Bioabsorbable organic bioelectronic device

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 their use in the treatment of diseases such as cancer, cardiovascular diseases, infections or neurodegenerative diseases; wherein the polymer of formula (I) and one or more compounds of formula (II) or formula (III) are represented by the following structure: and A, E, Z, Z ', R1, R2, R3, R3', R4, R4 ', R5, R6, R7, R8, R9, R10, n, m, a, y and q are as defined in the specification.

Description

Technical Field

[0001] This invention relates to a composition comprising a polymer of formula (I) and one or more compounds of formula (II) or (III), and its use in treating diseases (e.g., cancer, cardiovascular disease, infection, immune modulation, pain, or neurodegenerative diseases) and in energy storage. Technical Background

[0002] Bioelectronics is an interdisciplinary research field at the intersection of biology and electronics. Bioelectronic devices, or bioelectronic medicine, hold promise for complementing traditional therapies in the treatment of non-chronic diseases such as immunotherapy, pain, and cancer. Traditional drugs typically target only biochemical processes, while bioelectronics focuses on addressing functional impairments in biological circuits. Furthermore, existing bioelectronic devices face the challenge of being invasive, requiring surgical implantation, removal, or both, and primarily addressing chronic diseases. These devices often require surgical removal after disease remission. Various strategies have been employed to design electrodes that can seamlessly connect to structures in the animal nervous system, most of which rely on external signals such as electric fields, chemicals including proteins, or genetic engineering. For example, pioneering work in the prior art focuses on the in vivo electropolymerization of a conductive polymer formed in the rodent brain—specifically, the formation of a poly(3,4-ethylenedioxythiophene) (PEDOT):PSS structure by templated 3,4-ethylenedioxythiophene (PEDOT):PSS on poly(styrene sulfonate) (PSS). However, the resulting polymer mixture protruded from the injection electrode in a huge cloud-like shape, failing to achieve successful cell integration.

[0003] In light of the above, there is a need for improved bioelectronic devices that can provide minimally invasive methods and new ways of treating diseases, especially in the brain, avoiding the limitations of traditional craniotomy or rigid electrode implantation. Invention Overview

[0005] In view of the above, one objective of this technology is to provide a self-organizing substrate-free organic electrode that has the potential to meet the aforementioned shortcomings and criteria, and to meet the need for seamless integration into dynamic biological systems, which is impossible for conventional rigid solid-state electronic devices.

[0006] Another objective is to provide a general method for generating bioelectronic devices that does not depend on specific external or endogenous triggers.

[0007] Another objective is to provide compositions or pharmaceutical compositions that are biocompatible, i.e., well-tolerated in humans.

[0008] Another objective is to provide a composition or pharmaceutical composition comprising a conductive structure that can be precisely positioned in a target region.

[0009] Another objective is to provide a composition that can be assembled into a bioabsorbable electrode from, for example, water-dispersible nanoparticles in and around the brain, tumors, heart tissue, and vascular system.

[0010] Another objective is to provide a composition that can act as an electrode in situ and elicit a controlled cellular response in a target organ (e.g., brain, heart, tumor, spinal cord) upon delivery of external stimuli.

[0011] Another objective is to provide electroactive organic polymers that can be generated in vivo in a spatially controlled manner using different methods (e.g., enzymatic, photochemical (i.e., photoinduced), electrochemical controlled processes, or combinations thereof).

[0012] Another object of this disclosure is to provide a composition that does not cause tissue damage or cellular and organ toxicity after biological absorption.

[0013] Another objective is to provide a composition or pharmaceutical composition that can be assembled in vivo within cellular structures, is fully integrated, and is a transient organic bioelectronic device that can be used for non-chronic treatment.

[0014] Another objective is to provide a composition that can be easily and inexpensively synthesized from commercially available and affordable materials.

[0015] Therefore, in order to achieve at least one of the above-described objectives and others that are obvious from the following description, the independent claims define a composition or pharmaceutically acceptable salt thereof, a pharmaceutical composition or kit thereof for treating or preventing diseases (e.g., cancer, cardiovascular disease, infection or neurodegenerative disease); the use of the composition or pharmaceutically acceptable salt thereof; and a method for treating cancer, cardiovascular disease, infection or neurodegenerative disease. Preferred variations of the inventive concept will be apparent from the dependent claims.

[0016] According to a first aspect, a composition is provided comprising a polymer of formula (I) and one or more compounds of formula (II) or formula (III), wherein the polymer of formula (I) is represented by the following structure:

[0017]

[0018] One or more compounds of formula (II) or formula (III) are represented by the following structures:

[0019]

[0020] in

[0021] Each A is selected from H, Na, K, Li, Ca, Mg, Sr, and Ba; E is selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl, aryl, heteroaryl, -(CH2CH2O) q CH2CH2OH, wherein C 1-6 Alkyl groups are optionally prefixed with -N3, -OH, -SO3A, or -N(C) 1-6 alkyl)2、-NH + (C 1-6 alkyl)2 and -N + (C 1-6 One or more substitutions in alkyl group 3; R1 is selected from H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, aryl, heteroaryl, wherein the C 1-6 Alkyl groups are optionally surrounded by one or more -N(C) groups. 1-6 alkyl)2、-NH + (C 1-6 alkyl)2 and -N + (C 1-6 Alkyl)3-substituted; each Z is selected from 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, heterocyclic, heteroaryl and Si(C) 1-6 Alkyl)3, the C 1-20 Alkyl, aryl, heterocyclic, and heteroaryl groups are optionally substituted with one or more R5 or R6 groups; R3, R3', R4, and R4' are each selected from H and -(CH2). y -Z-(R2), when R3 and R3' are C 1-6 When alkoxy groups are present, they form heterocycles with the atoms to which they are attached, optionally substituted with one or more R5 or R2 groups; when R4 and R4' are C 1-6 When alkoxy groups are present, they, together with the atoms to which they are attached, form heterocycles that are optionally substituted with one or more R5 or R2 groups; R5 is selected from H, C. 1-6 Alkyl, C 2-6 alkenyl and C 2-6 Alkyne group; Z' is a 6- or 7-membered heterocycle; each R6 is selected from -SO3A, -CO2A, -CO2(R9), -OH, -O(R9), halogen, -N3, -NH2, -NH(R9), -NHC(O)(R9), -N(C1-6 Alkyl)2, -N + (C 1-6 Alkyl group 3, -(OCH2CH2) q -(R8), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, aryl, heteroaryl, heterocyclic, ferrocene, -C(O)NH(C) 1-6 alkyl), wherein the -N + (C 1-6 alkyl)3 and -C(O)NH(C 1-6 The alkyl group is optionally substituted with one or more R7 groups, and the C2 group is... 1-6 Alkyl, heterocyclic, heteroaryl, and aryl groups are bound by one or more R7, R9, or R6 groups. 10 Substitution; R7 is selected from -SO3A, -CO2A, and -(OCH2CH2). q -(R8), -NH2, -NHC(O)-(R9), -OC(O)-(R9), aryl, guanidinyl, -C(O)NH(C 1-6 alkyl), wherein the guanidine group and -C(O)NH(C 1-6 Alkyl groups are optionally substituted with one or more R8 groups; the R8 groups are selected from C10. 1-6 Alkyl, C 1-6 Alkoxy, -OH, -NH2, -NH(C) 1-6 Alkyl groups, -N3, -OC(O)-(R9), and -C(O)NH(C 1-6 alkyl), wherein the C 1-6 Alkyl groups and -C(O)NH(C) 1-6 The alkyl group is optionally substituted with one or more R9s; R9s are selected from C10. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, heterocyclic, ferrocene, -B(OH)2, -CO2A and -CO2(C 1-6 alkyl), wherein the C 1-6 Alkyl, aryl, heterocyclic and -CO2(C 1-6 Alkyl) optionally enclosed by one or more R 10 Replace; R 10 Selected from oxygen, -SO3A, -NH2, -CO2A, -OH, -P(O)(OH)2, C 1-6 Alkyl, C 1-6 Alkyl, aryl, and heteroaryl; wherein n is 4-32; m is 0-10; a is 1-5; y is 0-16; q is 0-15; or a pharmaceutically acceptable salt thereof.

[0022] According to another aspect, a composition of the first aspect or a pharmaceutically acceptable salt thereof is provided for the treatment or prevention of disease.

[0023] According to another aspect, a composition of the first aspect or a pharmaceutically acceptable salt thereof is provided for the treatment or prevention of diseases selected from cancer, cardiovascular diseases, infections, and neurodegenerative diseases.

[0024] According to another aspect, the use of the composition of the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer, cardiovascular disease, infection or neurodegenerative disease is provided.

[0025] According to another aspect, the use of the composition of the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating spinal cord injuries, such as spinal cord injury, wherein the medicament induces the regeneration of nerves in the spinal cord is provided.

[0026] According to another aspect, a method for treating cancer, cardiovascular disease, infection, or neurodegenerative disease is provided, comprising administering to a patient in need a therapeutically effective amount of the composition according to the first aspect or a pharmaceutically acceptable salt thereof.

[0027] According to another aspect, a pharmaceutical composition is provided comprising the composition according to the first aspect or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, and / or excipient.

[0028] According to another aspect, a pharmaceutical composition is provided comprising a therapeutically effective amount of the composition according to the first aspect 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 modulators.

[0029] According to another aspect, a hydrogel is provided that comprises the composition according to the first aspect, which is used for treating or preventing diseases.

[0030] According to another aspect, a kit is provided comprising the composition according to the first aspect, which is used for treating or preventing disease. Invention Details

[0032] This invention focuses on a composition for a novel modality of intratumoral electrotherapy that combines redox processes, ionic currents, or a combination of both. The invention relates to a composition that can self-assemble into a biocompatible and bioabsorbable transient conductive electrode. The fluid properties of this conductive structure indicate that it can be implanted via minimally invasive techniques and seamlessly integrated into and around the tumor. Furthermore, the ability to alter electrode properties post-implantation facilitates more precise targeting of mechanisms associated with dysfunction of biocircuits in cancer. This soft electrode can be used as a standalone therapeutic agent or in conjunction with irreversible electroporation (IRE) techniques such as NanoKnife. In the latter case, the tumor-integrated soft electrode will facilitate the delivery of treatment throughout the tumor by the NanoKnife electrode. Therefore, this disclosure focuses on a universal method independent of specific external or endogenous triggers to assemble compositions into bioabsorbable, high-performance electrode structures implanted via minimally invasive methods, for example, within the central nervous system (CNS).

[0033] Objects within the body, such as nerves and tissues, can vary greatly in size and shape, affecting the interaction between medical devices like electrodes and these structures. For example, nerve bundles and ganglia can be highly irregular in shape and size, making it difficult to achieve standard, one-size-fits-all electrodes due to fit and adaptation issues. Implantation of traditional electrodes typically requires invasive surgery, involving large incisions to directly access the target area. This process can lead to severe trauma, bleeding, and inflammatory responses, potentially causing connective tissue growth that can interfere with electrode function. Furthermore, the shape of traditional electrodes is often determined by the manufacturing process, limiting their flexibility in adapting to various anatomical features within the central and peripheral nervous systems. For instance, flat electrodes manufactured using silicon wafer technology or rod-shaped electrodes designed for deep brain stimulation may generate uneven and imprecise electric fields, posing a challenge to effectively stimulating the intended target without affecting adjacent areas. Moreover, surgical implantation of these electrodes may trigger further tissue irritation and inflammation over time, potentially reducing electrode effectiveness and requiring additional medical intervention. In addition, surgical implantation carries economic and health risks that may deter patients from choosing these treatment methods. Given these challenges, there is a clear need for an innovative electrode design that can be introduced into the body in a less invasive manner, such as through injection. Ideally, this new type of electrode would be able to closely adhere to and encapsulate the target nerve or tissue, thereby generating a more effective and uniform electric field, minimizing trauma, and providing more stable and longer-lasting therapeutic effects.

[0034] The concept of the invention will now be described more fully with reference to the accompanying drawings and embodiments showing preferred variations thereof.

[0035] This disclosure relates to a composition or a pharmaceutically acceptable salt thereof comprising a polymer of formula (I) and one or more compounds of formula (II) or (III), wherein the polymer of formula (I) and one or more compounds of formula (II) or (III) are represented by the following structure:

[0036]

[0037] Among them, 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.

[0038] In the conceptualization process of materials design, we anticipate that the polymer of formula (I), upon injection into tissue, will induce self-aggregation and form a conductive backbone. Subsequently, one or more trimers of formula (II) or (III) in the mixture (smaller than the polymer backbone) will diffuse within and out of the backbone. Applying external stimuli, such as low potential, light, or enzymes, can functionalize the entire volume of the polymer backbone, with the trimers attaching to and extending from the backbone, thereby altering the properties of the structure. This approach precisely positions the conductive backbone to the target site and promotes its broader diffusion into surrounding tissues and cells. Because trimers with different functional groups can be used during the functionalization process, control over chemical properties is possible.

[0039] However, the inventive concept can be embodied in many different forms and should not be construed as limited to the variations described herein; rather, these variations are provided for comprehensiveness and completeness and to fully communicate the scope of this disclosure to those skilled in the art. Details of this disclosure are set forth in the appended specification. Although similar or equivalent methods and materials to those described herein may be used in practice or testing of this disclosure, only exemplary methods and materials are described hereafter. Other features, objects, and advantages of this disclosure will be apparent from the specification and claims. In the specification and appended claims, the singular form includes the plural form unless the context clearly requires otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] While a single feature may be contained in different variants, these features may also be combined in other ways, and inclusion in different variants does not imply that combinations of features are not feasible. In the context of this disclosure, the term “a (or an)” does not exclude plurals. The term “optional substitution” should be understood to mean that a given chemical moiety (e.g., an alkyl group) may (but is not required to) be bonded to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group may be a fully saturated alkyl chain (i.e., a pure hydrocarbon). Alternatively, the same optionally substituted alkyl group may have substituents other than hydrogen. For example, it may be bonded to a halogen atom, a hydroxyl group, or any other substituent described herein at any position along the chain. Thus, the term “optional substitution” means that a given chemical moiety may contain other functional groups, but does not necessarily contain any other functional groups. Suitable substituents for optional substitution of said groups will be further defined and described below.

[0041] 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 selected from H and C. 1-6 Alkyl group, -(CH2CH2O) q CH2CH2OH, C substituted with -N3 1-6 Alkyl groups, C substituted with -SO3A 1-6 Alkyl groups and those with -SO3A and -N + (C 1-6 alkyl)3-substituted C 1-6 Alkyl group, and R1 is preferably selected from H and C. 1-6 Alkyl groups, such as methyl groups and those with -N + (C 1-6 alkyl)3-substituted C 1-6 Alkyl group. When m is 1, E is preferably H, and R1 is preferably selected from H and C. 1-6 Alkyl groups, such as methyl groups. When m is 0, R1 is preferably selected from H and -N groups. + (C 1-6 alkyl)3-substituted C 1-6 alkyl.

[0042] In one embodiment, the 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 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), formula (III-a), formula (III-b), formula (III-c), and formula (III-d) are represented by the following structures:

[0043] Or a pharmaceutically acceptable salt thereof, wherein 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 of formula (II-a), (III-a), (III-b), (III-c) and (III-d).

[0044] The substituent R2 in any of the aforementioned structures may be selected from the following groups:

[0045] -N3, -Cl, or their pharmaceutically acceptable salts.

[0046] In one embodiment, the composition of this disclosure comprises a copolymer represented by structure (I-1).

[0047] Where p is 1-12; and one or more compounds represented by the following structures (II-1) and (II-2):

[0048] Or its pharmaceutically acceptable salt.

[0049] Compositions comprising a polymer of formula (I) and one or more compounds of formula (II) or (III) or pharmaceutically acceptable salts thereof, for the treatment or prevention of diseases such as cancer, cardiovascular disease, infection, and neurodegenerative diseases. Alternatively, the present invention provides a method of treating cancer, cardiovascular disease, infection, or neurodegenerative disease comprising administering a therapeutically effective amount of the composition or a pharmaceutically acceptable salt thereof to a patient in need.

[0050] A significant advantage of this new invention is its ability to be applied directly to the target area without the need for traditional surgical methods, such as cutting tissue with a scalpel or scissors. This method greatly reduces or eliminates damage to the target area and its surroundings. The invention uniquely adjusts to the precise contours of the target area, effectively achieving a customized fit by shaping around the target shape. This is achieved through a multifunctional 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 method ensures optimal electrical connection and secures the device in place by enhancing mechanical adhesion to the target, thus providing a stable and effective solution.

[0051] Furthermore, the solidified electrodes of this disclosure can be placed in hard-to-reach locations within the body via injection, where surgeons may be reluctant to place existing technology devices, such as ganglia of the sympathetic nervous system or nerves of the CNS or PNS, near major blood vessels, located medially within the body and difficult to access linearly from outside. This invention utilizes a composition according to the first aspect to introduce a novel bioelectronic approach for cancer treatment, the composition comprising compounds of formulas (I), (II), and / or (III). This composition can be used to directly target cancer cells, disrupting their growth and proliferation. By integrating such polymers into bioelectronic devices, targeted therapy can be performed, disrupting cancer activity at the molecular level, thus providing a new avenue for combating various types of cancer, potentially with fewer side effects than conventional therapies. Typically, the cancers are selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors.

[0052] For neurodegenerative diseases, the composition according to the first aspect can target and modulate neuronal function and degenerative pathways. By integrating this composition into a bioelectronic system, it is possible to influence cellular processes involved in diseases such as Alzheimer's and Parkinson's, thereby potentially slowing disease progression or alleviating symptoms through targeted electronic intervention. Typically, the neurodegenerative diseases are selected from traumatic brain injury, spinal cord injury, peripheral nervous system injury, and motor neuron diseases.

[0053] For cardiovascular diseases, the composition according to the first aspect can interact with biological processes that lead to heart and vascular diseases. By integrating this composition into a bioelectronic system, the technology can precisely control heart rhythm, blood flow, and vascular health, potentially treating or managing conditions such as arrhythmias, hypertension, and atherosclerosis. This method represents a breakthrough step forward in the application of bioelectronic technology in cardiovascular interventions. Typically, the cardiovascular diseases are selected from coronary artery diseases (e.g., angina pectoris, heart attack), heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmias, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral artery disease, thromboembolic diseases, and venous thrombosis.

[0054] The composition according to the first aspect can also be used to control infection. By targeting bioelectronic interactions within pathogens or infected host cells, the composition can inhibit viral or bacterial replication or modulate the body's immune response to these pathogens. This approach opens new avenues for treating infections, particularly those resistant to conventional drugs, through bioelectronic means. Typically, the infections are selected from viral, bacterial, parasitic, and fungal infections.

[0055] In terms of immune modulation, the composition according to the first aspect can be used to modulate the activity of the immune system. This bioelectronic application has the potential to treat autoimmune diseases, reduce inflammation, or enhance immune responses against pathogens and tumors. By modulating the electronic signals of the immune system, this technology provides a novel pathway to influence immune-related diseases and conditions. Therefore, the composition according to the first aspect can be used to modulate the immune system activity as described above.

[0056] Furthermore, the composition according to the first aspect can be used in pain management applications, which involve using the composition in a bioelectronic device to target neural signals that transmit pain. This method provides a novel strategy for managing chronic pain conditions by blocking or modulating pain signals before they reach the brain, offering a potentially effective and non-pharmacological alternative to traditional pain treatments. Therefore, the composition according to the first aspect can be used for pain management as described above.

[0057] This composition or a pharmaceutically acceptable salt thereof can be used to prepare medicaments for treating cancer, cardiovascular disease, infection, or neurodegenerative diseases, such as medicaments for treating cancer, cardiovascular disease, infection, or neurodegenerative diseases. Typically, the cancers are selected from brain cancer, glioblastoma, neuroblastoma, prostate cancer, breast cancer, and solid tumors, and the neurodegenerative diseases are selected from traumatic brain injury, spinal cord injury, peripheral nervous system injury, and motor neuron disease.

[0058] The composition or a pharmaceutically acceptable salt thereof can also be used to prepare a medicament for treating spinal cord injuries, such as spinal cord rupture, wherein the medicament induces the regeneration of nerves in the spinal cord.

[0059] In addition, a pharmaceutical composition is provided comprising the composition or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, and / or excipient. The pharmaceutical composition may also comprise another anticancer agent selected from alkylating agents, antimetabolites, anticancer camptothecin derivatives, plant-derived anticancer agents, antibiotics, enzymes, platinum coordination compounds, tyrosine kinase inhibitors, hormones, hormone antagonists, monoclonal antibodies, interferons, and biological response modulators.

[0060] Alternatively, a hydrogel or kit comprising the composition or a pharmaceutically acceptable salt thereof is provided, said hydrogel or kit being used to treat or prevent disease. The kit may have different portions or compartments such that a compound of formula (I) or a pharmaceutically acceptable salt thereof is located in a first portion of the kit, and one or more compounds of formula (II) or (III) or a pharmaceutically acceptable salt thereof are located in a second portion of the kit.

[0061] By selecting conductive elements, the specific mechanical and structural properties of the formed electrode can be altered to match the characteristics of the target tissue. 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) is very close to that of agarose (38 ± 5 kPa). This is several orders of magnitude lower than the reported PEDOT:PSS hydrogel (2–20 MPa) and falls within the range of human cardiac tissue (10–200 kPa). The increased elastic modulus (with a shear modulus of 0.57 ± 0.1 kPa compared to 0.5–1 kPa in brain tissue) compared to our previously reported injectable and bioabsorbable electrodes for brain tissue confirms the additional viscosity required for dynamic cardiac tissue compared to brain tissue, and a better tissue match between the material and the corresponding tissue.

[0062] Furthermore, the implementation of this disclosure does not require significantly higher costs than conventional surgery, nor does it incur the associated risks of general anesthesia and infection. Pain specialists familiar with the placement of pharmacological nerve blocks can perform this invention, whether or not ultrasound or angiography is used for visualization.

[0063] This invention, as an electrical system for body tissue, offers another significant advantage over existing technologies: superior quality. The wires or needle tips, or flat or smooth metal contacts, have only a small surface area, thus injecting current capacitively. In one embodiment of this disclosure, the conductive hydrogel can cover a large area, such as after brain tumor surgery. Charge injection in the implanted electrode can consist of both capacitive and resistive current transmission. In body tissue, in some cases, the optimal way to inject current is via capacitive charge injection, which does not lead to irreversible chemical reactions; in other cases, it may be preferable to inject and remove electrons via redox reactions; in some cases, a combination of capacitive charge injection and redox reactions is preferred.

[0064] The present invention includes a variety of material-specific physical parameters, including but not limited to in vivo curing, from flexible to rigid and / or rigid post-curing, different conductivity, and the ability to perform mechanical interface in a location near the target organ in vivo, thereby providing additional stress and / or strain relief to the organ and the cured electrode after placement.

[0065] Unlike existing electrodes (whose microscopic surface structure and macroscopic shape are both formed in vitro), the electrodes disclosed herein acquire both their microscopic surface structure and macroscopic shape in vivo: adapting to the target in a manner similar to casting a mold around an arm or leg. This is achieved through one or more processes for fabricating electrodes in or outside a living organism. While the electrodes can be formed entirely or partially in vivo, they can also be formed in vitro in a contactless manner.

[0066] Once the developed (cured) electrodes are set, they can be strategically positioned to bind tightly to blood vessels, thereby stimulating or inhibiting signaling pathways within the vessel wall. This innovative approach allows for the direct injection of liquid compounds around blood vessels to control blood flow, potentially tightening or loosening the vessels to modulate blood delivery to organs, tissues, or skin. This modulation can be used to enhance circulation or limit heat loss as needed. In specific applications, this technology can target blood vessels supplying tumors, aiming to restrict or block blood flow to these areas. This method effectively deprives growth of essential nutrients and oxygen, potentially slowing or even reversing unwanted cell proliferation. The process involves delivering the mixture into or around the vessel wall using a catheter and establishing electrical contact from the outside of the vessel via connected wires. Alternatively, the electrode compound can be injected from a distance, gradually approaching the vessel to form a ring around it. This ring is formed by puncturing the vessel wall from the inside out and can be a complete or segmented encirclement. The wire assemblies of individually inserted electrodes then establish the necessary electrical connections to precisely target areas inside the body or just under the skin, thereby enhancing the effectiveness and specificity of the treatment.

[0067] Once the developed (solidified) electrodes are set up, they can be strategically positioned to contact blood vessels, enabling them to stimulate or inhibit signaling pathways within neurons of the CNS or PNS, since the distance between the capillary system and neurons is less than 100 micrometers.

[0068] In some embodiments, this disclosure can be placed within, near, or around an organ, particularly specific structures of the organ such as internal blood vessels or neurons, or the inner or outer wall of the organ, to enable electrical stimulation or blocking of signal transmission within the organ, the nerve innervation of the organ (e.g., the bladder), or blood supply. Organ activity can be altered by increasing or decreasing neural communication in and out of the organ, and the growth and activity of certain organs can be upregulated or downregulated by allowing more or less blood to enter the organ, for example, in the cases of the intestines, liver, lungs, or kidneys, which are the body's exchange systems, utilizing a network of fine blood vessels interwoven with other vessels that add or extract chemicals in the form of dissolved gases or liquids. The present invention provides an efficient way to access organs, for example, by injecting a liquid mixture into the outer wall of an organ near a nerve innervation point.

[0069] Beyond medical treatment, this disclosure also covers energy storage applications, wherein the unique properties of the compositions of the first aspect can be used in bioelectronic devices. This includes creating more efficient, biocompatible batteries or capacitors for medical implants and other bioelectronic devices, highlighting the broad potential of these compounds in the healthcare and technology fields. Therefore, the use of the compositions according to the first aspect can be for energy storage applications, such as bioelectronic devices or other biocompatible devices. For example, a bioelectronic device could be a biocompatible battery or capacitor for use in medical implants.

[0070] definition

[0071] The term "C" used in this article 1-6 "Alkyl" refers to straight-chain and branched saturated hydrocarbon groups having 1 to 6 carbon atoms, while the term "C" refers to... 1-20 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 20 carbon atoms. The term "C"... 1-6 "Alkoxy" refers to OC 1-6 Alkyl, wherein "C" 1-6 The use of "alkyl" is as described above. The term "C" 3-6 "Cycloalkyl" refers to a cyclic saturated hydrocarbon group having 3 to 6 carbon atoms. The term "halogen" refers to fluorine, chlorine, bromine, or iodine. The term "C"... 2-6 "Alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing 2-6 carbon atoms. An "alkenyl" group contains at least one double bond. The double bond in an alkenyl group can be non-conjugated or conjugated with another unsaturated group. The term "C"... 2-6"Alynyl group" refers to a straight-chain or branched unsaturated hydrocarbon group containing 2-6 carbon atoms. The "alkynyl group" contains at least one triple bond in the chain.

[0072] Unless otherwise explicitly 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. When containing two aromatic rings (bicyclic, etc.), the aromatic rings of an aryl group can be connected at a single point (e.g., biphenyl) or fused (e.g., naphthyl).

[0073] As used herein, the term "heteroaryl" refers to a monocyclic aromatic group consisting of one to three carbon atoms substituted with one or more heteroatoms (e.g., nitrogen, oxygen, and / or sulfur). Examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, thiopheneyl, pyrroleyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiazolyl, pyridinyl, triazolyl, triazinyl, pyridazinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl.

[0074] As used herein, the term "heterocyclic group" refers to a cyclic group of carbon atoms in which one to three carbon atoms are substituted by one or more heteroatoms (e.g., nitrogen, oxygen, and / or sulfur). Examples of heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, piperidinyl, piperazineyl, morpholinyl, and dioxaneyl.

[0075] The term "curing" as used in this article refers to a chemical process, including but not limited to polymerization, crosslinking, precipitation and / or self-organization, gelation, or other phase transitions, to become conductive, retaining its shape when subjected to the shear forces expected by a living organism under non-extreme conditions. Curing processes can occur substantially instantaneously, within seconds or minutes, or over longer periods.

[0076] "Injection" refers to the introduction of material into body tissue by: (a) using a needle or needle-like dispensing device without any incision outside the needle; (b) a catheter in a blood vessel or other body structure with a lumen; (c) a pump in a laparoscopic device inserted through a small incision; (d) using a hole formed by a separate incision; or (e) an auger system that delivers injectable material within a lumen, expressing it from the lumen to the vicinity, interior, or surrounding of an interface target. Another term, "injection," refers to the injection of an electrode solution into the body or organ through contact with the surface of the body or organ using a needleless jet injector to form a conductive structure within the body or organ.

[0077] Using needle-based, needleless jet injectors, intravascular catheters, and laparoscopic methods to place liquid mixtures, resulting in the formation of solidified electrodes, allows for novel organ connections via back surgery, similar to the ability to connect intercostal nerves and ganglia of the autonomic nervous system.

[0078] "Patient" or "subject" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or a non-human primate, such as a monkey, chimpanzee, baboon, or rhesus monkey.

[0079] When used in conjunction with a compound, “effective amount” refers to the amount that is effective in treating or preventing disease in the subjects described herein.

[0080] As used in this disclosure, the term "carrier" includes carriers, excipients, and diluents, and refers to a material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, for carrying or transporting a drug from one organ or part of a subject to another organ or part of a body.

[0081] For the subject, the term "treatment" refers to the improvement of at least one symptom of the subject's disease. Treatment includes curing, improving, or at least partially alleviating the disease.

[0082] Unless otherwise stated, the term “barrier” as used in this disclosure means the terms disease, ailment or ailment, and is used interchangeably with them.

[0083] As used in this disclosure, the terms “administration,” “application,” or “administration” mean the direct administration of a pharmaceutically acceptable salt of the disclosed compound or composition to a subject, or the administration of a prodrug derivative or analog of the compound or composition or a pharmaceutically acceptable salt of the compound or composition to a subject, wherein the prodrug derivative or analog can form an equivalent amount of the active compound in the subject’s body.

[0084] Based on the substituents present in 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), these compounds can form salts within the scope of this disclosure. Salts of said compounds suitable for pharmaceutical use refer to salts in which the counterion is pharmaceutically acceptable.

[0085] According to the invention, suitable salts include salts formed with organic or inorganic acids or bases. Specifically, according to the invention, suitable salts formed with acids include salts formed with inorganic acids, strong organic carboxylic acids (e.g., unsubstituted or halogenated alkylcarboxylic acids of 1 to 4 carbon atoms, e.g., saturated or unsaturated dicarboxylic acids, e.g., hydroxycarboxylic acids, e.g., amino acids); or salts formed with organic sulfonic acids (e.g., unsubstituted or halogenated (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, hydroxyethanesulfonic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid, glutamic acid, lysine, and arginine.

[0086] 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 formed with organic bases such as dicyclohexylamine, N-methyl-D-glucosamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-, or tri-alkylamines (e.g., ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl, or dimethylpropylamine) or mono-, di-, or tri-hydroxyalkylamines (e.g., monoethanolamine, diethanolamine, or triethanolamine). Furthermore, corresponding internal salts can also be formed.

[0087] The compounds disclosed herein can be used alone or in the form of pharmaceutical compositions for prevention and / or treatment. Although the active ingredients can be administered alone, they can also be present in pharmaceutical compositions. Therefore, this disclosure provides a pharmaceutical composition comprising a polymer of formula (I) and one or more compounds of formula (II) or (III), and pharmaceutically acceptable diluents, excipients, and / or carriers. The pharmaceutical compositions of this disclosure can take the form of pharmaceutical compositions as described below.

[0088] The polymer can be selected from one of the following different types of polymers: 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 shown in the compound of formula (Ia), p is 1 to 12, preferably 5 to 12, a is 1 to 5, E is preferably H, and R1 is H or C. 1-6Alkyl groups, such as methyl groups. Other examples include compounds of formula (Ib-1) and formula (Ic-1). Compounds of formula (Ib-1) where A is sodium are designated as compound (I-1), and are referred to herein as PEDOT-S derivative A5 or PEDOT-S(A5). Compounds of formula (Ic-1) are referred to herein as Ok-PEDOT-S. Any of the foregoing examples of compounds with m=1 can be converted to equivalent compounds, where E is selected from -(CH2CH2O). q CH2CH2OH (q=0-15), C substituted with -N3 1-6 Alkyl groups, C substituted with -SO3A 1-6 Alkyl groups and those with -SO3A and -N + (C 1-6 alkyl)3-substituted C 1-6 Alkyl groups. These compounds are referred to as PEDOT-SE in this paper.

[0089] Furthermore, the polymer in formula (I) can be a homopolymer. In this case, m is 0, and R1 is selected from H and -N. + (C 1-6 alkyl)3-substituted C 1-6 Alkyl. Examples of polymers of formula (I) with m=0 include compounds (Id) where R1 is hydrogen, and compounds where R1 is -N + Me2(R 11 ) replaced by C 1-6 Alkyl compounds (Ie), wherein R 11 Selected from H and C 1-6 Alkyl groups, r = 4-30, a = 1-4, b = 1-3. The compound (Ie) is referred to herein as PEDOT-SA.

[0090] Therefore, examples of polymers of formula (I) with m=1 include the following:

[0091]

[0092] Therefore, examples of polymers of formula (I) with m=1 include the following:

[0093]

[0094] The polymer of formula (I) can be a self-doped, water-soluble, and mixed ion-electron conductor of poly(3,4-ethylenedioxythiophene)butoxy-1-sulfonate (PEDOT-S) derivatives. Among the PEDOT-S derivatives, A5 is unique because this polymer self-assembles in an agarose gel casting containing physiological buffer to form a highly conductive hydrogel (1-5 S cm⁻¹). -1PEDOT-S can also remain effective for several months. On average, PEDOT-S derivatives contain small polymers, i.e., oligomers of 7-8 monomers. Therefore, PEDOT-S derivatives (e.g., A5) are smaller than antisense oligonucleotide drugs containing about 20 nucleotides. The polymers of formula (I) are expected to have better bioabsorption performance than PEDOT:PSS, where the PSS moiety is a large polymer of 200-300 monomers, M... n Approximately 70,000 g / mol. The polymer of formula (I) can be in the form of highly water-dispersible nanoparticles. Furthermore, the polymer of formula (I) can be a self-doped p-type conductive copolymer.

[0095] One or more compounds of formula (II) or formula (III) can be more specifically represented as compounds of formulas (II-a) and (III-a)-(III)-d as previously described. These compounds may also be referred to as trimers of ETE-R derivatives, EEE-R derivatives, or TET-R derivatives, where “E” represents the monomeric structure of 3,4-ethylenedioxythiophene (EDOT) and “T” represents the monomeric structure of thiophene. The term “R” in these trimer structures indicates that the trimer may optionally be substituted by one or more substituents, which are not explicitly indicated by the substitution “R” but only by 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: 0.3–0.5 V and 1.2 V, respectively, which is crucial for minimizing tissue damage during electropolymerization. In addition to structural modifications, i.e., the shape and function of the compound, and increased in vivo stability, the measured electrical conductivity is two to three orders of magnitude higher than that of tissue. For example, ETE-Rs can provide selected functionalizations and facilitate access to neurons that are inaccessible to initially formed electrodes. Some examples of ETE-Rs include the previously mentioned sodium 4-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thieno-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). The next section will present other examples of ETE-Rs, as well as EEE-R and TET-R derivatives.

[0096] ETE-R derivatives can be incorporated into a solution (e.g., an aqueous solution) of a polymer (I) (e.g., A5). 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 range from 1 to 100 g / ml. This solution can be injected into the brain and electrofunctionalized to form a hybrid ion- and electron-conducting hydrogel with different properties, depending in part on the R substituents on one or more ETEs. The term "hydrogel" refers to a biphasic material comprising a porous, permeable solid (e.g., an insoluble three-dimensional network of a natural or synthetic polymer) and at least 10% by weight or volume of interstitial fluid, which is entirely or primarily composed of water. Thus, a hydrogel can be described as a polymer network structure capable of absorbing large amounts of water.

[0097] The term "hybrid ionic-electron conductive hydrogel" refers to an electrode that conducts both ions and electrons.

[0098] The effect of conductive hydrogels is transient; the initial inflammation in the brain caused by injection subsides without leaving tissue damage caused by electrodes. Therefore, ETE-R provides selected functionalization and facilitates reaching neurons that are inaccessible to existing electrode techniques.

[0099] The term "transient bioelectronic device" or "transient organic bioelectronic device" refers to a bioabsorbable device or composition that disappears from the environment after a planned period of time and leaves minimal and harmless traces after disposal. Depending on the composition's structure, the bioabsorption process, i.e., the degradation of the composition in vivo, can be 1-5 days, 1-14 days, 1-60 days, 1-120 days, 1-240 days, or even up to 1 year. The in vivo polymerization method for the composition according to the first aspect of this disclosure can be electropolymerization, photopolymerization, i.e., photoinduced polymerization, such as visible light, preferably blue (450-495 nm) and / or green (495-570 nm) visible light, ultraviolet light, and / or infrared light, or enzymatic polymerization, i.e., polymerization using enzymes, such as endogenous catalases and peroxidases, such as horseradish peroxidase (HRP), myeloperoxidase (MPO), and lactoperoxidase (LPO).

[0100] Methods for synthesizing compounds and compositions

[0101] The compounds and compositions disclosed herein can be prepared by a variety of methods, including standard chemical methods. Suitable synthetic routes are shown below.

[0102] Polymers of formula (I) and compounds of formulas (II)-(II) can be prepared by methods known in the field of organic synthesis, as illustrated in part by the following synthetic schemes. In the following schemes, it is well understood that, in accordance with general principles or chemical principles, protecting groups are used where necessary for sensitive or reactive groups.

[0103] The compounds described herein can be made from commercially available starting materials or synthesized using known organic, inorganic, and / or enzymatic methods.

[0104] Another aspect of this disclosure provides a method for preparing polymers of formula (I) and compounds of formulas (II)-(II) or pharmaceutically acceptable salts thereof, wherein, unless otherwise stated, all substituents are as defined herein. The method comprises:

[0105] (i) Preparation of polymers of formula (I-1):

[0106]

[0107]

[0108] Where R1 is as defined above; E is C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl, aryl, heteroaryl, -(CH2CH2O) q CH2CH2OH, wherein C 1-6 Alkyl groups are optionally prefixed with -N3, -OH, -SO3A, or -N(C) 1-6 alkyl)2、-NH + (C 1-6 alkyl)2 and -N + (C 1-6 One or more substitutions in alkyl group 3; LG is a leaving group, such as halogen, toluenesulfonate group, methanesulfonate group; and x is 2 or 3.

[0109] 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); PEDOT-BuSA (compound I-2). The polymers of formula (Ib) or (Ic) are 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)), and the polymers of formula (I) optionally further substituted can be synthesized by reacting the polymer of formula (Ib) with an alkane sulpholactone (e.g., 1,3-propane sulpholactone or 1,4-butane sulpholactone, optionally substituted with R1) or E-LG as shown above.

[0110] For example, polymers I-2 and Id are synthesized according to the following reaction scheme:

[0111]

[0112] (ii) Preparation of compounds of formulas (II)-(III):

[0113]

[0114] Structures 200, 201, 300, and 301 are commercially available or synthesized according to known literature methods, such as monobromination or dibromination of the corresponding thiophene precursors, optionally further functionalized with boric acid or borate esters (e.g., bis(pinacolyl)diboron). Compounds of formula (II) or (III) can be synthesized from structures 200, 201, 300, and 301 using the Suzuki coupling reaction or any other alternative method known to those skilled in the art, according to the following scheme.

[0115] Non-limiting examples of ETE-R derivatives prepared using one or more of the steps shown include:

[0116]

[0117] -ETE-R derivatives: ETE-S (compound II-1), ETE-PC (compound II-2), ETE-BuSultone (compound II-3), ETE-BuSA (compound II-4),

[0118]

[0119]

[0120]

[0121]

[0122] In one embodiment, the ETE derivative may 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.

[0123] As an example, compound II-4 was synthesized according to the following reaction scheme:

[0124]

[0125] Non-limiting examples of EEE-R or EPE-R derivative compounds prepared using one or more of the steps shown include:

[0126] EEE-R derivatives or EPE-R derivatives

[0127] -EEE-R derivatives:

[0128]

[0129] In one embodiment, the EEE derivative may be selected from one or more of III-2, III-5, and III-6.

[0130] -EPE-R derivatives:

[0131]

[0132] Non-limiting examples of TET-R derivative compounds prepared using one or more of the steps shown include:

[0133]

[0134] -TET-R derivatives:

[0135]

[0136]

[0137]

[0138] As an example, compound III-32 was synthesized according to the following reaction scheme:

[0139]

[0140] The polymer of formula (I) self-assembles to form conductive electrodes in tissues.

[0141] Low-concentration agarose gels, when combined with physiological buffer (Ringer's solution), mimic divalent ions in brain tissue, enabling the polymer to self-assemble into a hydrogel that is stable in the Ringer's solution-agarose gel for extended periods, exhibiting higher conductivity than the surrounding environment. Therefore, this polymer (e.g., A5) can be used as an injectable in vivo electrode.

[0142] The zebrafish tail fin is a model system for limb regeneration and neuropathy, and therefore highly dynamic. It is transparent, allowing for direct optical contact with injected polymers. A5 (20 mg / mL) -1 The A5 was injected between the rays of the zebrafish's caudal fin. To facilitate the assembly of the soft electrodes within the fin, a 25% Ringer's solution was used to dissolve A5. When injected into the zebrafish brain, a formulation composition with a higher ionic strength than Milli-Q water was not required. However, this illustrates the adaptability of A5, allowing the nanoparticle formulation of the present invention to be matched to specific tissues and enabling injection into areas with both lower and higher ionic strengths. Immediately after injection, a coherent, deep blue structure was formed and was visually visible between the ray.

[0143] Electrical properties of A5 in peripheral tissues: Preliminary measurements indicate that the resistivity of A5 gel is significantly lower than that of the reference sample, suggesting higher conductivity. Specifically, the measured resistivity for A5 was 0.16 MΩ, compared to 0.32 MΩ for the control group. After drying, the resistivity of A5 further decreased to 0.02 MΩ, demonstrating enhanced conductivity, while the resistivity of the reference increased to 1 MΩ. These findings highlight the superior conductivity of A5, attributed to the more compact arrangement of its oligomers after drying.

[0144] Biodegradability and biocompatibility: A5 exhibited transient and bioabsorbable properties in a zebrafish model. When applied to the caudal fin, the conductive structures of A5 partially degraded after one week and completely degraded after four weeks without affecting zebrafish behavior or causing fin damage, demonstrating its excellent biocompatibility and leaving behind healthy tissue after degradation.

[0145] Brain Tissue Application and Injection Method: A5 nanoparticles designed for brain tissue application exhibit unique adaptability. A columnar injection technique using a 30 μm diameter cannula was employed to minimize tissue damage and effectively avoid vascular rupture. No adverse effects were observed up to nine days post-injection, demonstrating the safety of this method and the compatibility of A5 with fragile brain tissue.

[0146] Inflammatory response and healing: Initial post-injection observations showed an inflammatory response that subsided within 9 days, indicating that the inflammation was caused by the injection process rather than A5 itself. This is crucial for demonstrating the biocompatibility and safety of this polymer for human use.

[0147] Conductivity in Brain Tissue: Despite technical challenges such as variations in electrode contact resistance and complex biological interactions, A5 maintains conductivity in brain tissue. Under bright-field microscopy, a clear conductive pattern is visible in A5, and a linear current-voltage relationship is observed at microscale distances. This property highlights the potential of A5 for bioelectronic applications, even in the challenging environment of brain tissue.

[0148] Comparative conductivity and technical challenges: Although the in vitro conductivity of A5 exceeds 30 S cm⁻¹ -1 However, due to biological sequestration and other factors, in vivo measurements within the brain showed low conductivity. Nevertheless, even under these conditions, A5 exhibited good resistance and a clear current-voltage dependence, demonstrating its potential for application in bioelectronic devices.

[0149] Electrofunctionalization of the polymer of formula (I)

[0150] The use of column injection techniques allows for crucial control over the placement and patterning of soft electrodes within tissues, potentially yielding better results than assembly controlled by enzyme gene expression. To enhance modularity of polymers of formula (I) (e.g., A5), an electrode-tissue interface has been developed as an auxiliary method to achieve flexibility in seamlessly extending the electrode into tissues by increasing surface area, functionality, and further protrusion. Co-injection of the aforementioned ETE-R derivative (e.g., ETE-S) with, for example, A5 allows for the localization of the soft electrode to the target site. Due to the concentration gradient and electrostatic repulsion between the negatively charged ETE-S and the negatively charged A5, the former diffuses from the formed A5 electrode. After a time delay, electropolymerization is performed using A5 as the electrode, attaching ETE-S to the A5 surface and embedding it into the A5 backbone.

[0151] In addition to increasing surface area and tissue reach, different substituents (I) on ETE-R derivatives can alter the properties of the final polymeric electrode, as illustrated herein by examples such as ETE-S and the zwitterionic ETE-phosphatidylcholine (ETE-PC). Furthermore, mixtures of different trimers (e.g., ETE, EEE, or TET) with different R groups can be used to tune the desired properties of the composition. The high solubility required for injectable electrodes dictates that the nanoparticles forming the soft electrodes must be highly water-soluble, capable of self-assembling upon injection into tissue, and subsequently bioresorbed without damaging the tissue. Therefore, this modular approach leverages the unique polymer properties and then adds soluble trimers in situ to customize the electrode-tissue interface, rather than redesigning oligomers of the formula (I) (e.g., A5).

[0152] As an example, polymers of formula (I), such as PEDOT-S polymer A5 (I-1), form the backbone of the electrode. A5 can be combined with trimers (e.g., ETE-PC-R such as II-24, II-25, II-26, or II-45) to form an injectable pre-electrode solution. Once injected and electrofunctionalized, the properties of the electrode surface are primarily determined by trimer-R. By sequentially loading various polymer blends into a syringe, different properties can be produced along the length of the electrode. For example, the combination of A5 and ETE-PC may produce an electrode with surface insulation, a mechanism derived from ion-repellent membrane filtration technology, where changes in electron energy within the core have minimal impact on ions. Therefore, when the electrode is coated with ETE-PC, cellular ion activation (bioelectricity) is significantly reduced. Conversely, when A5 is mixed with trimer-R (where R represents a redox medium, such as TEMPO, ferrocene, acetyl eugenol, or catechol), the resulting electrode segment contains a redox medium. This setting can be customized (by selecting a redox medium that matches the target redox process) to influence specific redox reactions inside and around the tumor.

[0153] The modular concept of auxiliary A5 was evaluated in an in vitro agarose gel (0.5%) casting containing Ringer's solution, simulating brain tissue. A5 (20 mg / mL) was used... -1 Dissolve in ETE-S or ETE-PC (40 mg / mL) -1No precipitation occurred. The dark solution was injected into the agarose gel using a Hamilton syringe, at which point the polymer (A5) immediately aggregated. Diffusion of the ETE-R derivative from the injection path of polymer A5 was monitored using UV light (365 nm). After 2 hours, the ETE-R had diffused from A5 approximately twice the thickness of the A5 hydrogel electrode. A5 was then used as a connecting electrode, and ETE-R was electropolymerized under an applied bias of 1.5 V. Electropolymerization was achieved even with potential contact resistance. The applied bias could be further optimized, but this bias was chosen for its high tolerance to changes in contact resistance during electropolymerization.

[0154] The increased thickness of the formed A5 electrode confirmed the successful polymerization of ETE-S. Further image analysis revealed dendritic structures growing from the A5 core. The electropolymerized region also showed increased conductivity. During electropolymerization, one contact point was on the A5 (and the other in the agarose), where the relatively high resistance ensured a limited and constant current. Conversely, by simultaneously contacting both electrodes on the A5 during electropolymerization, the decrease in resistance directly translated into an increase in current. The current increased by an order of magnitude during the 12-minute polymerization process. No changes in the A5 core geometry were observed other than dendrite formation. The decrease in resistance was attributed to the insertion of polymerized ETE-S between the A5 nanoparticles. Although electropolymerization was optically observed by the darkening of the A5 upon application of voltage, no corresponding increase in current was recorded within the first 5 minutes. The initial incubation time was likely caused by one or more resistive bottlenecks limiting the current. Once these bottlenecks were eliminated through electropolymerization, a gradual increase in current was observed as electropolymerization proceeded uniformly along the A5. Keeping the two ends of A5 in contact allows for cyclic voltammetry measurements, and the results show that electropolymerization increases the current by 100 times.

[0155] Electropolymerized A-ETE-R in agarose was used as the basis for evaluating mechanical properties. The brain is very soft, with a shear modulus of approximately 0.5–1 kPa, which poses a challenge for matching conventional inorganic electrodes. Electropolymerized A5-ETE-PC closely resembles brain tissue, with a static shear modulus of 0.57 ± 0.1 kPa. A5-ETE-S was electropolymerized in agarose gel incorporating living cells to evaluate biocompatibility. Lung adenocarcinoma cells (A549 cell line) were molded into the agarose gel, labeled with and without contrast-enhancing DiI (lipophilic dye) cell markers. After injection of the A5-ETE-S solution, one end of the polymer electrode was contacted, and the grounded counter electrode was held within the agarose (outside the A5). During electropolymerization, newly formed ETE-S (sodium salt of II-1) dendritic structures extend from A5 (I-1) and achieve cell contact. In some cases, the electrode embeds into the cell, creating tight contact without causing any observable harmful effects, such as loss of cell integrity. Tight connections between the electrode and cell have been demonstrated to be necessary for efficient and precise low-voltage electrostimulation-recording. The toxicity of A5 and ETE-R was also evaluated using extreme dilution assays, with concentrations up to 1 mg / mL. -1 After one day of exposure to A5 or ETE-PC, neither A5 nor ETE-PC showed cytotoxicity. On the other hand, ETE-S exhibited some toxicity at high concentrations. Toxicity studies covered a wide range, with injected ETE-R exceeding 1000 times the amount injected in in vivo (approximately 200 μg vs 400 ng), and significant dilution occurring upon tissue injection. No cytotoxicity was observed at the amounts used in in vivo experiments due to any of the compounds. Therefore, the in vitro experiments of this invention conclude that A5-mediated electropolymerization enables flexible surface modification, close cell contact, and significantly reduced electrical resistance.

[0156] In vivo electropolymerization

[0157] Transferring the above method to an in vivo setting imposed strict limitations on the experimental setup: 1) small-diameter injection capillaries were required to avoid vascular rupture; 2) A5, ETE-R, and A5-ETE-R all needed to be highly soluble and biocompatible; 3) the applied voltage and current for electropolymerization needed to be kept low to avoid damaging brain tissue; and 4) the process needed to be rapid to avoid anesthesia-related injury. A5-ETE-S solution was injected into the brain of anesthetized zebrafish using a 30 μm diameter capillary pre-coated with 50 nm iridium. After injection, ETE-S was allowed to diffuse into the tissue for 1 minute. The coated capillary was then used as a bias electrode to establish seamless contact with the injected A5. The counter electrode was placed on the skin at the zebrafish's nostrils, allowing electropolymerization to proceed in the sedated fish at a low current (e.g., 1–3 μA) simulating an agarose setting. The procedure, injection, and electropolymerization lasted approximately 10 minutes, after which the zebrafish awoke and exhibited normal behavior after another 5–10 minutes. Typically, no rapid lurching or abnormal swimming patterns were observed, nor were any buoyancy or balance difficulties indicative of brain injury, or stress behaviors caused by discomfort (e.g., pain). No adverse events occurred, indicating the effectiveness of this minimally invasive method and good fish tolerance.

[0158] Histological staining of sagittal brain sections (30 μm thick) containing A5-ETE-S revealed tight contact between cells and polymers at the polymer-cell interface. Imaging showed that A5-ETE-S injection extended deep into the brain between the cerebellar body (C) and optic tectum (OT), with ETE-S dendrites extending radially from A5 into the granular and molecular layers of C and the superficial layer of OT. Similar to the in vitro cell-agarose model, A5-ETE-S encapsulated neurons, some even completely surrounded by conductive polymer electrodes, without any visible damage to the cells. This highlights the advantages of gel-like microstructured electrodes, which allow for the exchange of metabolites and ions through the electrodes, thereby maintaining cellular homeostasis.

[0159] Inflammatory response to A5 injection: Initial observations after A5 injection revealed that the inflammatory response induced by mechanical injection subsided within a few days. This response was attributed to tissue oxidation caused during the injection process.

[0160] Introduction of the auxiliary module (ETE-R): This study investigated the effect of combining ETE-R (electroplated trimer) with A5 on tissue integrity. This did not exacerbate tissue damage or inflammation.

[0161] Comparison of inflammatory responses between ETE-S and ETE-PC: Two variants, A5-ETE-S (sulfonate-functionalized) and A5-ETE-PC (phosphatidylcholine), were tested. For ETE-S, the initially significant inflammatory response observed around the injection site began to decrease significantly within 7–9 days, while for ETE-PC, it completely subsided within 3 days, indicating a rapid healing process.

[0162] Effects of electropolymerization voltage: The electropolymerization / electrofunctionalization process is crucial for the formation of flexible, conductive dendritic electrodes and was carefully evaluated to ensure it did not induce further oxidative stress or tissue damage. Results confirmed that the applied voltage for electropolymerization was safe and did not lead to further tissue oxidation.

[0163] Minimally Invasive Approach and Conductive Dendritic Electrodes: This study highlights the minimally invasive nature of this method, suitable for placing flexible conductive dendritic electrodes in brain tissue. The method combines A5 with an electropolymerized trimer without causing additional tissue damage and is compatible with normal fish behavior, demonstrating its potential for neurological applications.

[0164] A5-ETE-R electrical properties in zebrafish brain

[0165] Functionalization with ETE-R improved the conductivity of A5. For fish undergoing electropolymerization, under the same applied bias voltage, a current more than 10 times higher was observed for, for example, ETE-S and ETE-PC (resistance 5-10 MΩ). Conductivity over long electrode distances could also be plotted, allowing for the derivation of conductivity values. Based on the increase in the diameter of the conductive polymer after electropolymerization, the conductivity for A5-ETE-S and A5-ETE-PC is estimated to be approximately 3 S cm⁻¹. -1 This is more than most organizations (<10). -2 S cm -1 The conductivity was 2 to 3 orders of magnitude higher. Even with the conductive polymer present in the brain for 7 days, some fish swam around and exhibited normal fish behavior. For A5-ETE-S and A5-ETE-PC, the different polymers were clearly observed in the brains of two-thirds of the fish, but their conductivity was lower than in the one-day experiment. A5-ETE-PC exhibited a linear voltage-dependent current (resistance approximately 1 GΩ) in the low nA range, indicating that the polymer still exhibits long-distance conductivity. Interestingly, A5-ETE-PC showed higher conductivity at 7 days, suggesting that this modification of the polymer makes it more stable, thus different trimers have different properties.

[0166] Method Overview: The disclosed method involves injecting a mixture of A5 and ETE-S into the brain of zebrafish, followed by electropolymerization. This process creates soft electrodes within the brain tissue, which are then analyzed for their ability to stimulate specific brain regions and alter neuronal activity.

[0167] Experimental Procedure: Casper mutant adult zebrafish were used, genetically modified to express the GCaMP6f calcium indicator. This allowed action potentials to be visualized with increased green fluorescence, providing a non-invasive method for tracking neural activity. The presence of the A5-ETE-S electrode was crucial for transmitting electrical impulses and inducing long-distance neuronal firing in the brain.

[0168] Spatial specificity and toxicity testing: Experiments showed that the conductive polymer can target specific brain regions without causing acute cellular toxicity. The addition of PTZ (a GABAA receptor antagonist) demonstrated the ability to stimulate regions not directly adjacent to the electrodes, highlighting the potential of this method for precise neurostimulation.

[0169] External Contact Approach: A novel method that allows polymer electrodes to make external contact within the brain. By preserving a portion of the microcapillary for injection, external contact can be established with soft electrodes, thereby promoting brain activity without directly interfering with brain tissue. This technology represents a significant advance for clinical applications, providing a non-invasive method for interacting with internal bioelectronic devices.

[0170] Functional and Transient Bioelectronic Devices: The disclosed bioelectronic devices are not only functional but also well-tolerated in the brain, thus providing a promising avenue for transient bioelectronic therapy. Highly water-dispersible nanoparticles formed using thiophene oligomers (A5) enable the generation of stable soft electrodes upon interaction with endogenous ions, thereby eliminating the need for specific electrode formation triggering conditions.

[0171] Therefore, this disclosure relates to functional and well-tolerated organic bioelectronic devices for the brain. To meet the needs of transient bioelectronic therapy, implantation is performed using minimally invasive injection techniques, and the resulting structure is bioabsorbable. Bioabsorbability is a desirable property, for example, in electrotherapy for cancer treatment, thus replacing reparative surgery. This can be achieved by using polymers of formula (I), such as thiophene oligomers (A5), which form nanoparticles. These nanoparticles have high water dispersibility, allowing them to exist in high concentrations in solution without aggregation. However, by injecting them into tissues and interacting with endogenous ions, stable soft electrodes are formed. Therefore, no specific triggering conditions are required. The nanoparticle solution can also be ion-matched to the strength of endogenous ions in very different tissues, as demonstrated by establishing conductive structures, for example, in the zebrafish tail fin and brain. This makes this disclosure applicable to a wide range of tissues and across species. Furthermore, since the nanoparticles contain oligomers of the same size as conventional drugs, they are bioabsorbable.

[0172] The unique property of polymers of formula (I) (e.g., A5) to produce functional flexibility at the electrode-tissue interface enables the development of a modular approach. This modular approach utilizes the aforementioned properties and then adds a soluble trimer, namely one or more compounds of formula (II) or (III), which are in situ attached to the polymer to customize the electrode-tissue interface. Electropolymerization of trimers with low oxidation potentials in situ increases conductivity, forms tight junctions with cells, and functionalizes the polymer electrode. This has been demonstrated using, for example, trimers ETE-S and ETE-PC, the latter exhibiting higher long-term stability and lower toxicity. Furthermore, electropolymerization has been shown not to cause additional oxidative damage to brain tissue. This modular approach opens up possibilities for one or more compounds of formula (II) or (III) with different functional groups and low oxidation potentials, as well as potentially other trimers.

[0173] Despite the small size of the zebrafish brain, a key challenge—accessing soft neural electrodes to achieve efficient external interaction—was overcome. Through external contact, electrical impulses were applied to modulate neuronal signals in live brain slices excised from a fish implanted with bioelectronic devices.

[0174] The methods and workflows described here are general and not limited to zebrafish. For larger brains, such as rodents and primates, these procedures are more direct, especially regarding external connections.

[0175] In summary, this disclosure relates to in vivo assembled, fully integrated, bioabsorbable electronic devices in the nervous system and other tissues that can be used for non-chronic treatment.

[0176] Example 1

[0177] Evaluation of A5-ETE-S agarose mold

[0178] Add A5 (20 mg mL) -1 ) and ETE-S (40mg mL) -1 The solution in H2O was injected into an agarose mold (0.5% agarose Ringer's solution). ETE-S diffusion was monitored at 365 nm using a UV lamp. After 2 hours, one gold-coated tungsten electrode was attached to the A5 aggregate (the other to the agarose mold) to electropolymerize the ETE-S to achieve 100% coverage. ETE-S electropolymerization was performed using a Keithley 2612B at 1.5 V. The agarose was imaged using a bright-field microscope (10x and 40x objectives).

[0179] The conductivity of A5 and A5-ETE-S in agarose was measured using a two-ended apparatus in which a 25 μm gold-coated tungsten microprobe (Signatone, Gilroy, CA) was attached to a polymer embedded in the agarose. By scanning the applied potential and recording the current generated at different distances, the conductivity could be estimated using a transmission line model.

[0180] MTT testing

[0181] MTT assay was performed to determine the in vitro toxicity of A5, ETE-S, and ETE-PC. Briefly, HLF-1 (2 x 10⁶ cells per well) was used. 4 Cells were seeded in 96-well flat-bottom microplates and allowed to grow for 24 hours. Cells were then treated with A5, ETE-S, or ETE-PC (0-1000 μg / ml). -1 Treat for 24 hours. After treatment, add 200 μL of MTT (0.5 mg / mL) to each well. -1 The sample was incubated at 37°C for 4 hours. After incubation, 200 μL of isopropanol was added to dissolve the formazan crystals. The optical density of the formazan solution (as a measure of viable cells) was obtained at 570 nm using a microplate reader (Spark Cyto, Tecan). The formazan signal (viable cell count) was normalized based on control samples not exposed to A5 or ETE-R. For each setting, three biological replicates and three technical replicates were performed. Each well contained up to 200 μg of our compound (1 mg / mL). -1 200 μL), which can be compared with typical in vivo injection of 400 ng (40 mg / mL) -1 , 10nL) for comparison.

[0182] In vivo - tail fin test

[0183] Prior to microinjection, administer fish-grade tricaine (ethyl 3-aminobenzoate methanesulfonate; 0.2 mg / mL) -1 Anesthetize the fish until gill movement ceases and the fish becomes unresponsive to vibrations caused by tapping near the tricaine container. Place the anesthetized fish laterally on a plate containing 1% agarose in E3 medium (already solidified) (Agarose, LE, analytical grade, Promega). Place a damp paper towel on the fish to prevent drying, but leave the caudal fin exposed. Transfer the culture plate to a microinjection device and inject between the caudal fin rays using a 30 μm diameter, angled-tipped glass capillary (catalog number BM100T-15; angled, straight, shortened + fire-polished tip, from Biomedical-Instruments GMBH). The total injection volume between each fin is estimated to be in the range of 100 nL. After injection, revive the fish directly by rinsing the gills with fresh aquarium water and transfer them to a post-operative aquarium for observation.

[0184] In vivo - brain. Surgery and microinjection

[0185] Prior to surgery and microinjection, fish were given tricaine (ethyl 3-aminobenzoate methanesulfonate; 0.2 mg / mL). -1 Anesthetize the fish until gill cover movement ceases and the fish becomes unresponsive to tail pinching. During the procedure, the anesthetized fish is placed in a mold made of damp paper towels for stability. A small hole is then created in the parietal bone, directly above the cerebellum and adjacent to the left side of the midline, using the tip of a 30G needle. The fish, along with the paper towel mold, is then transferred to a microinjection device. A 30μm diameter capillary tube with an angled tip (catalog number BM100T-15; angled, straight, shortened + fire-polished end, from Biomedical-Instruments GMBH) filled with polymer solution (see below) is inserted through the small hole at the top of the skull to a depth of 700μm. Three injections are then performed: at depths of 700μm, 500μm, and 300μm, respectively. The total injection volume is estimated to be 10nL. After injection, the fish is either revived directly by rinsing the gills with fresh aquarium water and then transferred to a postoperative aquarium for observation, or subjected to electropolymerization.

[0186] In vivo - brain. Electropolymerization

[0187] When electropolymerization is performed after polymer injection, a counter electrode is placed on the skin of one nostril, and an iridium-coated polymer-containing capillary is used as the electrode. Injection is performed as described above; after injection, the capillary is left in the brain. After 1 minute (allowing the polymer solution to diffuse), electropolymerization of the injected polymer is performed by applying 1.5V (approximately 1-3μA current) to the electrode for 5 minutes using a Keithley source meter (Keithley Instruments). One side of a 30μm diameter glass injection capillary (catalog number BM100T-15; beveled, straight, shortened + fire-polished end, from Biomedical-Instruments GMBH) is pre-coated with 50nm Ir in a Quorum sputtering machine (QT 150, Quorum technologies) to form a conductive capillary with a maintained back optical channel for verification of liquid level before injection.

[0188] When the experiment's out-of-water time exceeded 10 minutes, the fish were cannulated and a Peri-Star Pro peristaltic pump (World Precision Instruments) was used to deliver 0.1 mg / mL of [a substance / material]. -1 Tricaine was used to fill the aquarium water into the gill chambers. Initially, we conducted experiments with diffusion and electropolymerization times exceeding 5 minutes. This did improve polymer diffusion and polymerization, but it also had a more negative impact on the fish.

[0189] Polymer formulations for microinjection

[0190] The following polymer formulation is intended for intracerebral microinjection (completely dissolved in Millipore water): A5 (20 mg / mL) -1 A5 (20mg / mL) -1 )+ETE-S (40mg mL) -1 ); A5(20mg mL-1)+ETE-PC(40mg mL -1 ).

[0191] Post-experimental tissue processing

[0192] Following polymer injection, regardless of whether electropolymerization was performed, the fish recovered as described above and were then transferred to aquariums for different post-injection survival times. Fish were euthanized at 1 hour, 2 hours, 3 hours, 4 hours, 1 day, 2 days, 3 days, 7 days, 8 days, or 9 days for histological examination or conductivity measurements. Euthanasia was achieved by immersion in ice water for 10 minutes followed by decapitation. The brain was removed directly without fixation and frozen in dry ice in a TissueTek OCT (brand name), or processed immediately for redox staining (see below), or the skull was opened and the head (jaws removed) was fixed overnight in 4% paraformaldehyde in 0.1M phosphate buffer.

[0193] Freshly frozen brain tissue was cryosectioned along the sagittal plane in a Cryostar NX70 cryostat (section thickness 20-50 μm, depending on subsequent processing). The sections were then fixed onto Superfrost Gold microscope slides for microscopic observation or onto interdigitated gold electrodes for conductivity measurement.

[0194] The paraformaldehyde-fixed brain was removed from the skull, rinsed with phosphate-buffered saline (PBS), cryoprotected with PBS containing 25% (w / v) sucrose, and frozen on dry ice in a TissueTek OCT. The brain was then cryosectioned along the sagittal plane (section thickness 30–50 μm, depending on subsequent processing). The sections were fixed onto Superfrost Gold slides for further processing.

[0195] Electrical Measurement

[0196] Brain slices coated with polymer were placed on interdigital Au electrodes connected to a Keithley source table. Two of the interdigital electrodes were brought into contact using an external microelectrode. The applied voltage was scanned and the resulting current was recorded. This procedure was repeated for all interdigital electrode leads coated with the conductive polymer. The distance between adjacent electrodes was 15 μm, and the width was 2.5 mm.

[0197] Injection and brain slice preparation

[0198] As described above, a mixture of A5 and ETE-S was microinjected into the brains of adult zebrafish Casper mutants (Tg(elav3:GCaMP6f)) and electropolymerized therein. In these experiments, capillaries were severed directly above the top of the skull, leaving the tips exposed on the brain surface. One day after injection, the fish were euthanized and decapitated by immersion in ice-cold aquarium water. The brains were rapidly dissected and embedded in 3% low-melting-point agarose dissolved in normal zebrafish Ringer's solution. The blocks were cooled on metal plates and then transferred to NMDG cutting solution on ice for trimming and fixation for vibratory sectioning. 300 and 400 μm sagittal sections containing capillary tips and polymer electrodes in the tissue were excised from each brain, transferred to HEPES recovery solution, and allowed to reach room temperature (approximately 24°C). The vibratory sections were then transferred to artificial (zebrafish) cerebrospinal fluid (aCSF), examined for GFP positivity, and prepared for electrical stimulation and Ca2+ stimulation. 2+ Imaging.

[0199] Electrical stimulation of brain slices

[0200] A 10 μm tungsten microelectrode (Signatone, Gilroy CA) was brought into contact with A5 in the brain slice, either directly or via an injection capillary that in turn contacted A5. A Grass S48 stimulator (Astro Med) was used to provide square-wave voltage pulses at the following settings: 2 sequences per second, 200 ms sequence duration, 20 pulses per second, and 2 ms pulse duration. The voltage pulse amplitude input to the Grass stimulator ranged from 6 to 14 V. High losses (including contact resistance, stray currents in the buffer around the tissue slice, and poor impedance matching) necessitated a relatively high stimulation voltage. No significant bubble formation was observed around the electrode (unlikely at higher input power).

[0201] 3D imaging

[0202] Samples were imaged in chambers filled with DBE. Cleared brain slices embedded in agarose were imaged on an UltraMicroscope II (LaVision Biotec) equipped with an sCMOS camera (Andor Neo, model 5.5-CL3) and 4x objectives (LaVision LVMI-FluoR4x / 0.3). Two laser configurations (488nm and 640nm) and the following emission filters were used: 525 / 50 for visualization of intrinsic background (vascularity) and ETE trimers, and 680 / 30nm for visualization of neurons (Neurotrace 640 / 660). 3D volumetric acquisition stacks were obtained using an Imspector Pro 64 (LaVision Biotec) with a 3μm z-step. The image stacks were stitched together using Arivis Vision 4D 3.5.0 (Arivis AG) to visualize the brain slices in 3D. The presented film was compiled in Final Cut Pro 10.4.3 (Apple Inc.).

[0203] Mechanical measurement

[0204] Inject 3 μl of A5 [20 mg mL] using a Hamilton syringe. -1 +ETE-PC [40mg ml] -1 0.6% agarose was shaped in Ringer buffer. A5-ETE-PC was electropolymerized at 1.75V for 20 minutes. A cross-section was cut and placed on a Biomomentum Mach-1 mechanical testing instrument. Testing was performed in indentation mode using a 0.5mm diameter spherical indenter at a speed of 0.01mm / s. Indentation depths were 0.15, 0.3, and 0.45mm. The test protocol included the following steps:

[0205] -Contact (0.1gf)

[0206] - Wait 10 minutes for the body to recover from contact.

[0207] -3 stress-relaxation

[0208] - Three sine wave tests were performed at 0.1, 1, and 4 Hz respectively.

[0209] Agarose and A5-ETE-PC are difficult to distinguish at 0.1 Hz. At 1 Hz, A5-ETE-PC has a lower modulus. Based on the composition of the modulus, agarose exhibits a higher elastic response, while A5-ETE-PC may become more viscous.

[0210] Example 2: Biocompatible 3D Flexible Electrode for Electrotherapy of Glioblastoma

[0211] Enzymatic polymerization of ETE-PC

[0212] ETE-PC solutions (500 μg / ml) were prepared in DPBS (Thermo Fischer, Gibco, catalog number 14190-250) and mixed with different concentrations of hydrogen peroxide (H2O2) (0.001-0.01%) and horseradish peroxidase (HRP) (Merck, Sigma Aldrich, P8375-25KU) (5 U / ml in DPBS). The polymerization status was assessed by measuring absorbance at 350 nm and 780 nm to detect polymerized and unpolymerized ETE-PC, as the color of the ETE-PC solution changed from pale yellow to black. The maximum absorbance of polymerized and unpolymerized ETE-PC was determined using UV-Vis spectrophotometry via a Spark Cyto (Tecan) multi-mode microplate reader.

[0213] The maximum absorbance of unpolymerized and polymerized ETE-PC was determined to be 350 nm and 780 nm, respectively, by wavelength scanning using UV-Vis spectroscopy. Enzymatic polymerization results showed that, as indicated by the color change of the ETE-PC solution, HRP can successfully polymerize ETE-PC in the presence of H2O2. Furthermore, the H2O2 concentration required for HRP-mediated ETE-PC polymerization was as low as 0.001%. The absorbance of polymerized and unpolymerized ETE-PC is presented as a line graph, showing a decrease in absorbance for unpolymerized ETE-PC and an increase in absorbance for polymerized ETE-PC. Moreover, HRP saturated the polymerization of ETE-PC at 0.002% H2O2. These data indicate 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. These data suggest that HRP can utilize H2O2 secreted by cancer cells to polymerize ETE-PC in the tumor microenvironment, creating the possibility of manipulating the tumor microenvironment for cancer electrotherapy.

[0214] Enzymatic polymerization of ETE-PC in the presence of cancer cells

[0215] The enzymatic polymerization of cancer cell-mediated ETE-PC was examined using 2D and 3D glioblastoma models.

[0216] For the 2D model, U87 glioblastoma cells were seeded at a density of 10,000 cells per well in 96-well plates in phenol red-free (PR-free) complete DMEM medium (ThermoFisher, Gibco, Cat no. 21063045) and incubated at 37°C in a CO2 incubator for 24 hours. After 24 hours, the cells were treated with HRP (5 U / ml in DPBS) and ETE-PC (100 μg / ml in PR-free DMEM), alone or in combination. The cells were incubated for 72 hours, and then at 24, 48, and 72 hours, the ETE-PC polymerization was observed using a bright-field microscope (10x objective) and UV-Vis spectrophotometry (Spark Cyto (Tecan) multi-plate reader, 350 nm and 780 nm).

[0217] Cells treated with an ETE-PC / HRP mixture showed a black deposit of polymerized ETE-PC at 24 hours, which gradually deepened at 48 and 72 hours. Cells treated with HRP or ETE-PC alone did not show any polymerization. UV-Vis spectroscopy results validated these observations, showing that the absorbance of unpolymerized ETE-PC decreased over time, while the absorbance of polymerized ETE-PC increased.

[0218] In the case of the 3D model, to simulate brain tissue, we used a low-concentration agarose gel casting (0.5% [agarose, low gel temperature, Sigma Aldrich, catalog number A9414-25G] in PR-free DMEM) embedded with U87 cells or spheroids. Briefly, 100 μl of a suspension of U87 cells or spheroids in PR-free DMEM was mixed with 1% agarose (100 μl in PR-free DMEM) to obtain a 0.5% agarose concentration, and then added to the wells of a 96-well plate. The cells were normalized by incubating them in a CO2 incubator at 37°C for 24 hours. The cells / spheroids were then treated individually with ETE-PC (100 μg / ml in PR-free DMEM) or in the presence of HRP (5 U / ml in PBS) and incubated for 72 hours. After 72 hours, images were taken using a bright-field microscope (10x objective).

[0219] As observed by the black deposits around the cells / spheres and throughout the pores, both cells and spheres exhibited ETE-PC polymerization in the presence of HRP. These results indicate that, once injected, ETE-PC and HRP are able to diffuse into tumor tissue and polymerize therewith with the aid of H2O2 present in the tumor microenvironment.

[0220] Electrofunctionalization of ETE-PC on A5 and fabrication of A5 / ETE-PC 3D flexible electrodes in agarose.

[0221] To prepare A5 / ETE-PC 3D flexible electrodes, 200 μl of 0.5% agarose (agarose, low gel temperature, Sigma Aldrich, catalog number A9414-25G) was added to the wells of an 8-well chamber slide. Once the agarose had gelled, 20 μl of A5 / ETE-PC solution (20 mg / ml A5 and 40 mg / ml ETE-PC in PR-free DMEM) was added to one side of the well, and the ETE-PC was allowed to diffuse laterally into the agarose for 2 hours. After 2 hours, A5 was electrofunctionalized with ETE-PC to form a flexible electrode by applying a 2 V bias relative to a gold counter electrode (Keithley Source Table 2612B, Keithley Instruments) for 30 minutes. The electrode formation was examined using a bright-field microscope (10x objective).

[0222] Upon application of the A5 / ETE-PC mixture, A5 rapidly transformed into a thick gel, while ETE-PC began to diffuse into the agarose. High-magnification images after electrofunctionalization showed that the dendritic structure of ETE-PC branched out from A5, confirming the formation of the A5 / ETE-PC flexible electrode.

[0223] After electrode formation, electrochemical impedance spectroscopy (EIS) was performed using an Autolab PGSTAT204 potentiostat (Metrohm) to measure impedance in the frequency range of 1 Hz to 100 kHz in 10 Hz increments. Impedance measurements on agarose gel without a flexible electrode were used as a control.

[0224] EIS studies showed that the use of flexible electrodes significantly reduced the impedance of agarose-based electrical systems, indicating that the A5 / ETE-PC flexible electrode can enhance the conductivity of electrical systems. Furthermore, A5 / ETE-PC electrodes were prepared around U87 cells and spheres for in vitro experiments. For this purpose, U87 cells were seeded at a density of 20,000 cells per well in wells of chambered slides and incubated in DMEM medium at 37°C and 5% CO2 for 24 hours. After 24 hours, 0.5% agarose (200 μl) was added to the cells to form a layer, and the A5 / ETE-PC mixture was applied from one side of the well. ETE-PC was allowed to diffuse for 2 hours and then electrofunctionalized to prepare the electrodes. Cells were stained with calcein-AM for identification. In the case of spheres, U87 spheres prepared using the drop method were mixed with 0.5% agarose and added to the wells of chambered slides to create a 3D model of agarose-embedded spheres. The A5 / ETE-PC electrode was prepared as described above. Results showed that dendritic soft electrodes branching from the A5 cell and spheroids were successfully synthesized. No harmful morphological changes were observed in the cells and spheroids, indicating the electrode's biocompatibility. These results provide us with a functional 3D cancer model for evaluating the capabilities of the A5 / ETE-PC electrode in cancer electrotherapy.

[0225] A5 / ETE-PC electrode performs irreversible electroporation on cancer cells.

[0226] The efficacy of the A5 / ETE-PC flexible electrode in cancer electrotherapy was examined by irreversible electroporation (IRE) of a 3D in vitro model (cells and spheroids) of glioblastoma in an agarose (agarose, low gel temperature, Sigma Aldrich, catalog number A9414-25G) mold. Briefly, 100 μl of a suspension of U87 cells or spheroids in PR-free DMEM was mixed with 1% agarose (100 μl in PR-free DMEM) to obtain a 0.5% agarose concentration, which was then added to the wells of an 8-well chamber slide (μ-Slide 8well). high ibiTreat (ibidi). Cells were normalized by incubating them in a CO2 incubator at 37°C for 24 hours. After 24 hours, A5 / ETE-PC electrodes were prepared in an agarose mold containing cells / spheres as described above.

[0227] For IRE, a high-voltage pulsed electric field (MicroPulser, Bio-Rad) was applied to the cells / spheres using an A5 / ETE-PC electrode. Specifically, the cells were treated with a series of pulsed electric fields (200-800 V / cm) by applying three sets of 1 ms long pulses (50 pulses per set). For comparison, a similar treatment was performed using a conventional gold needle electrode with the same setup. Untreated samples served as controls. After treatment, 100 μl of the live cell staining dye calcein-AM (ThermoFisher, Invitrogen, catalog number C3100MP) (2 μM in PR-free DMEM) was added to the chamber, and the cells were incubated for 30 minutes before being examined for live cells using a fluorescence microscope.

[0228] The results showed that, as determined by the reduction in green fluorescence, IRE using gold electrodes induced significant cell death at 600 and 800 V. However, IRE using A5 / ETE-PC electrodes exhibited almost complete cell killing at 200 V alone in a cell- and spheroid-based glioblastoma model. This indicates a significant improvement in IRE efficacy when using A5 / ETE-PC electrodes. Traditional solid needle electrodes cover a small area around them, resulting in the need for higher voltages to cover large amounts of tissue, which can lead to side effects. Conversely, due to the flexibility and wide coverage of A5 / ETE-PC electrodes, they can cover a larger area, thus potentially requiring lower-intensity electric fields to perform effective IRE. These results open the possibility of significantly more effective electrotherapy via IRE using A5 / ETE-PC electrodes. However, these results need to be replicated in vivo first.

[0229] Cytotoxicity assessment of ETE-PC in normal lung fibroblasts

[0230] The in vitro toxicity of ETE-PC in normal human lung fibroblasts (HLF-1) was investigated. Briefly, HLF-1 cells were cultured at 2 x 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 μg / ml in 96-well plates and grown for 24 hours. Afterward, the cells were treated with different concentrations of ETE-PC (5-1000 μg / ml) for 24 hours. Following 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 each well and incubated for 30 minutes. Live cells were then detected using fluorescence imaging (Spark Cyto cell imager, Tecan).

[0231] The results showed that, compared with the control group, even at concentrations as high as 1000 μg / ml, the calcein-AM fluorescence (green) in cells treated with ETE-PC remained unchanged. Phase-contrast imaging revealed no harmful changes in HLF-1 cell morphology. This indicates that ETE-PC is highly biocompatible with normal cells, making it suitable for therapeutic applications.

[0232] In vivo fabrication of A5 / ETE-PC flexible electrode in U87 tumor

[0233] A mixture of A5 / ETE-PC (20 / 40 mg / ml) was injected into U87 tumors on CAM (chicken embryo chorioallantoic membrane). ETE-PC was first electrofunctionalized onto A5 by applying a 1.2 V bias for 2.5 minutes. Then, ETE-PC was allowed to diffuse into the tumor tissue for 5 minutes, followed by a second electrofunctionalization by applying a 3 V bias for 15 minutes. After electrofunctionalization, the tumors were collected and fixed in 4% paraformaldehyde at 4°C 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). Frozen tumor and liver sections (10–50 μm thick) were sectioned using a Cryostar NX70 cryostat and fixed onto Superfrost Gold microscope slides for microscopic examination. Images were taken using a brightfield microscope (using 4x, 10x, and 20x objectives). During the experiment, the tumor was examined by EIS before and after the flexible electrode was fabricated inside the tumor.

[0234] EIS data showed a significant decrease in tumor impedance after injection of A5 / ETE-PC into the tumor followed by electrofunctionalization. This indicates the successful fabrication of A5 / ETE-PC electrodes in the tumor and their ability to significantly enhance conductivity within tumor tissue. Microscopic images of tumor sections confirmed the assembly of the flexible electrode within the tumor. Well-formed dendritic structures growing from the A5 nucleus were observed, indicating successful electrofunctionalization 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 while significant electrofunctionalization of ETE-PC has occurred, it has not yet reached its optimal state. Therefore, different conditions can be used for electrofunctionalization to optimize the fabrication of flexible electrodes in the tumor.

[0235] Electrode formation and distribution in tissues, tumors, and around cancer cells

[0236] Preliminary studies on capsular formation and tissue penetration were conducted using a mouse brain model. In this experiment, a cavity was created and a nanoparticle solution was injected, which, as expected, formed a capsular layer in which dendrites extended into the tissue. To link this to treatment, a protocol simulating a postoperative scenario was designed, embedding GBM U87 cells and spheroids in an agarose gel. A5 / ETE-PC was added on top of the agarose gel and allowed to diffuse into the agarose; subsequent electrofunctionalization generated dendrites. These dendritic structures created close contact with and embedded the cells and spheroids, which demonstrated increased efficacy during IRE compared to gold electrodes.

[0237] This further demonstrates electrotaxis, i.e., cells actively move toward the electrode layer in response to an electric field. This may be beneficial for treatment to stop invasive cells, bring them closer to the electrode, and eliminate them.

[0238] The term “abscopal,” meaning “far from the target site,” was coined after documenting tumor remission outside the radiation field in patients with metastatic cancer. Since then, researchers have published nearly 50 case reports of abscopal effects on various cancers following radiotherapy. This represents a considerable number of cases, considering that approximately 50-60% of cancer patients receive radiation at some point in their disease. Abscopal effects are caused by an immune response against the untreated tumor. However, the rarity of abscopal effects highlights the difficulties required to elicit a significant immune response. Five key events have been associated with the effective initiation of T cells: (I) release of TAAs, (II) release of damage-associated molecular patterns (DAMPs), (III) uptake and processing of TAAs by antigen-presenting cells (APCs), (IV) presentation of antigens by APCs to naive T cells, and finally, (V) activation and proliferation of cancer-specific CD8+ T cells, thereby reversing immunosuppression in the tumor environment. Notably, mild infections have been observed more frequently in patients exhibiting abscopal effects. The role of infection-driven inflammation as a promoter of distal effects is exciting and warrants further investigation. We believe that modular electrotherapy (combining variations in pulse sequences with redox patterns) holds promise for unlocking distal effects.

[0239] IREs have shown promising efficacy in cancer treatment, but their effectiveness is limited to tumor cells within a 60-100 μm radius of the electrode. To overcome this limitation, we propose integrating redox modulation into soft electrodes that can be implanted inside and around the tumor to target specific redox responses. IREs and redox modulation represent two distinct cancer treatment approaches. Both have shown potential to activate the immune system to fight cancer cells, with synergistic effects observed in the activation of immune cells, antigen presentation, and the production of cytokines and chemokines, satisfying all five key elements. IREs release DAMP and TAA, which stimulate the activation of immune cells (e.g., macrophages such as microglia, dendritic cells (DCs), and T cells), which can be achieved through controlled electroporation to release cellular contents over an extended period, allowing APCs time to phagocytose and present antigens. Some released TAAs are unstable (e.g., mRNA) and are rapidly eliminated; however, electroporation can facilitate release from cancer cells and uptake by APCs, enabling lateral intercellular metastasis. Furthermore, IREs enhance the activity of NK cells, enabling them to recognize and kill tumor cells without prior sensitization. Additionally, redox reactions modulate the intracellular redox environment and tumor microenvironment, indirectly activating NK cells through the release of cytokines IL2, IL12, and IL15 from immune cells. Other redox targets include key molecular components such as glutathione (GSH) and redox-sensitive cysteine ​​residues in target proteins. These components play a crucial role in maintaining intracellular redox homeostasis. They can influence key signaling pathways responsible for cancer cell proliferation, survival, and metastasis, such as the nuclear factor κB (NF-κB) and STAT3 pathways. By targeting increased ROS production in cancer cells (cancer cells are more susceptible to oxidative stress than healthy cells), we can disrupt natural defense mechanisms against ROS and RNS by reducing GSH levels. Another approach is to target tumor cell metabolism by consuming essential amino acids such as cysteine, tyrosine, and lysine. Redox regulation in the tumor microenvironment can also influence the polarization state of tumor-associated macrophages (e.g., microglia), shifting them from a pro-tumorigenic M2 phenotype to an anti-tumorigenic M1 phenotype (this simplified model is used here for clarity), thereby enhancing the anti-tumor immune response and inhibiting tumor growth. These effects complement IRE-induced DAMP release, demonstrating the synergistic effect of IREs and redox responses in activating the immune system. Another potential target is the tumor vasculature, which is crucial for tumor growth and metastasis. Supplementing IREs through redox targeting can enhance the efficacy of cancer therapy by combining their advantages: targeting the intracellular redox environment, the tumor microenvironment, and the immune system for more effective and targeted cancer treatment.

[0240] Example 3: In-situ assembly of a bioabsorbable, injectable cardiac stimulator

[0241] background

[0242] In cases of cardiac arrest or dangerous arrhythmias, the standard approach to restoring a heartbeat typically involves applying electrical stimulation using a defibrillator, implanted pacemaker, or both. A compact, bioresorbable, injectable cardiac stimulator (BICS), about the size of a pen, can serve as a less invasive and lighter alternative to traditional defibrillators and pacemakers, which require open surgery for implantation. BICS is intended for short-term use, particularly in hard-to-reach rural areas, designed to avoid the challenges of transporting bulky equipment or performing open surgery. It is particularly suitable for providing temporary cardiac stimulation in remote clinical settings until the patient can be transferred to a facility equipped with a permanently implanted device. Imaging guidance, such as ultrasound, ensures precise placement, similar to interventional techniques like pericardiocentesis. However, in emergency, life-threatening situations, such as in war zones or remote areas where advanced imaging tools may be unavailable, anatomical landmarks can be used to guide the device into place.

[0243] The implanted hydrogel has reportedly exhibited a conductivity slightly below 14 mS / cm, approximately twice that of the surrounding tissue (6 mS / cm). Blood conductivity has been reported to be between 10 and 20 mS / cm, consistent with the hydrogel's conductivity. Most existing injectable conductive hydrogels are used for passive applications, such as cardiac patches, or have been reported as not showing external connectivity; their conductivity is typically in the low mS / cm range. -1 However, no hydrogels for cardiac stimulation have been reported in the prior art. Developing injectable electrodes for cardiac applications presents numerous challenges beyond simply injecting sufficient energy for cardiac stimulation. These challenges include: the formed electrode needs to adhere firmly to the surface of the beating heart without affecting its natural synchronous movement, and match the elasticity and stiffness of the cardiac tissue, regardless of the polymer's overall location on the heart. The electrode formulation should be administered via fine capillaries for minimally invasive purposes. Therefore, the formulation must be highly soluble for injection, yet readily aggregate in vivo to form conductive structures that attach to the beating heart and create external connections. These critical adhesion-softness and solubility-aggregation dualities are essential and challenging criteria in material design. Furthermore, the hydrogel should be bioabsorbable and non-toxic after achieving its purpose.

[0244] Given the high complexity of developing BICS, evaluating criteria solely through in vitro methods is challenging. Zebrafish (Danio rerio) provide a viable model for studying the effects and interactions of various substances in organisms. Zebrafish are increasingly used to replicate human cardiac pathology, including arrhythmias, because their heart rate (120 bpm in zebrafish vs. 60 bpm in humans) is closer to that of mice (600 bpm), potentially making them a better model for this purpose. While there are differences between the zebrafish and human hearts—for example, the zebrafish heart has two chambers (one atrium and one ventricle), while the human heart has four—key aspects of cardiac electrophysiology are conserved between the two species. A model that is anatomically very similar to the human heart is the chicken embryo heart model, a 3R in vivo model with the advantages of an exploratory model.

[0245] Therefore, 8-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thieno-3-yl)ethoxy)-1-(trimethylammonium)octane-4-sulfonate (ETE-BuSA), a zwitterionic thiophene trimer, is considered suitable for in vivo application. ETE-BuSA is mixed with poly(3,4-ethylenedioxythiophene)butoxy-1-sulfonate (PEDOT-S, A5) to form a highly water-soluble mixture, denoted as proBICS. proBICS is injected into the pericardial cavity using a small-diameter capillary tube, where it self-organizes into a hybrid ionic-electron conductive hydrogel—BICS—around the heart, and is used to stimulate the hearts of zebrafish and chicken embryos. Furthermore, the conductive hydrogel extends beyond the pericardial cavity and is placed on the skin as an external contact point to transmit external stimulation. This conductive hydrogel is designed to be temporary and will not damage the electrodes. Furthermore, the animals that received the implant did not exhibit any behavioral changes during or after the bio-absorption process, and their offspring did not show any developmental or behavioral problems.

[0246] Synthesis of ETE-BuSA

[0247] The synthesis of ETE-BuSA began with the coupling of 2-(2,5-dibromothiophene-3-yl)ethane-1-ol with EDOT pinacol boronic acid ester. This step, using a palladium catalyst PEPPSI-IPr, yielded ETE-OH in 48% yield; this step was subsequently repeated on a larger scale (11 g), increasing the yield to 74%, demonstrating good scalability. Next, ETE-OH was alkylated with dibromobutane in the presence of a tetrabutylammonium bromide (TBAB) catalyst to yield ETE-BuBr in 75% yield. The synthesis continued with the deprotonation of 1,4-butanesulfonyl lactone in anhydrous tetrahydrofuran (THF) at -78 °C using n-butyllithium (n-BuLi) followed by electrophilic quenching with ETE-BuBr to form ETE-BuSultone. Finally, ETE-BuSultone underwent ring-opening with trimethylamine to yield the target molecule, ETE-BuSA. Dehydrated ETE-BuSA powder is easy to handle and can be stored in a regular refrigerator.

[0248] Synthesis of 2-(2,5-bis(2,3-dihydrothiopheno[3,4-b][1,4]dioxin-5-yl)thiophen-3-yl)ethane-1-ol (ETE-OH)(II-10). Under a nitrogen atmosphere, 2-(2,5-dibromothiophen-3-yl)ethanol (1.54 g, 5.4 mmol) was dissolved in a two-necked flask containing 50 mL of anhydrous THF. EDOT borate (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). Subsequently, 15 mL of degassed water was added to the reaction mixture, and the mixture was purged with nitrogen for 30 min. The mixture was then heated to 85 °C and maintained for 6 h. The reaction progress was monitored using TLC (40% EtOAc in pentane). After cooling to room temperature, the reaction mixture was filtered through a short silica gel pad and then washed with THF and EtOAc. The organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography using a gradient of EtOAc in pentane (0→80%) to give a yellow foam (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). 13C NMR (151MHz, CD3CN) δ143.2,142.9,139.6,139.1,138.4,134.6,127.7,126.1,112.1,110.1,100.0,97.9,66.1,65.9,65.6,65.5,62.6,33.6.

[0249] Synthesis of 5,'5'-(3-(2-(4-bromobutoxy)ethyl)thiophene-2,5-diyl)bis(2,3-dihydrothiopheno-[3,4-b][1,4]dioxin)(ETE-BuBr)(II-55). (According to published methods for EDOT-BuBr) 4 The synthesis of ETE-BuBr was carried out by adding a solution of ETE-OH (1.0 g, 2.45 mmol, 1 equivalent) in 20 mL of DCM to a mixture of 1,4-dibromobutane (3.5 mL, 29.3 mmol, 12 equivalent) and tetrabutylammonium bromide (TBAB) (239 mg, 0.74 mmol, 0.3 equivalent) in 10 mL of DCM. The mixture was stirred for 15 minutes, and then 30 mL of 50 wt% NaOH aqueous solution was added. The resulting biphasic system was then stirred vigorously overnight. The reaction progress was monitored using TLC (40% EtOAc in pentane). The reaction mixture was diluted with 100 mL of water and added to the reaction mixture, and the product was extracted with three 100 mL aliquots 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 EtOAc in pentane (0→40%) to give the product as a viscous yellow liquid (1 g, yield 75.3%). 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=6.3,4.0,2.3H z,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). 13C 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.

[0250] Synthesis of 3-(4-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thieno-3-yl)-ethoxy)butyl)-1,2-oxothione-butane-2,2-dioxide (ETE-Busultone) (II-53). Under an inert nitrogen atmosphere, n-butyllithium (2.5M hexane solution, 1.85 mmol, 740 μL) was added dropwise to a solution of 1,4-butanesulfonyl lactone (170 μL, 1.66 mmol) in 6 mL of anhydrous THF at -78 °C. The mixture was stirred at -78 °C for 30 min. Subsequently, a solution of ETE-BuBr (897 mg, 1.65 mmol) in 6 mL of anhydrous THF was added, resulting in the immediate formation of a pale red solution. The reaction mixture was stirred at -78 °C for another 30 min, and then the cooling bath was removed, allowing the reaction to proceed overnight at room temperature. The reaction was quenched with a small amount of water, and the solvent was removed under reduced pressure. The residue was redissolved in 100 mL of water and extracted with two 100 mL aliquots of EtOAc. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using a gradient of EtOAc in pentane (0->100%). A yellow, foamy compound (393 mg, 40% yield) was given. 1 H NMR(600MHz,CD3CN)δ7.13(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=5.4,3.1,1.1Hz,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).

[0251] Synthesis of 8-(2-(2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thieno-3-yl)ethoxy)-1-(trimethylammonium)octane-4-sulfonate (ETE-BuSA)(II-4). Trimethylamine (1.3 mL, 5.46 mmol, 14.8 equivalents) was added as a 4.2 M ethanol solution to a 15 mL pressure-resistant tube containing a degassed solution of ETE-BuBr (0.220 g, 0.37 mmol, 1 equivalent) in 1.5 mL anhydrous acetonitrile. The tube was heated to 85 °C overnight. After cooling to room temperature, the crude solution was transferred to a small flask, and the solvent was removed under reduced pressure to give the compound as a viscous yellow liquid with pale yellow foam. The residue was separated by column chromatography (EtOAc:MeCN:MeOH:H2O = 3:1:1:1) to give a fluffy 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 CNMR(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] + For C 29 H 40 Calculated value of NO8S4: 658.1637, experimental value: 658.1649.

[0252] In vitro studies

[0253] proBICS, containing a mixture of A5 and ETE-BuSA, was injected into agarose, forming a dark structure inside and around the injection site. Using an external electrode to contact the proBICS and applying a low voltage, ETE-BuSA was attached to the A5, forming a stable gel electrode within the agarose. The formation of BICS can be observed through structural darkening, and at higher magnification, dendritic structures extending into the agarose interior are revealed, similar to the reaction mechanism of other trimers.

[0254] Electropolymerization significantly improved the specific capacitance of BICS, reaching 30 to 40 F / cm in vitro. 3 This improvement is crucial because it directly relates to the device's charge retention capability, thereby enhancing its overall performance and efficiency according to the invention. The reduced impedance due to electropolymerization is another parallel advantage, meaning easier electron / ion movement within the BICS structure, which is essential for fast and efficient charge-discharge processes and for enabling it to function as a transistor. The reduced impedance is related to the proven volume expansion of BICS.

[0255] After confirming that the injectable proBICS solution could be stabilized in situ via electropolymerization and possessed excellent electrical properties, its mechanical properties were explored. The static and dynamic properties of BICS in agarose gel were investigated using a mechanical indenter, by gently pushing / pulling in or 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 cyclic strain testing. BICS exhibited values ​​very close to those of natural cardiac tissue. These results highlight the ability of BICS to be seamlessly integrated and manipulated in cardiac applications.

[0256] In vitro isolation of zebrafish hearts provides a controlled platform for understanding the relationship between BICS and cardiac tissue. After resection, the heart continued to beat for over an hour. Application of proBICS followed by electropolymerization showed no significant damage to the heart, and the maintenance of the beating frequency further confirms the biocompatibility of the material.

[0257] Contacting the BICS (with the counter electrode located in the surrounding buffer) on the heart and using it to deliver an external voltage pulse resulted in an increase in the heart rate. Initially, the isolated heart beat at its natural frequency of 0.8 Hz. After applying an external electrical stimulation of 2 Hz, the heart's heart rate adjusted to match this frequency, demonstrating its responsiveness to external electrical stimulation from the BICS in contact with it. Upon cessation of stimulation, the heart naturally returned to its initial heart rate of 0.8 Hz.

[0258] In vivo studies

[0259] ProBICS microinjection is performed on anesthetized fish using a metal-coated capillary. Upon retraction of the injection capillary, additional proBICS is injected, forming a continuous structure extending from the pericardium to the surface, where a further proBICS patch is deposited. A metal-coated microinjection capillary is placed on the patch and connected to an external power source to drive electrofunctionalization (with the counter electrode placed beneath the fish). Once functionalized, BICS forms around the heart, and depending on the assay method used, the fish is either awakened, anesthetized, or euthanized.

[0260] In vivo electrochemical analysis provided conclusions consistent with in vitro findings. Specifically, electrofunctionalization of proBICS within the pericardium resulted in a significant decrease in impedance, indicating a shift towards lower frequencies. This change suggests an increase in the capacitance of BICS relative to its pre-functionalized pro-BICS state, highlighting an overall improvement in the material's electrochemical behavior.

[0261] One of the most common and least invasive methods for monitoring cardiac activity is the use of an electrocardiogram (ECG). Due to their small size, ECGs recorded from zebrafish are typically obtained using three electrodes (compared to 12 electrodes for most human ECGs).

[0262] ECG recording of zebrafish was initiated. This allowed for comprehensive analysis of the beating curves of hearts with BICS and comparison with control groups. Before recording the zebrafish ECG, they must be immobilized with an anesthetic (e.g., tricaine, a sodium channel blocker). Tricaine is the only FDA-approved anesthetic for this purpose, and although it may affect the zebrafish's heart rate, it is widely used in the research community for zebrafish anesthesia. In this study, the depth of anesthesia and duration of the procedure were carefully controlled to minimize the potential effects of tricaine. On the other hand, to ensure the stability and reproducibility of the ECG sampling results, appropriate high-pass and low-pass filters must be implemented, particularly a filter with 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 and characterized using an isoelectric baseline within a 2 mV range, effectively minimizing the impact of noise. Recording the ECG of zebrafish is challenging due to their small size and the potential for low signal-to-noise ratios caused by electrode placement. To address this issue, a literature-based procedure was employed. The significant similarity between zebrafish and normal human ECGs in R-wave and T-wave concordance enhances the clinical relevance of the zebrafish heart model as a substitute for understanding human cardiac electrophysiology. Similar to human ECG recordings (which are susceptible to noise from various sources such as power line artifacts, electrode contact noise, and muscle motion artifacts), previous studies have outlined the challenges of interpreting adult zebrafish ECG signals. 8 These challenges include significant differences in waveform morphology and interference-induced QT and QT. C Variations in intervals. Differences in signal morphology and amplitude among different fish species can be attributed to inherent differences in their cardiac physiology, body shape, and variations in electrode placement, despite efforts to consistently place the electrodes in the same location. The raw ECG signal from zebrafish is very similar to that of human ECG, exhibiting distinct peaks such as the P wave, QRS complex, and T wave, which are identifiable without signal processing. On the other hand, given that the BICS is a conductive structure, its extensive coverage on the heart can lead to P wave masking by interference. The conductive properties of the BICS generate electrical signals that interfere with P wave detection, making it difficult to distinguish from background noise. To gain a deeper understanding, ECG recordings were also performed from a BICS patch applied to the fish's skin. The results confirmed previous observations, but the P wave remained discernible. Therefore, to determine the impact on the ECG spectrum, the time difference between two consecutive R peaks was analyzed. This approach allows for a precise assessment of the effects of the BICS patch on heart rhythm, providing a more nuanced understanding of its influence on cardiac function.

[0263] When the heart is stimulated with injected BICS, the low-voltage electrical signal (0.1mV) from the heart is masked by the relatively high stimulation voltage (4V). Frequency overlap makes the stimulation pulses impossible to filter out. Therefore, attention turned to a mechanical method for recording the heartbeat—echocardiography.

[0264] A small spherical indenter was used to detect stress and relaxation at the top of the thoracic cavity, allowing for tracking of cardiac motion while stimulation electrodes remained on the BICS patch. The cycloplegic resonator results correlated with ECG results by displaying the QRST complex. Comparative analysis of the primary ECG showed that the ECG and cycloplegic resonator signals were dynamically similar, with the cycloplegic resonator offering the advantage of recording heartbeats without electrodes and leads. When recording the stimulation period in vivo with the cycloplegic resonator, the heartbeat signal was visible, clearly following the stimulation peak at 2 Hz. Fourier transform of the obtained results revealed that the natural heartbeat increased from approximately 60 bpm to 120 bpm (2 Hz), and the QRST complex was clearly visible. Interestingly, some fish exhibited arrhythmias, which could be corrected using the BICS stimulator. Inhomogeneous rhythms, extra beats, and absent beats were observed in the spectrum. During BICS stimulation, the arrhythmic heartbeats were synchronized, and no further signs of arrhythmia appeared. Arrhythmias recurred after the stimulation cycle ended. Controlled experiments conducted on fish without BICS highlight the necessity for the heart to receive external stimulation from BICS, thus external pulses have no effect on heartbeat.

[0265] BICS Concept and Design BICS is envisioned as a compact, bioresorbable, and injectable device that can be used as an alternative to traditional surgically implantable defibrillators and pacemakers. Designed for short-term use, especially in remote or hard-to-access areas, BICS provides a less invasive solution that can deliver temporary cardiac stimulation before permanent treatment is scheduled.

[0266] Injection and placement methods In emergency situations, BICS can be accurately placed using image guidance such as ultrasound or anatomical landmarks, minimizing the need for open surgery and allowing its use in challenging conditions such as war zones or remote areas.

[0267] Materials and Formulation Challenges Developing injectable electrodes for cardiac applications that adhere to the heart surface without interfering with cardiac function is a significant challenge. An electrode-promoting formulation (a mixture of A5 and ETE-BuSA) addresses these challenges by exhibiting high solubility, ease of injection, and the formation of a conductive structure in vivo that matches the elasticity and stiffness of cardiac tissue. Another mixture (EEE-COOH, ETE-S, and A5) demonstrates a combination of two trimers with A5, all of which have undergone electrofunctionalization and enzymatic polymerization.

[0268] Zebrafish and chicken embryo models Due to their anatomical and physiological similarities to the human heart system, zebrafish and chicken embryos have been used in in vivo studies, providing a feasible model for evaluating the efficacy and biocompatibility of BICS.

[0269] In vitro and in vivo studies Studies have shown that BICS can effectively stimulate cardiac activity, with injected proBICS forming a stable gel electrode and showing no significant damage to cardiac tissue. In vivo studies further validated the effectiveness and biocompatibility of BICS in stimulating heartbeat, with no behavioral changes observed in animals after bioabsorption.

[0270] Mechanical and electrochemical analysis Mechanical compatibility with cardiac tissue was demonstrated using parameters such as Young's modulus and stress relaxation. In vivo electrochemical analysis showed that impedance decreased and capacitance increased after electropolymerization, indicating improved material properties.

[0271] ECG and Echocardiography Studies: ECG recordings of zebrafish provide insights into cardiac activity influenced by BICS, with adjustments in heart rate observed upon electrical stimulation. Echocardiography offers a mechanical method for recording the heartbeat, confirming the findings of ECG and highlighting the ability of BICS to correct arrhythmias.

[0272] Example 4: Formation of photopolymers in conductive bioelectronic devices

[0273] background

[0274] The present invention relates to the spatial control of the formed conductive polymers or polymer electrodes. Specifically, the present invention relates to the specific targeting and improved 3D control of tissue structures. Spatially controlled polymerization is achieved using photopolymerization with or without a photomask. This disclosure provides an optimized oxidation potential for the compositions of the first aspect, enabling the photocatalytic formation of conductive bioelectronic devices in a spatially controlled manner in live zebrafish.

[0275] A5-EEE-S solution

[0276] Add 20 μL of surfactant solution to 0.8 mg EEE-S. Add 1 μL of 10 mM rose benzene (rose Bengal) from MQW to this solution. Oxygenate the solution by bubbling oxygen into it. Add the oxygenated EEE-S solution to a vial containing 0.2 mg A5. Sonicate the solution for 1 minute. The final concentrations of the solution correspond to 40 mg / ml EEE-S, 10 mg / ml A5, and 0.4 mM rose benzene. Prepare solutions fresh for each experiment and use them immediately after preparation.

[0277] Photopolymerization in microtiter plates

[0278] A solution of the trimer (2 μL, 20 mg / mL, in MilliQ water), an optional photocatalyst (1 μL, 10 mM, in MilliQ water for rose red; in DMSO for SIR-COOH), and MilliQ water were added to a 96-well microtiter plate with a black transparent bottom to obtain 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 certain period of time. The absorbance spectrum (280–1000 nm) was then recorded (Tecan SparkCyto 400).

[0279] Catalyst Support

[0280] Photopolymerization was carried out in microtiter plates according to the general method, using EEE-COONa(III-5) (20 mg / mL in MilliQ water) and 1, 4, 13, 40 and 113 mol% rose red (10 mM in DMSO).

[0281] Photopolymerization using agarose molds

[0282] Wavelength-specific photopolymerization

[0283] Using a Hamilton syringe, administer 2 μL of the trimer solution, along with 15 μL of LEEE-COONa (20 mg / mL) in MilliQ water. -1 Prepared with or without 1 μL of rose red (10 mM in DMSO), the solution was injected in two parallel lines into an agarose mold (0.5% agarose in Ringer's solution). The agarose mold was transferred to a microscope setup (Nikon ECLIPSEFN1) and, using a 4x / 0.10 Nikon objective lens, irradiated with green light for 5 minutes, followed by UV light (385 nm) for 5 minutes. Imaging was performed using the same objective lens.

[0284] The conductivity of A5 and A5-ETE-S in agarose was measured using a two-ended apparatus, in which a 25 μm Au-coated tungsten microprobe (Signatone, Gilroy, CA) was attached to a polymer embedded in the agarose. The conductivity was estimated using a transmission line model by scanning the applied potential and recording the current generated across different distances.

[0285] Space-controlled light aggregation

[0286] Add EEE-COONa (5μL, 20mg mL) -1Add 0.5% agarose in Ringer's solution to the surface of an agarose mold (in MilliQ water), and dry the solution to form a trimer thin layer. Insert two 3D-printed photomasks into the optical path (ND filter slot). Transfer the agarose mold to a microscope setup (Nikon ECLIPSE FN1), and then irradiate with UV light (385nm) for 5 minutes under mask 1, followed by 5 minutes under mask 2, using a 20x / 0.45 Nikon objective. Image was then taken using a 4x / 0.10 Nikon objective.

[0287] Patterning and electrical measurement

[0288] Add 2 μL of A5-EEE-S solution (see Preparation of A5-EEE-S Solution) to the surface of the slide. Place a 1 x 2 x 0.5 cm agarose mold on top of the trimer solution (0.5% agarose in Ringer's solution). Insert the 3D-printed photomask into the optical path (ND filter slot). Transfer the agarose mold to the microscope setup (Nikon ECLIPSE FN1) and illuminate it for 15 minutes under green light (561 nm) using a 20 x 0.45 Nikon objective. Image using 4 x 0.10 and 20 x 0.45 Nikon objectives. Remove the patterned agarose from the slide and wash with 3 ml of MilliQ water.

[0289] The agarose pattern was then placed on an interleaved Au electrode connected to a Keithley 2612B source meter (Keithley Instruments). The agarose pattern faced the Au electrode. Two interleaved electrodes were contacted using external microelectrodes. The applied voltage was scanned, and the generated current was recorded. This process was repeated for all interleaved electrode leads covered with the conductive polymer. The distance between adjacent electrodes was 15 μm, and the width was 2.5 mm.

[0290] In vitro photopolymerization

[0291] The trimer solution contains 15 μL of EEE-COONa (20 mg / mL) -1 In MilliQ water) and 1 μL of rose red (10 mM in DMSO). Tricaine (ethyl 3-aminobenzoate methanesulfonate; 0.2 mg / mL) was added prior to microinjection and photopolymerization. -1Juvenile fish (Casper mutant (Tg(elav3:GCaMP6f)) on a nacreous background) were euthanized until they ceased activity and showed no response to vibrations from tapping near a tricaine container for at least 10 minutes. The juveniles were placed laterally on a plate containing 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in E3 medium (already solidified). The plate was then transferred to a microinjection apparatus, and a 30 μm diameter capillary with a beveled tip (catalog number BM100T-15; beveled, straight, shortened + fire-polished end, from Biomedical-Instruments GMBH) containing a trimer solution was inserted into the ventricle. The total injection volume was estimated at 1 nL. After injection, the juveniles were transferred to a microscope (Nikon ECLIPSE). Using FN1, the head was irradiated with green light for 15 minutes using a 4x / 0.10 Nikon objective. The photopolymerization reaction was confirmed by bright-field imaging and UV (385nm) using the same objective.

[0292] In vitro patterning procedure on the resected brain-dura complex.

[0293] 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-dura mater complex using a 10 μL syringe (Hamilton Company). The injected sample was placed in a plate containing 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in Ringer's medium (already cured), with the injection site facing ventrally. The plate was then transferred to a photopolymerization apparatus, and a photomask was inserted into the optical path / ND filter slot of the microscope. The pattern was focused onto the sample surface and illuminated with 541 nm light for 15 minutes. Imaging was performed using a 4x / 0.10 Nikon objective.

[0294] In vivo photopolymerization - tail fin patterning / photolithography

[0295] Adult wild-type zebrafish (Danio rerio) AB were used for patterning experiments. Prior to the patterning procedure, the fish were anesthetized with tricaine medium (final concentration 0.2 mg / mL) until gill cover movement ceased and the fish did not respond to vibrations caused by tapping near the tricaine container. The anesthetized fish were placed sideways in a container of 1% agarose (Agarose, LE, analytical grade, Promega Corporation) in Ringer's medium (already hardened). A damp paper towel was placed over the fish to prevent drying, but the caudal fin was left exposed. The caudal peduncle was carefully lifted with tweezers to slide a glass plate (slide data) under the caudal fin. The caudal fin was blotted dry with a paper towel. The fish was gently lifted from the caudal peduncle using a pipette containing 3 μL of freshly prepared EEE-S:A5 mixture to apply the mixture to the fish. The pipette tip was rotated towards the caudal fin, squeezing the mixture between the caudal fin and the slide. The plate was then transferred to the photopolymerization device, and the photomask was inserted into the optical path / ND filter slot of the microscope. The pattern was focused onto the sample surface and irradiated with 550nm light for 15 minutes. After patterning, excess material was washed off the tail fin. The gills were rinsed with fresh aquarium water to revive the fish, and it was then transferred to a post-operative aquarium for observation.

[0296] result

[0297] One objective was to identify compounds of formulas (I), (II), and / or (III) that, upon photopolymerization, form conductive organic polymers while maintaining optimal water solubility and biocompatibility for in vivo applications. The compounds (i.e., trimers) and conditions were efficiently evaluated using a microtiter-based approach, employing fluorescence microscopy with an LED light source covering the visible light region from far UV to red. The far UV wavelength (385 nm) matched well with the absorbance peaks of trimers, such as ETE and EEE, in the 350–400 nm range for efficient activation. Trimer conversion and product formation were monitored by spectrophotometric analysis (absorbance scanning). However, the ETE trimer did not form the same type of product (e.g., spectra) upon light exposure compared to enzymatic conditions using HRP and H2O2. A relatively large absorbance peak similar to that of the trimer was observed at 350–400 nm, which may indicate low conversion efficiency. However, the peak did not decrease as the reaction time increased, indicating that the product still has three conjugated thiophene units, but the structural changes are unknown.

[0298] Trimers EEE-S, EEE-COONa, and EEE-PC were synthesized. Redox analysis showed that the redox potential of the EEE scaffold was indeed lower than that of ETE. Furthermore, exposure to natural light in the laboratory caused both the dry powder and aqueous solution of the EEE trimer to darken in color, indicating its high reactivity; therefore, the EEE trimer needs to be stored at -80°C. When exposed to far-UV light (385 nm), these trimers formed a deep blue-green solution within 5 minutes, a characteristic feature of PEDOT, with its spectrum overlapping with the enzymatic reaction and a broad peak around approximately 600 nm. This provides a good starting point for further evaluation, but even far-UV light is incompatible with biological systems, can damage tissues, and faces limited penetration due to light scattering and absorption. Ideally, longer wavelengths of green or red light could be used to mitigate these effects. For example, photocatalysts have been used to achieve longer wavelength photopolymerization in tissues.

[0299] Furthermore, photopolymerization of EEE-COONa under green light using a catalytic amount of rose red yielded similar results within 5 minutes as with the uncatalyst-free process using far-UV light. This is encouraging for in vivo zebrafish experiments, as reaction time is crucial. High reaction efficiency was also observed at 1% catalyst loading, with the reaction completing almost within 5 minutes, and 100% conversion was achieved for loadings of 4% and above.

[0300] Furthermore, the search for photocatalysts that can be used with red light to further improve in vivo applications is also a goal. SIR-COOH is a relatively new analogue of the commonly used fluorescent dye rhodamine, in which the bridging oxygen of rhodamine is replaced by a dimethylsilyl group. Interestingly, replacing O with Si(Me)₂ causes a 70-100 nm redshift in both excitation and emission spectra while maintaining the original brightness. Therefore, SIR-COOH exhibits an excitation peak at 650 nm in the lower range of the first NIR window in tissues (approximately 650-1000 nm). SIR-COOH has recently been used in bioorthogonal photoclick reactions for cell-based applications, further supporting its suitability for in vivo applications, but its application in oxidative photopolymerization has not been previously evaluated. SIR-COOH functions as a photocatalyst under 621 nm red light similarly to rose red, leading to the conversion of the EEE-COONa trimer. However, this reaction is less efficient than the reaction using rose red, and some trimer remains after 5 minutes. For the EEE trimer, the absorbance spectra of the enzymatic and photocatalytic reactions overlapped quite well, but an additional peak spectrum was observed at 800 nm when using SIR-COOH as a catalyst, a peak not seen when using rose red or far-UV photocatalysis without a catalyst. Further analysis of the reaction mixtures from SIR-COOH and far-UV conditions using Maldi-MS analysis revealed that dimers formed in both cases, but trimers formed only when using SIR-COOH as a photocatalyst. This was observed in both EEE-S and EEE-COONa. It is speculated that the peak at 600 nm represents the hexamer and the peak at 800 nm represents the nonamer. In this case, red light at 621 nm in the SIR-COOH catalytic reaction can excite the dimer, and SIR-COOH activates the monomer, resulting in a monomer-dimer reaction that can form the nonamer. Adding more trimer to the completed reaction and then further exposing it to red light resulted in an increased peak at 800 nm compared to the peak at 600 nm, which may support a dual activation mechanism in the SIR-COOH catalytic reaction. When the trimer concentration increased from 0.4 mg / mL to 4 mg / mL, the reaction efficiency decreased, and most of the trimer remained after 5 minutes. The trimer itself absorbs light, therefore the conversion depends on the length (i.e., cross-section) of the light path in the trimer solution, which can be well illustrated by reducing the volume in the pores, thus achieving complete conversion after 5 minutes. These preliminary solution studies indicate that EEE-PC exhibits the worst stability in aqueous solution compared to EEE-S and EEE-COONa, therefore these trimers (EEE-S and EEE-COONa) may be preferred in further photopolymerization studies.

[0301] Detailed list of implementation plans

[0302] Item 1. A composition comprising a copolymer of formula (I):

[0303]

[0304] Where n is 2-25 and m is 0-1n;

[0305] And compounds of formula (II) and / or compounds of formula (III):

[0306]

[0307] Where Z is selected from -O- or -CH2-; each y is an integer selected from 0, 1, 2, 3, and 4, and

[0308] Each R 1 It is selected from optionally substituted phosphates, optionally substituted phosphate esters and their derivatives, optionally substituted amines and optionally substituted amides.

[0309] Item 2. The composition according to Item 1 further comprises an aqueous solvent.

[0310] Item 3. The composition according to Item 2, wherein the aqueous solvent is water.

[0311] Item 4. The composition according to any one of items 1-3, wherein the compound of formula (III) is not present.

[0312] Item 5. The composition according to any one of items 1-3, wherein the compound of formula (II) is not present.

[0313] Item 6. The composition according to any one of items 1-5, wherein R 1 Choose freely substituted phosphatidylcholine, substituted phosphatidylamide, or substituted C. 1-10 Alkyl sulfonates, optionally substituted alkoxylated sulfonates, optionally substituted C 1-10 Alkylamines, optionally substituted alkoxylated amines, optionally substituted C 1-10 Alkyl carboxylic esters and optionally substituted alkoxylated carboxylic esters.

[0314] Item 7. The composition according to Item 6, wherein R 1 It is alkoxylated, while it is ethoxylated.

[0315] Item 8. The composition according to any one of items 1-5, wherein R 1 It is phosphatidylcholine.

[0316] Item 9. The composition according to any one of items 1-5, wherein R 1It is phosphatidylcholine substituted with ethoxylated azide.

[0317] Item 10. A composition according to any one of items 1-4, wherein the compound of formula (II) is selected from...

[0318]

[0319]

[0320] Item 11. A composition according to any one of items 1-3 and 5, wherein the compound of formula (III) is selected from...

[0321]

Claims

1. A composition comprising a polymer of formula (I) and one or more compounds of formula (II) or (III), in The polymer of formula (I) is represented by the following structure: One or more compounds of formula (II) or formula (III) are represented by the following structures: And among them Each A is selected from H, Na, K, Li, Ca, Mg, Sr, and Ba; E is selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl, aryl, heteroaryl, -(CH2CH2O) q CH2CH2OH, wherein C 1-6 Alkyl groups are optionally prefixed with -N3, -OH, -SO3A, or -N(C) 1-6 alkyl)2、-NH + (C 1-6 alkyl)2 and -N + (C 1-6 One or more substitutions in alkyl group 3; R1 is selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, aryl, heteroaryl, wherein the C 1-6 Alkyl groups are optionally surrounded by one or more -N(C) groups. 1-6 alkyl)2、-NH + (C 1-6 alkyl)2 and -N + (C 1-6 Alkyl)3-substituted; Each Z is selected from key, -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, heterocyclic, heteroaryl and Si(C) 1-6 Alkyl)3, the C 1-20 Alkyl, aryl, heterocyclic and heteroaryl groups are optionally substituted with one or more R5 or R6 groups; R3, R3', R4, and R4' are each selected from H and -(CH2). y -Z-(R2), When R3 and R3' are C 1-6 When alkoxy groups are present, they together with the atoms to which they are attached form heterocycles that are optionally substituted with one or more R5 or R2 groups; When R4 and R4' are C 1-6 When alkoxy groups are present, they together with the atoms to which they are attached form heterocycles that are optionally substituted with one or more R5 or R2 groups; R5 is selected from H and C. 1-6 Alkyl, C 2-6 alkenyl and C 2-6 alkynyl group; Z' is a 6- or 7-membered heterocyclic ring; Each R6 is selected from -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 group 3, -(OCH2CH2) q -(R8), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, aryl, heteroaryl, heterocyclic, ferrocene, -C(O)NH(C) 1-6 alkyl), wherein the -N + (C 1-6 alkyl)3 and -C(O)NH(C 1-6 The alkyl group is optionally substituted with one or more R7 groups, and the C2 group is... 1-6 Alkyl, heterocyclic, heteroaryl, and aryl groups are bound by one or more R7, R9, or R6 groups. 10 replace; R7 is selected from -SO3A, -CO2A, and -(OCH2CH2). q -(R8), -NH2, -NHC(O)-(R9), -OC(O)-(R9), aryl, guanidinyl, -C(O)NH(C 1-6 alkyl), wherein the guanidine group and -C(O)NH(C 1-6 Alkyl groups are optionally substituted with one or more R8 groups; R8 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, -OH, -NH2, -NH(C) 1-6 Alkyl groups, -N3, -OC(O)-(R9), and -C(O)NH(C 1-6 alkyl), wherein the C 1-6 Alkyl groups and -C(O)NH(C) 1-6 Alkyl groups are optionally substituted with one or more R9 groups; R9 is selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, heterocyclic, ferrocene, -B(OH)2, -CO2A and -CO2(C 1-6 alkyl), wherein the C 1-6 Alkyl, aryl, heterocyclic and -CO2(C 1-6 Alkyl) optionally enclosed by one or more R 10 replace; R 10 Selected from oxygen, -SO3A, -NH2, -CO2A, -OH, -P(O)(OH)2, C 1-6 Alkyl, C 1-6 Alkyl, aryl, and heteroaryl; and n is 4-32; m is 0-10; a is 1-5; y is 0-16; q is 0-15, Or its pharmaceutically acceptable salt.

2. The composition according to claim 1, wherein m is 0 or 1, and E is selected from H and C. 1-6 Alkyl group, -(CH2CH2O) q CH2CH2OH, C substituted with -N3 1-6 Alkyl groups, C substituted with -SO3A 1-6 Alkyl groups and those with -SO3A and -N + (C 1-6 alkyl)3-substituted C 1-6 Alkyl group, and R1 is selected from H, C 1-6 Alkyl groups, such as methyl groups and those with -N + (C 1-6 alkyl)3-substituted C 1-6 alkyl.

3. The composition according to claim 1 or 2, wherein m is 1, E is H, and R1 is selected from H and C. 1-6 Alkyl groups, such as methyl groups.

4. The composition according to claim 1 or 2, wherein m is 0 and R1 is selected from H and -N + (C 1-6 alkyl)3-substituted C 1-6 alkyl.

5. The composition according to any one of claims 1-4, wherein the composition comprises a polymer of formula (I) and one or more compounds of formula (II-a), (III-a), (III-b), (III-c), and (III-d) represented by the following structures. as well as Or its pharmaceutically acceptable salt.

6. The composition according to any one of claims 1-5, wherein R2 is selected from... Or its pharmaceutically acceptable salt.

7. The composition according to any one of claims 1-6, wherein the composition comprises a polymer represented by the following structure. Where p is 1-12; and one or more compounds represented by the following structures Or its pharmaceutically acceptable salt.

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

9. The composition or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, for the treatment of a disease selected from cancer, cardiovascular disease, infection or neurodegenerative disease.

10. The composition of 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, peripheral nervous system injury, 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, myocarditis, aortic aneurysm, peripheral artery disease, thromboembolic disease, and venous thrombosis; and the infection 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 1-7 in the preparation of a medicament for treating cancer, cardiovascular disease, infection or neurodegenerative disease.

12. Use of the composition of any one of claims 1-7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament 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, peripheral nervous system injury, 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, myocarditis, aortic aneurysm, peripheral artery disease, thromboembolic disease, and venous thrombosis; and the infection is selected from viral infection, bacterial infection, parasitic infection, and fungal infection.

13. Use of the composition of any one of claims 1-7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating spinal cord injury, such as spinal cord injury, wherein the medicament induces the regeneration of nerves in the spinal cord.

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

15. The method of 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, peripheral nervous system injury, 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, myocarditis, aortic aneurysm, peripheral artery disease, thromboembolic disease, and venous thrombosis; and the infection is selected from viral infection, bacterial infection, parasitic infection, and fungal infection.

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

17. A pharmaceutical composition comprising a therapeutically effective amount of the composition according to any one of claims 1-7 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 modulators.

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

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

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

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