TWI867920B - Adhesive hydrogel electrode - Google Patents

Abstract

This invention provides an adhesive hydrogel electrode, which comprises an insulating layer, a conductive part, and a gel layer. The conductive part covers a portion of the surface of the insulating layer, with one end of the conductive part forming the electrode unit, and the conductive part itself being a rough and porous mesh structure. The gel layer covers both the insulating layer and the conductive part and is a porous structure filled with one or more payloads. Overall, the adhesive hydrogel electrode possesses characteristics of high biocompatibility and conductivity.

Description

Adhesive hydrogel electrode

The present invention relates to the field of gel electrode technology, and in particular to an adhesive hydrogel electrode for use in biological organisms.

In the field of neural interface technology, implantable neural interface technology has made significant progress in the application of neuroprosthetics and is used to treat various nervous system diseases and injuries. However, traditional neural interfaces have limitations in compatibility and functionality with biological tissues. For example, traditional electrode materials such as gold, silver or titanium, although they have good conductivity, may produce some rejection when in contact with soft tissues due to their rigidity and possible biological reactions.

The biohybrid neural interfaces (BHNIs) technology developed in recent years is a highlight in enhancing the application of neural interfaces in organisms. BHNIs mainly use microelectrode arrays (MEAs) for electrophysiological recording and electrical stimulation. However, most microelectrode arrays are usually composed of metal electrodes and different substrate materials, such as silicon, polyimide, poly(p-xylene) or polydimethylsiloxane (PDMS). After being implanted into the organism, the above-mentioned substrate materials are prone to expose the metal electrodes, which may cause mechanical damage to the organism and hinder the differentiation of cells in the organism. Therefore, due to the incompatibility of biological and electrical systems, there are relatively few related research and development on devices using biohybrid neural interfaces.

In addition, in the past, gel electronic devices often have insufficient flexibility in their rigid materials and cannot be flexibly attached to irregularly shaped surfaces (such as muscles). Some gel electronic devices even have some deformations that affect their normal functions.

In summary, how to improve the compatibility between devices using biohybrid neural interfaces and biological bodies, and further provide detection or treatment applications, has become a problem to be solved in the relevant technical field.

The main purpose of the present invention is to provide an adhesive hydrogel electrode with an improved hydrogel microelectrode array, which combines biocompatibility, biodegradability, and excellent electrical conductivity and mechanical stability. Through the innovative application of double-crosslinked gelatin-based electroconductive hydrogels (ECHs), the present invention can improve the accuracy and efficiency of electrophysiological signal monitoring and electrical stimulation of nerve cells and tissues. In addition, the present invention provides a method of using an adhesive hydrogel electrode, which can be applied to supportive treatment of peripheral nerve injury, and together with electrical stimulation and payloads, it helps regenerate and repair nerve tissue. This invention has broken through the limitations of existing BHNIs technology and provides a more effective and biocompatible solution for neuroscience research and clinical applications.

According to the above purpose, the present invention provides an adhesive hydrogel electrode, comprising: an insulating layer, a conductive part and a gel layer. The conductive part covers the surface of the insulating layer, and one end of the conductive part is an electrode unit, and the conductive part is a rough and porous mesh structure. The gel layer covers the insulating layer and the conductive part, and the gel layer is a porous structure, and one or more loads are filled in the porous structure.

The electrode unit is composed of multiple microelectrodes, each with a diameter of 150 to 300 microns.

Among them, the materials of several microelectrodes include methacrylic anhydride gelatin, poly(3,4-ethylenedioxythiophene) and graphene oxide.

The electrode unit also includes a reference electrode and a working electrode, which are used to detect the potential difference between the reference electrode and the working electrode, and the distance between the reference electrode and the working electrode is more than 1 mm.

Furthermore, the distance between the reference electrode and the working electrode of the electrode unit is 1~3 mm.

Wherein, the insulating layer is selected from the group consisting of polylactic acid, polyvinyl alcohol, polyacrylic acid, polyvinyl pyrrolidone and polyacrylate.

The gel layer includes a first polymer compound containing glutamine, a second polymer compound containing lysine, and glutaminase.

The carrier is selected from the group consisting of stem cells, stem cell secretions, drugs, growth factors, cytokines, nucleic acid substances, antibodies, antibody fragments, peptides, proteins and gene therapy agents.

The present invention also provides a method for using an adhesive hydrogel electrode, which includes the following steps: placing the adhesive hydrogel electrode on a biological part; connecting the adhesive hydrogel electrode to a power supply through a connector connected to the other end of the conductive part of the adhesive hydrogel electrode; and transmitting electrical stimulation to the conductive part through the power supply.

Among them, the species corresponding to the biological parts are mammals.

Among them, at least part of the biological part is nerve damage.

The present invention also provides a use of an adhesive hydrogel electrode, which is used to improve diseases or symptoms related to nerve damage.

Wherein, the disease or condition is sciatica, carpal tunnel syndrome, post-traumatic neuropathy, spinal cord injury, multiple sclerosis, sequelae of stroke or peripheral neuropathy.

Compared with the previous technology, the adhesive hydrogel electrode provided by the present invention can claim the following effects: multifunctional electrode structure, high conductivity and biocompatibility, optimized electrical signal receiving mode, biodegradability and mechanical stability, can be applied to the filling and release of loads, excellent electrical stimulation effect, good biocompatibility and cell growth environment, can promote nerve damage recovery, reduce invasiveness and surgical requirements, etc.

The following text will provide a detailed description through specific embodiments and the attached drawings, so that it will be easier to understand the purpose, technical content, features and effects achieved by the present invention.

100: Adhesive hydrogel electrode

120: Insulation layer

140: Conductive part

142: Electrode unit

160: Gel layer

200: Connector

FIG1 is a schematic diagram of the first structure of the adhesive hydrogel electrode of the embodiment provided by the present invention; FIG2 is a schematic diagram of the second structure of the adhesive hydrogel electrode of the embodiment provided by the present invention; FIG3a is a flow chart of the patterning process of the conductive part; FIG3b is the effect of the graphene oxide concentration on the spatial resolution of PDGO; FIG3c is an electron microscope image of the patterned PDGO; FIG4 is an electron microscope image of the end point of the electrode unit; FIG5 is an electron microscope image of the conductive part of each component; FIG6 is an electron microscope image of the conductive part of each component; The conductivity test of the electrical part; Figure 7a is the signal reception test with a double microelectrode distance of 3 mm; Figure 7b is the signal reception test with a double microelectrode distance of 2 mm; Figure 7c is the signal reception test with a double microelectrode distance of 1 mm; Figure 7d is the frequency power of the sciatic nerve electrical signal received by each group of double microelectrode distances; Figure 7e is the main frequency of the sciatic nerve electrical signal received by each group of double microelectrode distances; Figure 7f is the electrical response test of each group of double microelectrode distances and current amplitudes of different electrical stimuli; Figure 8a is the stretching of the adhesive hydrogel electrode Figure 8a is an impedance test of the adhesive hydrogel electrode in a twisted state; Figure 8c is an impedance test of the adhesive hydrogel electrode in a bent state; Figure 9 is a degradation test of the adhesive hydrogel electrode; Figure 10a is a photo of the actual adhesive hydrogel electrode; Figure 10b is a photo of the adhesive hydrogel electrode actually applied to the sciatic nerve; Figure 11 is an immunostaining image of nerve cells growing on the insulating layer, the conductive part, and the gel layer; Figure 12 is a photo of the recovery process of the damaged nerve; Figure 13a is a photo of each therapeutic use evaluation group Analysis of electrical signals of nerve cells 0-14 days after treatment; Figure 13b is a statistical graph of nerve cell recovery in each treatment use assessment group; Figure 13c is an immunostaining graph of nerve cell repair results in each treatment use assessment group; Figure 13d is a statistical graph of MAP2 expression in nerve cells in each treatment use assessment group; Figure 14a is the result of S100β expression in nerve cells in each treatment use assessment group; and Figure 14b is the result of cytokine expression in nerve cells in each treatment use assessment group.

The embodiments of this invention will be further explained below with the help of the relevant drawings. As far as possible, the same reference numerals in the drawings and the manual represent the same or similar components. In the drawings, the shapes and thicknesses may be exaggerated for the sake of simplicity and convenience. It is understood that the components not specifically shown in the drawings or described in the manual have the form known to the ordinary technicians in the relevant technical field. The ordinary technicians in this field can make various changes and modifications based on the content of this invention.

According to one embodiment, it is related to an adhesive hydrogel electrode structure 100.

Please refer to Figures 1 and 2 at the same time. Figure 1 is a first structural schematic diagram (side view) of the adhesive hydrogel electrode of the embodiment provided by the present invention. Figure 2 is a second structural schematic diagram (3D schematic diagram) of the adhesive hydrogel electrode of the embodiment provided by the present invention.

In this embodiment, the adhesive hydrogel electrode 100 includes: an insulating layer 120, a conductive part 140 and a gel layer 160. The conductive part 140 covers the surface of the insulating layer 120, and one end of the conductive part 140 is an electrode unit 142, and the conductive part 140 is a rough and porous mesh structure. The gel layer 160 covers the insulating layer 120 and the conductive part 140, and the gel layer 160 is a porous structure, and one or more loads are filled in the porous structure.

Regarding the conductive part 140, the preparation of the conductive hydrogel (ECHs) uses gelatin methacryloyl (GelMA) and graphene oxide (GO) as the main raw materials in this embodiment.

In brief, methacrylic anhydride gelatin is dissolved in phosphate buffered saline (PBS), and then poly(3,4-ethylenedioxythiophene) (PEDOT) or (3,4-ethylenedioxythiophene) (EDOT) is added for cross-linking reaction to produce PDGMA (GelMA-doped PEDOT). On the other hand, graphene oxide is chemically modified with methacrylic anhydride gelatin to produce GOGMA (GelMA-modified graphene oxide). Finally, PDGMA and GOGMA were mixed and UV-cured to form highly porous electrically conductive hydrogels (ECHs), namely PDGO (Poly(3,4-ethylenedioxythiophene)doped with Gelatin Methacryloyl Graphene Oxide).

The chemical modification for preparing GOGMA is a chemical reaction between the molecules of methacrylic anhydride gelatin and the surface functional groups of graphene oxide (mostly carboxyl groups), thereby fixing methacrylic anhydride gelatin on the surface of graphene oxide. The reaction involves covalent bonding or strong electrostatic interaction.

The following is a specific process for preparing PDGMA:

(1) Dissolve methacrylic anhydride gelatin or gelatin in phosphate buffer (PBS, pH 7.4).

(2) Add methacrylic anhydride (MA) to prepare a mixed solution.

(3) The mixed solution is dialyzed to remove unreacted methacrylic anhydride and other by-products. The solution can be freeze-dried for storage, or the freeze-dried powder can continue the preparation process.

(4) Prepare a 10% methacrylic anhydride gelatin solution at 50°C and add 9.4 mL of deionized water (Distillation-Distillation H2O, _ddH2O ).

(5) FeCl ₃ ． 6H ₂ O and EDOT (Fe ³⁺ ：EDOT=3：1 mol mol ^-1 ) were added to the methacrylic anhydride gelatin solution, and then stirred at 50°C overnight for oxidative polymerization.

(6) Obtain a dark blue product, wash it three times with water/ethanol (1:9), and then dry it at 55°C overnight to obtain PDGMA.

The following is a specific process for preparing GOGMA:

(1) 1 g of graphite flakes were pre-oxidized with 1 g of P ₂ O ₅ , 1 g of K ₂ S ₂ O ₈ and 3 mL of H ₂ SO ₄ and then incubated at 80°C for 6 hours.

(2) After washing with deionized water and filtering, the pre-oxidized graphite was dried in an oven at 60°C overnight.

(3) The pre-oxidized graphite powder was added to 23 mL H ₂ SO ₄ for further oxidation, and KMnO ₄ was slowly added, and finally stirred at 35°C for 2 hours.

(4) After 2 hours, add 140 mL of distilled water to terminate the reaction and obtain a graphite mixture.

(5) Add 30% hydrogen peroxide solution (H ₂ O ₂ ) to the graphite mixture until the color of the graphite mixture changes from black to yellow.

(6) Finally, the mixture was centrifuged at 11000 rpm for 30 minutes to remove excess H ₂ O ₂ , thereby obtaining a graphene oxide solution.

(7) GelMA was added to the graphene oxide solution (GO:GelMA=1:10 gg ^-1 ) to form a 1% GOGMA solution.

The following is a specific process for preparing PDGO: Mix 1.68% PDGMA and 1% GOGMA in 1 ml of deionized water and stir at 50°C to obtain a PDGO solution.

According to another embodiment, it was found through experiments that the concentration of graphene oxide affects the process and results of PDGO patterning.

Please refer to Figures 3a to 3c. Figure 3a is the process of the patterning process of the conductive part; Figure 3b is the result of the effect of graphene oxide concentration on the spatial resolution of PDGO; Figure 3c is an electron microscope image of the patterned PDGO. As shown in Figure 3a, based on the photocuring property of PDGO, PDGO can be coated on the mask, and then after UV exposure, the uncured PDGO can be stripped with water at about 50°C to realize the patterning process of PDGO. As shown in Figure 3b, it is found through experiments that the increase in the concentration of graphene oxide in PDGO can increase the cross-linking density of the conductive hydrogel that leads to the conductive part 140, thereby improving the spatial resolution of the formed pattern. However, as shown in Figure 3b, too high a concentration of graphene oxide (greater than 1.8%) will make the pattern outline of PDGO rough, so 1% graphene oxide can provide the highest pattern resolution of PDGO, and its line width can reach 30 microns. As shown in Figure 3c, due to the plasticity of PDGO, any pattern can be formed according to the required process. Figure 3c demonstrates a star-shaped 3D microporous network structure.

The principle of PDGO's photocuring properties includes: GelMA is made by introducing methacryloyl groups (MA) into gelatin molecules. Its methacryloyl groups contain unsaturated carbon-carbon double bonds, and its double bonds will undergo cross-linking reactions under appropriate light conditions (especially UV light), thereby achieving polymerization and curing.

Please refer to Figure 4, which is an electron microscope image of the end of the electrode unit. Figure 4 shows the special structure of PDGO with high roughness and high porosity.

Please refer to Figure 5 again, which is an electron microscope image of the conductive part of each component. In order to illustrate that the PDGO obtained by the above preparation process has a special structure with significantly higher roughness and higher porosity compared to methacrylic anhydride gelatin (GelMA), PDGMA, and GOGMA. It can be seen from Figure 5 that PDGO has a more porous and dense (providing high roughness) structure under the same unit area.

Please refer to Figure 6 again, which shows the conductivity test of the conductive part of each component. Further, in order to confirm that the high roughness and high porosity structure of PDGO can help mass/charge transfer. Figure 6 shows the analysis of the conductivity of methacrylic anhydride gelatin (GelMA), GOGMA, PDGMA, and PDGO in dehydration (dehydration, D) and hydration (H) states. In the dehydrated state, the conductivity of PDGO and PDGMA is the same, but higher than that of methacrylic anhydride gelatin and GOGMA. In the hydrated state, the conductivity of PDGO is significantly higher than that of methacrylic anhydride gelatin, GOGMA, and PDGMA. Considering that the adhesive hydrogel electrode 100 may be implanted in a living body, the conductive portion 140 needs to be kept in a hydrated state. Therefore, in practical applications, PDGO has the best conductive properties.

According to another embodiment, the adhesive hydrogel electrode 100 provided in this embodiment has the complex functions of receiving electrical signals (such as physiological signals) and electrical stimulation. When applied to a biological body, how to confirm that the electrical stimulation signal is actually applied to the target position requires minimizing the noise received by the adhesive hydrogel electrode 100.

In summary, PDGO is a composite material that combines conductive polymer PEDOT, highly biocompatible methacrylic anhydride gelatin, and functionalized graphene oxide. The characteristics of PDGO include: rough and porous mesh structure, good conductivity, high biocompatibility, high mechanical strength and elasticity, high chemical stability, allowing cell adhesion and growth, and high plasticity. Among them, the rough and porous structure is a special structure of PDGO in terms of physical structure, which helps mass/charge transfer and enhances the adhesion and growth of neural stem cells.

Please refer to Figures 7a~7c. Figure 7a is an electrical signal reception test with a distance between two microelectrodes of 3 mm; Figure 7b is an electrical signal reception test with a distance between two microelectrodes of 2 mm; Figure 7c is an electrical signal reception test with a distance between two microelectrodes of 1 mm.

As shown in Figures 7a to 7c, three sets of two-electrode units 142 with different electrode spacings (D1=3mm; D2=2mm; D3=1mm) were selected to test and record the results of the compound nerve action potential (CNAP) of the induced sciatic nerve. When the electrode spacing is 3mm (D1), the best CNAP record with the highest amplitude potential can be obtained, and the signal received when no electrical stimulation is performed is also significantly smaller. This indicates that the spatial relative arrangement of the two electrode units 142 has a significant impact on the quality of the received electrical signal. Furthermore, all electrode spacings (D1, D2, and D3) in Figures 7a to 7c can actually measure CNAP correctly.

Please refer to Figures 7d~7f. Figure 7d shows the frequency power of the sciatic nerve electrical signal received by each group of double microelectrode spacing; Figure 7e shows the main frequency of the sciatic nerve electrical signal received by each group of double microelectrode spacing; Figure 7f shows the electrical response test of each group of double microelectrode spacing and current amplitude of different electrical stimulation.

As shown in Figure 7d, the CNAP and noise in Figures 7a~7c are further processed by Fast Fourier Transform (FFT). According to the results of frequency power accumulation, all electrode spacings (D1, D2, and D3) can correctly measure CNAP compared to the unstimulated control group, but the signal-to-noise ratio (SNR) of the electrode spacing of 3mm (D1) is excellent, which can clearly highlight the strength of CNAP. As shown in Figure 7e, compared with the electrode spacing of 2mm (D2) and 1mm (D3), the CNAP received by the electrode spacing of 3mm (D1) has the widest spectrum, up to 1000Hz.

Furthermore, when applying electrical stimulation with different current amplitudes, using electrical stimulation with a larger current can obtain CNAPs with a higher amplitude. As shown in Figure 7f, with different current amplitudes (1, 3, and 5 mA) and electrode spacing (D1=3mm; D2=2mm; D3=1mm), it is also shown that a larger electrode spacing can produce more significant electromyography (EMG). The results of the current amplitudes of 3 and 5 mA are helpful as key parameters for the treatment of peripheral nerve injury, because when treating peripheral nerve injury, the choice of current amplitude is crucial to achieve effective nerve and muscle stimulation. Choosing the appropriate current amplitude can maximize the treatment effect while avoiding damage to nerves or surrounding tissues.

Regarding the gel layer 160, the gel layer 160 needs to be a porous structure in order to be filled with a load. When the gel layer 160 is applied to a biological body, it also needs to have porosity, high biocompatibility, and sufficient viscosity and elasticity in order to cover the target part of the biological body. Therefore, the gel layer 160 can be, for example, a hydrogel formed by at least two polymer compounds through an amide bond. The first polymer compound can be, for example, a polymer compound containing glutamic acid, such as: a group consisting of gelatin, gelatin methacryloyl (GelMA), collagen, and poly (glutamic acid) (PGA). The second polymer compound may be, for example, silk protein, collagen, wool protein, cotton protein, keratin, egg white protein, gluten, soy protein, and casein. The weight ratio of the first polymer compound to the second polymer compound may be, for example, 1:0.16 to 1:0.5.

Furthermore, to catalyze the formation of amide bonds, the gel layer 160 also includes a catalyst having glutaminase, and the catalyst accounts for 0.2 to 0.8 weight percent.

The biological part may be, for example, a tissue (such as muscle, nerve, skin) or an organ (such as heart, spinal cord), and is not limited to humans or animals.

Regarding the insulating layer 120, a material that can form a hydrogen bond with the surface of the gel layer 160 is selected to make the gel layer 160 adhere to the insulating layer 120. Applicable materials for the insulating layer 120 include: Polylactic Acid (PLA), Polyvinyl alcohol (PVA), Poly (acrylic acid) (PAA), Polyvinylpyrrolidone (PVP) and polyurethane (PU). The above materials can be prepared as an insulating suspension and then cured to form the insulating layer 120.

In summary, the manufacturing method of the adhesive hydrogel electrode 100 may be, for example:

(1) Apply an insulating suspension onto a substrate (such as a substrate with a photomask), and solidify the insulating suspension to form an insulating layer.

(2) Apply a conductive material (such as PDGO) on the insulating layer and solidify the conductive material to form a conductive part.

(3) Apply the first polymer compound and the second polymer compound on the insulating layer and add corresponding catalysts to form a gel layer.

(4) Separate the adhesive hydrogel electrode from the substrate.

According to another embodiment, the adhesive hydrogel electrode 100 of this embodiment has been tested to have excellent resistance to physical deformation.

Please refer to Figures 8a to 8c. Figure 8a is an impedance test of the adhesive hydrogel electrode in a stretched state; Figure 8b is an impedance test of the adhesive hydrogel electrode in a twisted state; Figure 8c is an impedance test of the adhesive hydrogel electrode in a bent state. As shown in Figures 8a to 8c, the impedance of the electrode unit 142 (150 microns and 300 microns) at 1 kHz in different mechanical deformation states (stretching, twisting, and bending) and different diameter sizes is used to evaluate the mechanical stability of the adhesive hydrogel electrode 100.

The results show that the electrochemical properties of smaller electrode diameters (150 microns) are more significantly affected by mechanical stimulation than those of larger electrode diameters (300 microns). For electrodes with a diameter of 150 microns, the impedance increases to 5.2 kilohms when stretched uniaxially from 0% to 33%, to 54.8 kilohms when twisted to 720°, and to 17.7 kilohms when folded to 135°. For electrodes with a diameter of 300 microns, the increase in impedance in response to mechanical stimulation was less than that for electrodes with a diameter of 150 microns. However, after the mechanical stimulation disappeared, the impedance was able to return to its original level. No permanent effects of mechanical stimulation on the adhesive hydrogel electrode 100 were found.

According to the above results, the adhesive hydrogel electrode 100 that has undergone various degrees of mechanical deformation still meets the electrochemical performance requirements of neural recording standards and can continuously obtain electrical signals.

Since the adhesive hydrogel electrode 100 provided in this embodiment is biodegradable, the need for invasive and expensive removal surgery is reduced.

Please refer to Figure 9. Figure 9 shows the degradation test of the adhesive hydrogel electrode.

To simulate the hydrolysis process of the adhesive hydrogel electrode 100, the adhesive hydrogel electrode 100 was placed in PBS for an in vitro degradation experiment. As shown in FIG9 , although the adhesive hydrogel electrode 100 lost more than 45% of its original weight on the 7th day in PBS, the gel layer 160 degraded according to the volume erosion mechanism, so that the entire adhesive hydrogel electrode 100 structure was maintained and used for electrochemical measurements. After 28 days of in vitro degradation, the adhesive hydrogel electrode 100 still had a CSC of 1.99 microcoulombs per square centimeter and an impedance of 1.6±0.8 kilohms compared to the original value, and it still had the ability to receive electrical signals and perform electrical stimulation.

Therefore, this embodiment further provides a method for using an adhesive hydrogel electrode, which includes the following steps:

(1) Place the adhesive hydrogel electrode 100 on the biological part.

(2) Connect the power supply through the connector 200 connected to the other end of the conductive part 140 of the adhesive hydrogel electrode 100.

Please refer to Figure 10a. Figure 10a is a photo of an actual adhesive hydrogel electrode. As shown in Figure 10a, one end of the conductive part 140 is the electrode unit 142, and the connector 200 at the other end is used to connect to a power supply and/or an electrical signal receiver.

(3) Transmitting electrical stimulation to the conductive part through the power supply and/or receiving electrical signals from the biological part through the electrical signal receiver.

Please refer to Figure 10b. Figure 10b is a photo of the adhesive hydrogel electrode actually applied to the sciatic nerve. As shown in Figure 10b, the adhesive hydrogel electrode 100 can be wrapped around the sciatic nerve, and electrically stimulate the peripheral muscles or the sciatic nerve, and collect the electrical signals of the sciatic nerve. The electrical stimulation can refer to the embodiment of Figure 7f, for example, it can be set to a current of 3 to 5 mA. In the embodiment of Figure 10b, experimental rats (Sprague-Dawley, SD) belonging to mammals are used as test subjects.

According to the above embodiments, the conductive part 140 and the gel layer 160 in the adhesive hydrogel electrode 100 have a porous structure. And the material itself has excellent biocompatibility.

Please refer to Figure 11. Figure 11 is an immunostaining image of nerve cells growing on the insulating layer, the conductive part, and the gel layer. Figure 11 uses immunofluorescence (IF). The control group is nerve cells cultured on a general plastic cell culture dish.

As shown in Figure 11, the red fluorescent HB9 (HLXB9) is a transcription factor that is often used as a marker for mature motor neurons. In neuronal cell research, the expression of HB9 is often used to confirm the presence and degree of differentiation of motor neurons. HB9 staining is often used to identify and study cell populations specific to motor neurons; the green fluorescent Tuj1 (Beta III Tubulin) is a microtubule protein that is widely present in neurons, especially in their axons. Tuj1 staining is used to mark neurons and their protrusions (especially axons) and is an important indicator of neuronal differentiation and maturation. The blue fluorescent DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that can specifically bind to adenine and thymine in the double helix structure of DNA. It is often used to visualize and count cell nuclei because it fluoresces blue under UV light. DAPI staining is widely used in a variety of cell biology and histology studies for labeling cell nuclei and counting cells.

Therefore, as can be seen from Figure 11, nerve cells grow well in the gel layer (GS-MTG) and the conductive part (PDGO), indicating that it has good biocompatibility. However, nerve cells cannot grow well in the insulating layer (PLA), which is also an expected result. If nerve cells enter the insulating layer, the insulating layer will lose its insulating function, thereby affecting the ability of the conductive layer to perform electrical stimulation or receive electrical signals.

The GS-MTG is an abbreviation of gelatin (abbreviated G) + silk protein (abbreviated S) + microbial transglutaminase (abbreviated MTG).

According to another embodiment, on the premise that the adhesive hydrogel electrode provided in the above embodiment has good biocompatibility, this embodiment will further provide an explanation of the use of the adhesive hydrogel electrode as a treatment.

Since the conductive part and gel layer of the adhesive hydrogel electrode are porous structures, the conductive part provides a good medium for charge transfer. Furthermore, its porous structure can also be used as a space filled with one or more loads.

In the embodiments of Figures 12, 13a, 13b, 13c, 13d, 14a, and 14b below, experimental rats (Sprague-Dawley, SD) belonging to mammals are used as test subjects.

Please refer to Figure 12. Figure 12 is a photo of the recovery process of damaged nerves. Day 0 (upper left); Day 1 (upper right): nerve damage; Day 1 (lower left): placement of adhesive hydrogel electrodes; Day 7 (lower right): nerve tissue recovery state.

As shown in Figure 12, this embodiment first creates a sciatic nerve injury model. In this embodiment, the adhesive hydrogel electrode is divided into five groups for testing, including a control group, a MEA group (adhesive hydrogel electrode), a MEA/NPC group (adhesive hydrogel electrode with stem cell transplantation), a MEA/NPC group (adhesive hydrogel electrode with electrical stimulation), and a MEA/NPC/ES group (adhesive hydrogel electrode with stem cell transplantation and electrical stimulation).

The NPC is the full English name for neuron progenitor cell, which is a type of neural precursor cell (stem cell).

Please refer to Figures 13a~13b. Figure 13a is an analysis of the electrical signals of the treatment purpose assessment groups for 0~14 days after the treatment of nerve cells; Figure 13b is a statistical graph of the recovery of nerve cells for the treatment purpose assessment groups. As shown in Figure 13a, the nerve signals are recorded through the adhesive hydrogel electrode to monitor the recovery of nerve injury. It can be seen that the CNAP amplitude of all groups decreased significantly on the first day after the injury, indicating that the function of the damaged nerve has been damaged. Although the adhesive hydrogel electrode can no longer provide signal information after 14 days of implantation due to the degradation of its own structure.

However, significant differences in peak amplitudes between groups were found on day 7. Specifically, the degree of recovery was defined based on the normal CNAP signal before injury. As shown in Figure 13b, combined treatment (MEA/NPC/ES group) showed a recovery rate of up to 60% on day 7, which was significantly higher than other groups, demonstrating the usefulness of combined treatment.

Please also refer to Figures 13c~13d. Figure 13c is an immunostaining diagram of the results of neuronal cell repair in each treatment evaluation group; Figure 13d is a statistical diagram of the expression of MAP2 in neurons in each treatment evaluation group. Figure 13c uses immunofluorescence staining. The control group is the damaged nerve without any treatment. The green fluorescent MAP2 in Figure 13c is a microtubule protein, a cytoskeletal protein widely present in neurons, especially in dendrites, and can be used as a medium for evaluating neuronal cell recovery; the red fluorescence is HB9; the blue fluorescence is DAPI.

As shown in Figures 13c~13d, in the early stage of wound healing (day 7), the MEA/ES group showed more significant MAP2 expression than the MEA/NPC group. However, in the later stage (day 28), the MAP2 expression of the MEA/NPC group began to increase rapidly, even reaching a level comparable to that of the MEA/ES group. The above results indicate that electrical stimulation helps accelerate nerve repair, and the NPC cells provided by the adhesive hydrogel electrode only show the function of nerve repair after differentiation. In addition, the combined treatment (MEA/NPC/ES group) showed higher MAP2 expression than the MEA/ES group during the nerve recovery process, further proving that electrical stimulation can further promote the synergistic effect of NPC cells in differentiating into functional neurons.

Please refer to Figure 14a. Figure 14a shows the results of each treatment evaluation group on the expression of S100β in nerve cells. By observing the expression of specific myelin protein (S100β), the axonal neurogenesis of the damaged nerve can be further evaluated. The quantitative analysis results of Figure 14a clearly show that the expression of S100β in the MEA/NPC/ES group is significantly higher than that in other groups, proving that electrical stimulation and NPC cells can accelerate myelin proliferation and restore the synergistic effect of nerve cells to normal function.

Please refer to Figure 14b again. Figure 14b shows the results of the cytokine expression of nerve cells in each treatment evaluation group.

To investigate whether the fragments after the degradation of the adhesive hydrogel electrode have a negative impact on the neural tissue. The acute immune response was evaluated on the 28th day after nerve injury. The quantitative analysis results in Figure 14b clearly show that the expression of the relevant inflammatory factor markers (CD68, iNOS, Iba1) in each group is correlated with the expression of MAP2 and S100β. That is, the fragments after the degradation of the adhesive hydrogel electrode not only do not induce the production of inflammatory factors, but also further promote myelin hyperplasia (increase in S100β expression) and promote neural cell differentiation (increase in MAP2 expression).

In summary, the above examples have demonstrated the efficacy of NPC cells combined with electrical stimulation in improving nerve damage.

Furthermore, the payload may be, for example, a therapeutic payload, and the therapeutic payload may further include: stem cell secretions, drugs, growth factors, cytokines, nucleic acid substances, antibodies or antibody fragments, peptides, proteins, and gene therapy agents. The charge of the conductive part and the gel layer can be used to control the preservation and release of specific charged molecules through electrical stimulation, thereby achieving the purpose of precise treatment. Possible applications may include: combining GS-MTG with PEDOT or other conductive polymers to improve its response to electrical stimulation, and electrical stimulation can change the electric field distribution inside the gel layer, thereby affecting the migration and release of charged drug molecules.

In addition, the load may be some substances that change the physical or chemical properties of the adhesive hydrogel electrode, and its properties may be, for example, pH value, heat sensitivity, light sensitivity, conductivity, magnetism, etc.; The substance may be, for example, a catalyst, a pH adjuster, a heat-sensitive material, a light-sensitive material, a conductive particle (such as metal nanoparticles or carbon-based materials), a magnetic material, etc.

The above is only an example to illustrate the best implementation method of this creation, and is not intended to limit the scope of implementation. All simple substitutions and equivalent changes made based on the scope of patent application of this creation and the content of the patent specification are within the scope of patent application of this creation.

100: Adhesive hydrogel electrode

120: Insulation layer

140: Conductive part

142: Electrode unit

160: Gel layer

200: Connector

Claims (7)

An adhesive hydrogel electrode comprises: an insulating layer; a conductive part covering the surface of the insulating layer, and one end of the conductive part is an electrode unit, and the conductive part is a rough and porous mesh structure, wherein the electrode unit is composed of a plurality of microelectrodes, and each of the microelectrodes has a diameter of 150 to 300 microns; and a gel layer covering the insulating layer and the conductive part, and the gel layer is a porous structure, and the porous structure is filled with one or more loads. As described in claim 1, the adhesive hydrogel electrode, wherein the materials of the micro-electrodes include methacrylic anhydride gelatin, poly (3,4-ethylenedioxythiophene) and graphene oxide. As described in claim 1, the adhesive hydrogel electrode, wherein the electrode unit further includes a reference electrode and a working electrode, which are used to detect the potential difference between the reference electrode and the working electrode, and the reference electrode and the working electrode are spaced at a distance of more than 1 mm. As described in claim 3, the adhesive hydrogel electrode, wherein the reference electrode and the working electrode of the electrode unit are spaced 1 to 3 mm apart. The adhesive hydrogel electrode as described in claim 1, wherein the insulating layer is selected from the group consisting of polylactic acid, polyvinyl alcohol, polyacrylic acid, polyvinyl pyrrolidone and polyacrylate. The adhesive hydrogel electrode as described in claim 1, wherein the gel layer comprises a first polymer compound containing glutamic acid, a second polymer compound containing lysine, and a glutaminase. The adhesive hydrogel electrode as described in claim 1, wherein the carrier is selected from the group consisting of stem cells, stem cell secretions, drugs, growth factors, cytokines, nucleic acid substances, antibodies, antibody fragments, polypeptides, proteins and gene therapy agents.