Bioabsorbable organic bioelectronics

Abstract

The present invention relates to a composition comprising a polymer of formula (I) and one or more compounds of formula (II) or formula (III), and to its use in the treatment of diseases (e.g., cancer, cardiovascular disease, infectious disease, or neurodegenerative disease). The polymer of formula (I) and one or more compounds of formula (II) or formula (III) have the following structure [Formula 1] Represented by JPEG2026513146000041.jpg60159, A, E, Z, Z', R1, R2, R3, R3', R4, R4', R5, R6, R7, R8, R9, R 10 n, m, a, y, and q are as defined in the specification.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a polymer of formula (I) and one or more compounds of formula (II) or formula (III), and to its use in the treatment of diseases (e.g., cancer, cardiovascular disease, infection, immunomodulation, pain, or neurodegenerative diseases) and in energy storage. [Background technology]

[0002] Bioelectronics is a field of research that fuses biology and electronics. Bioelectronic devices, or bioelectronic drugs, have the potential to complement conventional therapies in immunotherapy, pain management, and treatments that are not long-term, such as cancer. While conventional drugs typically address only biochemical processes, bioelectronics address dysfunctions of bioelectric circuits. Furthermore, conventional bioelectronic devices face the problem of being invasive, requiring surgical implantation, removal, or both, and primarily focusing on chronic diseases. Such types of devices usually require surgical removal after the disease has subsided. Various strategies have been developed in designing electrodes that seamlessly connect to the nervous system structure of animals, but most of these strategies rely on external factors such as electric fields, chemicals such as proteins, or genetic engineering. For example, pioneering research presented in the prior art concerns conductive polymers, which are formed in the brains of rodents. Specifically, poly(3,4-ethylenedioxythiophene)(PEDOT):PSS structures were formed in the mouse brain by in vivo electropolymerization of 3,4-ethylenedioxythiophene (EDOT) monomer using polystyrene sulfonic acid (PSS) as a template. However, the formed polymer mixture did not successfully integrate into the cells and protruded from the injection electrode as a clumpy cloud.

[0003] Therefore, there is a need for improved bioelectronics that can minimize invasive approaches and offer new methods for treating brain diseases in particular. Such methods would allow us to avoid the limitations of conventional craniotomy and rigid electrode implantation. [Overview of the Initiative]

[0004] Therefore, one objective of this technology is to provide a substrate-free, self-assembled organic electrode. Such an electrode may address the aforementioned shortcomings and criteria, and furthermore, it may meet the requirement for seamless integration into dynamic biological systems in ways that are impossible with conventional, inflexible solid-state electronics.

[0005] Another objective is to provide a general approach to generating bioelectronics that are independent of specific external or endogenous triggers.

[0006] Another objective is to provide a composition or pharmaceutical composition that is biocompatible, that is, well-tolerated in the human body.

[0007] Another objective is to provide a composition or pharmaceutical composition that includes a conductive structure that can be positioned precisely in a target area.

[0008] Another objective is to provide compositions that can constitute bioabsorbable electrodes from water-dispersible nanoparticles, for example, in the brain, in and around tumors, in and around cardiac tissue, and on and around the vascular system.

[0009] Another objective is to provide compositions that can function as electrodes in insights and, when relaying external stimuli, can induce controlled cellular responses in target organs, such as the brain, heart, tumors, and spinal cord.

[0010] Another objective is to provide electroactive organic polymers that can be produced by spatial control in vivo using different modalities (e.g., enzymatic processes, photochemical processes, i.e., photo-inducible processes, electrochemically controlled processes, or combinations thereof).

[0011] Another object of this disclosure is to provide compositions that do not cause tissue damage, cytotoxicity, or organ toxicity after bioresorption.

[0012] Another objective is to provide compositions or pharmaceutical compositions that can be assembled in vivo within cellular structures, fully integrated, and transient organic bioelectronics that can be used for non-long-term treatments.

[0013] Another objective is to provide compositions that are easy and inexpensive to synthesize from readily available commercially available materials.

[0014] Accordingly, in order to achieve at least one of the above-mentioned objectives and other objectives that may become apparent from the following description, a composition or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a kit for use in the treatment or prevention of diseases such as cancer, cardiovascular disease, infection, or neurodegenerative disease is defined in an independent claim, as well as the uses of the composition or a pharmaceutically acceptable salt thereof, and a method for treating cancer, cardiovascular disease, infection, or neurodegenerative disease is defined in an independent claim. Various preferred embodiments of the concept of the present invention will become apparent from the dependent claims.

[0015] In a first embodiment, a composition comprising a polymer of formula (I) and one or more compounds of formula (II) or formula (III), or a pharmaceutically acceptable salt thereof, is provided, wherein the polymer of formula (I) is represented by the following structure: [ka] One or more compounds of formula (II) or formula (III) are represented by the following structure: [Chemical formula] In the formula, each A is selected from H, Na, K, Li, Ca, Mg, Sr, and Ba, and E is H, C + , 1~6 , 2~6 , y , 1~6 , 1~6 , + , - , + , 1~6 , 1~6 , 1~6 , 1~6 , 1~20 , 3~6 , 1~20 , 2~6 , 1~6 , 1~6 alkyl, C 2~6 alkenyl, C 2~6 alkynyl, C 3~6 cycloalkyl, aryl, heteroaryl, -(CH2CH2O) q CH2CH2OH, and here C 1~6 alkyl may be substituted with one or more of -N3, -OH, -SO3A, -N(C 1~6 alkyl)2, -NH + (C 1~6 alkyl)2, and -N + (C 1~6 alkyl)3. R1 is selected from H, C 1~6 alkyl, C 2~6 alkenyl, C 2~6 alkynyl, C 3~6 cycloalkyl, aryl, heteroaryl, and here C 1~6 alkyl may be substituted with one or more of -N(C 1~6 alkyl)2, -NH + (C 1~6 alkyl)2, and -N + (C 1~6 alkyl)3. Each Z is selected from a bond, -O-, -OP(O)(O - )O-, -OP(O)(OH)O-, -OC(O)-, -C(O)O-, -OC(O)NH-. Each R2 is selected from H, C 1~20 alkyl, aryl, heterocyclyl, heteroaryl, and Si(C 1~6 alkyl)3. C 1~20 alkyl, aryl, heterocyclyl, and heteroaryl may be substituted with one or more R5 or R6. Each of R3, R3’, R4, and R4’ is selected from H and -(CH2) y -Z-(R2), and R3 and R3’ are C 1~6When they are alkoxys and together with the atoms to which they are each bonded, they form a heterocycle which may be substituted with one or more R5 or R2 atoms, and R4 and R4' are C 1~6 When they are alkoxys and together with the atoms to which they are bonded, they form a heterocycle which may be substituted with one or more R5 or R2 atoms. R5 is H, C 1~6 Alkyl, C 2~6 Alkenyl and C 2~6 Selected from alkynyl groups, Z' is a 6-membered or 7-membered heterocycle, and each R6 is -SO3A, -CO2A, -CO2(R9), -OH, -O(R9), halogen, -N3, -NH2, -NH(R9), -NHC(O)(R9), -N(C 1~6 Alkyl)2,-N + (C 1~6 Alkyl)3,-(OCH2CH2) q -(R8), C 1~6 Alkyl, C 2~6 Alkenil, C 2~6 Alkinyl, aryl, heteroaryl, heterocyclyl, ferrosenyl, -C(O)NH(C 1~6 Selected from alkyl, where -N + (C 1~6 Alkyl)3 and -C(O)NH(C 1~6 The alkyl group may be substituted with one or more R7 groups, and C 1~6 Alkyl, heterocyclyl, heteroaryl, and aryl compounds may contain one or more R7, R9, or R 10 It is replaced with -SO3A, -CO2A, -(OCH2CH2) q -(R8), -NH2, -NHC(O)-(R9), -OC(O)-(R9), aryl, guanidinyl, -C(O)NH(C 1~6 Selected from alkyl, where guanidinyl and -C(O)NH(C 1~6 The alkyl group may be substituted with one or more R8s, where R8 is C 1~6 Alkyl, C 1~6 Alkoxy, -OH, -NH2, -NH(C 1~6 Alkyl), -N3, -OC(O)-(R9), and -C(O)NH(C 1~6Selected from alkyl, where C 1~6 Alkyl and -C(O)NH(C 1~6 The alkyl group may be substituted with one or more R9 groups, where R9 is C 1~6 Alkyl, C 2~6 Alkenil, C 2~6 Alkynyl, aryl, heteroaryl, heterocyclyl, ferrocenyl, -B(OH)2, -CO2A, and -CO2(C 1~6 Selected from alkyl, where C 1~6 Alkyl, aryl, heterocyclyl, and -CO2(C 1~6 Alkyl) is one or more R 10 It may also be replaced with R 10 These are oxo, -SO3A, -NH2, -CO2A, -OH, -P(O)(OH)2, C 1~6 Alkyl, C 1~6 Selected from alkoxy, aryl, and heteroaryl groups, where n is 4-32, m is 0-10, a is 1-5, y is 0-16, and q is 0-15.

[0016] In another embodiment, a composition of the first embodiment or a pharmaceutically acceptable salt thereof is provided for use in the treatment or prevention of a disease.

[0017] In another embodiment, a composition of the first embodiment or a pharmaceutically acceptable salt thereof is provided for use in the treatment or prevention of a disease, the disease being selected from cancer, cardiovascular disease, infectious disease, and neurodegenerative disease.

[0018] In another embodiment, the use of the composition of the first embodiment or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of cancer, cardiovascular disease, infection, or neurodegenerative disease is provided.

[0019] In another aspect, the use of the composition of the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating spinal cord injuries such as spinal cord injury, the drug inducing nerve regeneration in the spinal cord.

[0020] In another embodiment, a method is provided for treating cancer, cardiovascular disease, infection, or neurodegenerative disease, the method comprising administering a therapeutically effective amount of the composition according to the first embodiment or a pharmaceutically acceptable salt thereof to a patient in need of treatment.

[0021] In another embodiment, a pharmaceutical composition is provided, comprising a composition according to the first embodiment or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, and / or additive.

[0022] In another embodiment, a pharmaceutical composition is provided, comprising a therapeutically effective amount of the composition according to the first embodiment or a pharmaceutically acceptable salt thereof, and another anticancer agent selected from alkylating agents, antimetabolites, anticancer camptothecin derivatives, plant-derived anticancer agents, antibiotics, enzymes, platinum coordination complexes, tyrosine kinase inhibitors, hormones, hormone antagonists, monoclonal antibodies, interferons, and biological response modifiers.

[0023] In another embodiment, a hydrogel comprising the composition according to the first embodiment is provided for use in the treatment or prevention of a disease.

[0024] In another embodiment, a kit comprising the composition according to the first embodiment is provided for use in the treatment or prevention of a disease. [Modes for carrying out the invention]

[0025] The concept of this invention focuses on compositions that can be used in novel forms of intratumor electrotherapy incorporating redox processes, ionic currents, or a combination of both. This invention relates to compositions that can self-assemble transient conductive electrodes that are biocompatible and bioabsorbable. Because these conductive structures have fluid properties, they can be implanted using minimally invasive techniques and seamlessly integrated within and around tumors. Furthermore, the ability to modify electrode properties after implantation facilitates more precise targeting of mechanisms associated with dysfunction of bioelectric circuits in cancer. These flexible electrodes can be used as a standalone therapeutic agent or in combination with irreversible electroporation (IRE) methods such as nanoknife. In the latter case, the flexible electrodes integrated into the tumor help the nanoknife electrodes deliver treatment throughout the tumor. Therefore, this disclosure focuses on a general approach to assembling bioabsorbable, high-performance electrode structures by compositions within the central nervous system (CNS), for example, implanted using minimally invasive methods, without relying on specific external or endogenous triggers.

[0026] Internal organs such as nerves and tissues can vary greatly in size and shape, which affects how medical devices such as electrodes interact with them. For example, nerve bundles and ganglia can have very irregular shapes and sizes, and standard, uniform electrodes pose problems due to fit and adaptability issues. Conventional electrodes often require invasive surgical implantation. Such surgery involves making large incisions to directly access the target area. This process can lead to significant trauma, bleeding, and inflammatory responses. As a result, connective tissue may grow, interfering with the function of the electrode. Furthermore, the shape of conventional electrodes is often determined by the fabrication process, which limits the flexibility of electrodes to accommodate the diverse anatomical features found within the central and peripheral nervous systems. For example, flat electrodes fabricated using silicon wafer technology, or rod-shaped electrodes designed for deep brain stimulation, can produce uneven and inaccurate electric fields. This is problematic when trying to effectively stimulate the intended target without affecting adjacent areas. Moreover, surgical placement of such electrodes can induce further tissue irritation and inflammation over time. This could reduce the effectiveness of the electrodes, potentially necessitating further medical intervention. There are also concerns about the financial and health risks associated with surgical implantation. This could deter patients from choosing such treatment. These challenges clearly highlight the need for innovative electrode designs that can be introduced into the body using less invasive methods, such as injection. Ideally, such new types of electrodes would be shaped to conform to the target nerve or tissue, encapsulating it, creating a more effective and uniform electric field, minimizing trauma, and resulting in more stable long-term outcomes.

[0027] The concept of the present invention will be further explained below with reference to the attached schemes and figures illustrating various preferred embodiments of the concept.

[0028] This disclosure relates to compositions comprising a polymer of formula (I) and one or more compounds of formula (II) or formula (III), or to pharmaceutically acceptable salts thereof. Here, the polymer of formula (I) and one or more compounds of formula (II) or formula (III) are represented by the following structures. [ka] In the formula, A, E, Z, Z', R1, R2, R3, R3', R4, R4', R5, R6, R7, R8, R9, R 10 n, m, a, y, and q are as described above.

[0029] In the conceptual design of the material, it was anticipated that the polymer of formula (I), upon injection into tissue, would induce self-aggregation and the formation of conductive structures. Subsequently, one or more trimers of formula (II) or (III) in the mixture, being smaller than the polymer backbone, would diffuse from within to outside the backbone. Upon application of external stimuli such as low potential, light, or enzymes, the entire volume of the polymer backbone would be functionalized with trimers that adhere to and extend outward from it, thereby altering the properties of the polymer structure. This method allows for precise placement of conductive structures at target sites and facilitates broader diffusion into surrounding tissues and cells. Because trimers with different parts can be used during functionalization, this method allows for control over chemical properties.

[0030] However, the concept of the present invention can be embodied in many different forms and should not be construed as being limited to the various embodiments shown herein. Rather, such various embodiments are shown for the sake of thoroughness and completeness and to fully convey the scope of this disclosure to those skilled in the art. Details of this disclosure are shown in the following description. Exemplary methods and materials are described herein, but similar or equivalent methods and materials can be used in the implementation or testing of this disclosure. Other features, purposes, and advantages of this disclosure will become apparent from this specification and the claims. Unless otherwise clearly indicated in the context, singular nouns and plural nouns are also included herein and in the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would be generally understood by those skilled in the art to which the present invention pertains.

[0031] While individual features may be included in different embodiments, such features can be combined in other ways, and their inclusion in different embodiments does not mean that features cannot be combined. In this disclosure, the terms "a" and "an" are not plural. The term "may be substituted" is understood to mean that a given chemical part (e.g., an alkyl group) may (but does not have to) be bonded to other substituents (e.g., heteroatoms). For example, a may be substituted alkyl group may be a fully saturated alkyl chain (i.e., a pure hydrocarbon). Alternatively, the same may be substituted alkyl group may have substituents other than hydrogen. For example, it may be bonded at any position along the chain to a halogen atom, a hydroxyl group, or any other substituent described herein. Thus, the term "may be substituted" means that a given chemical part may contain other functional groups, but does not necessarily have further functional groups. Preferred substituents to be used to optionally substitute the groups described are further defined and described below.

[0032] In the polymer of formula (I), the number of repeating units is represented by n and m, where n is 4 to 32, preferably 5 to 12, and m is 0 to 10, preferably 0 to 3. In one embodiment, m is 0 or 1, and E is preferably H, C 1~6 Alkyl, -(CH2CH2O) q CH2CH2OH, C substituted with -N3 1~6 Alkyl, C substituted with -SO3A 1~6 Alkyl, as well as SO3A and -N + (C 1~6 C substituted with alkyl)3 1~6 Selected from alkyl groups, R1 is preferably H, methyl, or other C groups. 1~6 Alkyl and -N + (C 1~6 C substituted with alkyl)3 1~6 Selected from alkyl groups. When m is 1, E is preferably H, and R1 is preferably H and C such as methyl. 1~6 Selected from alkyl groups. When m is 0, R1 is preferably H and -N + (C 1~6 C substituted with alkyl)3 1~6 Selected from alkyl groups.

[0033] In one embodiment, one or more compounds of formula (II) or formula (III) are one or more compounds of formula (II-a), formula (III-a), formula (III-b), formula (III-c), and formula (III-d), and therefore the composition comprises a polymer of formula (I) and one or more compounds of formula (II-a), formula (III-a), formula (III-b), formula (III-c), and formula (III-d). The compounds of formula (II-a), (III-a), (III-b), (III-c), and (III-d) have the following structure [ka] A salt of which is represented by or is pharmaceutically acceptable, where Z, R2, R3, R3', R4, R4', R5, R6, R7, R8, R9, R 10, y, and q are as described above. Therefore, the composition comprises a copolymer of formula (I) and one or more compounds from formulas (II-a), (III-a), (III-b), (III-c), and (III-d).

[0034] The substituent R2 in any one of the aforementioned structures can be selected from the following: [ka]

[0035] In one embodiment, the composition of the present disclosure comprises a copolymer represented by the following structure (I-1), [ka] (wherein p is 1 to 12) One or more compounds represented by the following structures (II-1) and (II-2) [ka] or a pharmaceutically acceptable salt thereof.

[0036] A composition comprising a polymer of formula (I) and one or more compounds of formula (II) or (III), or a pharmaceutically acceptable salt thereof, is used for the treatment or prevention of diseases (e.g., cancer, cardiovascular disease, infection, and neurodegenerative disease). Alternatively, a method for treating cancer, cardiovascular disease, infection, or neurodegenerative disease is provided, the method comprising administering a therapeutically effective amount of the composition or a pharmaceutically acceptable salt thereof to a patient in need of treatment.

[0037] A key advantage of this new invention is that it can be administered directly to the target area without requiring conventional surgical methods such as cutting tissue with a scalpel or scissors. This method significantly reduces or avoids damage to the target area and its surroundings. The invention effectively provides a custom fit by uniquely conforming to the precise contour of the target area and forming around the shape of the target. This is achieved by a versatile system that utilizes a combination of external forces (such as electronic and light energy) and internal processes (such as enzymatic reactions) to shape and adapt to the target. This approach ensures optimal electrical connectivity and secures the device in place through strong mechanical bonding to the target, resulting in a stable and effective solution.

[0038] Furthermore, the hardened electrodes of this disclosure can be placed by injection in hard-to-reach locations within the body where surgeons are reluctant to place prior art devices by selective general surgery. Such locations are, for example, ganglia of the sympathetic chain or nerves of the CNS or PNS, adjacent to major blood vessels, and located inside the body in a way that makes direct access from outside the body difficult. The present invention introduces a novel bioelectronic approach to cancer treatment by utilizing compositions according to a first embodiment comprising compounds of formula (I), (II), and / or (III). Using these compositions, cancer cells can be directly targeted to inhibit their growth and proliferation. By incorporating these polymers into bioelectronic devices, it is possible to administer targeted therapies that inhibit cancer activity at the molecular level. This provides a new means of combating various types of cancer with fewer potential side effects than conventional therapies. Typically, cancers are selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors.

[0039] In the case of neurodegenerative diseases, the composition according to the first embodiment can target and modulate neuronal function and degenerative pathways. By incorporating this composition into a bioelectronic system, it can act on cellular processes involved in diseases such as Alzheimer's disease and Parkinson's disease. This may slow disease progression or alleviate symptoms through targeted electronic intervention. Typically, neurodegenerative diseases are selected from traumatic brain injury, spinal cord injury, trauma to the peripheral nervous system, and motor neuron diseases.

[0040] In the case of cardiovascular disease, the composition according to the first embodiment can interact with the underlying biological processes of heart and vascular diseases. By incorporating this composition into a bioelectronic system, the technology can provide precise control over heart rhythm, blood flow, and vascular health. This may allow for the treatment or management of conditions such as arrhythmias, hypertension, and atherosclerosis. This method represents a groundbreaking step in the use of bioelectronics for interventions in the cardiovascular system. Typically, cardiovascular diseases are selected from coronary artery disease (e.g., angina pectoris, heart attack), heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmias, congenital heart disease, valvular heart disease, cardiitis, aortic aneurysm, peripheral artery disease, thromboembolism, and venous thrombosis.

[0041] The compositions according to the first embodiment are also applicable to the control of infectious diseases. By targeting bioelectronic interactions within the cells of pathogens or infected hosts, the compositions can inhibit the replication of viruses or bacteria or modulate the body's immune response to these pathogens. This approach opens new doors for treating infectious diseases, particularly those resistant to conventional drugs, using bioelectronic means. Typically, infectious diseases are selected from viral infections, bacterial infections, parasitic infections, and fungal infections.

[0042] With regard to immunomodulation, the composition according to the first embodiment can be used to modulate the activity of the immune system. This bioelectronic application has potential for the treatment of autoimmune diseases, reduction of inflammation, or enhancement of immune responses to pathogens and tumors. By modulating the electronic signals of the immune system, this technology provides a novel pathway for acting on immune-related diseases and illnesses. Therefore, the composition according to the first embodiment can be used to modulate the activity of the immune system as described above.

[0043] Furthermore, the composition according to the first embodiment can be used for pain management applications. This pain management application involves using the composition within a bioelectronic device to target nerve signals that transmit pain. This method provides a novel strategy for addressing chronic pain conditions by interfering with or modulating pain signals before they reach the brain. This provides an effective and potentially non-pharmacological treatment that can replace conventional pain treatments. Thus, the composition according to the first embodiment can be used for pain management as described above.

[0044] The composition or its pharmaceutically acceptable salts can be used in the preparation of agents for the treatment of cancer, cardiovascular disease, infection, or neurodegenerative disease (e.g., agents for the treatment of cancer, cardiovascular disease, infection, or neurodegenerative disease). Typically, cancers are selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors, and neurodegenerative diseases are selected from traumatic brain injury, spinal cord injury, trauma to the peripheral nervous system, and motor neuron diseases.

[0045] The composition or its pharmaceutically acceptable salts can be used to prepare drugs for the treatment of spinal cord injuries, such as spinal cord injury, in which the drugs induce nerve regeneration in the spinal cord.

[0046] The present invention also provides a pharmaceutical composition comprising the above composition or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, and / or additive. The pharmaceutical composition may also contain another anticancer agent selected from alkylating agents, antimetabolites, anticancer camptothecin derivatives, plant-derived anticancer agents, antibiotics, enzymes, platinum coordination complexes, tyrosine kinase inhibitors, hormones, hormone antagonists, monoclonal antibodies, interferons, and biological response modifiers.

[0047] Alternatively, a hydrogel or kit comprising the above composition or a pharmaceutically acceptable salt thereof is provided. The hydrogel or kit can be used for the treatment or prevention of a disease. The kit may have different parts or fractions, so that the compound of formula (I) or a pharmaceutically acceptable salt thereof may be in the first part of the kit, and one or more compounds of formula (II) or formula (III) or a pharmaceutically acceptable salt thereof may be in the second part of the kit.

[0048] The specific mechanical and structural properties of the electrode to be formed can be modified to match the properties of the target tissue by selecting conductive elements. The formation process involves introducing electrical energy, light, enzymatic reactions, or a combination thereof. For example, the measured Young's modulus of BICS (32 ± 6 kPa) was in good agreement with that of agarose (38 ± 5 kPa). This is several orders of magnitude lower than that reported for PEDOT:PSS hydrogel (2–20 MPa) and within the range of human cardiac muscle tissue (10–200 kPa). This modulus is higher than that of the injectable bioabsorbable electrode for brain tissue previously reported by the inventors (which had a stiffness modulus of 0.57 ± 0.1 kPa compared to 0.5–1 kPa for brain tissue). Such a higher modulus ensures the additional viscosity required for cardiac tissue, which is more dynamic than brain tissue, and such materials are better suited to each respective tissue.

[0049] Furthermore, implementing this disclosure does not involve the significant costs associated with general surgery, nor does it involve the risks associated with general anesthesia and infection. The present invention can be implemented by a pain specialist familiar with the placement of pharmacological nerve blocks, with or without the use of ultrasound or angiography as a means of visualization.

[0050] The present invention also has another distinct advantage over the prior art in that it has superior quality as an electrical system for body tissue. Wire or needle tips, or flat or smooth metal contacts, have a small surface area for capacitively injecting current. In one embodiment of the present disclosure, a conductive hydrogel can be applied to a large area, for example, after brain tumor surgery. Charge injection at implanted electrodes can consist of both capacitive and resistive current transfer. In body tissue, in some situations, the best method for injecting current is via capacitive charge injection, which does not result in irreversible chemical reactions. However, in other situations, the injection and removal of electrons in redox reactions may be preferable. In some situations, a combination of capacitive charge injection and redox reactions is preferred.

[0051] The present invention includes various material-specific physical parameters. Such parameters include, but are not limited to, curing within a body, curing at various conductivity levels from a flexible state to a hard and / or rigid state after curing, and curing in a way that allows for mechanical connection with a nearby location in the body adjacent to the target organ, thereby further reducing stress and / or strain on both the organ and the cured electrode after placement.

[0052] In contrast to prior art electrodes in which microscopic surface structures and macroscopic shapes are formed ex vivo, the electrodes disclosed herein are conferred both microscopic surface structures and macroscopic shapes in vivo by conforming to a target, similar to how a cast molds itself around an arm or leg. This is achieved by one or more processes for manufacturing the electrode in vivo, either inside or on the outside of a living body. The electrode may be formed entirely or partially inside the body, or externally without contact.

[0053] Once deployed (hardened), electrodes can be strategically positioned to engage with blood vessels. This allows the electrodes to stimulate or inhibit signaling pathways within the vessel wall. This innovative approach enables the direct injection of a liquid compound surrounding a vessel to manage blood flow. This could potentially constrict or relax the vessel to regulate blood supply to organs, tissues, or skin. This regulation could promote circulation or limit heat loss as needed. In specific applications, this technology could target blood vessels supplying tumors and aim to restrict or stop blood flow to such vascular regions. Such methods could effectively halt the increase of essential nutrients and oxygen, slowing or, in some cases, reversing undesirable cell proliferation. Such processes involve delivering the mixture either inside or around the vessel wall using a catheter and establishing electrical contact from outside the vessel via a connected wire. Alternatively, the electrode compound can be administered from a distance, gradually approaching the vessel and forming a ring around it. This ring, created by perforating the vessel wall from the inside out, can completely or partially surround the vessel. Next, the wire component of the separately inserted electrode establishes the necessary electrical connection to the precise target area inside the body or just beneath the skin, improving the effectiveness and specificity of the treatment.

[0054] Once deployed (hardened), electrodes can be strategically positioned to engage with blood vessels. This allows for the stimulation or inhibition of signaling pathways within neurons in the CNS or PNS, when the distance to the capillary system neurons is less than 100 μm.

[0055] In some embodiments, this disclosure allows for the placement of liquid mixtures within, near, or around specific structures of an organ, such as internal blood vessels or neurons, or within, near, or around the inner or outer wall of an organ, thereby enabling electrical stimulation or blockage of signal transmission in the organ, enabling innervation or blood supply to an organ, such as the bladder. Organ activity can be altered by increasing or decreasing nerve transmission into or out of the organ. The growth and activity of some organs can be controlled upward or downward by increasing or decreasing blood flow into the organ, such as in the case of the intestines, liver, lungs, or kidneys (which are the body's exchange systems that utilize a fine network of blood vessels intertwined with other vessels that add or extract chemicals in the form of dissolved gases or liquids). The present invention enables an efficient method of contacting an organ, for example, by injecting a liquid mixture into the outer wall of the organ near a point of innervation.

[0056] Beyond medical treatments, this disclosure also encompasses energy storage applications. In such applications, the inherent properties of the compositions of the first embodiment can be utilized in bioelectronic devices. This includes creating more efficient and biocompatible batteries or capacitors for medical implants and other bioelectronic devices. This demonstrates that the compounds have broad applications in both medical and scientific and technological fields. Accordingly, compositions of the first embodiment can be used in energy storage applications such as bioelectronic devices or other biocompatible devices. Bioelectronic devices can be, for example, biocompatible batteries or capacitors for medical implants.

[0057] definition When used herein, "C 1~6 The term "alkyl" refers to both straight-chain saturated hydrocarbon groups and branched-chain saturated hydrocarbon groups having 1 to 6 carbon atoms, and "C 1~20 The term "alkyl" refers to a straight-chain saturated hydrocarbon group or a branched-chain saturated hydrocarbon group having 1 to 20 carbon atoms. 1~6 "alkoxy" is OC 1~6 It means an alkyl group, and here "C 1~6 The term "alkyl" is used as described above. 3~6 The term "cycloalkyl" refers to a cyclic saturated hydrocarbon group having 3 to 6 carbon atoms. The term "halogen" refers to fluorine, chlorine, bromine, or iodine. 2~6 The term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon containing 2 to 6 carbon atoms. An "alkenyl" group contains at least one double bond in its chain. The double bond of the alkenyl group may be unconjugated or conjugated with another unsaturated group. 2~6 The term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon containing 2 to 6 carbon atoms. The "alkynyl" group contains at least one triple bond in its chain.

[0058] Unless otherwise specifically defined, the term "aryl" refers to a cyclic aromatic hydrocarbon group having one to three aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. If it contains two aromatic rings (e.g., bicyclic), The aromatic rings of the aryl groups may be bonded at a single point (e.g., biphenyl) or they may be fused together (e.g., naphthyl).

[0059] As used herein, the term “heteroaryl” means a monocyclic aromatic group of carbon atoms in which one to three carbon atoms are substituted with one or more heteroatoms such as nitrogen, oxygen, and / or sulfur. Examples of monocyclic heteroaryl groups include, but are not limited to, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrizolyl, and pyrimidinyl.

[0060] As used herein, the term “heterocyclyl” means a monocyclic group of carbon atoms in which one to three carbon atoms are substituted with one or more heteroatoms such as nitrogen, oxygen, and / or sulfur. Examples of heterocyclyl groups include, but are not limited to, tetrahydrofuryl, tetrahydropyranyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, and dioxanyl.

[0061] In this specification, the term “curing” refers to a chemical process that includes, but is not limited to, polymerization, crosslinking, precipitation, and / or self-assembly, gelation, or other phase transitions to become a conductive material. Such conductive materials retain their shape when subjected to expected shear forces in living organisms under non-harsh conditions. Curing can be substantially instantaneous, may occur over seconds or minutes, or may occur over a longer period of time.

[0062] "Injection" means introducing into body tissue by any of the following: (a) a dispenser with a needle or needle-like structure that does not require an incision other than a needle incision; (b) a catheter placed inside a blood vessel or inside another body structure having a lumen; (c) a pump via a laparoscopic device inserted through a small incision; (d) a hole formed by another incision; or (e) an auger system that delivers injectable material into a lumen (from the lumen, the material is delivered near, in, or around an interface target). In other words, injection means injecting an electrode solution by contacting the surface of the human body or organ using a needleless jet injector to form a conductive structure within the body or organ.

[0063] Approaches using needles to deploy a liquid mixture that provides a hardening electrode, approaches using needleless jet injectors, approaches using intravascular catheters, and laparoscopic approaches enable connections to intercostal nerves and autonomic nervous system ganglia, as well as new surgical approaches to connect to organs from the dorsal side.

[0064] "Patient" or "subject" means a mammal (e.g., human, mouse, rat, guinea pig, dog, cat, horse, cattle, pig, or non-human primate (e.g., monkey, chimpanzee, baboon, or rhinoceros)).

[0065] When used in relation to a compound, "effective amount" means an amount that is effective in treating or preventing a disease in a subject as described herein.

[0066] Where used in this disclosure, the term "carrier" refers to a carrier, an additive, and Diluents are included, and liquid or solid fillers, diluents, additives, solvents, or encapsulating materials, This means a material, composition, or medium involved in transporting or delivering a drug from one organ or part of the body to another organ or part of the body.

[0067] With respect to the subject, the term "treatment" refers to improving at least one symptom of the disorder in the subject. Treatment includes curing, improving, or at least partially relieving the disorder.

[0068] As used in this disclosure, the term “disability” is used interchangeably with the terms “disease,” “illness,” or “disease” unless otherwise indicated.

[0069] When used in this disclosure, the terms “administer,” “dosing,” or “administer” refer to direct administration to a pharmaceutically acceptable salt of the disclosed compound or composition, or to administration to a prodrug derivative or analogue of the compound or a pharmaceutically acceptable salt of the compound or composition that can form an equivalent amount of the active compound in the body of the subject.

[0070] Depending on the substituents present in the compounds of formulas (I), (I-1), (Ia), (Ib), (Ic), (Id), and (Ie), formulas (II) and (IIa), and formulas (III), (III-a), (III-b), (III-c), and (III-d), the compounds may form salts within the scope of this disclosure. Salts of compounds suitable for pharmaceutical use are those in which the counterions are pharmaceutically acceptable.

[0071] Preferred salts according to this disclosure include those formed by organic or inorganic acids or bases. In particular, preferred salts formed by acids according to this disclosure include salts formed by mineral acids, salts formed by strong organic carboxylic acids (e.g., unsubstituted or, for example, halogen-substituted 1-4 carbon atom alkane carboxylic acids, saturated or unsaturated dicarboxylic acids, hydroxycarboxylic acids, amino acids), or salts formed by organic sulfonic acids (e.g., unsubstituted or, for example, halogen-substituted (C1-C4) alkylsulfonic acids or arylsulfonic acids). Pharmaceutically acceptable acid addition salts include those formed from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, isethionic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid, and glutamic acid, lysine, and arginine.

[0072] Examples of pharmaceutically acceptable base salts include ammonium salts, alkali metal salts (e.g., potassium and sodium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and salts with organic bases (e.g., dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono, di, or tri lower alkylamines (e.g., ethylamine, tert-butylamine, diethylamine, diisopropylamine, triethylamine, tributylamine, or dimethylpropylamine), or mono, di, or trihydroxy lower alkylamines (e.g., monotriethanolamine, diethanolamine, or triethanolamine)). Corresponding internal salts can be further formed.

[0073] The compounds of the present disclosure can be used for prevention and / or treatment, either as such or in the form of pharmaceutical compositions. The active ingredient can be administered alone, but it can also be present in a pharmaceutical composition. Accordingly, the present disclosure provides a pharmaceutical composition, which comprises a polymer of formula (I), one or more compounds of formula (II) or formula (III), and a pharmaceutically acceptable diluent, additive, and / or carrier. The pharmaceutical composition of the present disclosure can take the forms of the pharmaceutical compositions described below.

[0074] The polymer can be one of different types of polymers selected from the group consisting of homopolymers, alternating copolymers, random copolymers, and block copolymers. In the polymer of formula (I), the number of repeating units is represented by n and m, where n is 4 to 32, preferably 5 to 12, and m is 0 to 10, preferably 0 to 3. When m is 1 as in the compound of formula (I-a), p is 1 to 12, preferably 5 to 12, a is 1 to 5, E is preferably H, and R1 is H or C 1~6 alkyl such as methyl. Further examples are the compounds of formula (I-b-1) and formula (I-c-1). The compound of formula (I-b-1) where A is sodium is represented as compound (I-1) and is referred to herein as PEDOT-S derivative A5 or PEDOT-S(A5). The compound of formula (I-c-1) is referred to herein as Ok-PEDOT-S. Any of the aforementioned examples of the compound with m = 1 can be converted into an equivalent compound selected from -(CH2CH2O) q CH2CH2OH (q = 0 to 15), C 1~6 alkyl substituted with -N3, C 1~6 alkyl substituted with -SO3A, and C + (C 1~6 alkyl)3-substituted C 1~6 alkyl. These compounds are referred to herein as PEDOT-S-E.

[0075] Furthermore, the polymer of formula (I) may be a homopolymer. In this case, m is 0, and R1 is H and -N+ (C 1~6 alkyl)3-substituted C 1~6 alkyl is selected from. Examples of the polymer of formula (I) where m = 0 include compound (I-d) where R1 is hydrogen, and also compound (I-e) where R1 is -N + Me2(R 11 )-substituted C 1~6 alkyl, where R 11 [[ID=1,3]]is selected from H and C 1~6 alkyl, r is 4 - 30, a is 1 - 4, and b is 1 - 3. Compound (I-e) is referred to herein as PEDOT-SA.

[0076] Examples of the polymer of formula (I) where m = 1 include the following.

Chemical formula

[0077] Examples of the polymer of formula (I) where m = 1 include the following.

Chemical formula

[0078] <G The polymer of formula (I) can be a self-doped type water-soluble mixed ion-electron conductor of poly(3,4-ethylenedioxythiophene) butoxy-1-sulfonate (PEDOT-S) derivatives. Among the PEDOT-S derivatives, A5 etc. are unique. Such polymers self-assemble in an agarose gel prepared with a physiological buffer solution to form a highly conductive hydrogel (1 - 5 S / cm -1This is to generate ). Furthermore, PEDOT-S is stable for several months. On average, PEDOT-S derivatives are low molecular weight polymers, i.e., oligomers of 7-8 monomers. Therefore, a PEDOT-S derivative, for example A5, is smaller than an antisense oligonucleotide drug containing about 20 nucleotides. The polymer of formula (I) is expected to have better bioavailability than PEDOT:PSS. In PEDOT:PSS, the PSS portion is M n It is a large polymer of 200-300 monomers with a molecular weight of approximately 70,000 g / mol. The polymer of formula (I) may be in the form of nanoparticles highly dispersed in water. Furthermore, the polymer of formula (I) may be a self-doped p-type conductive copolymer.

[0079] One or more compounds of formula (II) or formula (III) can more specifically be the compounds of formulas (II-a) and (III-a)-(III)-d above. Such compounds can also be called trimers of ETE-R derivatives, trimers of EEE-R derivatives, or trimers of TET-R derivatives, where "E" represents the monomer structure of 3,4-ethylenedioxythiophene (EDOT) and "T" represents the monomer structure of thiophene. In such trimer structures, the term "R" means that the trimer may be substituted with one or more substituents. "R" does not specify the substituents, but indicates the type of substitution. The trimers, e.g., ETE-R, EEE-R, or TET-R, are designed to have lower oxidation potentials than the EDOT monomer (0.3-0.5V and 1.2V, respectively). This is important to minimize damage to the tissue during electropolymerization. In addition to structural modifications (i.e., improvements in the morphology and functionality of the compound, as well as in vivo stability), conductivity two to three orders of magnitude higher than that of tissue was measured. For example, ETE-R can result in optimal functionalization, facilitating access to neurons that would not have been reached with the initially formed electrodes. Some examples of ETE-R include the aforementioned sodium 4-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thiophene-3-yl)ethoxy)butane-1-sulfonate (compound II-1, referred to herein as ETE-S) and compound II-2 (referred to herein as ETE-PC). Other examples of EEE-R and TET-R derivatives, as well as ETE-R, are shown in the following sections.

[0080] ETE-R derivatives may be mixed with a solution of polymer (I) (e.g., A5), such as an aqueous solution. Other solvents, such as organic solvents like acetone, acetonitrile, butanone, dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, glycerol, polyethylene glycol (PEG-400), and propylene glycol, can also be used. The concentration of the polymer in the solution can be in the range of 1 to 100 g / ml. By injecting this solution into the brain and electrochemically functionalizing it, a mixed ion-conducting hydrogel can be formed. The mixed ion-conducting hydrogel has different properties depending in part on the R substituents on one or more ETEs. The term "hydrogel" refers to a two-phase material comprising a mixture of a porous and permeable solid (e.g., an insoluble three-dimensional network of natural or synthetic polymers) and at least 10% by weight or 10% by volume of interstitial fluid composed entirely or mainly of water. Thus, a hydrogel can be described as a polymer network structure capable of absorbing large amounts of water.

[0081] The term "mixed ion-electron conductive hydrogel" means that the electrode in question conducts both ions and electrons.

[0082] The conductive hydrogel is transient, and the initial inflammation in the brain caused by the injection disappears, leaving no residual tissue damage from the electrodes. Therefore, ETE-R provides optimal functionalization and facilitates the reach of neurons that would not have been accessible with conventional electrodes.

[0083] The terms “transient bioelectronics” or “transient organic bioelectronics” mean a device or composition that is bioabsorbable, i.e., disappears from the environment after a predetermined time and leaves only minimal and harmless traces after disappearance. Depending on the structure of the composition, the bioabsorption process, i.e., the in vivo degradation of the composition, may be within the range of 1 to 5 days, 1 to 14 days, 1 to 60 days, 1 to 120 days, 1 to 240 days, or at most 1 year. The in vivo polymerization method of the composition according to the first aspect of this disclosure may be electropolymerization, photopolymerization (i.e., polymerization induced by light such as visible light, preferably blue (450 to 495 nm) and / or green (495 to 570 nm) visible light, ultraviolet light, and / or infrared light), or enzymatic polymerization (i.e., polymerization by endogenous catalase and peroxidase enzymes, e.g., horseradish peroxidase (HRP), myeloperoxidase (MPO), and lactoperoxidase (LPO)).

[0084] Methods for synthesizing compounds and compositions The compounds and compositions of this disclosure can be prepared by a variety of methods, including standard chemical reactions. A preferred synthetic route is shown in the following scheme.

[0085] The polymer of formula (I) and the compounds of formulas (II) to (II) can be prepared by methods known in the art of organic synthesis, as partially shown by the following synthesis scheme. In the scheme described below, as is well understood, protecting groups for sensitive or reactive groups are used as necessary in accordance with general principles or general chemistry.

[0086] The compounds described herein can be prepared from commercially available starting materials or synthesized using known organic, inorganic, and / or enzymatic processes.

[0087] Another aspect of this disclosure provides a method for preparing polymers of formula (I) and compounds of formulas (II) to (II) or pharmaceutically acceptable salts thereof, where all substituents are as defined herein unless otherwise specified. The method is: (i) The preparation of a polymer of formula (I-1), [ka] In the formula JPEG2026513146000012.jpg63159, R1 is as defined above, and E is C 1~6 Alkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~6 Cycloalkyl, aryl, heteroaryl, -(CH2CH2O) q CH2CH2OH, where C 1~6 Alkyl groups include -N3, -OH, -SO3A, and -N(C 1~6 Alkyl)2,-NH + (C 1~6 Alkyl)2, and -N + (C 1~6 It may be substituted with one or more alkyl)3 groups, LG is a leaving group such as a halide, tosylate, or mesylate, and x is 2 or 3.

[0088] Non-limiting examples of polymers of formula (I) prepared using one or more of the steps described above include PEDOT-S(A5) (compound I-1), Ok-PEDOT-S (compound Ic-1), compound Id, PEDOT-SA (compound Ie), and PEDOT-BuSA (compound I-2). Polymers of formula (Ib) or (Ic) were synthesized according to Mousa, A. Het al. (Method Matters: Exploring Alkoxysulfonate-Functionalized Poly(3,4-ethylenedioxythiophene) and Its Unintentional Self-Aggregating Copolymer toward Injectable Bioelectronics. Chemistry of Materials 34, 2752-2763 (2022)). In some cases, the polymer of formula (I) which is further substituted can be synthesized by reacting the polymer of formula (Ib) with an alkanesultone (for example, 1,3-propanesultone or 1,4-butanesultone which may be substituted with R1 or the above E-LG).

[0089] As an example, polymers I-2 and Id were synthesized according to the following reaction scheme. [ka]

[0090] (ii) Preparation of compounds of formulas (II) to (III): [ka] Structures 200, 201, 300, and 301 can be synthesized according to commercially available or known literature procedures (e.g., monobromination or dibromination of their respective thiophene precursors) and may be further functionalized with boronic acids or boronic acid esters such as bis(pinacolato)diborone. Compounds of formula (II) or (III) can be synthesized from structures 200, 201, 300, and 301 according to the following scheme using Suzuki coupling or any other alternative method known to those skilled in the art.

[0091] Non-limiting examples of ETE-R derivative compounds prepared using one or more of the steps shown include: [ka] ETE-R derivatives: ETE-S (compound II-1), ETE-PC (compound II-2), ETE-Bu sultone (compound II-3), ETE-BuSA (compound II-4), [ka] JPEG2026513146000017.jpg234159JPEG2026513146000018.jpg141159JPEG2026513146000019.jpg120159JPEG2026513146000020.jpg193159

[0092] In one embodiment, the ETE derivative can be selected from one or more of II-1, II-2, II-14, II-22, II-23, II-24, II-25, II-28, II-30, II-37, II-39, II-40, II-43, II-44, and II-45.

[0093] As an example, compound II-4 was synthesized according to the following reaction scheme. [ka]

[0094] Non-limiting examples of EEE-R derivative or EPE-R derivative compounds prepared using one or more of the steps shown include: [ka] EEE-R derivative: [ka]

[0095] In one embodiment, the EEE derivative can be selected from one or more of III-2, III-5, and III-6. EPE-R derivative: [ka]

[0096] Non-limiting examples of TET-R derivative compounds prepared using one or more of the steps shown include: [ka] TET-R derivative: [ka] JPEG2026513146000027.jpg182159JPEG2026513146000028.jpg140159

[0097] As an example, compound III-32 was synthesized according to the following reaction scheme. [ka]

[0098] The polymer of formula (I) self-assembles to form conductive electrodes within the tissue. Low-concentration agarose gels prepared with physiological buffer (Ringer's solution) mimic the divalent ions of brain tissue and exhibit significantly higher conductivity than the surrounding environment. This allows polymers to self-assemble into long-term stable hydrogels within the Ringer's solution-agarose gel. Therefore, polymers (e.g., A5) can be used as injectable in vivo electrodes.

[0099] The zebrafish caudal fin is a model system for limb regeneration and neurological disorders, and is therefore highly dynamic. It is transparent, allowing direct light access to the injected polymer. A5 (20 mg mL) -1 A5 was injected between the fin rays of the caudal fin of a zebrafish. To facilitate the assembly of the flexible electrode within the fin, A5 was dissolved in 25% Ringer's solution. When injecting into the brain of a zebrafish, it is not essential to use a prepared composition with a higher ionic strength compared to Milli-Q water. However, this demonstrates the adaptability of A5, allowing the nanoparticle formulation of the composition of the present invention to be adapted to specific tissues and to be injected into areas of lower and higher ionic strength. Immediately after injection, a dark blue coherent structure was formed, which could be visually observed between the fin rays.

[0100] Electrical properties of A5 in peripheral tissue: Initial measurements revealed that the resistance of the A5 gel was significantly lower than that of the reference sample, indicating higher conductivity. Specifically, the measured resistance was 0.16 MΩ for A5, compared to 0.32 MΩ for the control. Upon drying, the resistance of A5 further decreased to 0.02 MΩ, increasing conductivity, while the resistance of the reference increased to 1 MΩ. These findings indicate that the superior conductivity of A5 is due to the more closely aligned oligomers upon drying.

[0101] Biodegradation and Biocompatibility: In a zebrafish model, A5 exhibited transient and bioabsorbable properties. When applied to the tail fin, the conductive structure of A5 partially degraded after one week and completely after four weeks, demonstrating excellent biocompatibility without affecting the behavior of zebrafish or causing fin damage, leaving healthy tissue after degradation.

[0102] Application and injection method to brain tissue: The A5 nanoparticles designed for application to brain tissue demonstrated unique adaptability. To minimize tissue damage and effectively avoid vascular rupture, a column injection method using a 30 μm diameter cannula was employed. No adverse effects were observed for up to 9 days after injection, demonstrating the safety of this method and the suitability of A5 for fragile brain tissue.

[0103] Inflammatory response and healing: Observation after the initial injection revealed an inflammatory response, but this resolved within 9 days, indicating that the inflammation was caused by the injection process rather than A5 itself. This is important in demonstrating the biocompatibility of the polymer and its safety for human application.

[0104] Conductive properties in brain tissue: Despite technical challenges such as the possibility of variable electrode contact resistance and complex biological interactions, A5 maintained its conductive properties within brain tissue. Visible under a bright-field microscope, A5 formed a clear conductive pattern, and a linear current-voltage relationship was observed at microscale distances. This performance clearly demonstrates the potential of A5 for bioelectronics applications even in the challenging environment of brain tissue.

[0105] Conductivity Comparison and Technical Challenges: The in vitro conductivity of A5 is 30 Scm. -1 Although it exceeded the stated value, in vivo measurements in the brain revealed that conductivity was lower due to biological isolation and other factors. Nevertheless, even under these conditions, A5 exhibited favorable resistance values ​​and a clear current-voltage dependence, demonstrating its applicability in bioelectronics.

[0106] Electrochemical functionalization of polymer of formula (I) By using column injection, a more fundamental control over the position and pattern of flexible electrodes within the tissue is possible than with assemblies controlled by the genetic expression of enzymes. To add modularity to the polymer of formula (I) (e.g., the A5 electrode-tissue interface), an auxiliary method was developed that allows for flexibility, functionality, and further protrusion for seamless extension into the tissue of the electrode surface area. The aforementioned ETE-R derivative (e.g., ETE-S) was injected co-injected with A5, for example, to position the flexible electrode at the target site. Due to the concentration gradient and electrostatic repulsion between the negatively charged ETE-S and the negatively charged A5, the former diffused from the formed A5 electrode. Later, by electropolymerization using A5 as the electrode, ETE-S adhered to the surface of A5 and entered the A5 framework.

[0107] In addition to increasing surface area and tissue reachability, different substituents I on the ETE-R derivative, such as ETE-S and zwitterionic ETE-phosphatidylcholine (ETE-PC), will alter the properties of the final polymer electrode, as illustrated herein. Furthermore, mixtures of different trimers, such as ETE, EEE, or TET, having different R groups, may be used to tune the desired properties of the composition. High solubility is required for the implantable electrode, so requirements are placed on the nanoparticles forming the flexible electrode. Specifically, the nanoparticles are desirable to be highly water-soluble, and furthermore, to be able to self-assemble when injected into tissue and subsequently bioabsorbable without damaging the tissue. Thus, instead of redesigning the oligomers that form the polymer of formula (I) (e.g., A5), this modular approach leverages unique polymer properties and customizes the electrode-tissue interface by adding soluble trimers in situ.

[0108] As an example, polymers of formula (I), such as PEDOT-S polymer A5(I-1), form the electrode skeleton. A5 can be mixed with trimers such as ETE-PC-R (e.g., II-24, II-25, II-26, or II-45) to produce an injectable precursor electrode solution. Once injected and electrochemically functionalized, the properties of the electrode surface are primarily determined by the trimer-R. By sequentially loading various polymer blends into a syringe, distinct attributes can be obtained along the electrode length. For example, a combination of A5 and ETE-PC may result in an electrode with surface insulation. This is a mechanism rooted in the ion repulsion membrane filtration method, where the effect of changes in the core's electronic energy on ions is minimized. Therefore, when an electrode is coated with ETE-PC, the activation (bioelectricity) of cellular ions is significantly reduced. In contrast, when A5 is mixed with trimer-R, where R is a redox mediator such as TEMPO, ferrocene, acetosyringic acid, or catechol, the resulting electrode segment contains the redox mediator. This configuration can be customized to influence specific redox reactions within and around the tumor (by selecting a redox mediator that matches the targeted redox process).

[0109] The concept of the auxiliary A5 module was evaluated in an in vitro agarose gel (0.5%) prepared with Ringer's solution to mimic brain tissue. A5 (20 mg mL) -1 ) without forming a precipitate, ETE-S or ETE-PC (40 mg mL -1The polymer (i.e., A5) was dissolved in agarose. When the dark-colored solution was injected into agarose using a Hamilton syringe, the polymer immediately formed aggregates. The diffusion of the ETE-R derivative from the injection site of polymer A5 was monitored using UV light (365 nm). After 2 hours, ETE-R had diffused from A5 over a distance of approximately twice the thickness of the A5 hydrogel electrode. Next, A5 was used as the connecting electrode, and ETE-R was electropolymerized with an applied bias of 1.5 V. This allowed for electropolymerization even in the event of potential contact resistance. The applied bias can be further optimized, but it was chosen to have a high tolerance for variations in contact resistance during electropolymerization for further use.

[0110] The thickness of the formed A5 electrode increased, confirming the successful polymerization of ETE-S. Further image analysis revealed a dendritic structure growing from the A5 core. The electropolymerized region also exhibited higher conductivity. During electropolymerization, if one electrode is in contact with A5 (while the other is in agarose), a limited constant current is ensured due to the relatively high resistance in the agarose. Alternatively, if both electrodes are in contact with A5 during electropolymerization, the decrease in resistance can be directly reflected in the increase in current. The current increased by an order of magnitude during polymerization over a 12-minute time course. No changes in shape were observed other than the dendritic formation of the A5 core. The decrease in resistance was thought to be due to the polymerization of ETE-S interposed between the A5 nanoparticles. Electropolymerization was optically observed by the darkening of A5 when voltage was applied, but no corresponding increase in current was observed for the first 5 minutes. The initial incubation time may be due to a bottleneck caused by one or more resistances limiting the current. Once such a bottleneck was eliminated by electropolymerization, a gradual increase in current was observed as the electropolymerization continued uniformly along A5. By maintaining contact between both ends of A5, periodic voltammetry measurements became possible, and it was revealed that the current increased 100-fold due to electrolytic polymerization.

[0111] A-ETE-R, electropolymerized in agarose, was used as a base for evaluating its mechanical properties. Brain tissue is very soft, with a stiffness modulus of approximately 0.5–1 kPa, making it difficult to adapt to conventional inorganic electrodes. Electropolymerized A5-ETE-PC had a static stiffness modulus of 0.57 ± 0.1 kPa, which closely mimicked brain tissue. Biocompatibility of A5-ETE-S was evaluated by electropolymerizing it in an agarose gel incorporating living cells. Lung adenocarcinoma cells (A549 cell line) were molded and placed in the agarose gel with or without contrast-enhancing DiI (lipophilic staining) cell labeling. After injection of the A5-ETE-S solution, one end of the polymer electrode was brought into contact with the agarose, while the grounded counter electrode was maintained in the agarose (outside A5). During electropolymerization, the newly formed dendritic structure of ETE-S (sodium salt of II-1) extended from A5(I-1), achieving cell contact and, in some cases, embedding cells, resulting in close contact with the cells, without any adverse effects such as loss of cell integrity being observed. It was revealed that close connection between the electrode and cells is necessary for efficient and accurate recording of low-voltage electrical stimulation. Furthermore, the toxicity of A5 and ETE-R was evaluated using limiting dilution assays, with a maximum dose of 1 mg / mL. -1 After 1 day of exposure to A5 or ETE-PC, neither A5 nor ETE-PC showed cytotoxicity. On the other hand, ETE-S showed some toxicity at high concentrations. This toxicity test was widespread, reaching up to more than 1000 times the amount of ETE-R injected during the in vivo experiment (approximately 200 μg vs. 400 ng). High dilution was performed before injection into tissue. No cytotoxicity was observed from either compound at the amounts used in the in vivo setting. Therefore, as a conclusion from in vitro experiments based on the concept of the present invention, A5-mediated electropolymerization enables flexible surface modification, close contact with cells, and a significant reduction in electrical resistance.

[0112] In vivo electropolymerization When the above approach is moved to an in vivo setting, the following strict constraints are imposed on the experimental setup: 1) use of small-diameter injection capillaries to avoid vascular rupture, 2) A5, ETE-R, and A5-ETE-R must all be highly soluble and biocompatible, 3) the applied voltage and current used for electropolymerization must be kept low to avoid damaging brain tissue, and 4) the procedure must be performed rapidly to avoid anesthetic damage. A5-ETE-S solution was injected into the brains of anesthetized zebrafish using 30 μm diameter capillaries pre-coated with 50 nm iridium. After injection, ETE-S was allowed to diffuse into the tissue for 1 minute. Next, the coated capillary was used as a bias electrode to establish seamless contact with the injected A5. By placing the counter electrode in the skin of the zebrafish in the nostril, electropolymerization at low currents (e.g., 1-3 μA), mimicking the agarose setting, was possible in sedated fish. This procedure, injection, and electropolymerization were continued for approximately 10 minutes, and after a further 5-10 minutes, the fish became alert and exhibited normal behavior. Typically, no rapid movements or abnormal swimming patterns were observed, nor were there any buoyancy and balance problems indicating brain damage due to discomfort (e.g., pain) or stress-related behaviors. The absence of adverse events indicates that the minimally invasive approach was sound and well tolerated by the fish.

[0113] Histological staining of sagittal brain sections (30 μm thick) containing A5-ETE-S revealed close contact between cells and polymer at the polymer-cell interface. Imaging showed that A5-ETE-S injection extended deep into the brain between the cerebellar body (C) and tectum (OT), and that radially extending ETE-S dendritic structures from A5 reached the granular and molecular layers of C and the surface of OT. Similar to the in vitro cell-agarose model, A5-ETE-S enveloped neurons, with some neurons completely surrounded by conductive polymer electrodes, and no cell damage was observed. This demonstrates the effectiveness of gel-like microstructure electrodes in enabling the exchange of metabolites and ions via electrodes, thereby maintaining cellular homeostasis.

[0114] Inflammatory response to A5 injection: Initial observations after A5 injection revealed an inflammatory response due to the mechanical injection, but this resolved within a few days. This reaction was due to tissue oxidation caused by the injection process.

[0115] Introduction of the auxiliary module (ETE-R): The study investigated the effect of incorporating ETE-R (electropolymer trimer) into A5 on tissue integrity. This did not exacerbate tissue damage or inflammation.

[0116] Comparison of inflammatory responses with ETE-S and ETE-PC: Both A5-ETE-S (sulfonate functional) and A5-ETE-PC (phosphatidylcholine) were tested. The initial significant inflammatory response observed near the injection site began to decrease significantly within 7-9 days with ETE-S, and completely disappeared within 3 days with ETE-PC, demonstrating a rapid healing process.

[0117] Effects of electrolytic polymerization voltage: The electrolytic polymerization / electrochemical functionalization process, essential for the formation of flexible conductive dendritic electrodes, was carefully evaluated to ensure that it does not induce further oxidative stress or damage to the tissue. The results confirmed that the voltage applied for electrolytic polymerization is safe and does not cause further tissue oxidation.

[0118] Minimally Invasive Method and Conductive Dendritic Electrodes: This study demonstrates that the method is minimally invasive and suitable for introducing flexible conductive dendritic electrodes into brain tissue. This approach, combining A5 with an electrolytically polymerized trimer, did not cause further tissue damage, did not disrupt normal fish behavior, and demonstrated potential for neurological applications.

[0119] Electrical properties of A5-ETE-R in the brain of zebrafish The conductivity of A5 was improved by functionalization with ETE-R. In fish subjected to electropolymerization, for example, both ETE-S and ETE-PC showed currents more than 10 times higher (resistance of 5-10 MΩ) under the same applied bias. Furthermore, it was possible to map conductivity over long distances of electrodes, enabling estimation of conductivity values. Corresponding to the increase in the diameter of the conductive polymer during electropolymerization, the conductivity increased to approximately 3 Scm in both A5-ETE-S and A5-ETE-PC. -1 It was estimated that this was the case for most organizations (<10 -2 Scm -1 The conductivity was 2 to 3 orders of magnitude higher than that of the other polymer. In addition, some fish swam around for 7 days with the conductive polymer still in their brains and exhibited normal fish behavior. In A5-ETE-S and A5-ETE-PC, the respective polymers were clearly detected in the brains of 2 out of 3 fish, but the conductivity was lower than in the 1-day experiment. A5-ETE-PC showed a linear voltage-dependent current in the low nA region (resistance of approximately 1 GΩ). Therefore, the presence of a polymer that still exhibits conductivity over long distances was revealed. Interestingly, the high conductivity of A5-ETE-PC over 7 days indicates that modification of this polymer makes it more stable, and that different trimers confer different properties.

[0120] Method Summary: The method of this disclosure comprises injecting a mixture of A5 and ETE-S into the brain of a zebrafish, followed by electropolymerization. This process results in the formation of flexible electrodes within the brain tissue. The flexible electrodes are then analyzed for their ability to stimulate specific brain regions and alter neuronal activity.

[0121] Experimental Procedure: Adult zebrafish of the Casper mutant strain, genetically modified to express the GCaMP6f calcium indicator, were used. This allowed for the visualization of action potentials as an increase in green fluorescence. This provided a non-invasive method for tracking neural activity. The presence of the A5-ETE-S electrode was essential for delivering electrical pulses over long distances within the brain to induce neuronal firing.

[0122] Spatial specificity and toxicity testing: Experiments revealed that the conductive polymer can target specific brain regions without causing acute toxicity to cells. The addition of PTZ (GABAA receptor antagonist) demonstrated the ability to stimulate areas not directly adjacent to the electrode, indicating that this method can enable precise neural stimulation.

[0123] External Contact Method: A novel approach enabled external contact of polymer electrodes within the brain. By leaving a portion of the microcapillary used for injection in place, external contact with the flexible electrode could be established. This allowed for the regulation of brain activity without direct intervention of brain tissue. This technology represents a significant advance for clinical applications and provides a non-invasive method for interacting with bioelectronics in the body.

[0124] Functional and Transient Bioelectronics: The bioelectronics of this disclosure are not only functional but also well-tolerated in the brain, providing a promising means of transient bioelectronic therapy. By forming highly water-dispersible nanoparticles using thiophene oligomers (A5), it is possible to form flexible electrodes that are stable against interactions with endogenous ions, eliminating the need for specific triggers for electrode formation.

[0125] Thus, this disclosure relates to functional and well-tolerated organic bioelectronics in the brain. To meet the demand for transient bioelectronic therapy, implantation was performed using a minimally invasive injection method. The resulting structure was bioabsorbable. Bioabsorbability is a desirable property, for example, in the treatment of cancer by electrotherapy, eliminating the need for corrective surgery. This was made possible by using polymers of formula (I), such as thiophene oligomers (A5), which form nanoparticles. Such nanoparticles are highly dispersed in water, thus allowing them to be present in solution at high concentrations without aggregation. On the other hand, by injecting the nanoparticles into tissue and then allowing them to interact with endogenous ions, stable flexible electrodes are formed. Therefore, no specific trigger is required. For example, as demonstrated by establishing conductive structures in the tail fin and brain of zebrafish, it was also possible to harmonize the nanoparticle solution with the endogenous ion intensity and ions in completely different tissues. Thus, this disclosure can be commonly applied across several tissues and heterogeneous species. Furthermore, since the nanoparticles contain oligomers, which are the size of conventional drugs, the nanoparticles are bioabsorbable.

[0126] The unique properties of polymers of formula (I) (e.g., A5), which provide flexible functionality at the electrode-tissue interface, have enabled the development of auxiliary modular approaches. This modular approach utilizes the above properties and customizes the electrode-tissue interface by adding soluble trimers (i.e., one or more compounds of formula (II) or formula (III) that bond to the polymer in situ (insights)). Electropolymerization of trimers with low oxidation potential at insights increased conductivity, formed close connections to cells, and established functional group modification of the polymer electrode. This was demonstrated, for example, using trimers ETE-S and ETE-PC. In the demonstration, the latter showed higher long-term stability and lower toxicity. It was also found that electropolymerization did not cause further oxidative damage to brain tissue. This modular approach opens up possibilities for one or more compounds of formula (II) or formula (III) with different functional groups, as well as for other trimers with low oxidation potential.

[0127] Despite the small size of the zebrafish brain, the crucial challenge of enabling flexible neural electrode contact for efficient external interaction was overcome. In living brain sections excised from fish implanted with bioelectronics, neuronal signaling was regulated by applying electrical pulses via external contact.

[0128] The methodology and workflow presented here are general and not limited to zebrafish. The procedure may be easier in larger brains (e.g., rodents and primates), particularly in terms of external connectivity.

[0129] In summary, this disclosure relates to bioabsorbable electronics that are assembled in vivo and fully integrated within the nervous system and other tissues, which can be applied to treatments that are not long-term. [Examples]

[0130] Example 1 Evaluation of A5-ETE-S agarose type A5 (20 mg mL) -1 ) and ETE-S (40 mg mL) -1 A H2O solution of () was injected into an agarose mold (0.5% agarose in Ringer's solution). The diffusion of ETE-S was monitored using a UV lamp at 365 nm. After 2 hours, one end of the Au-coated W electrode was connected to the A5 aggregate (the other end was in the agarose mold), and ETE-S was electropolymerized to obtain 100% coverage. ETE-S was electropolymerized using 1.5 V (Keithley 2612B). The agarose was imaged using a bright-field microscope (10x and 40x objective lenses).

[0131] The conductivity of A5 and A5-ETE-S in agarose was measured using a two-terminal method. A 25 μm gold-coated tungsten microprobe (Signatone, Gilroy, CA) was connected to a polymer embedded in agarose. By sweeping the applied potential and recording the resulting current over different distances, conductivity could be estimated using a transmission line model.

[0132] MTT assay To determine the in vitro toxicity of A5, ETE-S, and ETE-PC, MTT cell viability assays were performed. Briefly, HLF-1 (2 × 10⁻¹⁰) 4 Cell wells -1 The cells were seeded in a 96-well flat-bottom microplate and allowed to grow for 24 hours. The cells were treated with A5, ETE-S, or ETE-PC (0-1000 μg ml). -1 The sample was treated with ) for 24 hours. After treatment, 200 μL of MTT (0.5 mg mL) was used. -1The compound (1 mg mL) was added to each well and incubated at 37°C for 4 hours. After incubation, 200 μL of isopropanol was added to dissolve the formazan crystals. As a measure of viability, the optical density of the formazan solution was measured at 570 nm using a microplate reader (Spark Cyto, Tecan). The formazan signal (number of viable cells) was normalized to a control sample not exposed to A5 or ETE-R. The experiment was repeated three times for both the organism and the method. Each well contained up to 200 μg of the inventors' compound (1 mg mL). -1 It contained 200 μL. This is equivalent to 400 ng (40 mg mL). -1 This can be compared to a typical in vivo injectable of 10 nL.

[0133] In vivo caudal fin assay Before microinjection, the fish was treated with tricaine (ethylmethanesulfonate 3-aminobenzoate, 0.2 mg mL). -1 The fish were anesthetized with tricaine. Anesthesia was continued until the gill cover movement ceased and the fish became unresponsive to vibrations caused by tapping near the tricaine container. The anesthetized fish were placed on their sides on a plate filled with 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in solidified E3 medium. The fish were covered with damp tissue paper to prevent drying, but the caudal fin was left exposed. Next, the plate was transferred to a microinjection apparatus and injected into the interray region of the caudal fin using a glass capillary with a 30 μm diameter bevel tip (catalog no. BM100T-15. Bevel, straight, shortened + flame-finished end, obtained from Biomedical-Instruments GMBH) filled with polymer solution. The total injection volume per interray region was estimated to be within the range of 100 nL. After injection, the fish were directly allowed to recover by washing their gills with fresh aquarium water, and then moved to the treated aquarium for observation.

[0134] In vivo brain surgery and microinjection Before surgery and microinjection, treat the fish with Tricaine (ethylmethanesulfonate 3-aminobenzoate, 0.2 mg mL). -1 The fish were anesthetized with ) until opercular movement ceased and the fish no longer reacted when its tail fin was pinched. For the surgery, the anesthetized fish were placed in a mold made of damp tissue paper to stabilize them. Next, a small hole was made in the parietal bone just above the cerebellar body and just to the left of the midline using the tip of a 30G needle. Then, the fish in the tissue paper mold was transferred to a microinjection apparatus and a capillary with a 30 μm diameter bevel tip (catalog number BM100T-15. Bevel, straight, shortened + flame-finished end, obtained from Biomedical-Instruments GMBH) filled with polymer solution (see below) was inserted through the hole in the parietal bone to a depth of 700 μm. Three injections were then performed, one injection at depths of 700 μm, 500 μm, and 300 μm, respectively. The total injection volume was estimated to be 10 nL. After injection, the fish were directly allowed to recover by washing their gills with fresh aquarium water, and then transferred to the post-treatment aquarium for observation, or subjected to electropolymerization.

[0135] In vivo brain electropolymerization. When electropolymerization was performed following polymer injection, the counter electrode was placed in the skin of one nostril, and the iridium-coated polymer-containing cavilary functioned as the electrode. The injection was performed as described above. After injection, the cavilary was left in place in the brain. After 1 minute, the polymer solution was diffused, and then the injected polymer was electropolymerized by applying 1.5V (current of approximately 1-3μA) to the electrode for 5 minutes using a Keithley source meter (Keithley Instruments). One end of a 30μm diameter glass injection capillary (catalog no. BM100T-15, bevel, straight, shortened + flame-finished end, available from Biomedical-Instruments GmbH) was pre-coated with 50nm Ir in a Quorum sputtering apparatus (QT150, Quorum technologies) to obtain a conductive cavilary that maintained light access from the back side to confirm the liquid level before injection.

[0136] If the experiment is conducted outside of water for longer than 10 minutes, use a Peri-Star Pro peristaltic pump (World Precision Instruments) to dispense 0.1 mg mL -1 Aerated aquarium water containing tricaine was supplied to the fish via intubation to perfuse the gill chambers. Initially, the inventors conducted experiments with diffusion and electropolymerization durations longer than 5 minutes, respectively. This improved polymer spreading and polymerization, but also increased adverse effects on the fish.

[0137] Polymer formulations for microinjection The following polymer formulations were used for microinjection into the brain (all dissolved in Millipore water): A5 (20 mg mL) -1 ), A5 (20 mg mL -1 ) + ETE-S (40 mg mL) -1 ), A5(20mgmL-1)+ETE-PC(40mgmL -1 ).

[0138] Tissue treatment after the experiment With or without electropolymerization, after injecting the polymer, the fish were allowed to recover as described above and then transferred to an aquarium to survive for different periods after injection. The fish were killed for histological examination or conductivity measurement after 1 hour, 2 hours, 3 hours, 4 hours, 1 day, 2 days, 3 days, 7 days, 8 days, or 9 days. The fish were euthanized by immersion in ice water for 10 minutes and then decapitated. The brain was directly excised without fixation to freeze on dry ice in TissueTek OCT®. Alternatively, the brain was immediately treated for redox staining (see below). Alternatively, the parietal bone was opened and the head (excluding the jaw) was fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer.

[0139] Frozen sections (with section thicknesses of 20-50 μm depending on subsequent processing) were prepared from fresh, frozen brain tissue using a Cryostar NX70 cryostat. The sections were mounted on Superfrost Gold microscope slides and observed under a microscope. Additionally, sections were mounted on comb-type gold electrodes for conductivity measurement.

[0140] Brains fixed with paraformaldehyde were excised from the skull, rinsed with phosphate-buffered saline (PBS), cryoprotected in PBS containing 25% (w / v) sucrose, and frozen on dry ice in TissueTek OCT. Frozen sections of sagittal sections (with section thicknesses of 30-50 μm depending on subsequent processing) were prepared from the brain. The sections were mounted on Superfrost Gold slides and further processed.

[0141] Electrical measurement Brain sections containing polymer were placed on comb-type Au electrodes connected to a Keithley source meter. Two of the comb electrodes were brought into contact using an external microelectrode. The applied voltage was swept, and the resulting current was recorded. This was repeated for all comb electrode leads relative to the conductive polymer. The distance between adjacent electrodes was 15 μm, and the width was 2.5 mm.

[0142] Injection and preparation of brain sections A mixture of A5 and ETE-S was microinjected into adult zebrafish Casper mutants (Tg(elav3:GCaMP6f)) as described above and electropolymerized in the brain. For this experiment, the capillary was cut directly above the parietal bone, leaving its tip superficially in the brain. One day after injection, the fish were euthanized by immersion in ice-cold aquarium water for 10 minutes and then decapitated. The brains were rapidly dissected and embedded in 3% low-melting-point agarose dissolved in standard Ringer's solution for zebrafish. The blocks were cooled on a metal plate, transferred to NMDG cutting solution on ice, trimmed, and mounted for vibratome sectioning. 300 μm and 400 μm sagittal sections with the capillary tip and polymer electrode in the tissue were cut from each brain, transferred to HEPES recovery solution, and allowed to reach room temperature (approximately 24°C). Next, the vibratome sections were transferred to artificial (zebrafish) cerebrospinal fluid (aCSF), and tested for GFP positivity, followed by electrical stimulation and Ca 2+ I prepared for imaging.

[0143] Electrical stimulation in brain sections A 10 μm tungsten microelectrode (Signatone, Gilroy CA) was brought into direct contact with A5 in the brain section, or into contact with A5 via the injected cavitary. A square voltage stimulation pulse was supplied using a Gass S48 stimulator (Astro Med) with settings of 2 trains per second, a train duration of 200 ms, 20 pulses per second, and a pulse duration of 2 ms. The magnitude of the voltage pulses regulated by the Gass stimulator was in the range of 6–14 V. A moderately high stimulation voltage is required due to significant losses, including contact resistance, stray currents in the buffer surrounding the tissue section, and poor impedance matching. Extensive bubble formation around the electrode, which could occur with high input power, was not observed.

[0144] 3D imaging Samples were imaged in a chamber filled with DBE. Cleared brain sections embedded in agarose were imaged using an Ultra Microscope II (LaVision Biotec) equipped with an sCMOS camera (Andor Neo, Model 5.5-CL3) and a 4x objective lens (LaVision LVMI-Fluor 4x / 0.3). Two laser configurations (488nm and 640nm) were used with the following emission filters: a 525 / 50 filter for endogenous background (vascular) and ETE trimer visualization, and a 680 / 30nm filter (Neurotrace 640 / 660) for neuron visualization. The stack was acquired using ImspectorPro64 (LaVision Biotec) with a 3μm z-step, and the 3D volume was determined. This image stack was stitched together, and the brain sections were visualized in 3D using Arivis Vision 4D 3.5.0 (Arivis AG). The rendered footage was compiled using Final Cut Pro 10.4.3 (Apple Inc.).

[0145] mechanical measurements 0.6% agarose molded in Ringer's buffer, with 3 μl of A5 [20 mg ml] -1 ]+ETE-PC[40mgml -1 The material was injected using a Hamilton syringe. A5-ETE-PC was electropolymerized at 1.75V for 20 minutes. A cross-section was cut and placed in a Biomomentum Mach-1 mechanical tester. The test was performed in indentation mode using a 0.5 mm diameter spherical indenter at a speed of 0.01 mm / s. Indenter depths: 0.15, 0.3, and 0.45 mm. The test profile consisted of the following steps. -Contact (0.1gf) - Wait 10 minutes to recover from contact. - 3 stress relief sessions - Three sine wave tests (0.1, 1, and 4 Hz, respectively) Agarose and A5-ETE-PC could not be distinguished at 0.1 Hz. At 1 Hz, A5-ETE-PC had a lower elastic modulus. Looking at the components of the elastic modulus, agarose showed a higher elastic response, while A5-ETE-PC may have been more viscous.

[0146] Example 2: Biocompatible 3D Flexible Electrode for Electrotherapy of Glioblastoma Enzymatic polymerization of ETE-PC Solutions of ETE-PC (500 μg / ml) were prepared in DPBS (Thermo Fischer, Gibco, catalog no. 14190-250), mixed with hydrogen peroxide (H2O2) at different concentrations (0.001-0.01%), and horseradish peroxidase (HRP) (Merck, Sigma Aldrich, P8375-25KU) (5 U / ml in DPBS) was added. Polymerization was investigated by detecting polymerized and unpolymerized ETE-PC by observing the color change of the ETE-PC solution from pale yellow to black, and by measuring the absorbance at 350 nm and 780 nm. The maximum absorbance of polymerized and unpolymerized ETE-PC was determined by wavelength scanning using UV-Vis spectrophotometric analysis with a Spark Cyto (Tecan) multimode plate reader.

[0147] Wavelength scanning using UV-Vis spectroscopy determined that the maximum absorbances of unpolymerized ETE-PC and polymerized ETE-PC were at 350 nm and 780 nm, respectively. Enzymatic polymerization revealed that HRP successfully polymerizes ETE-PC in the presence of H2O2, as indicated by the color change of the ETE-PC solution. Furthermore, the H2O2 concentration required for HRP-mediated polymerization of ETE-PC was as low as 0.001%. A line graph showing the absorbance of polymerized ETE-PC compared to unpolymerized ETE-PC showed a decrease in absorbance for unpolymerized ETE-PC and an increase for polymerized ETE-PC. Additionally, HRP-mediated polymerization of ETE-PC saturated at 0.002% H2O2. These data suggest that HRP can effectively polymerize ETE-PC in the presence of very low concentrations of H2O2. Tumor cells secrete large amounts of H2O2 compared to normal cells. This data suggests that ETE-PC can be polymerized within the tumor microenvironment using H2O2 secreted by cancer cells via HRP. This indicates the potential for manipulating the tumor microenvironment for electrotherapy of cancer.

[0148] Enzymatic polymerization of ETE-PC in the presence of cancer cells We investigated the enzymatic polymerization of ETE-PC mediated by cancer cells using 2D and 3D glioblastoma models.

[0149] In the 2D model, U87 glioblastoma cells were seeded in 96-well plates at a density of 10,000 cells per well in phenol red-free (PR-free) complete DMEM (Thermo Fisher, Gibco, catalog number 21063045) and placed in a CO2 incubator for 37 days. o Cells were held in 1C for 24 hours. After 24 hours, cells were treated with either HRP (5 U / ml in DPBS) or ETE-PC (100 μg / ml in PR-free DMEM), either alone or in combination. Cells were incubated for 72 hours. Polymerization of ETE-PC was then observed at 24, 48, and 72-hour intervals using a bright-field microscope (10x objective lens) and UV-Vis spectrophotometric analysis (350 nm and 780 nm using a Spark Cyto (Tecan) multimode plate reader).

[0150] Cells treated with an ETE-PC / HRP mixture produced a black precipitate of polymerized ETE-PC at 24 hours, which became increasingly darker at 48 and 72 hours. Cells treated with HRP or ETE-PC alone did not undergo polymerization. UV-Vis spectroscopy results supported these observations, showing that the absorbance of unpolymerized ETE-PC decreased over time, while the absorbance of polymerized ETE-PC increased.

[0151] For 3D models, low-concentration agarose gels (0.5% agarose in PR-free DMEM, low gelation temperature, Sigma Aldrich, catalog number A9414-25G) embedded with U87 cells or spheroids were used to mimic brain tissue. Briefly, 100 μL of U87 cell or spheroid suspension in PR-free DMEM was mixed with 1% agarose (100 μL in PR-free DMEM) to a 0.5% agarose concentration and added to the wells of a 96-well plate. The cells were acclimatized by holding them in a 37°C CO2 incubator for 24 minutes. The cells / spheroids were then treated either with ETE-PC (100 μg / ml in PR-free DMEM) alone or with ETE-PC (100 μg / ml in PR-free DMEM) in the presence of HRP (5 U / ml in PBS) and incubated for 72 hours. After 72 hours, images were acquired using a bright-field microscope (10x objective lens).

[0152] As observed by the black precipitate around the cells / spheroids and throughout the wells, both cells and spheroids underwent polymerization of ETE-PC in the presence of HRP. These results suggest that, after injection, ETE-PC and HRP can diffuse into the tumor tissue and polymerize there with the help of H2O2 present in the tumor microenvironment.

[0153] Electrochemical functionalization of A5 with ETE-PC and preparation of A5 / ETE-PC three-dimensional flexible electrodes in agarose To prepare the A5 / ETE-PC three-dimensional flexible electrode, 200 μl of 0.5% agarose (low gelation temperature, Sigma Aldrich, catalog number A9414-25G) was added to the wells of an 8-well chamber slide. Once the agarose gelled, 20 μl of A5 / ETE-PC solution (20 mg / ml of A5 and 40 mg / ml of ETE-PC in PR-free DMEM) was added to one side of the well, and the ETE-PC was diffused laterally in the agarose for 2 hours. After 2 hours, A5 was electrochemically functionalized with ETE-PC, and the flexible electrode was formed by applying a 2V bias (Keithley Sourcemeter 2612B, Keithley Instruments) to the Au counter electrode for 30 minutes. Electrode formation was observed by bright-field microscopy (10x objective lens).

[0154] After application of the A5 / ETE-PC mixture, A5 rapidly formed a thick gel, while ETE-PC began to diffuse into the agarose. High-magnification images after electrochemical functionalization show a dendritic structure of ETE-PC branching from A5, supporting the formation of an A5 / ETE-PC flexible electrode.

[0155] After electrode formation, impedance was measured by performing electrochemical impedance spectroscopy (EIS) in 10Hz increments over the frequency range of 1Hz to 100kHz using an Autolab PGSTAT204 potentiostat (Metrohm). Impedance measurement was performed against a control agarose gel without flexible electrodes.

[0156] EIS experiments revealed a significant decrease in impedance in agarose-based electrical systems when flexible electrodes were used. This suggests that A5 / ETE-PC flexible electrodes can enhance the conductivity of electrical systems. Furthermore, to conduct in vitro experiments, A5 / ETE-PC electrodes were prepared around U87 cells and spheroids. To do this, U87 cells were seeded into chamber slide wells at a density of 20,000 cells per well and incubated in DMEM medium in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, 0.5% agarose (200 μl) was added to the cells to create a layer, and the A5 / ETE-PC mixture was applied from one side of the well. ETE-PC was diffused for 2 hours and electrochemically functionalized to prepare the electrodes. Cells were stained with calcein-AM for identification. In the case of spheroids, U87 spheroids prepared by the suspension method were mixed with 0.5% agarose and added to the wells of a chamber slide to create a 3D model of spheroid-embedded agarose. The A5 / ETE-PC electrode was prepared as described above. The results showed that a dendritic, flexible electrode branching from A5 surrounding the cells and spheroids was successfully synthesized. No harmful changes were observed in the morphology of the cells and spheroids, suggesting the biocompatibility of the electrode. These results provided a functional 3D cancer model for evaluating the capabilities of the A5 / ETE-PC electrode in cancer electrotherapy.

[0157] Irreversible electroperforation of cancer cells using A5 / ETE-PC electrodes The efficacy of A5 / ETE-PC flexible electrodes in cancer electrotherapy was investigated by performing irreversible electroporation (IRE) in a 3D in vitro model of glioblastoma in agarose (low gelation temperature, Sigma Aldrich, catalog number A9414-25G). Briefly, 100 μL of U87 cell or spheroid suspension in PR-free DMEM was mixed with 1% agarose (100 μL in PR-free DMEM) to obtain a 0.5% agarose concentration, and the mixture was then placed in an 8-well chamber slide (μ-Slide 8 well highThe cells were added to the ibiTreat (ibidi) chamber. o C's CO 2 The electrodes were kept in an incubator for 24 minutes to allow them to acclimate. After 24 hours, the A5 / ETE-PC electrodes were prepared in cells / spheroids containing agarose as described above.

[0158] To perform IRE (Integrated Resonance), a high-voltage pulsed electric field was applied to cells / spheroids using an A5 / ETE-PC electrode (MicroPulser, Bio-Rad). Specifically, cells were treated by applying a series of pulsed electric fields (200-800 V / cm) in three sets of 1 ms long pulses (50 pulses each). For comparison, the same treatment was performed using a conventional Au needle electrode with the same settings. Untreated samples were used as a control. After treatment, 100 μl of the live cell staining dye calcein-AM (Thermo Fisher, Invitrogen, catalog number C3100MP) (2 μM in PR-free DMEM) was added to the chamber and incubated for 30 minutes, after which live cells were observed by fluorescence microscopy.

[0159] These results revealed that IRE using Au electrodes induced significant cell death at 600V and 800V, as measured by a decrease in green fluorescence. On the other hand, IRE using A5 / ETE-PC electrodes showed a nearly complete cell death effect from 200V alone in both cell and spheroid-based glioblastoma models. This indicates a significant increase in the IRE effect when using A5 / ETE-PC electrodes. Conventional solid needle electrodes require higher voltages to treat large amounts of tissue due to their limited pericellular reach, potentially leading to side effects. In contrast, A5 / ETE-PC electrodes, due to their flexibility and wide reach, cover a larger area and therefore require only low-intensity electric fields to perform effective IRE. These results open up the potential of A5 / ETE-PC electrodes for significantly more effective electrotherapy with IRE. However, the results need to be replicated in vivo first.

[0160] Cytotoxicity evaluation of ETE-PC in normal lung fibroblasts In vitro toxicity testing of ETE-PC in normal human lung fibroblasts (HLF-1) was conducted. Briefly, HLF-1 cells were placed in a 96-well plate at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a specified density and allowed to grow for 24 hours. Subsequently, the cells were treated with ETE-PC (5-1000 μg / ml) at various concentrations for 24 hours. After treatment, 200 μl of the live cell staining dye calcein-AM (Thermo Fisher, Invitrogen, catalog number C3100MP) (2 μM in PR-free DMEM) was added to the wells, incubated for 30 minutes, and then the live cells were observed by fluorescence cell imaging (Spark Cyto cell imaging system, Tecan).

[0161] The results revealed that even in cells treated with a high concentration of 1000 μg / ml of ETE-PC, the calcein-AM fluorescence (green) did not change compared to the control. Phase-contrast imaging showed no harmful changes in the morphology of HLF-1 cells. This suggests that ETE-PC is highly biocompatible with normal cells and suitable for therapeutic use.

[0162] Preparation of A5 / ETE-PC flexible electrodes in U87 tumors in vivo A mixture of A5 / ETE-PC (20 / 40 mg / ml) was injected into U87 tumors of CAM (chicken chorioalulia). First, ETE-PC was electrochemically functionalized on A5 by applying a bias of 1.2V for 2.5 minutes. Next, a second electrochemical functionalization was performed by diffusing ETE-PC into the tumor tissue for 5 minutes and applying 3V for 15 minutes. After electrochemical functionalization, the tumors were harvested and treated with 4% paraformaldehyde. oThe tissues were fixed in 1C for 24 hours, washed with PBS, and then incubated in 30% sucrose solution for cryoprotection. The tissues were frozen on dry ice in a TissueTek OCT (Fisher Scientific: epredia Neg-50). The frozen tumor and liver sections were cryosectioned (10–50 μm thick) using a Cryostar NX70 cryostat and mounted on Superfrost Gold microscope slides for microscopy. Images were acquired using bright-field microscopy (with 4x, 10x, and 20x objective lenses). During the experiment, EIS was performed on the tumors before and after the preparation of flexible electrodes within the tumors.

[0163] EIS data revealed a significant decrease in impedance in the tumor after electrochemical functionalization of A5 / ETE-PC by injection. This indicates successful A5 / ETE-PC electrode preparation within the tumor and demonstrates its ability to significantly increase conductivity within tumor tissue. Microscopic images of tumor sections revealed that the flexible electrode was assembled within the tumor. A well-formed dendritic structure growing from the A5 core was observed, indicating successful electrochemical functionalization of ETE-PC. However, fluorescence imaging revealed the presence of unpolymerized ETE-PC, as indicated by the green fluorescence of the trimer. This suggests that significant electrochemical functionalization of ETE-PC occurred, but was not optimal. Therefore, electrochemical functionalization can be performed using different conditions to optimize the preparation of the flexible electrode in the tumor.

[0164] Formation and distribution of electrodes around tissues, tumors, and cancer cells Preliminary experiments on coating formation and tissue permeability were conducted using a mouse brain model. In these experiments, cavities were formed and coated with a nanoparticle solution. As expected, a coating was formed with dendritic structures extending into the tissue. To link this to a procedure designed to simulate a post-surgical scenario, GBM U87 cells and spheroids were embedded in an agarose gel. A5 / ETE-PC was added to the top and diffused into the agarose. Subsequent electrochemical functionalization led to the formation of dendritic structures. These dendritic structures formed close contact and embedded the cells and spheroids, demonstrating higher efficacy during IRE compared to gold electrodes.

[0165] Furthermore, electrotaxis was revealed, indicating that cells actively move toward the electrode layer in response to the electric field. This could be advantageous for therapies that stop infiltrating cells, bring them closer to the electrodes, and remove them.

[0166] The term "abscopal" (meaning "away from the target") was coined after the literature showed that tumors in patients with metastatic disease could go into remission outside the radiation field. Since then, researchers have published approximately 50 case reports on the abscopal effect for various cancers after radiotherapy. This is a significant number of cases, considering that approximately 50-60% of cancer patients receive radiotherapy at some point during their disease. The abscopal effect arises from an immune response to tumors that are not directly treated. However, the rarity of the abscopal effect strongly suggests that there are challenges to overcome in eliciting a significant immune response. Five key events associated with effective T cell priming are: (I) TAA release, (II) DAM release, (III) TAA uptake and processing by antigen-presenting cells (APCs), (IV) antigen presentation to naive T cells by APCs, and (V) activation and proliferation of cancer-specific CD8+ T cells (which reverses immunosuppression in the tumor environment). In particular, mild infections were more frequently observed in patients who exhibited the abscopal effect. The role of inflammation due to infection as an abscopal effect-promoting factor is noteworthy and warrants further investigation. We believe that modular electrotherapy (various forms of pulse sequences combined with redox modalities) produces the expected abscopal effect.

[0167] While IRE has shown promising results in cancer treatment, its effectiveness is limited to tumor cells in close proximity to the electrode at 60-100 μm. To overcome this limitation, the inventors propose incorporating a redox modality into a flexible electrode. The flexible electrode can be implanted in or around the tumor to target specific redox reactions. IRE and redox modulation represent two distinct approaches to cancer treatment. Both methods demonstrate some potential to activate the immune system to fight cancer cells, exhibiting synergistic effects in immune cell activation, antigen presentation, and cytokine and chemokine production, fulfilling all five key elements. IRE releases DAMP and TAA, which stimulate the activation of immune cells such as macrophages (e.g., microglia), dendritic cells (DCs), and T cells. This can be achieved through controlled electroporation, releasing cellular contents over extended periods, giving APCs time to take up and present antigens. Some of the released TAA is unstable (e.g., mRNA) and is rapidly eliminated. However, electroporation can facilitate both the release from cancer cells and the uptake by APCs during lateral intercellular transfer. Furthermore, IRE enhances NK cell activity, allowing NK cells to recognize and kill tumor cells without prior sensitization. In addition, redox reactions regulate the intracellular redox environment and tumor microenvironment, indirectly activating NK cells through cytokines IL2, IL12, and IL15 released from immune cells. Other redox targets include key molecular components in target proteins, such as glutathione (GSH) and redox-sensitive cysteine ​​residues. These components play a crucial role in maintaining intracellular redox homeostasis. They can act on important signaling pathways involved in cancer cell proliferation, survival, and metastasis (e.g., the nuclear factor kappa B (NF-κB) and STAT3 pathways). By targeting increased ROS production in cancer cells, which are more susceptible to oxidative stress than healthy cells, and thereby reducing GSH levels, the natural defense mechanisms against ROS and RNS can be weakened.Another approach involves targeting tumor cell metabolism by depleting essential amino acids such as cysteine, tyrosine, and lysine. Furthermore, redox regulation in the tumor microenvironment can act on the polarization state of tumor-associated macrophages (e.g., microglia), shifting them from a protumorogenic M2 phenotype to an antitumorogenic M1 phenotype (for clarity, this overly simplified model is used herein), thereby enhancing the antitumor immune response and suppressing tumor growth. These effects complement IRE-induced DAMP release, revealing a synergistic effect between IRE and redox reactions in immune system activation. Another possible target is the tumor vascular structure, essential for tumor growth and metastasis. By supplementing IRE with redox-targeting modalities, their combined strengths can be enhanced to improve the effectiveness of cancer therapy. This targets the intracellular redox environment, the tumor microenvironment, and the immune system to achieve more effective and targeted cancer treatment.

[0168] Example 3: Assembly of a bioabsorbable, injectable cardiac stimulator at Insights background In cases of cardiac arrest or dangerous arrhythmias, common approaches to restoring heartbeat typically involve electrical stimulation through the use of a defibrillator, pacemaker implantation, or both. Pen-sized, compact, bioabsorbable, and injectable cardiac stimulators (BICS) may offer a less invasive, lighter alternative to conventional defibrillators and pacemakers, which require open surgery for implantation. BICS aims to avoid the challenges of transporting bulky equipment and performing open surgery, particularly for short-term use in remote locations that are difficult to access. BICS is especially well-suited for temporarily stimulating the heart in remote clinical scenarios until the patient can be transferred to a facility capable of permanent implantation. Precise placement can be ensured using imaging guidance such as ultrasound, similar to pericardiocentesis interventions. On the other hand, in emergency, life-threatening situations, such as in combat zones or remote locations where advanced imaging tools may not be available, anatomical landmarks can be used to guide the device into place.

[0169] The conductivity of the implanted hydrogel was reported to be slightly below 14 mS / cm, which was about twice that of the surrounding tissue (6 mS / cm). Blood conductivity has been reported to be in the range of 10–20 mS / cm, which is consistent with the conductivity of the hydrogel. Most conventional in vivo injectable conductive hydrogels are used passively (e.g., cardiac patches) or reported without external connections, and such conductive hydrogels generally have low mS / cm conductivity. -1It exhibits conductivity. However, the hydrogels reported in prior art have not been shown to be usable for cardiac stimulation. In addition to injecting sufficient energy for cardiac stimulation, there are several challenges in developing injectable electrodes for cardiac use. Such challenges include the need for the formed electrode to adhere firmly to the surface of the beating heart without disrupting the heart's natural synchronous motion, and to match the elasticity and stiffness of cardiac tissue regardless of the overall arrangement of polymers on the heart. The precursor electrode formulation is preferably administered through a narrow cavitary to minimize invasiveness. Therefore, the formulation must be highly soluble for injection, while readily agglomerating into a conductive structure in vivo to adhere to the beating heart and form an external connection. This crucial duality of adhesion-flexibility and solubility-aggregation is a critical and challenging material design criterion. Furthermore, after fulfilling its purpose, the hydrogel is required to be bioabsorbable and non-toxic during this process.

[0170] Given the intricate complexity of BICS development, evaluation using only in vitro methods presents a challenging criterion. Zebrafish (Danio reio) provide a viable model for studying the effects and interactions of various materials in vivo. Zebrafish are increasingly being used to replicate human cardiac pathology, including arrhythmias. This is because zebrafish have a heart rate closer to that of humans (120 bpm in zebrafish, 60 bpm in humans) than that of mice (600 bpm) (potentially making them a better model in this respect). While there are differences between the zebrafish heart and the human heart, such as the zebrafish having a two-ventricle heart (one atrium and one ventricle) compared to the human heart having a four-ventricle heart, important aspects of cardiac electrophysiology are conserved across both species. An anatomically similar model to the human heart is the chicken embryonic heart model, a 3R in vivo model. This offers the advantages of an exploratory model.

[0171] Thus, 8-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thiophene-3-yl)ethoxy)-1-(tri-methylammonio)octane-4-sulfonate (ETE-BuSA) (a zwitterionic thiophene trimer) was considered suitable for in vivo applications. ETE-BuSA, mixed with poly(3,4-ethylenedioxythiophene)butoxy-1-sulfonate (PEDOT-S, A5), formed a highly water-soluble mixture called precursor BICS. Precursor BICS, injected into the pericardial cavity using a small-diameter cavilery, self-assembled around the heart into a mixed ion-electron-conductive hydrogel-BICS. This was used to stimulate the hearts of zebrafish and chicken embryos. Furthermore, the conductive hydrogel was extended out of the pericardial cavity and placed on the skin as an external contact point to relay external stimuli. The conductive hydrogel, designed as a transient device, leaves no damage from the electrodes. Furthermore, animals using this implant showed no behavioral changes during or after bioresorption, and their offspring showed no developmental or behavioral problems.

[0172] ETE-BuSA synthesis The synthesis of ETE-BuSA was initiated by coupling 2-(2,5-dibromothiophen-3-yl)ethane-1-ol with EDOT pinacol boronate ester. This step was performed using the palladium catalyst PEPPSI-IPr, yielding ETE-OH in 48% yield. Later, this step was repeated on a larger scale (11 g) to obtain a higher yield of 74%, demonstrating good scalability. Next, ETE-OH was alkylated using dibromobutane in the presence of tetrabutylammonium bromide (TBAB) catalyst to obtain ETE-BuBr in 75% yield. The synthesis was continued by deprotonating 1,4-butanesultone with n-butyllithium (n-BuLi) in anhydrous tetrahydrofuran (THF) at -78°C, and quenching with ETE-BuBr acting as an electrophile, resulting in the formation of ETE-Busultone. Finally, ETE-Bu-sultone was ring-opened with trimethylamine to obtain the target molecule, ETE-BuSA. After dehydration, the ETE-BuSA powder was easy to handle and could be stored in a regular freezer.

[0173] Synthesis of 2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thiophene-3-yl)ethane-1-ol (ETE-OH)(II-10) Under a nitrogen atmosphere, 2-(2,5-dibromothiophene-3-yl)ethanol (1.54 g, 5.4 mmol) was dissolved in a two-necked flask packed with anhydrous THF (50 mL). EDOT boronic acid ester (3.34 g, 12.5 mmol) was added to the solution, followed by PEPPSI-iPr (0.183 g, 0.27 mmol) and KF (1.87 g, 32.2 mmol). Next, 15 mL of degassed water was added to the reaction mixture, which was then purged with nitrogen for 30 minutes. The mixture was then heated to 85°C for 6 hours. The reaction progress was monitored using TLC (40% toluene in pentane). After cooling to room temperature, the reaction mixture was filtered through a short silica pad and then washed with THF and toluene. The organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography using a pentane-toluene toluene gradient (0→80%) to obtain a yellow foamy substance (1.05 g, 48% yield). 1 H NMR(600MHz,CD3CN)δ7.12(s,1H),6.46(s,1H),6.32(s,1H),4.34-4.30(m,2H),4.27(ddd,J=5.5,3.1,1.2Hz,2 H),4.23(tdd,J=3.9,3.3,2.1Hz,4H),3.70(td,J=6.9,5.6Hz,2H),2.85(t,J=6.9Hz,2H),2.72(t,J=5.7Hz,1H). 13 C NMR(151MHz,CD3CN)δ143.2,142.9,139.6,139.1,138.4,134.6,127.7,126.1,112.1,110.1,100.0,97.9,66.1,65.9,65.6,65.5,36.6.

[0174] Synthesis of 5,5'-(3-(2-(4-bromobutoxy)ethyl)thiophene-2,5-diyl)bis(2,3-dihydrothieno-[3,4-b][1,4]dioxin)(ETE-BuBr)(II-55) (ETE-BuBr is EDOT-BuBr) 4(The compound was synthesized according to the publicly available procedure.) A solution of ETE-OH (1.0 g, 2.45 mmol, 1 equivalent) in 20 mL of DCM was added to a mixture of 1,4-dibromobutane (3.5 mL, 29.3 mmol, 12 equivalents) and tetrabutylammonium bromide (TBAB) (239 mg, 0.74 mmol, 0.3 equivalents) in 10 mL of DCM. The mixture was stirred for 15 minutes, after which 30 mL of 50 wt% NaOH aqueous solution was added. The resulting two-phase system was then vigorously stirred overnight. The progress of the reaction was tracked using TLC (40% ethyl acetate in pentane). The reaction mixture was diluted with 100 mL of water, and the product was extracted three times using 100 mL of DCM. The organic layer was dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by column chromatography using a gradient of ethyl acetate in pentane (0 → 40%) to obtain the product as a viscous yellow liquid (1 g, 75.3% yield). 1 H NMR(600MHz,CD3CN)δ7.12(s,1H),6.43(s,1H),6.29(s,1H),4.32-4.29(m,2H),4.24 (td,J=3.6,2.0Hz,2H),4.21(ddt,J=6.3,4.0,2.3Hz,4H),3.58(t,J=6.7Hz,2H),3.44(t,J=6 .8Hz,2H),3.40(t,J=6.2Hz,2H),2.87(t,J=6.7Hz,2H),1.90-1.81(m,2H),1.65-1.58(m,2H). 13 C NMR(151MHz,CD3CN)δ143.15,142.86,139.57,139.01,138.34,134.56,127.61,126.15,112.14 ,110.06,100.01,97.92,70.96,70.36,66.07,65.84,65.55,65.44,35.27,30.67,30.52,29.03.

[0175] Synthesis of 3-(4-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thiophen-3-yl)-ethoxy)butyl)-1,2-oxatian 2,2-dioxide (ETE-Bu sultone) (II-53) Under an inert nitrogen atmosphere, at -78°C, n-BuLi (2.5 M, 1.85 mmol, 740 μL) was added dropwise to a solution of 1,4-butanesultone (170 μL, 1.66 mmol) in 6 mL of anhydrous THF. The mixture was stirred at -78°C for 30 minutes. Subsequently, a solution of ETE-BuBr (897 mg, 1.65 mmol) in 6 mL of anhydrous THF was added, immediately producing a pale red solution. The reaction mixture was stirred further at -78°C for 30 minutes, after which the cooling bath was removed and the reaction was allowed to proceed overnight at room temperature. The reaction was stopped using a small amount of water, and the solvent was removed under reduced pressure. The residue was redissolved in 100 mL of water and extracted twice with 100 mL of siRNA. The organic layer was dried over anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using a gradient (0→100%) of siRNA in pentane. The title compound was obtained as a yellow foamy substance (393 mg, 40% yield). 1 H NMR(600MHz,CD3CN)δ 7.13(s,1H),6.46(s,1H),6.32(s,1H),4.42(ddd,J=8.9,3.4,1.7Hz,2H),4.34 -4.31(m,2H),4.26(ddd,J=5.4,3.1,1.1 Hz,2H),4.25-4.20(m,4H),3.60(t,J=6.7Hz,2H),3.40(t,J=6.0Hz,2H),3.06(dddd,J=11.1,7.7 ,5.7,3.8Hz,1H),2.89(t,J=6.7Hz,2H),1.92-1.71(m,4H),1.57-1.44(m,4H),1.44-1.36(m,1H).

[0176] Synthesis of 8-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thiophen-3-yl)ethoxy)-1-(trimethylammonio)octane-4-sulfonate (ETE-BuSA)(II-4) A 4.2 M ethanol solution of trimethylamine (1.3 mL, 5.46 mmol, 14.8 equivalents) was added in a 15 mL pressure tube to a degassed solution of ETE-BuBr (0.220 g, 0.37 mmol, 1 equivalent) in 1.5 mL of anhydrous acetonitrile. This tube was heated overnight to 85°C. After cooling to room temperature, the crude solution was transferred to a small flask, and the solvent was removed under reduced pressure to obtain the title compound as a pale yellow foam in a viscous yellow liquid. The residue was subjected to column chromatography (Â:MeCN:MeOH:H2O 3:1:1:1) to obtain a cottony yellow solid (145 mg, 60% yield). 1 H NMR(800MHz,CD3OD)δ7.15(s,1H),6.46(s,1H),6.32(s,1H),4.37-4.32(m,2H),4.30-4.26(m,2H),4.26-4 .21(m,4H),3.65(td,J=6.7,3.1Hz,2H),3.48(t,J=6.0Hz,2H),3.28(dt,J=12.2,6.0Hz,1H),3.20(dd,J=12 .1,5.4Hz,1H),3.07(s,9H),2.91(t,J=6.7Hz,2H),2.68(tt,J=8.1,4.2Hz,1H),1.98(tdd,J=15.4,8.3,4.4 Hz,3H),1.75(dddd,J=14.7,9.4,7.4,5.5Hz,1H),1.69-1.60(m,2H),1.60-1.52(m,3H),1.49-1.43(m,1H). 13 C NMR(201MHz,CD3OD)δ143.59,143.26,139.77,139.20,138.37,135.22,128.01,126.24,112.80,110.62,100. 15,97.84,71.49,67.77,66.42,66.17,65.89,65.76,60.21,53.56,31.16,30.78,30.74,27.60,24.84,21.41. HRMS(ESI)m / z:[M+H] + C 29 H 40 Calculated value for NO8S4: 658.1637, measured value: 658.1649.

[0177] Exvivo trial A precursor BICS containing a mixture of A5 and ETE-BuSA was injected into agarose to form a dark-colored structure within and around the injection channel. By using an external electrode to contact the precursor BICS and applying a low voltage, ETE-BuSA adhered to A5, forming a stable gel electrode inside the agarose. The formation of BICS could be observed from the darkening of the structure, and at higher magnification, dendritic structures extending within the agarose were revealed, similar to the reactions of other trimers.

[0178] Electropolymerization significantly increases the specific volume of BICS, reaching 30-40 F / cm² in vitro. 3 The value reached [value]. This improvement is significant because it directly relates to the capacity of the charge-holding device, thereby improving the overall performance and efficiency of the device in accordance with the concept of the present invention. The decrease in impedance due to electrolytic polymerization is another parallel advantageous result. This means that electrons / ions move more easily within the BICS structure, which is essential for a fast and efficient charging / discharging process while operating as a transistor. The decrease in impedance is associated with the observed expansion of the BICS volume.

[0179] We demonstrated that an injectable precursor BICS solution could be stabilized in Insights with excellent electrical properties by electropolymerization, and then investigated its mechanical properties. Using a mechanical indenter, the static and dynamic properties of BICS were examined in an agarose gel by gentle indentation / extrusion or by applying sinusoidal indenter displacement. Notably, the mechanical compatibility of BICS with cardiac tissue was quantitatively demonstrated by measuring parameters such as Young's modulus, stress relaxation, and cycle strain. BICS showed values ​​that closely matched those of intrinsic cardiac tissue. These results demonstrate the remarkable ability of BICS for seamless integration and manipulation in cardiac applications.

[0180] By ex vivo extraction of zebrafish hearts, a controlled environment is provided to investigate the relationship between BICS and cardiac tissue. The hearts can continue to beat for more than one hour after resection. No damage to the heart was observed, and the maintained heart rate further supported the biocompatibility of the material.

[0181] When a BICS on the heart was brought into contact with the counter electrode in the surrounding buffer and used as a relay for an external voltage pulse, the heart rate increased. Initially, the ex vivo heart was beating at a natural frequency of 0.8 Hz. When an external electrical stimulation of 2 Hz was applied, the heart rate adjusted to match this frequency, demonstrating the heart's responsiveness to external electrical stimulation from the contacting BICS. After the stimulation was stopped, the heart spontaneously returned to its original heart rate of 0.8 Hz.

[0182] In vivo testing A small amount of precursor BICS was injected into anesthetized fish using a metal-coated cavilary. Additional precursor BICS was injected as the injecting cavilary was withdrawn, forming a continuous structure extending from the pericardium to the deposited surface of the additional precursor BICS portion. A metal-coated microinjection capillary was placed in the additional precursor BICS portion and electrochemical functionalization was performed by connecting it to an external voltage source (counter electrode placed beneath the fish). After functionalization, BICS was generated around the heart, and the fish were either awakened, kept anesthetized, or euthanized, depending on the assay they were to be used in.

[0183] In vivo electrochemical analysis provided insights consistent with in vitro findings. Specifically, electrochemical functionalization of precursor BICS within the pericardium resulted in a significant decrease in impedance and a shift to lower frequencies. This change suggests an increase in the capacitance of BICS compared to its precursor BICS state before electrochemical functionalization, and such a change indicates an overall improvement in the electrochemical behavior of the material.

[0184] One of the most common and least invasive methods for monitoring cardiac activity is by using an electrocardiogram (ECG). Because zebrafish are small, ECGs recorded from them are typically acquired using three electrodes (compared to 12 electrodes for most human ECGs).

[0185] ECG recording was initiated from zebrafish. This allowed for a comprehensive analysis and comparison of cardiac rate profiles using BICS compared to a control group. A crucial step before initiating ECG recording from zebrafish was to immobilize the fish using a paralyzing agent such as tricaine, a sodium channel blocker. Tricaine is the only FDA-approved anesthetic for this purpose and is widely used in the zebrafish anesthesia research community despite its potential impact on zebrafish heart rate. In this study, the depth of anesthesia and duration of the procedure were carefully controlled to minimize the potential effects of tricaine. Meanwhile, ensuring stable and reproducible results in ECG sampling requires the implementation of appropriate high-pass and low-pass filters, particularly at a sampling frequency of 1 kHz. By using a 0.3 Hz high-pass filter, a 1 kHz low-pass filter, and a 50 Hz notch filter, the raw ECG signal was successfully acquired, characterized by an isoelectric baseline within a 2 mV range, effectively minimizing the impact of noise. Recording electrocardiograms (ECGs) from zebrafish presents challenges due to the fish's small size and the potential for low signal-to-noise ratios depending on electrode placement. To address this, we adopted a procedure based on existing literature. A notable similarity in R-wave and T-wave agreement exists between normal zebrafish ECGs and normal human ECGs, highlighting the clinical importance of using zebrafish cardiac models as substitutes to understand the electrophysiology of the human heart. Similar to human ECG recordings, which are susceptible to noise from various sources including electrical system interference, electrode contact noise, and muscle movement, previous studies have outlined the challenges of adult zebrafish ECG signals.8 Such challenges include considerable variation in waveform morphology, as well as interference-induced variations in QT and QTc intervals. Variations in signal morphology and amplitude between different fish can cause inherent differences in cardiac physiology, body shape, and electrode placement, despite attempts to consistently place electrodes in the same position each time. Raw ECG signals from zebrafish reflect raw human ECG signals well, exhibiting distinguishable peaks such as P waves, QRS complex, and T waves that are identifiable without signal processing. On the other hand, if a given BICS is a conductive structure, the spread of the BICS over the heart can lead to situations where the P wave becomes obscured by interference. The conductive properties of BICS can generate electrical signals that interfere with P wave detection, which can make it difficult to distinguish the P wave from background noise. To gain deeper insights, ECG recordings were also performed from BICS patches attached to the skin of fish. The results confirmed previous findings, but the P wave remained distinguishable. Therefore, to identify the impact on the ECG spectrum, we focused on analyzing the time difference between two consecutive R peaks. This approach allowed us to accurately assess the effect of the BICS patch on cardiac rhythm and provide a more subtle understanding of its effects on cardiac function.

[0186] When the heart was stimulated using injected BICS, the low-voltage electrical signal from the heart (0.1mV) was masked by an equally high stimulation voltage (4V). Because the frequencies overlapped, it was impossible to filter and extract the stimulation pulses. Therefore, we focused on cardiac oscillometry, a mechanical method for recording heartbeats.

[0187] By using small spherical piezoresistors to detect stress and relaxation at the apex of the chest cavity, it became possible to track heart movements while the stimulating electrodes were placed on the BICS patch. The results of the vibration measurement correlated with the results of the ECG by showing the QRST complex. As a result of the comparative analysis of the main electrocardiograms, the dynamic similarity between the electrocardiogram and the heart vibration measurement signal became clear. On the other hand, heart vibration measurement brought the advantage of being able to record heartbeats without electrodes and wires. When the in vivo stimulation period was recorded by vibration measurement, the heartbeat signal could be seen, which clearly followed the 2 Hz stimulation peak. Fourier transformation of the obtained results led to the conclusion that the original heart rate of about 60 bpm increased to 120 bpm (2 Hz) and the QRST complex became higher. Interestingly, some fish experienced irregular heart rhythms, arrhythmias, which could be restored using the BICS stimulation device. In the spectrum, non-uniform rhythms, extra beats, and missing beats were observed. During BICS stimulation, the arrhythmic heartbeats were synchronized and did not show signs of arrhythmia. After the stimulation cycle, the heart became arrhythmic again. Control experiments performed on fish without BICS strongly indicated the need for the heart to be externally stimulated by BICS, and the external pulses did not affect the heartbeat of the heart.

[0188] The concept and design of BICS: BICS, envisioned as a compact and bioabsorbable injectable device, functions as an alternative to conventional surgically implanted defibrillators and pacemakers. Specifically designed for short-term use, especially in remote or hard-to-access areas, BICS provides a less invasive solution for temporarily stimulating the heart until permanent treatment can be arranged.

[0189] Methods of injection and placement: BICS can be accurately placed using image guidance such as ultrasound or anatomical landmarks in emergencies. This minimizes the need for open surgery and enables use in difficult situations such as battlefields and border areas.

[0190] Problems of Materials and Formulations: There are significant challenges in fabricating injectable electrodes for the heart that adhere to the heart surface without interrupting the heart's movement. The precursor electrode formulation, which is a mixture of A5 and ETE-BuSA, has high solubility for injection and addresses such challenges by forming an in vivo conductive structure that conforms to the elasticity and rigidity of heart tissue. Another mixture (EEE-COOH, ETE-S, and A5) is a combination of two trimers and A5 (both electrochemical functionalization and enzymatic polymerization).

[0191] Models of Zebrafish and Chicken Embryos: Zebrafish and chicken embryos are utilized in in vivo tests because they are anatomically and physiologically similar to the human heart system. This provides a feasible model for evaluating the effectiveness and biocompatibility of BICS.

[0192] Ex vivo and In vivo Tests: Tests revealed the ability of BICS to effectively stimulate heart activity. The injected precursor BICS formed stable gel electrodes, and no damage to heart tissue was observed. In in vivo tests, the effectiveness of BICS in stimulating heartbeats and its biocompatibility were further confirmed. In the tests, animals showed no change in behavior after biodegradation.

[0193] Mechanical and Electrochemical Analyses: Mechanical compatibility with heart tissue was clarified through parameters such as Young's modulus and stress relaxation. Electrochemical analysis in vivo revealed a decrease in impedance and an increase in capacitance after electrolytic polymerization. This indicates an improvement in material properties.

[0194] ECG and Heart Vibrometry Tests: Insights into the effect of BICS on heart activity were obtained from zebrafish ECG recordings, and an adjustment of the heartbeat frequency was observed during electrical stimulation. Heart vibrometry provides a mechanical method for recording heartbeats, corroborates the ECG findings, and reveals the ability of BICS to correct arrhythmias.

[0195] Example 4: In Vivo Formation of Conductive Bioelectronics by Photopolymerization background The concept of the present invention relates to the spatial control of a formed conductive polymer or polymer electrode. In particular, the concept of the present invention relates to specifically targeting tissue structure and improving 3D control. Polymerization with spatial control has been carried out using photopolymerization with or without a photomask. This disclosure provides a composition of a first embodiment in which the oxidation potential is optimized. The composition enables the formation of conductive bioelectronics in living zebrafish by a photocatalytic reaction with spatial control.

[0196] A5-EEE-S solution 20 μL of surfactant solution was added to 0.8 mg of EEE-S. 1 μL of 10 mM rose bengal in MQW was added to this solution. The solution was oxidized by bubbling oxygen through it. The oxidized EEE-S solution was added to a vial containing 0.2 mg of A5. The solution was sonicated for 1 minute. The final concentrations of the solution were 40 mg / ml of EEE-S, 10 mg / ml of A5, and 0.4 mM rose bengal. A fresh solution was prepared for each experiment and used immediately after preparation.

[0197] Photopolymerization in microtiter plates A solution of trimer (2 μL, 20 mg / mL in Milli-Q water), optionally photocatalyst (1 μL, 10 mM in Milli-Q water for Rose Bengal, 10 mM in DMSO for SIR-COOH), and Milli-Q water was added to a 96-well microtiter plate with a black transparent bottom to a total volume of 100 μL. The solution was irradiated with light (UV 385 nm, green 550 nm, or red 621 nm, D-LEDI Nikon) for a predetermined time. The absorbance spectrum (280-1000 nm) was then recorded (Tecan SparkCyto 400).

[0198] Catalyst filling Following general procedures, photopolymerization was performed in microtiter plates using EEE-COONa(III-5) (20 mg / mL in Milli-Q water) with 1, 4, 13, 40, and 113 mol% rose bengal (10 mM in DMSO).

[0199] Photopolymerization using agarose type Wavelength-specific photopolymerization Trimer solution (2 μL, 15 μL of EEE-COONa (MilliQ water 20 mg mL) -1 Using a Hamilton syringe, 1 μL of rose bengal (prepared with or without 10 mM in DMSO) was injected into agarose mold (0.5% agarose in Ringer's solution) as two parallel lines. The agarose mold was transferred to a microscope (Nikon ECLIPSE FN1), and then irradiated with green light for 5 minutes, followed by UV light (385 nm) for 5 minutes, using a 4× / 0.10 Nikon objective lens. Imaging was performed using the same objective lens.

[0200] The conductivity of A5 and A5-ETE-S in agarose was measured using a two-terminal method. A 25 μm gold-coated tungsten microprobe (Signatone, Gilroy, CA) was connected to a polymer embedded in agarose. By sweeping the applied potential and recording the resulting current over different distances, conductivity could be estimated using a transmission line model.

[0201] Spatially controlled photopolymerization EEE-COONa (5 μL, MilliQ water 20 mg mL) -1The agarose material (0.5% agarose in Ringer's solution) was added to the surface, and the solution was dried to form a thin layer of trimers. Two 3D printed photolithography masks were inserted into the optical path (ND filter slot). The agarose material was transferred to a microscope (Nikon ECLIPSE FN1), and then, using a 20× / 0.45 Nikon objective lens, it was irradiated with UV light (385nm) for 5 minutes with mask 1, followed by UV light (385nm) for 5 minutes with mask 2. Imaging was performed using a 4× / 0.10 Nikon objective lens.

[0202] Pattern and electrical measurement A5-EEE-S solution (2 μL, see Preparation of A5-EEE-S Solution) was applied to the surface of a glass slide. A 1 × 2 × 0.5 cm agarose mold (0.5% agarose in Ringer's solution) was placed on top of the trimer solution. A 3D printed photolithography mask was inserted into the optical path (ND filter slot). The agarose mold was transferred to a microscope (Nikon ECLIPSE FN1), and then irradiated with green light (561 nm) for 15 minutes using a 20 × / 0.45 Nikon objective lens. Imaging was performed using a 4 × / 0.10 Nikon objective lens and a 20 × / 0.45 Nikon objective lens. The patterned agarose was removed from the glass slide and washed with 3 ml of Milli-Q water.

[0203] Next, the agarose type was placed on a comb-type Au electrode connected to a Keithley source meter 2612B (Keithley Instruments). The pattern was oriented towards the Au electrode. Two of the comb-type electrodes were brought into contact using an external microelectrode. The applied voltage was swept and the resulting current was recorded. This was repeated for all the comb-type electrode leads relative to the conductive polymer. The distance between adjacent electrodes was 15 μm, and the width was 2.5 mm.

[0204] Photopolymerization in Exovivo The trimer solution is prepared by adding 20 mg / L of EEE-COONa (MilliQ) water to 15 μL of water. -1The solution contained ) and 1 μL of rose bengal (10 mM in DMSO). Before microinjection and photopolymerization, zebrafish larvae (Casper mutant (Tg(elav3:GCaMP6f)) in a nacre background were added, along with tricaine (ethyl methanesulfonate 3-aminobenzoate, 0.2 mg mL). -1 The fish were euthanized using ) . Before euthanasia, movement ceased, and within at least 10 minutes, the fish became unresponsive to vibrations caused by tapping near the tricaine container. The larvae were placed transversely on a plate filled with 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in solidified E3 medium. The plate was then transferred to a microinjection apparatus, and a capillary with a 30 μm diameter bevel tip (catalog number BM100T-15. Bevel, straight, shortened + flame-finished end, obtained from Biomedical-Instruments GMBH) filled with the trimer solution was inserted into the ventricle. The total injection volume was estimated to be 1 nL. After injection, the larvae were transferred to a microscope (Nikon ECLIPSE FN1), and the heads were irradiated with green light for 15 minutes using a 4× / 0.10 Nikon objective lens. Photopolymerization was confirmed by imaging using bright-field light and UV (385 nm) with the same objective lens.

[0205] Exovivo patterning was performed on the resected brain-dura complex. For patterning, 2 μL of A5-EEE-S solution (prepared as described above) was injected into the subdural space above the interhemispheric space of the brain-dural complex using a 10 μL microliter syringe (Hamilton Company). The injected sample was placed with the injection site facing downwards on a plate filled with 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in solidified Ringer's medium. The plate was then transferred to a photopolymerization apparatus, and a lithography mask was inserted into the optical path / ND filter slot of the microscope. The pattern was focused onto the surface of the sample, and 541 nm light was irradiated for 15 minutes. Imaging was performed using a 4x / 0.10 Nikon objective lens.

[0206] In vivo photopolymerization - caudal fin patterning / lithography Adult zebrafish (Danio reio) AB wild type were used for the patterning experiment. Before the patterning procedure, the fish were anesthetized with tricaine medium (final concentration 0.2 mg / mL). Anesthesia was continued until the gill cover movement stopped and the fish no longer responded to vibrations caused by tapping near the tricaine container. The anesthetized fish were placed on their sides on a plate filled with 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in solidified Ringer's medium. The fish were covered with damp tissue paper to prevent drying, but the caudal fin was left exposed. The caudal peduncle was carefully lifted with tweezers so that a glass plate (slide glass data) could be slid under the caudal fin. The caudal fin was dried by wiping it with a paper towel. 3 μL of freshly prepared EEE-S:A5 mixture was applied by slowly lifting the caudal peduncle of the fish with a pipette containing the mixture. The pipette tip was swirled towards the tail fin, and the mixture was extruded between the tail fin and the slide glass. The plate was then transferred to a photopolymerization apparatus, and a lithography mask was inserted into the microscope's light path / ND filter slot. The pattern was focused onto the surface of the sample, and 550 nm light was irradiated for 15 minutes. After patterning, excess material was washed off the tail fin. The fish were directly revived by washing the gills with fresh aquarium water and transferred to the post-treatment aquarium for observation.

[0207] result The purpose was to confirm that the compounds of formula (I), (II) and / or (III) form a conductive organic polymer after photopolymerization, and to confirm that the compounds of formula (I), (II) and / or (III) have water solubility and biocompatibility optimal for in vivo use. A fluorescence microscope equipped with an LED light source having wavelengths covering the visible light region from far UV to red was used to efficiently evaluate the compounds (i.e., trimers) and conditions using the microtiter plate method. The far UV wavelength (385 nm) well matches the peak absorbance of the ETE trimer and EEE trimer, for example, between 350 and 400 nm, for efficient excitation. The conversion of the trimer and the formation of the product were monitored by spectrophotometric analysis (absorbance scan). However, the ETE trimer did not form the same type of product (e.g., spectrum) by exposure to the product after the enzyme conditions using HRP and H2O2. There was a fairly large peak absorbance at 350 - 400 nm similar to the trimer, which may indicate ineffective conversion. However, this peak did not decrease even when the reaction time was lengthened, and still showed a product having three conjugated thiophene units but an unknown structural modification.

[0208] The trimers EEE-S, EEE-COONa, and EEE-PC were synthesized, and redox analysis revealed that the redox potential of the EEE scaffold is actually lower compared to ETE. Furthermore, both the dry powder and aqueous solution of the EEE trimer turned dark when exposed to natural light in the laboratory, indicating high reactivity. Therefore, the EEE trimer needs to be stored at -80°C. When exposed to far-UV light (385 nm), these trimers produced a deep blue-green solution within 5 minutes. This is typical for PEDOT, which has a overlapping spectrum with a broad peak around 600 nm for enzymatic treatment. This was a good starting point for further evaluation. However, even far-UV light is incompatible with the biological system, causing tissue damage, and also presents problems with limited transmittance due to light scattering and absorption. Ideally, longer wavelength green or red light should be used to mitigate these effects. To enable longer wavelength photopolymerization within the tissue, for example, a photocatalyst was used.

[0209] Furthermore, using a catalytic amount of rose bengal, EEE-COONa was photopolymerized under green light, achieving results similar to those without photocatalysis using far UV light within 5 minutes. This was encouraging for in vivo experiments using zebrafish, as reaction time is critical. The reaction was also efficient with a 1% catalyst packing, proceeding to near completion within 5 minutes, and 100% conversion was achieved with over 4% catalyst.

[0210] A further objective was to identify a photocatalyst that could be used with red light to further improve in vivo applications. SIR-COOH is a relatively new analogue of the commonly used fluorescent dye rhodamine, in which the cross-linking oxygen of rhodamine is replaced with a dimethylsilyl group. Interestingly, replacing O with Si(Me)2 shifts the excitation and emission spectra to red by 70-100 nm, while maintaining the original brightness. Thus, in the lower range of the first NIR (near-infrared) region (approximately 650-1000 nm) in tissues, the excitation maximum of SIR-COOH is 650 nm. SIR-COOH has recently been used in bioorthogonal photoclick reactions for cell-based applications, further confirming its suitability for in vivo applications, but its application to oxidative photopolymerization has not yet been evaluated. As a photocatalyst with 621 nm red light, SIR-COOH functioned similarly to rose bengal, resulting in the conversion of the EEE-COONa trimer. However, the reaction efficiency was lower compared to the reaction with rose bengal, and some trimer remained after 5 minutes. The absorbance spectra for the enzymatic and photoreactions were fairly consistent for the EEE trimer, but under conditions using SIR-COOH as a catalyst, there was an additional peak spectrum at 800 nm. This was not observed when the photoreaction was carried out with rose bengal or without a catalyst using far UV. Further analysis of the reaction mixtures from the SIR-COOH and far UV conditions using Maldi-MS revealed dimer formation in both cases, but trimer formation was revealed only when SIR-COOH was used as the photocatalyst. This was observed in both EEE-S and EEE-COONa. The peak at 600 nm was presumed to be the hexamer, and the peak at 800 nm was presumed to be the nonamer. In this case, the 621 nm red light in the SIR-COOH catalytic reaction could excite the dimer, and since SIR-COOH activates the monomer, it could potentially lead to a monomer-dimer reaction that can form a nonomer. When a trimer was added to an already completed reaction and then irradiated with red light again, the 800 nm peak increased in relation to the 600 nm peak.This supports the dual activation mechanism in the SIR-COOH catalytic reaction. Increasing the trimer concentration from 0.4 mg / mL to 4 mg / mL reduced the reaction efficiency, with most of the trimer remaining after 5 minutes. Since the trimer itself absorbs light, the conversion depends on the length of the optical path (i.e., the cross-section) in the trimer solution. This was well explained by the fact that complete conversion was achieved after 5 minutes by reducing the volume in the well. These initial studies in solution revealed that EEE-PC has the lowest stability in aqueous solution compared to EEE-S and EEE-COONa. This suggests that these trimers may be preferable for further photopolymerization studies.

[0211] Listing by Embodiment Item Item 1. Copolymer of the following formula (I), [ka] (In the formula, n is between 2 and 25, and m is between 0 and 1n.) Compounds of the following formula (II), and / or compounds of the following formula (III) [ka] (In the formula, Z is selected from -O- or -CH2-, and each y is an integer selected from the group consisting of 0, 1, 2, 3, and 4.) Each R 1 A composition comprising (selected from the group consisting of optionally substituted phosphates, optionally substituted phosphate esters and their derivatives, optionally substituted amines, and optionally substituted amides).

[0212] Item 2. The composition according to Item 1, further comprising an aqueous solvent.

[0213] Item 3. The composition according to item 2, wherein the aqueous solvent is water.

[0214] Item 4. A composition according to any one of items 1 to 3, wherein the compound of formula (III) is not present.

[0215] Item 5. A composition according to any one of items 1 to 3, wherein the compound of formula (II) is not present.

[0216] Item 6.R 1 However, phosphatidylcholine may be substituted, phosphatidylamine may be substituted, and C may be substituted. 1~10 Alkyl sulfonates, sulfonates which may be substituted and which may be alkoxylated, and C which may be substituted. 1~10 Alkylamines, amines which may be substituted and which may be alkoxylated, C which may be substituted 1~10 A composition according to any one of items 1 to 5, selected from the group consisting of alkyl carboxylates and carboxylates that may be substituted and may be alkoxylated.

[0217] Item 7.R 1 The composition described in item 6, wherein the composition is alkoxylated and ethoxylated.

[0218] Item 8.R 1 A composition according to any of items 1 to 5, wherein the composition is phosphatidylcholine.

[0219] Item 9.R 1 The composition according to any one of items 1 to 5, wherein the phosphatidylcholine is substituted with an ethoxylated azide.

[0220] Item 10. The compound of formula (II) above, [ka] A composition selected from the group consisting of items 1 to 4.

[0221] Item 11. The compound of formula (III) above, [ka] A composition selected from the group consisting of items 1 to 3 and 5.

Claims

1. A composition comprising a polymer of formula (I) and one or more compounds of formula (II) or formula (III), The polymer of formula (I) is represented by the following structure: 【Chemistry 1】 One or more compounds of formula (II) or formula (III) are represented by the following structure: 【Chemistry 2】 During the ceremony, Each A is selected from H, Na, K, Li, Ca, Mg, Sr, and Ba. E is H, C １～６ alkyl, C ２～６ alkenyl, C ２～６ alkynyl, C ３～６ cycloalkyl, aryl, heteroaryl, -(CH ２ CH ２ O) ｑ CH ２ CH ２ OH, and is selected from, where the C １～６ alkyl is -N ３ , -OH, -SO ３ A, -N(C １～６ alkyl) ２ , -NH ＋ (C １～６ alkyl) ２ , and -N ＋ (C １～６ alkyl) ３ and may be substituted with one or more of them. R １ However, H, C １～６ Alkyl, C ２～６ Alkenil, C ２～６ Alkinyl, C ３～６ Selected from cycloalkyl, aryl, and heteroaryl, where C １～６ Alkyl is -N(C １～６ Alkyl) ２ , -NH ＋ (C １～６ Alkyl) ２ , and -N ＋ (C １～６ Alkyl) ３ It is acceptable for one or more of them to be substituted, Each Z is bonded, -O-, -OP(O)(O － )O-, -OP(O)(OH)O-, -OC(O)-, -C(O)O-, -OC(O)NH- are selected, Each R ２ However, H, C １～２０ Alkyl, aryl, heterocyclyl, heteroaryl, and Si(C) １～６ Alkyl) ３ Selected from, the C １～２０ Alkyl, aryl, heterocyclyl, and heteroaryl are one or more R ５ or R ６ It may also be replaced with R ３ , R ３ ', R ４ , and R ４ Each of the following is H and -(CH ２ ) ｙ -Z-(R ２ ) are selected from, R ３ and R ３ 'But, C １～６ When an alkoxy is present and it is combined with the atom to which it is bonded, one or more R ５ or R ２ It forms a complex ring which may be substituted with, R ４ and R ４ 'But, C １～６ When an alkoxy is present and it is combined with the atom to which it is bonded, one or more R ５ or R ２ Form a heterogly ring which may be substituted, R ５ However, H, C １～６ Alkyl, C ２～６ Alkenyl and C ２～６ Selected from Alkinir, Z' is a 6-membered or 7-membered complex ring, Each R ６ is selected from -SO ３ A, -CO ２ A, -CO ２ (R ９ ), -OH, -O(R ９ ), halogen, -N ３ , -NH ２ , -NH(R ９ ), -NHC(O)(R ９ ), -N(C １～６ alkyl) ２ , -N ＋ (C １～６ alkyl) ３ , -(OCH ２ CH ２ ) ｑ -(R ８ ), C １～６ alkyl, C ２～６ alkenyl, C ２～６ alkynyl, aryl, heteroaryl, heterocyclyl, ferrocenyl, -C(O)NH(C １～６ alkyl), where said -N ＋ (C １～６ alkyl) ３ and -C(O)NH(C １～６ alkyl) may be substituted with one or more R ７ , and said C １～６ alkyl, heterocyclyl, heteroaryl, and aryl are substituted with one or more R ７ , R ９ , or R １０ . R ７ is -SO ３ A, -CO ２ A, -(OCH ２ CH ２ ), ｑ -(R ８ ), -NH ２ , -NHC(O)-(R ９ ), -OC(O)-(R ９ ), aryl, guanidinyl, -C(O)NH(C １～６ alkyl), where the guanidinyl and -C(O)NH(C １～６ alkyl) may be substituted with one or more R ８ s, R ８ However, C １～６ Alkyl, C １～６ Alkoxy, -OH, -NH ２ ,-NH(C １～６ Alkyl), -N ３ , -OC(O)-(R ９ ), and -C(O)NH(C １～６ Selected from alkyl, where C １～６ Alkyl and -C(O)NH(C １～６ Alkyl) is one or more R ９ It may also be replaced with R ９ However, C １～６ Alkyl, C ２～６ Alkenil, C ２～６ Alkinyl, aryl, heteroaryl, heterocyclyl, ferrocenyl, -B(OH) ２ , -CO ２ A, and -CO ２ (C １～６ Selected from alkyl, where C １～６ Alkyl, aryl, heterocyclyl, and -CO ２ (C １～６ Alkyl) is one or more R １０ It may also be replaced with R １０ However, oxo, -SO ３ A, -NH ２ , -CO ２ A, -OH, -P(O)(OH) ２ , C １～６ Alkyl, C １～６ Selected from alkoxy, aryl, and heteroaryl, n is between 4 and 32. m is between 0 and 10. a is 1 to 5, y is between 0 and 16. q is between 0 and 15. The aforementioned composition, or a pharmaceutically acceptable salt thereof.

2. m is 0 or 1, and E is H, C １～６ Alkyl, -(CH ２ CH ２ O) ｑ CH ２ CH ２ OH, -N ３ C replaced by １～６ Alkyl, -SO ３ C replaced by A １～６ Alkyl, and -SO ３ A and -N ＋ (C １～６ Alkyl) ３ C replaced by １～６ Selected from alkyl groups, R １ However, C such as H and methyl １～６ Alkyl and -N ＋ (C １～６ Alkyl) ３ C replaced by １～６ The composition according to claim 1, selected from alkyl groups.

3. m is 1, E is H, R １ However, H and methyl and other C １～６ A composition according to claim 1 or 2, selected from alkyl groups.

4. m is 0, R １ However, H and -N ＋ (C １～６ Alkyl) ３ C replaced by １～６ A composition according to claim 1 or 2, selected from alkyl groups.

5. The composition comprises a polymer of formula (I) and the following structure 【Transformation 3】 A composition according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, comprising one or more compounds from formulas (II-a), (III-a), (III-b), (III-c), and (III-d) represented by .

6. R ２ but, 【Chemistry 4】 【change】 A composition according to any one of claims 1 to 5, selected from the above, or a pharmaceutically acceptable salt thereof.

7. The above composition has the following structure 【Transformation 5】 The polymer represented by the following structure 【Transformation 6】 A composition according to any one of claims 1 to 6, comprising one or more compounds represented by, or a pharmaceutically acceptable salt thereof.

8. A composition according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease.

9. A composition according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease selected from cancer, cardiovascular disease, infectious disease, and neurodegenerative disease.

10. The composition according to claim 9, wherein the cancer is selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors; the neurodegenerative disease is selected from traumatic brain injury, spinal cord injury, trauma to the peripheral nervous system, and motor neuron disease; the cardiovascular disease is selected from coronary artery disease (e.g., angina pectoris, heart attack), heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, thromboembolism, and venous thrombosis; and the infectious disease is selected from viral infection, bacterial infection, parasitic infection, and fungal infection.

11. Use of the composition or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of a drug for treating cancer, cardiovascular disease, infection, or neurodegenerative disease.

12. The use of the composition or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of an agent for treating cancer, cardiovascular disease, infection, or neurodegenerative disease, wherein the cancer is selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors; the neurodegenerative disease is selected from traumatic brain injury, spinal cord injury, trauma to the peripheral nervous system, and motor neuron disease; the cardiovascular disease is selected from coronary artery disease (e.g., angina pectoris, heart attack), heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, thromboembolism, and venous thrombosis; and the infection is selected from viral infection, bacterial infection, parasitic infection, and fungal infection.

13. The use of a composition according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating spinal cord injury such as spinal cord injury, wherein the drug induces the regeneration of nerves in the spinal cord.

14. A method for treating cancer, cardiovascular disease, infection, or neurodegenerative disease, comprising administering a therapeutically effective amount of the composition or a pharmaceutically acceptable salt thereof described in any one of claims 1 to 7 to a patient in need of treatment.

15. The method according to claim 14, wherein the cancer is selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors; the neurodegenerative disease is selected from traumatic brain injury, spinal cord injury, trauma to the peripheral nervous system, and motor neuron disease; the cardiovascular disease is selected from coronary artery disease (e.g., angina pectoris, heart attack), heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, thromboembolism, and venous thrombosis; and the infectious disease is selected from viral infections, bacterial infections, parasitic infections, and fungal infections.

16. A pharmaceutical composition comprising a composition according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, and / or additive.

17. A pharmaceutical composition comprising a therapeutically effective amount of the composition according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and another anticancer agent selected from alkylating agents, antimetabolites, anticancer camptothecin derivatives, plant-derived anticancer agents, antibiotics, enzymes, platinum coordination complexes, tyrosine kinase inhibitors, hormones, hormone antagonists, monoclonal antibodies, interferons, and biological response modifiers.

18. A hydrogel comprising the composition according to any one of claims 1 to 7, for use in the treatment or prevention of a disease.

19. A kit comprising the composition according to any one of claims 1 to 7 for use in the treatment or prevention of a disease.

20. The kit according to claim 19, wherein the compound of formula (I) is present in the first part of the kit, and one or more compounds of formula (II) or formula (III) are present in the second part of the kit.

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