Noninvasive controllable flexible bioelectronic patch for treating diabetic erectile dysfunction

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a noninvasive controllable flexible biological electronic patch for treating diabetic erectile dysfunction. The erectile dysfunction related to diabetes is difficult to recover due to long-term damage of a cavernous body microenvironment, traditional oral administration or cavernous body injection treatment has poor targeting, non-uniform distribution and complication risk, and long-term curative effect is difficult to realize. The invention provides a flexible and attachable bioelectronic patch which is used for noninvasively, painlessly and controllably adjusting an erectile tissue microenvironment. The device synergistically promotes local vascular active factor expression and endothelial repair through mild electrical stimulation, realizes uniform transdermal delivery of drug molecules, shows high delivery efficiency and cell activity, and significantly improves cavernous body blood flow and cell functions. In an in-vitro cell model and a diabetic animal model, the device shows better vascular regeneration and function recovery effects than traditional medicine or injection therapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a non-invasive controllable flexible bio-electronic patch for treating diabetic erectile dysfunction. BACKGROUND

[0002] Diabetic erectile dysfunction is one of the common complications in diabetic men that is difficult to treat. Its pathological mechanism mainly originates from the impaired endothelial function of cavernosal blood vessels, leading to insufficient eNOS production and inducing oxidative stress, thereby destroying the relaxation of cavernosal smooth muscle and blood flow regulation. Current clinical treatment mainly relies on oral inhibitors and intracavernosal injection of drugs. However, these methods have limitations in efficiency, safety and controllability. For example, oral drugs rely on systemic circulation to deliver to penile tissue, which has poor targeting, which can limit efficiency and / or controllability, and increase the risk of systemic side effects; intracavernosal injection can directly act on the lesion and restore erectile function in the short term, but after the drug enters the cavernosal through single-point puncture, it only diffuses locally, uneven drug distribution not only reduces the overall efficacy, but also can cause sustained or abnormal erection due to local drug overdose, and under long-term repeated injection, it can induce cavernosal fibrosis, nodules and penile curvature. The pursuit of non-invasive and painless, uniform, controllable and efficient transdermal drug delivery strategy remains a long-term challenge.

[0003] The patent with publication number CN103550860A and the invention name of "Male sexual function therapeutic instrument with seven kinds of electric stimulation therapeutic effects" discloses treating dysfunction by acting on human body acupoints through electric pulse signals. However, this treatment method requires a large device, and in addition, the correct acupoint of the human body for the electric pulse device often needs to be operated by a professional, which reduces the patient's compliance.

[0004] In summary, there is an urgent need to provide a new strategy that can directly act on the affected area, is easy to use, and has high therapeutic effect. SUMMARY

[0005] The purpose of the present application is to provide a new type of medical device, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The present application specifically adopts the following technical solutions.

[0006] A non-invasive flexible electric stimulation patch, comprising two functional modules: (1) an electric stimulation drug delivery module and (2) a wireless transmission power supply module; The electric stimulation drug delivery module is composed of a substrate layer, a microelectrode array layer, a gel layer and a nanopore membrane layer. The substrate layer is a biodegradable flexible material for bearing and packaging. The microelectrode array layer is composed of multiple layers of electrodes, and includes multiple microelectrode pieces with consistent shapes and regular arrangement, which are connected by serpentine electrode lines, and the serpentine electrode lines have at least two S-shaped structures; each of the microelectrode pieces has at least two serpentine electrode lines; The gel layer includes a gel matrix with viscosity, hydroxyl and / or amino groups, and a drug to be electrically delivered; the microelectrode array layer is embedded in the gel layer; The material of the nanoporous membrane layer has ester groups capable of forming hydrogen bonds with the hydroxyl or amino groups of the gel matrix; the nanoporous membrane layer has a thickness of 10-50 μm, and a surface with a pore size structure of 500-1000 nm; The wireless power transmission module is connected to the microelectrode array layer through a lead wire to supply power to the electrically stimulating drug delivery module.

[0007] Further, the number of lead wires is two.

[0008] In some embodiments, the biodegradable flexible material is biodegradable plastic.

[0009] In some embodiments, the nanoporous membrane layer has a thickness of 10-20 μm.

[0010] Further, the material of the serpentine electrode lines is consistent with that of the lead wire.

[0011] Further, the material of the serpentine electrode lines is silver; or the material of the lead wire is silver.

[0012] Further, the microelectrode array layer is composed of Cr-Ag-Cr; the thickness of the Ag layer is 15-30 times the total thickness of the Cr layers (i.e., the thickness of two Cr layers stacked together).

[0013] Further, the shape of the microelectrode pieces includes triangle, quadrilateral, polygon or circle; the polygon is a planar figure composed of four or more line segments that are not on the same straight line, including pentagon, hexagon and others.

[0014] Further, the material of the gel matrix includes GelMA-chitosan; or the material of the nanoporous membrane layer includes polycarbonate membrane or polyethylene terephthalate (PET).

[0015] In the embodiments of the present application, the drug to be electrically delivered includes a drug for treating erectile dysfunction, but is not limited thereto. The non-invasive flexible electric stimulation patch of the present application can also contain other drugs suitable for local penetration administration for treating diseases.

[0016] In some embodiments, the nanopore diameter of the nanopore membrane layer ranges from 600 to 800 μm. The drug contained in the gel matrix can pass through the nanopores of the nanopore membrane layer into the skin of the target organ by electric delivery.

[0017] Further, the wireless transmission power supply module comprises a coil antenna and a rectification and regulation circuit block.

[0018] Further, the wireless transmission power supply module further comprises a flexible packaging structure.

[0019] Further, the rectification and regulation circuit adopts an NFC receiving end structure; and comprises a rectification module, a voltage stabilizing module and an energy storage capacitor.

[0020] In some specific embodiments, the coil antenna: copper wire or metal wire prepared on a flexible substrate, which can be made into a spiral or planar winding structure according to requirements; the rectification and regulation circuit: adopts a standard NFC receiving end structure, and comprises a rectification module, a voltage stabilizing module and an energy storage capacitor; the flexible packaging (optional): can be packaged using common biocompatible materials such as Parylene, PDMS or PI, but the function of the supply module is not dependent on specific materials.

[0021] The present application also provides a preparation method of the above-mentioned non-invasive flexible electric stimulation patch (device), comprising the following steps: S110: preparing an electric stimulation drug delivery module; S111: coating a biodegradable flexible material on a clean substrate to form a wet film, and then high-temperature curing; S112: coating photoresist on the cured flexible material, and moderately drying; S113: performing ultraviolet exposure through a pre-designed mask plate to pattern the photoresist, and blowing dry with nitrogen; S114: placing the sample treated in S113 in a magnetron sputtering instrument, vacuumizing to a background pressure, then introducing argon, maintaining a working gas pressure of 0.5 Pa, and performing silver target sputtering under the condition of substrate rotation and application of a sputtering voltage of 120 V; S115: after sputtering is completed, the sample is taken out of the chamber, the silver-coated flexible material film is peeled off from the substrate, and then a hydrogel containing a drug and a nanopore membrane are covered thereon; S120: preparing a wireless transmission power supply module on a transparent polyethylene terephthalate using a flexible printed circuit process; S130: assembling the electric stimulation drug delivery module and the wireless transmission power supply module to form the non-invasive flexible electric stimulation patch.

[0022] Beneficial technical effects: The application provides a new type of non-invasive flexible bioelectronic patch, FlexiStim patch, which can achieve non-invasive, painless, uniform and controllable drug delivery to target organs. The patch is soft, uses wireless power supply, is composed of thin and biocompatible layered materials, including Ecoflex substrate, serpentine interconnected silver microelectrode array and drug-loaded hydrogel. Among them, the hydrogel layer can form mechanical interlocking with the electrode layer and the nanomembrane layer, intermolecular interaction force, etc., so that the combination between layers is firm, and the presence of hydrogel can also neutralize the modulus between tissue and material, as a buffer layer, to protect the tissue and device from damage. The core design of the whole device is "nanopore-drug storage cavity-microelectrode" structure, and works under low amplitude pulse (<20V), realizing safe, efficient and uniform transdermal delivery of trace drugs. Experiments show that the delivery efficiency is as high as 95% and the cell viability is good. In the DMED model of mice, FlexiStim realizes the improvement of erectile function comparable to intracavernous injection with much lower drug dosage, activates the related signal pathways (EP2 / PKA / CREB) of promoting erection and inhibits the TGF-β / Smad pathway, thereby significantly reducing the fibrosis of the cavernous body. As a programmable nanodrug delivery platform, FlexiStim is not only suitable for drug delivery treatment of DMED, but also can be expanded to gene editing, protein and multi-drug synergistic delivery in the future, providing a new strategy for precise treatment of complex diseases with multiple mechanisms. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. In all the drawings, similar elements or parts are generally identified by similar reference signs. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without paying creative labor.

[0024] Figure 1 A schematic diagram of a wearable, non-invasive, painless, wireless drug delivery FlexiStim patch for the treatment of penile erectile dysfunction in one of the embodiments of the present application; Figure 2 A result graph of FlexiStim patch delivering PGE1 to relieve high glucose-induced smooth muscle and endothelial cell damage and improve cell function in one of the embodiments of the present application; Figure 3 A result graph of FlexiStim patch delivering PGE1 to promote cavernosal tissue repair and improve erectile function in DMED mice in one of the embodiments of the present application; Figure 4Figure showing the results of upregulation of the expression of erectile function related proteins in the corpus cavernosal tissue of DMED mice by PGE1 delivered by the FlexiStim patch in one of the embodiments of the present application; Figure 5 Figure showing the results of downregulation of the expression of fibrosis related genes and proteins in the corpus cavernosal tissue by FlexiStim-PGE1 compared to ICI-PGE1 in one of the embodiments of the present application; Figure 6 Figure showing the preparation process of the delivery module and the wireless transmission power supply module of the FlexiStim patch in one of the embodiments of the present application; Figure 7 Figure showing the circuit of the wireless transmission power supply module and the related modules of the FlexiStim patch in one of the embodiments of the present application; Figure 8 Figure showing the results of alleviation of high glucose induced damage to smooth muscle and endothelial cells and improvement of cell function by PGE1 delivered by the FlexiStim patch in one of the embodiments of the present application; Figure 9 Figure showing the results of downregulation of the expression of fibrosis related genes in the corpus cavernosal tissue by FlexiStim-PGE1 compared to ICI-PGE1 in one of the embodiments of the present application. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0026] In this document, “and / or” includes any and all combinations of one or more of the associated items.

[0027] In this document, “a plurality of” means two or more, that is, it includes two, three, four, five, and so on.

[0028] In this specification, the term “about” typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0029] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0030] Detailed description of the attached images: Figure 1 (a) A comparison of traditional intracavernosal injection and FlexiStim patch and their respective effects on related signal pathways; (b) A schematic diagram of the layered structure of the device; (c) A schematic diagram of the layered structure of the wireless transmission power supply module; (d) A schematic diagram of the delivery module of the device; (e) Optical images of the delivery module under natural conditions and simulated application scenarios under bending and twisting conditions; (f) Finite element analysis stress / strain simulation results under the corresponding optical images; (g) Resistance change test of FlexiStim patch after 1000 bending at 180°; (h) A comparison of the performance of several commonly used treatment methods in terms of non-invasiveness, treatment effect, treatment time, duration of action, local adverse reactions, and systemic adverse reactions.

[0031] Figure 2 (a) Representative images of PGE1 FITC expression in smooth muscle cells from the normal control group, PGE1 treatment group, and FlexiStim patch group; (b) Representative images of AM / PI staining in smooth muscle cells from each experimental group (green: live cells; red: dead cells), scale bar 100µm; (c) Smooth muscle cell viability detected by CCK-8 assay (n=3); (d) Real-time monitoring of smooth muscle cell proliferation curves from 0-48 h in the normal control group, HG group, HG-PGE1 treatment group, and HG-FlexiStim group using the IncuCyte live cell imaging system (n=3); (e) Representative scatter plot of smooth muscle cell apoptosis detected by flow cytometry (Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: normal cells); (f) Statistical analysis of apoptosis rate of smooth muscle cells in each group (n=3); (g) Ca2+ expression of smooth muscle cells. 2+Quantitative analysis of the fluorescence intensity of the probe (n = 3); (h) Schematic diagram of the damage of high glucose to smooth muscle cells and endothelial cells; (i) Representative images of immunofluorescence staining of the vascular formation ability of endothelial cells in each group (scale bar: 100 μm); (j) Quantitative analysis of the vascular formation ability (n = 3); (k) Quantitative analysis of the fluorescence intensity of the eNOS probe of endothelial cells in each group (n = 3). Statistical analysis: Data are expressed as mean ± standard error; n = 3 independent experiments; one-way ANOVA was used for comparison between groups.

[0032] Figure 3 : (a) Schematic diagram of the establishment of the DMED mouse model and the treatment process; (b) Real object display of the FlexiStim patch for delivering PGE1; (c) Distribution of FITC-labeled PGE1 in the control group (Control), ICI treatment group (ICI), and FlexiStim patch treatment group at 0 h, 3 h, and 6 h detected by the small animal live imaging system (green fluorescence); (d) Immunofluorescence staining images of penile corpus cavernosum tissue sections of mice in each group under a confocal microscope (blue: DAPI nuclear staining; green: FITC-labeled PGE1); (e) Representative images of the penile erection state of mice in each group after electrical stimulation of the corpus cavernosum nerve (scale bar: 5 mm). Experimental groups: normal control group (Control), DMED model group (DMED), DMED + ICI treatment group (DMED-ICI), and DMED + FlexiStim treatment group (DMED-FlexiStim); (f) Quantitative analysis of the penile erection angle of mice in each group after electrical stimulation (n = 6); (g) Typical real-time change curve of the intracavernous pressure (ICP) of mice in each group under electrical stimulation; (h) Statistical analysis of the maximum intracavernous pressure (ICPmax) of mice in each group (n = 6); Statistical analysis: Data are expressed as mean ± standard error (n = 6); one-way ANOVA was used for comparison between groups.

[0033] Figure 4 : (a) Representative images of immunofluorescence double staining of endothelial cells in the penile corpus cavernosum tissue (green: eNOS; red: vWF); scale bar: 30 μm; (b) : Quantitative analysis of the fluorescence intensity of α-SMA (n = 6); (c) Quantitative analysis of the fluorescence intensity of Desmin (n = 6); (d) Representative images of immunofluorescence double staining of smooth muscle cells in the corpus cavernosum tissue (green: α-SMA; red: Desmin). Scale bar: 30 μm; (e) Quantitative analysis of the fluorescence intensity of eNOS (n = 6); (f) Quantitative analysis of the fluorescence intensity of vWF (n = 6); (g) Western blot detection results of key proteins for erectile function (eNOS, α-SMA); (h) Relative quantitative analysis of eNOS protein expression (n = 6); (i) - Relative quantitative density of SMA (n=6); (j) Western blot results of PGE1 downstream signaling pathway proteins (EP2, PKA, CREB); (km) Relative quantitative analysis of EP2, PKA and CREB protein expression (as shown in the figure). -Tubulin was used as an internal reference, mean ± standard error, n=6); (n) Representative images of the corpus cavernosum tissue stained with Masson's trichrome (blue: collagen fibers; red: smooth muscle), overall structural scale bar: 500 μm; local magnified image scale bar: 50 μm; (o) Quantitative analysis of the smooth muscle / collagen ratio in the corpus cavernosum of each group to assess the degree of tissue fibrosis. Statistical analysis: All data are expressed as mean ± standard error (n=6); one-way ANOVA was used for comparisons between groups.

[0034] Figure 5 (a) Schematic diagram of transcriptome sequencing analysis of corpus cavernosum tissue in each group; (b) Principal component analysis (PCA) of corpus cavernosum tissue in each group (closer distances indicate higher similarity between samples; the horizontal axis represents the first principal component, and the vertical axis represents the second principal component. Different colors indicate different groups). (c) Heatmap of differentially expressed genes in each group (the horizontal axis represents genes, each column represents one sample; red indicates highly expressed genes, and blue indicates low-expressed genes). (d) Volcano plot of differentially expressed genes between the DMED-ICI and DMED-FlexiStim groups (x-axis: log2FC (DMED-ICI VS DMED-FlexiStim), y-axis: -log10 (pvalue). The two dashed lines represent 2-fold difference in expression; the horizontal dashed line represents the pvalue 0.05. Red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots indicate genes with no significant differential expression. (e) GO functional enrichment analysis of upregulated genes in the DMED-ICI group (top 5, bp, biological process, cc, cellular component, MF, molecular function); (f) GSEA enrichment analysis of the upregulated TGF-β signaling pathway in the DMED-ICI group; (g) Representative immunohistochemical staining images of Col1a1, Col3a1, and TGF-β in corpus cavernosum tissue (brown: positive signal); scale bar: 100. μm; (hj) Quantitative analysis of the proportion of immunohistochemically positive regions (n=6); (k) Western blot results of fibrosis-related proteins (Col1a1, Col3a1, TGF-β, MMP2, Smad5); (lp) Relative quantitative analysis of protein expression (with α-Tubulin as an internal reference, n=6); Statistical analysis: All data are expressed as mean ± standard error (n=6); One-way ANOVA was used for intergroup comparisons.

[0035] Figure 6 : Electric delivery module: the designed pattern is transferred to Eco-Flex by photolithography, the microelectrode area is covered with a layer of silver using magnetron sputtering, and the non-electrode area is cleaned to form a microelectrode array; wireless transmission power supply module: the three-layer composite sheet formed by PET with copper foil on both sides is cut into a circle, and a deep hole is punched using laser, then the deep hole is filled with copper by electroplating, and the copper foil is formed into a coil and a stimulating electrode by wet etching technology.

[0036] Figure 7 : (a) Hardware composition of wireless stimulation device; (b) Top and bottom layer wiring diagram of flexible printed circuit board (FPCB) for wireless stimulation; (c) Voltage waveform acting on tissue under different distances between transmitting coil and receiving coil; (d) Change of stimulation voltage amplitude under different coil distances.

[0037] Figure 8 : (a) Representative images of PGE1 FITC expression of endothelial cells in normal control group, PGE1 treatment group, and FlexiStim patch group; (b) Quantitative analysis of fluorescence intensity of AM / PI staining of smooth muscle cells (n = 3); (c) Representative scatter plot of flow cytometry for detecting apoptosis of endothelial cells in normal control group, PGE1 treatment group, and FlexiStim patch group; (d) Representative images of Ca 2+ probe staining; (e) Quantitative analysis of fluorescence intensity of ROS probe in each group; (f) Representative images of endothelial cell scratch test in each group (scale bar: 100 μm); (g) Quantitative analysis of endothelial cell scratch test in each group (n = 3); (h) Quantitative analysis of fluorescence intensity of eNOs probe of endothelial cells in each group (n = 3); (i) Quantitative analysis of NO level of endothelial cells in each group (n = 3). Statistical analysis: data are expressed as mean ± standard error; n = 3 independent experiments; one-way ANOVA was used for comparison between groups.

[0038] Figure 9 : (a) Wayne diagram showing the differentially expressed genes and their intersections between each pair of groups; (b) Expression heat map of fibrosis-related genes (horizontal represents genes, and each column represents a sample, red represents highly expressed genes, and blue represents lowly expressed genes); (c) Expression heat map of inflammation-related genes (horizontal represents genes, and each column represents a sample, red represents highly expressed genes, and blue represents lowly expressed genes).

[0039] Example 1 This example provides a design and working principle of a non-invasive flexible electric stimulation patch (hereinafter referred to as FlexiStim patch), as shown in Figure 1 .

[0040] Unlike the current clinical treatment of intracavernous injection, FlexiStim patch not only promotes the recovery of erectile function by activating the EP2 / PKA / CREB pathway, but also inhibits the TGF-β1 / Smad signaling pathway, thereby significantly reducing the progression of fibrosis caused by physical needle penetration of the corpus cavernosum (as shown in Figure 1 a). FlexiStim patch consists of two functional modules: (1) an electro-stimulation drug delivery module and (2) a wireless power module for providing electro-transfection ( Figure 1 b). Among them, the delivery module includes a four-layer structure. From the perspective of contacting the skin of the target organ, the outermost layer is a biodegradable plastic (Ecoflex) layer, which serves as a flexible substrate and device packaging, and can prolong the service life of the FlexiStim patch; the second layer is a microelectrode array layer, which includes a plurality of microelectrode pieces with consistent shape and regular arrangement, and the microelectrode pieces are connected by a silver serpentine electrode wire, which has at least two S-shaped structures; each of the microelectrode pieces has at least two serpentine electrode wires (the microelectrode array layer is a composite material: Cr / Ag / Cr, thickness: 5 μm / 150 μm / 5 μm, Cr as an adhesion layer); the third layer is a GelMA-chitosan hydrogel (100 μm thick), and a large number of hydrogen bonds are formed between the hydroxyl and amino groups of chitosan and the ester groups of Ecoflex, which ensures that the toughness of the hydrogel-Ecoflex interface is strong enough to prevent the inherent adhesion failure of FlexiStim, and its porous structure serves as a medium for storing payloads in liquid form, such as prostaglandin (PGE1); the innermost layer is a thin polycarbonate film (22 μm thick) with nano-pores on its surface, and the nano-pores (800 nm in diameter) can directly contact the target organ. The nano-pores strengthen the external electric field in the nano-pore-tissue juxtaposition configuration, thereby achieving efficient, safe and controllable drug delivery.

[0041] The wireless power module is connected to one side of the silver microelectrode array, which consists of an antenna coil and a rectifier circuit ( Figure 1 c). Inductive coupling between the transmitting coil and the receiving coil enables the modulation and amplification of signals from the transmitting end to be converted into pulse voltage at the receiving end ( Figure 7 ). Through the rectifier circuit, the electric pulse generated between the working electrodes can be adjusted according to the requirements of in vitro or in vivo delivery to provide a specific amplitude (e.g., 17V), frequency (e.g., 10 Hz) and duration (e.g., 5 ms) ( Figure 7The built-in Zener diode, plus the adjustment of the input power of the transmitter, can maintain the output voltage at a predetermined level under various conditions that can attenuate the transmission of radio frequency (RF) power, such as device bending and an increase in the distance between the transmitter and receiver antennas). During treatment, the FlexiStim patch delivers PGE1 transdermally, non-invasively, painlessly, and uniformly to the inside of the corpus cavernosum by applying a pulsed electric field Figure 1 d). The entire FlexiStim patch is soft and has a thin geometric structure, which enables it to achieve flexible, non-irritating close contact with the skin by a principle similar to that established by epidermal electronics.

[0042] The strain / stress distribution shown in the finite element analysis (FEA) results corresponding to the representative device optical image simulates the deformation of the FlexiStim patch under different mechanical deformations of the organ (penis) surface Figure 1 e, f). The maximum principal strain in the metal wire is not more than 3%. Note that the strain in most of the trace area is much lower (<0.5%) compared to the maximum strain. The FlexiStim patch can also withstand extreme deformations and has high mechanical durability. The ΔR / R of the FlexiStim patch remains constant after about 1000 bending cycles Figure 1 g).

[0043] To further illustrate the main advantages of the FlexiStim patch, the present experiment compared several existing treatment methods: Intracavernosal injection (ICI, intracavernosal injection), Oral PDE5Is (oral phosphodiesterase-5 inhibitors), Li-ESWT (low-intensity extracorporeal shock wave therapy), and the FlexiStim patch of the present application is more advantageous in the performance comparison of non-invasiveness, efficacy, onset time, duration of action, local adverse reactions, and systemic adverse reactions Figure 1 h).

[0044] The present embodiment also provides a specific example of a method for preparing a FlexiStim patch (see Figure 6 ).

[0045] Preparation of the wireless power module: The wireless power module was fabricated on a transparent polyethylene terephthalate (PET) film (thickness 100 pm) using flexible printed circuit (FPC) technology. Both sides of the double-sided copper-coated PET film (copper thickness 12 pm) were processed into a receiving coil (6 turns) and two serpentine wires (length 15 mm), respectively. The end of the serpentine wires was a one-side square electrode for connecting the drug delivery device and applying pulsed electrical stimulation. The one-side square electrode was gold-plated (thickness 200 nm; magnetron sputter, TRP450). Specifically, the copper-coated PET substrate film was first cleaned with deionized water and ethanol, then the substrate film was cut into the shape of the wireless power module, and a through-hole (diameter 100 pm) was formed by laser drilling. The copper foil on both sides was connected by the hole plating copper method. Subsequently, the substrate film was stacked with dry film by heating roller, exposed to ultraviolet light, and treated with developer (sodium carbonate). The bare copper foil on the PET was etched into a coil and a serpentine stimulation electrode using APS 100 copper etching solution. The remaining dry film was stripped using an alkaline solution (such as NaOH). Finally, the pad mask layer (i.e., the cover film) was punched by laser to ensure that the exposed surface was used to add electronic components and output pulsed voltage through the stimulation electrode. Then the cover film (thickness 12.5 pm) was used as an insulating layer and stacked on the double-sided graphic circuit by hot pressing. Two Schottky diodes (NSR02301MX4T5G), one Zener diode (GRM022D80G104ME15L), and three capacitors (180 pF, 4 pF, 0.1 uF) were added to the circuit using silver paste for final use.

[0046] Preparation of the drug delivery module: First, phase A and phase B of Eco-flex material were weighed and mixed in a 1:1 mass ratio, and stirred thoroughly until uniform. The mixture was then spin-coated on a clean glass substrate using a spin coater at a speed of 4600 rpm to form a wet film of about 20 μm in thickness, and then placed on a hot stage at 80 °C for 30 minutes to solidify. After solidification, photoresist was spin-coated on the Eco-flex substrate at a speed of 1000 rpm and soft-baked at 90 °C for 1 minute. Next, the photoresist was patterned by UV exposure through a pre-designed mask, and then developed in a developer for 20 seconds, rinsed with deionized water, and dried with nitrogen. Thereafter, the sample was placed in a magnetron sputtering instrument, and vacuumed to a base pressure. Argon gas was then introduced at a flow rate of 30 sccm, and the working pressure was maintained at 0.5 Pa. A silver target was sputtered on the substrate, which was rotating at a speed of 5 rpm, under a sputtering voltage of 120 V. The deposition time was 3 minutes. After sputtering, the sample was removed from the chamber, and the silver-coated Eco-flex film was gently peeled off from the glass plate and immersed in a photoresist stripper, with the aid of ultrasonic treatment at a power of 40 W for 20 minutes, to remove the photoresist and the silver film on the non-electrode area. Finally, a precise silver electrode pattern was formed on the surface of the Eco-flex. In addition, a hydrogel containing a drug and a nanopore membrane were coated thereon. After the completion of the electrode fabrication, the sample was rinsed with deionized water and dried with nitrogen, and then electrically connected to the wireless power supply module to assemble a complete device.

[0047] Example 2 In vitro drug delivery of the flexible electrical stimulation patch After the wireless transmission power supply module was turned on, the FlexiStim patch could deliver the drug contained therein to the cells. To evaluate the ability of the FlexiStim patch to deliver FITC-labeled PGE1 to smooth muscle cells (SMCs) and endothelial cells (ECs), the intracellular FITC fluorescence intensity was detected. PGE1 FITC The direct treatment group, like the negative control group, had almost no fluorescence detected in the cells; while the FlexiStim-PGE1 FITC The treatment group showed significant green fluorescence in both types of cells, confirming that the FlexiStim patch could successfully deliver FITC-labeled PGE1 to the cells. Figure 2 a). Further, live / dead cell staining was performed on SMCs using calcein-AM (green label live cells) and propidium iodide (PI, red label dead cells). Confocal microscope imaging showed that both the PGE1 direct treatment group and the FlexiStim-PGE1 treatment group maintained normal SMC morphology, and were comparable to the control group, mainly showing very little PI signal, thereby proving the high safety of the FlexiStim-PGE1 treatment group. Figure 2b). In addition, the cytotoxic effect of FlexiStim-PGE1 was quantitatively evaluated by Cell Counting Kit-8 (CCK-8) assay. Statistical analysis showed that FlexiStim did not exhibit significant cytotoxicity to SMCs compared with the control group (p>0.05) Figure 2 c), further confirming its biocompatibility. In addition, flow cytometry was further used to evaluate the toxicity of FlexiStim to endothelial cells (ECs), and the results showed that FlexiStim did not produce significant toxic effects on ECs Figure 8 c).

[0048] Establishment of high glucose group (HG) cell model: cells were placed in normal glucose (NG) 7mmol / L and higher glucose (HG) 32mmol / L conditions respectively, incubated at 37℃ for 48h, and high glucose medium incubation simulated diabetic cell damage.

[0049] Real-time proliferation analysis by Incucyte live cell analysis system showed that both PGE1 direct treatment and FlexiStim-PGE1 treatment significantly promoted SMCs proliferation compared with the high glucose group (HG) (p<0.05) Figure 2 d). Further detection of cell apoptosis rate by AnnexinV / PI double staining flow cytometry found that the apoptosis rate of smooth muscle cells in the high glucose group was significantly higher than that in the other three groups (p<0.05), while the apoptosis rate of PGE1 and FlexiStim-PGE1 treatment groups under high glucose conditions was significantly lower than that of the high glucose group (p<0.05) Figure 2 e-f). Further detection of Ca² + concentration. The results showed that the Ca² + level in the high glucose group was significantly increased (p<0.05 compared with the control group); while PGE1 direct treatment and FlexiStim-PGE1 treatment could significantly reduce the Ca² + level (p<0.05 compared with the high glucose group) Figure 2 g, Figure 8 d).

[0050] Under high glucose environment, capillary-like structure formation was significantly reduced compared with the NG group, while PGE1 and FlexiStim-PGE1 treatment could restore these angiogenesis-related parameters to near normal levels Figure 2 i-k). The results of scratch experiment showed that high glucose conditions would also damage the migration ability of endothelial cells Figure 8f,g), while FlexiStim-PGE1 treatment could significantly relieve this high glucose-induced migration inhibition. Further, we detected the ROS fluorescence probe intensity by flow cytometry, and the results showed that the ROS expression level of ECs in HG-treated group was significantly increased, and PGE1 and FlexiStim-PGE1 treatment could relieve this change, while FlexiStim-PGE1 treatment was significantly better Figure 8 e). Further, we detected the expression level of eNOS and nitrite (NO stable metabolite NaNO2) in ECs, and the results showed that the DAF-FM DA fluorescence intensity in HG-treated ECs was significantly decreased Figure 2 j, p<0.05); in addition, high glucose significantly inhibited the generation of NaNO2 in ECs (p<0.05 compared with the control group), while PGE1 and FlexiStim-PGE1 treatment could basically restore these key factors to normal Figure 8 i).

[0051] Example 3 Before the model was induced, there was no significant difference in body weight and fasting blood glucose between mice in each group. 7-week-old c57BL / 6J male mice were purchased, acclimatized for 1 week, and numbered, and their fasting blood glucose was measured. Before modeling, the mice were fasted for 16 hours, and streptozotocin STZ was dissolved in citrate buffer to prepare a 1% solution (freshly prepared), which was injected intraperitoneally at a dose of 120 mg / kg (i.e. 12 mL / kg). After injection, the mice were observed for 30 minutes, and then given sufficient feed and 10% sucrose solution (to prevent the death of mice due to hypoglycemia after STZ injection). Tail blood was collected at 24h, 72h, and 1 week after intraperitoneal injection of STZ to determine the body weight and random blood glucose. If the blood glucose was higher than 16.7 mmol / L for three times and was accompanied by the symptoms of "three more and one less", the mouse was determined to have successfully constructed a diabetic model, and was included in the experiment for further feeding. Other mice with substandard blood glucose were excluded from the experiment.

[0052] (2) The control group of mice was injected intraperitoneally with the corresponding volume of citrate buffer and fed with ordinary feed.

[0053] (3) APO test to screen DMED mice: After the success of the diabetic mouse model, apomorphine (100 pg / kg) was injected subcutaneously to screen ED mice for inclusion in the experiment. After injecting apomorphine, the control group could be observed to have an erection within 30 minutes; the diabetic group had no erection within 30 minutes after injecting apomorphine. After 28 days of successful modeling, the fasting blood glucose of the modeling group (DMED group, DMED-PGE1 group and DMED-FlexiStim group) mice was significantly increased (p<0.05), accompanied by a decrease in body weight. After 28 days of PGE1 and FlexiStim treatment, the trend of blood glucose index change continued to exist.

[0054] FlexiStim-PGE1 promotes corpus cavernosum tissue repair and improves erectile function Small animal live imaging results showed that FITC-labeled PGE1 significantly remained in the penile tissue at 0, 3 and 6 hours after radio stimulation, the same as the ICI effect ( Figure 3 c). In addition, immunofluorescence analysis showed that FITC-labeled PGE1 was successfully delivered to the corpus cavernosum tissue of mice after transdermal administration by FlexiStim. FlexiStim-mediated transdermal delivery resulted in uniform distribution of FITC-labeled PGE1 throughout the corpus cavernosum tissue. In contrast, after ICI injection, FITC-labeled PGE was mainly distributed at the injection site, and the fluorescence intensity gradually decreased in all directions. This proves that FlexiStim patch transdermal administration can deliver drugs more evenly to the inside of the target organ ( Figure 3 d). In addition, the experiment evaluated the safety of FlexiStim one month after in vivo delivery. Hematoxylin-eosin (H&E) histological staining of major organs such as heart, liver, spleen, lung and kidney showed no obvious pathological changes in the FlexiStim-PGE1 group.

[0055] Further evaluate its therapeutic potential for corpus cavernosum tissue repair in DMED mouse models. The results showed that: compared with the DMED group, DMED-ICI and DMED-FlexiStim administration both resulted in a significant increase in erection angle (both P<0.05), reaching a similar value to the healthy control group, but the erection angle of the DMED-FlexiStim administration group was closer to the healthy control group ( Figure 3 e-f). In addition, the results of erectile function detection showed that compared with the control group, the erectile function of DMED mice was significantly impaired (P<0.05). ICI and FlexiStim treatment can significantly improve the ICPmax of DMED mice ( Figure 3 g-h), and there was no significant statistical difference between the two treatment methods (P>0.05).

[0056] Immunofluorescence staining results showed that the expression levels of α-SMA and Desmin were significantly decreased in the DMED group (p<0.001), while the expression levels of α-SMA and Desmin were significantly increased in the DMED-ICI and DMED-FlexiStim groups, approaching the control group levels. Figure 4 ac). With DMED group (eNOS) + Compared to the DMED group (where cell counts were significantly reduced), both the DMED-ICI and DMED-FlexiStim groups showed significant upregulation of eNOS, reaching near-normal levels. Notably, compared to the DMED group, FlexiStim treatment showed a significant increase in the fluorescence intensity of eNOS and vWF (p<0.001). Figure 4 df). Furthermore, Western blot analysis showed that, compared to the DMED group, the protein expression of eNOS and α-SMA in the corpora cavernosa of both the DMED-ICI and DMED-FlexiStim groups was significantly increased. Figure 4 These results indicate that, compared to the DMED group, FlexiStim treatment restored the expression of smooth muscle markers α-smooth muscle actin (α-SMA) and desmin to near-healthy levels, with distribution and fluorescence intensity comparable to ICI treatment, suggesting that FlexiStim treatment can effectively improve corpus cavernosum smooth muscle damage. Regarding endothelial markers, FlexiStim treatment significantly increased eNOS expression, and the fluorescence intensity of vWF was approximately 1.34 times greater than in the DMED group, indicating that this drug delivery system can partially reverse endothelial dysfunction caused by hyperglycemia and promote angiogenesis and endothelial activity recovery.

[0057] Furthermore, Western blot analysis showed that, compared with the DMED group, the protein expression of EP2, PKA, and CREB in the corpus cavernosum of both the ICI-PGE1 group and the FlexiStim-PGE1 group was significantly increased, suggesting that ICI and FlexiStim treatment may improve erectile function in DMED mice through the EP2 / PKA / CREB pathway. Figure 4 jm).

[0058] The degree of corpus cavernosum fibrosis was assessed. Results showed that, compared to the control group, the smooth muscle (red) / collagen (blue) ratio was significantly lower in the DMED group (P<0.05), indicating that corpus cavernosum fibrosis was more severe in the DMED group. Compared to the DMED group, there was no statistically significant difference in the smooth muscle (red) / collagen (blue) ratio in the DMED-ICI group; however, the smooth muscle (red) / collagen (blue) ratio was significantly higher in the DMED-FlexiStim treatment group. Figure 4 no).

[0059] Further, transcriptome sequencing and analysis were performed on the corpus cavernosum tissue of mice from the DMED, DMED-ICI, and DMED-FlexiStim groups. Figure 5 a). First, principal component analysis was performed on the three groups of samples, and significant cluster differences were found among the three groups ( Figure 5 b); Differential gene analysis results showed that there were 2608 differentially expressed genes between the DMED-ICI group and the DMED-FlexiStim group, 351 differentially expressed genes between the DMED-FlexiStim group and the DMED group, and 2685 differentially expressed genes between the DMED-ICI group and the DMED group. Figure 9 a). Further cluster analysis of differentially expressed genes revealed significant differences in gene expression among the groups; compared with the DMED group and the DMED-FlexiStim group, the DMED-ICI group showed significantly upregulated fibrosis and inflammation-related genes, indicating that local injection is more likely to cause inflammation and tissue fibrosis. Figure 9 a,b).

[0060] To further clarify the differences in treatment efficacy between ICI-PGE1 and FlexiStim-PGE1, this study analyzed differentially expressed genes between the DMED-ICI and DMED-FlexiStim groups. The results showed that ICI-PGE1 treatment upregulated fibrosis-related genes such as MMP2, Col1a1, Col3a1, MMP9, Timp1, and Tgfbβ1. Figure 5 d). GO functional enrichment analysis showed that, compared to the ICI-PGE1 group, the genes downregulated by FlexiStim-PGE1 treatment were enriched in functional categories such as "collagen fibril organization" and "inflammatory response." Furthermore, KEGG pathway enrichment analysis revealed the "TGF-beta signaling pathway" (transforming growth factor-β signaling pathway). Figure 5 f) Pathways closely related to fibrosis and inflammation, such as the "TNF signaling pathway," were activated in the DMED-ICI group.

[0061] The immunohistochemical analysis results showed that the expression of COL1A1 and TGF-β in the cavernosal tissue of the DMED group was increased compared with the Control group, but the difference was not statistically significant; the expression of COL1A1 in the ICI-PGE1 group was significantly increased, and there was no significant difference between the FlexiStim-PGE1 group and the Control group. At the same time, the protein expression of COL3A1 increased in the DMED group, and the difference was statistically significant; the protein expression of COL3A1 in the ICI-PGE1 group was significantly increased, and there was no statistically significant difference between the FlexiStim-PGE1 group and the Control group. In addition, the Western blot results also showed that compared with the Control group, the protein expression of COL3A1, COL1A1, MMP2, TGF-β1 and SMAD5 in the DMED group increased, and the difference was statistically significant; in addition, compared with the Control group, the protein expression of COL3A1, COL1A1, MMP2, TGF-β1 and SMAD5 in the ICI-PGE1 group was significantly increased. The expression of the above indexes in the DMED-FlexiStim group was significantly lower than that in the DMED-ICI group, and tended to be closer to the Control group.

[0062] The experimental results of the transcriptome and protein level can be obtained: diabetic erectile dysfunction can cause tissue fibrosis, and intracavernosal injection of PGE1 can exacerbate the degree of fibrosis, which may be due to the activation of the TGF-β1 / SMAD pathway; compared with the ICI-PGE1 group, the activity of the pathway in the FlexiStim-PGE1 group is significantly down-regulated, which is the key to the better treatment effect of FlexiStim-PGE1.

[0063] It should be noted that in this paper, the terms "include", "contain" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or device. Without more limitations, the element defined by the statement "includes a" does not exclude the presence of another identical element in the process, method, article or device including the element.

[0064] The embodiments of the application are described above in conjunction with the drawings, but the application is not limited to the specific embodiments described above, which are only illustrative and not limiting, and those of ordinary skill in the art can make many forms under the inspiration of the application without departing from the purpose of the application and the scope protected by the claims.

Claims

1. A non-invasive flexible electrical stimulation patch, characterized in that, The non-invasive flexible electrical stimulation patch includes: (1) an electrical stimulation drug delivery module and (2) a wireless transmission power supply module; The electrostimulation drug delivery module is composed of a substrate layer, a microelectrode array layer, a gel layer, and a nanoporous membrane layer. The base layer is a biodegradable flexible material used for support and encapsulation; The microelectrode array layer is composed of multiple layers of electrodes. The microelectrode array layer includes multiple microelectrode sheets with the same shape and arranged regularly. The microelectrode sheets are connected by serpentine electrode lines. The serpentine electrode lines have at least two S-shaped structures. Each microelectrode sheet has at least two serpentine electrode lines. The gel layer comprises a viscous gel matrix with hydroxyl and / or amino groups and a drug to be delivered electrically; the microelectrode array layer is embedded in the gel layer; The nanoporous membrane layer is made of an ester group that can form hydrogen bonds with the hydroxyl or amino groups of the gel matrix; the nanoporous membrane layer has a thickness of 10-50 μm and a pore size structure of 500-1000 nm on its surface. The wireless transmission power supply module is connected to the microelectrode array layer via leads to supply power to the electrostimulation drug delivery module.

2. The non-invasive flexible electrical stimulation patch as described in claim 1, characterized in that, The serpentine electrode wire is made of silver; or, the lead wire is made of silver.

3. The non-invasive flexible electrical stimulation patch as described in claim 1, characterized in that, The microelectrode array layer is composed of Cr-Ag-Cr composite; the thickness of the Ag layer is 15 to 30 times the total thickness of the Cr layer.

4. The non-invasive flexible electrical stimulation patch as described in claim 1, characterized in that, The shape of the microelectrode sheet includes triangle, quadrilateral, polygon, or circle.

5. The non-invasive flexible electrical stimulation patch as described in claim 1, characterized in that, The material of the gel matrix includes GelMA-chitosan; or the material of the nanoporous membrane layer includes polycarbonate membrane or polyethylene terephthalate.

6. The non-invasive flexible electrical stimulation patch as described in claim 1, characterized in that, The drug to be delivered electrically includes drugs for treating erectile dysfunction.

7. The non-invasive flexible electrical stimulation patch as described in claim 6, characterized in that, The nanopore size of the nanoporous membrane is in the range of 600-800 μm.

8. The non-invasive flexible electrical stimulation patch as described in claim 1, characterized in that, The wireless transmission power supply module includes a coil antenna and a rectification and regulation circuit block.

9. The non-invasive flexible electrical stimulation patch as described in claim 8, characterized in that, The wireless transmission power supply module further includes a flexible packaging structure; or, the rectification and regulation circuit adopts an NFC receiver structure, including a rectification module, a voltage regulator module, and an energy storage capacitor.

10. The method for preparing the non-invasive flexible electrical stimulation patch according to any one of claims 1-9, characterized in that, Includes the following steps: S110: Fabrication of an electrically stimulated drug delivery module; S111: A biodegradable flexible material is coated onto a clean substrate to form a wet film, which is then cured at high temperature; S112: Coat the cured flexible material with photoresist and dry it appropriately; S113: Ultraviolet exposure is performed using a pre-designed mask to pattern the photoresist, followed by nitrogen drying; S114: Place the sample treated in S113 into a magnetron sputtering instrument, evacuate to the base pressure, then introduce argon gas to maintain the working pressure at 0.5 Pa. Apply a sputtering voltage of 120 V to sputter the silver target while the substrate is rotating. S115: After sputtering is completed, the sample is removed from the chamber, the silver-plated flexible material film is peeled off from the substrate, and then a drug-containing hydrogel and nanoporous membrane are coated on it. S120: A wireless transmission power supply module is fabricated on transparent polyethylene terephthalate using flexible printed circuit technology; S130: Assemble the electrostimulation drug delivery module and the wireless transmission power supply module to form the non-invasive flexible electrostimulation patch.

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