate for pre-baking, and then use a photolithography machine to expose it to ultraviolet light. This process needs to be operated on an ultraviolet exposure machine. After that, the silicon wafer after photolithography is post-baked and developed, and the pattern on the mask will be accurately copied to the photoresist on the silicon wafer. The mask effect is shown in the figure below. Figure 4As shown in A, the layout is drawn using the layout design software L-Edit;

[0072] (4) Etching and photoresist removal: Wet-etching the metal layer, wet-etching the gold layer and the chromium layer with potassium iodide solution and ammonium cerium nitrate solution respectively, and then removing the residual photoresist with nitrous acid to form a gold electrode;

[0073] (5) Peeling off the substrate layer: Gently peel off the substrate layer, and the flexible microelectrode array obtained on a single silicon wafer is complete. Figure 4 As shown in Figure B, the peeling process was completed in deionized water to reduce the possibility of patch collapse and the intact microelectrode array was cut into individual microelectrode arrays of appropriate size ( Figure 4 Middle C);

[0074] (6) Thermoplastic molding: Use 3D printing technology to make a hemispherical groove and a corresponding cover mold. Press the cut single microelectrode array into the mold, press it with the cover, put it in a forced air drying oven, set the temperature to 100°C, and take it out after two hours. A hemispherical groove will be formed in the center of the flexible electrode array.

[0075] The prepared flexible electrode structure is as follows Figure 5 The flexible microelectrode array adopts a "polymer-metal" structure, in which PET is used as a flexible substrate with a thickness of 50 μm and a length of 1.8×10 4 μm, width is 9×10 3 μm( Figure 5 In A), the substrate is closely connected and grown with regular rectangular interdigitated microelectrodes. Figure 5 As shown in B, its thickness is 0.08 μm, the width of each rectangular interdigitated microelectrode is 80 μm, and the distance between the two positive and negative rectangular electrodes is 200 μm. The radius of the groove is slightly larger than the radius of the eyeball axis of ordinary suckling mice, which is 1×10 3 μm.

[0076] Example 2

[0077] In order to verify that the flexible microelectrode array in this paper can achieve electrotransfection at different depths by controlling the voltage, three sets of voltage values ​​were set in the experiment, namely 5V, 10V, and 15V. At the same time, as a comparison, a simulation analysis (analysis software is COMSOL Multiphysics 5.5) was also performed on the traditional electrode chamber type with parallel plates at both ends (electroporation cup, Bio-Rad, catalog number 1652088), the voltage was 40V, and the spacing between the parallel plate electrodes at both ends was 4mm. In a uniform electric field: E = U / d (U is the potential difference between two points in a uniform electric field, and d is the distance between the two points along the field strength direction).

[0078] The first result to be compared is the electric field distribution simulation diagram. Figure 6 As shown in A, the electric field strength of the electrode cup (100V / cm) can electroporate the retina, so the colored area indicates the area that the flexible electrode can effectively affect. The color reference table is set in the range of 100V / cm to 600V / cm, which means that the electric field strength outside this range will not be displayed. Under the conditions of 5V, 10V and 15V, the depth of the effective electric field can reach about 80μm, 160μm and 200μm respectively. When conducting biological experiments, the retina attached to the lens is attached to the electrode with the lens side facing up. The relationship between the field strength and the depth is as follows: Figure 6 As shown in B, the direction is upward along the surface perpendicular to the lowest point of the flexible electrode. Therefore, it is shown that the flexible electrode of the present invention can realize the controllable depth of electrotransformation.

[0079] Example 3

[0080] In order to verify that the flexible microelectrode array of the present invention can realize the electrotransfer of retinal genes, an experiment was conducted to electrotransfer genes into the isolated neonatal rat retina.

[0081] 1. In vitro culture of neonatal mouse retina

[0082] This study used C57BL / 6 neonatal mice (P4, 1-3g, regardless of gender) for the experiment. The lens and retina were co-cultured in retinal culture medium (90% DMEM + 10% FBS + 1% P / S). Observation under a stereomicroscope showed that the retina could still maintain its shape and structure intact after 1 week of culture ( Figure 7 ).

[0083] 2. Using flexible electrodes for in vitro retinal gene electroporation

[0084] In this study, flexible electrodes were used to perform gene (EGFP plasmid) electroporation experiments in isolated neonatal rat retinas. The flexible electrode experimental group was divided into three groups according to the applied voltage: 5V, 10V, and 15V. The parameters of each group were the same except for the voltage. At the same time, an electrode cup group (voltage 40V) was set as a control. After 72 hours of retinal gene electroporation, retinal frozen sections and retinal flat mounts were performed and observed under a laser scanning confocal microscope, and the mean fluorescence intensity (MFIs) of the retinal flat mounts were quantitatively analyzed.

[0085] The results showed that the flexible electrode applied 5V voltage to transfer EGFP plasmid to the inner layer of the retina, 10V voltage to the deep layer of the retina, and 15V voltage to the entire retina. However, when using the electrode cup, 40V voltage could only transfer EGFP plasmid to the inner layer of the retina ( Figure 8 Middle A). Retinal flat mounts showed that the retinal structure remained relatively intact after electroporation using flexible electrodes ( Figure 8 (B, upper figure). After electroporation with flexible electrodes at 5V, 10V, and 15V, the entire retina expressed EGFP in the full field of view. The mean fluorescence intensity (MFIs) of retinal flat sheets was different under different voltages. The MFIs values ​​of the 0V, 5V, 10V, and 15V groups were 133.94±46.87, 4042.89±61.84, 4005.52±49.41, and 3455.87±274.56, respectively. Compared with the 0V group, the MFIs values ​​of the 5V, 10V, and 15V groups were significantly increased, and the difference was statistically significant (P<0.0001). There was no difference between the 5V and 10V groups. The 15V group was lower than the 5V and 10V groups, and the difference was statistically significant (P<0.05) ( Figure 8 Middle C).

[0086] 3. Evaluation of retinal tissue damage

[0087] Retinal tissue damage is a major consideration in determining whether the electroporation device can be used clinically. To evaluate tissue damage caused by in vitro electroporation, retinal HE staining and TUNEL staining were performed 30 minutes after retinal electroporation to evaluate retinal damage. HE staining was used to observe retinal histopathological changes, and TUNEL staining was used to detect cell apoptosis.

[0088] HE results showed that the retinal tissue after electroporation at 5V, 10V, and 15V of the flexible electrode had clear retinal stratification and a tight tissue structure ( Fig. 9 BD); After electroporation at 40V voltage in the traditional electrode cup, the retinal tissue structure was disordered and wavy, the RPE layer was damaged, and the number of ganglion cell layer cells was significantly reduced ( Fig. 9 Middle E).

[0089] DAPI staining of frozen retinal sections showed that the ganglion cell layer, inner nuclear layer, and outer nuclear layer of the retina after electroporation at 5V, 10V, and 15V voltages of the flexible electrode were clearly stratified and tightly structured. The retinal tissue after electroporation with the electrode cup at 40V voltage was unevenly arranged and had a disordered structure. TUNEL staining showed that when the traditional electrode cup was used at 40V voltage, most retinal tissue cells were apoptotic, and about 25% of the cells were apoptotic when observed using ImageJ software. In contrast, after electroporation with the flexible electrode, almost no apoptotic cells were found at 5V, and less than 1% of the cells were apoptotic at 10V and 15V ( Fig.10 ).

[0090] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0091] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A retinal or choroidal electrical transgenic stimulator, characterized in that: The retinal or choroidal electrical transgenic stimulator is used for electrically transducing the retina or choroid, and comprises: a flexible electrode, wherein the flexible electrode comprises a flexible substrate, wherein the flexible substrate has a concave portion, and a metal electrode layer is formed on at least the inner wall of the concave portion of the flexible substrate; The metal electrode layer has a hollow portion; The metal electrode layer comprises interdigitated electrodes; The metal electrode layer contains a microelectrode array formed by rectangular interdigitated microelectrodes; The thickness of the rectangular interdigital microelectrode is 0.08 μm, the width of each rectangular interdigital is 80 μm, and the distance between the rectangular interdigital fingers of adjacent positive and negative electrodes is 200 μm; The metal electrode layer includes: a gold layer and a chromium layer, and the chromium layer is formed between the gold layer and the flexible substrate.

2. The retinal or choroidal electrical transgenic stimulator according to claim 1, characterized in that: The ratio of thickness, length and width of the flexible substrate is (40-60): (1×10 4 ~2×10 4 ): (5×10 3 ~10×10 3 ).

3. The retinal or choroidal electrical transgenic stimulator according to claim 1, characterized in that: The flexible substrate is selected from polyethylene terephthalate.

4. The retinal or choroidal electrical transgenic stimulator according to claim 1, characterized in that: The concave portion is hemispherical.

5. The retinal or choroidal electrical transgenic stimulator according to claim 4, characterized in that: The radius of the hemisphere is 1 to 1.5 times the radius of the eyeball axis.

6. The retinal or choroidal electrical transgenic stimulator according to claim 1, characterized in that: The method for preparing the flexible electrode comprises: (1) forming a metal electrode layer on a flexible substrate; (2) The flexible substrate on which the metal electrode layer is formed is subjected to a molding process so as to form a concave portion on the flexible substrate, and the metal electrode layer is formed on at least an inner wall of the concave portion of the flexible substrate.

7. The retinal or choroidal electrical transgenic stimulator according to claim 6, characterized in that: Step (1) includes: (1-1) coating a flexible substrate material on a base material to form a flexible substrate on the base material; (1-2) first sputtering metal chromium on the flexible substrate to form a chromium layer, and then sputtering metal gold on the chromium layer to form a gold layer; (1-3) performing mask photolithography on the gold layer, and then etching and desizing to form a metal electrode layer on the flexible substrate, and finally peeling the flexible substrate from the base material.

8. The retinal or choroidal electrical transgenic stimulator according to claim 6, characterized in that: The molding process is performed by thermoplastic molding.

9. A retinal or choroidal electrical transgenic system, characterized in that: include: The retinal or choroidal electrical transgenic stimulator according to any one of claims 1 to 8; and A power supply device is connected to the metal electrode layer.

10. The system according to claim 9, characterized in that The power supply device is selected from an electroporator.

11. Application of flexible electrodes in the preparation of medical devices, characterized in that: The medical device is used to treat eye diseases; The medical device is used for gene electrotransfer to the retina or choroid; The flexible electrode comprises a flexible substrate, the flexible substrate has a concave portion, and a metal electrode layer is formed on at least an inner wall of the concave portion of the flexible substrate; The metal electrode layer has a hollow portion; The metal electrode layer comprises interdigitated electrodes; The metal electrode layer contains a microelectrode array formed by rectangular interdigitated microelectrodes; The thickness of the rectangular interdigital microelectrode is 0.08 μm, the width of each rectangular interdigital is 80 μm, and the distance between the rectangular interdigital fingers of adjacent positive and negative electrodes is 200 μm; The metal electrode layer includes: a gold layer and a chromium layer, and the chromium layer is formed between the gold layer and the flexible substrate.

12. The use of the flexible electrode in the preparation of medical devices according to claim 11, characterized in that: The eye disease is selected from retina or choroid related diseases; The medical device is selected from the retinal or choroidal electrical transgenic stimulator according to any one of claims 1 to 8 or the retinal or choroidal electrical transgenic system according to claim 9 or 10.

Images