Methods and materials for detecting and influencing the uptake and or release of biologically active substances by means of conductive hydrogels

Abstract

This invention relates to a method for detecting and influencing the absorption of bioactive substances in a hydrogel material (1) and / or the release of bioactive substances from the hydrogel material, wherein the hydrogel material is a polymer network formed by anionic and uncharged building blocks, the affinity of the polymer network for the bioactive substances can be configured by means of parameters defining the anionic building blocks, and the hydrogel material has a conductive component whose resistance and charge storage capacity depend on the interaction with the hydrogel building blocks and the bonding of the bioactive substances to the hydrogel material, wherein the conductive component is suitable for altering the anionic charge of the hydrogel material and its affinity for the bioactive substances by influencing the potential. In the method, the hydrogel material is contacted with a biofluid, wherein changes in the resistance and / or charge storage capacity of the hydrogel material are detected, and the absorption of bioactive substances into the hydrogel material or the release of bioactive substances from the hydrogel material into the biofluid is determined by means of the detected changes in resistance and / or charge storage capacity, and / or wherein the concentration of bioactive substances in the biofluid and / or in the hydrogel material is affected by the potential acting on the hydrogel material. The invention also relates to a suitable conductive hydrogel material.

Description

[0001] This invention relates to a method for influencing and detecting the absorption of bioactive substances in hydrogel materials and / or the release of bioactive substances from said hydrogel materials. The invention further relates to a conductive hydrogel suitable for performing said method.

[0002] Hydrogel-based material release and absorption systems hold great promise for applications in biotechnology, particularly in medicine, because hydrogels are similar to human physiological tissues in terms of water content and mechanical properties compared to many other biomaterials, and can enable the encapsulation and targeted release of various substances. By definition, hydrogels are highly hydrated, covalently or physically cross-linked polymers that allow substances to be reversibly bonded to the affinity polymeric building blocks of the hydrogel network via various non-covalent interactions, thereby enabling the targeted removal of these substances from biofluids or living tissues—that is, encapsulating them within the hydrogel or releasing them from the hydrogel into biofluids or living tissues. Such substances can be protein-based signaling molecules from the categories of cytokines, chemokines, hormones, neurotransmitters, and growth factors, or non-protein-based low-molecular-weight active ingredients, i.e., so-called small molecules. These small molecules perform one or more biological functions of the aforementioned protein-based signaling molecules. Alternatively, they can be non-protein-based chemical / medical active ingredients such as antimicrobial active ingredients, preservatives, and pigments. These substances are reversibly bound to affinity-imparting polymeric building blocks in the hydrogel through charge interactions and / or hydrophobic interactions or other specific chemical interactions such as hydrogen bridging. Hydrogel systems are known in the art where the physicochemical properties of the hydrogel for encapsulating and / or releasing certain substances must be fixed at formation. This is achieved through specific charge characteristics of the hydrogel network, for example, as known by WO 2018 / 162009 A2, or by selectively setting physical properties such as mesh width. It is also known that covalently coupled substances can be released in a controlled manner by enzyme triggers, light, or changing pH values. A disadvantage of such hydrogel networks is that reversible substance bonding is not possible. Therefore, controlled sequestration of substances and the resulting reduction of substances from biological fluids or tissue peripheries are impossible.

[0003] In known systems where affinity for a substance is reversibly achieved via non-covalent interactions, the affinity, and consequently the control over the encapsulation or release of the substance, is determined by its inherent network architecture (i.e., established during the formation of the hydrogel network). Subsequent modulation can only be achieved through the aforementioned external triggers such as light, pH changes, or enzyme cleavage.

[0004] In addition to inherently charged hydrogels, conductive hydrogels have also been described in biomedical research. These conductive hydrogels are primarily used as coatings for electrode materials, such as neural electrodes, to optimize their biocompatibility and charge injection. Due to their mechanical similarity to tissues and organs, the use of soft, hydrated polymeric materials produces less rejection compared to the use of metal electrodes. Furthermore, in the case of classic metal electrodes, the charge injection crucial for stimulating tissues and organs can only occur at the electrode surface. Attempts have been made to increase the surface area and thus the contact area between the electrode and the tissue fluid (biofluid) through nanostructuring, resulting in increased charge injection. Because the biofluid can permeate the entire volume of the material in the case of conductive hydrogels, the contact area is already significantly larger than that of metal electrodes of similar size without further structuring. This also results in a significantly improved charge injection. As a measure for enabling the conduction of charge into physiological solutions, the term charge storage capacity is used in the literature.

[0005] To prepare such conductive hydrogels, conductive materials such as carbon nanotubes and / or conductive polymers are used. In most cases, the conductive material is distributed in a hydrogel precursor solution and then polymerized and crosslinked in another component unrelated to the conductive material to form the hydrogel. It is worth mentioning, as a comprehensive characteristic, that the conductive component is mostly hydrophobic and therefore does not form a hydrogel.

[0006] Conductive organic polymers are increasingly used in industrial applications due to their diverse application possibilities and simple, cost-effective processing. These polymers operate according to semiconductor principles. The most important component is the conjugated π-electron system extending throughout the polymer backbone. These polymers inherently have low electrical conductivity. To generate free charge carriers and improve the molecular conductivity, charged molecules are needed to remove electrons from the polymer system (p-doping) or introduce electrons into the polymer system (n-doping). This process is called primary doping. The method used in most cases is p-doping. Doping and the resulting hydrophobic polymer interact with strongly charged hydrophilic molecules, further reducing the hydrophobicity of the polymer-dopant-complex and thus allowing these polymers to be used in aqueous solutions or hydrogels.

[0007] Besides primary doping, the arrangement of the conductive polymer chains relative to each other is also crucial. To transport charge carriers over long distances, carriers must be transferred between the conductive polymer chains. For this, the polymer chains need to be spatially close to each other while being uniformly distributed in volume. Aqueous solutions of conductive polymers and their corresponding dopants mostly form suspensions. The lack of interaction between the conductive polymer chains results in low conductivity. The required spatial proximity of the polymer chains can be achieved through structural reorganization (secondary doping), thereby further improving conductivity. This can be achieved, for example, by increasing the ion concentration and the associated charge shielding effect (characterized by Debye length).

[0008] An example of conductive poly-3,4-ethylenedioxythiophene (PEDOT) has been described by Zhenan Bao and other authors (DOI: 10.1038 / s41467-018-05222-4 and US20190390068A1). As is known from this literature, a weakly cross-linked hydrogel is first formed by physically winding a mixture of hydrophilic polystyrene sulfonate (PSS) and PEDOT (primary dopant) and using an ionic liquid (secondary dopant). The hydrogel is then mechanically stabilized by forming a pseudo-interpenetrating network of acrylate monomers through a free radical cross-linking reaction. A conductive hydrogel network is produced after polymerization (10.1038 / s41467-018-05222-4 and US20190390068A1), where the secondary polymer network serves only to stabilize and improve the mechanical properties of the material. The electrical properties of the hydrogel are derived solely from the primary non-covalent PEDOT:PSS hydrogel.

[0009] Another method for obtaining conductive hydrogels is known from US 9,299,476 B2. The method known from US 9,299,476 B2 results in a biopolymer-based hydrogel network with anionic functional groups, formed and deposited on the surface of an electrode. The primary network thus obtained is swollen or transferred into a solution containing 3,4-ethylenedioxythiophene (EDOT) monomer, and electropolymerization of the EDOT monomer to poly-3,4-ethylenedioxythiophene (PEDOT) is triggered by influencing the voltage. A pseudo-interpenetrating polymer network is generated through the ionic interaction of PEDOT with the anionic functional groups of the primary network. These anionic groups simultaneously act on PEDOT (primary dopant) in a p-doped manner and collectively produce a conductive hydrogel material. Based on the predetermined distribution of anionic groups covalently bonded in the hydrogel and the subsequent polymerization of PEDOT around the primary hydrogel network, hydrogels with high electrical conductivity can be obtained without secondary doping due to the uniform distribution of the PEDOT polymer chains.

[0010] Previously known hydrogel materials can encapsulate and / or release substances through inherently defined physicochemical characteristics, or, in the case of conductive hydrogel materials, exhibit limited moduliability and specificity for interactions with certain bioactive substances.

[0011] The object of this invention is to provide a method that can influence and determine the concentration of bioactive substances in or in the environment of a hydrogel material. Another object of this invention is to provide a conductive hydrogel whose electrical and physicochemical properties are moduloable and can enable the reversible encapsulation and / or release, particularly of substances carrying positively charged groups. Furthermore, this material should allow for the detection of substance bonding by means of characteristic changes in the resulting electrical properties.

[0012] This objective is achieved by a method having the features of claim 1 and a conductive hydrogel material having the features of claim 7. Improvements are given in the dependent claims. Use of the conductive hydrogel material is given in claims 19 to 22.

[0013] The present invention includes a method for detecting and influencing the absorption of bioactive substances in a hydrogel material and / or the release of bioactive substances from the hydrogel material, wherein the hydrogel material is defined as a polymer network formed by anionic and uncharged building blocks, the affinity of the polymer network for the bioactive substance can be configured by means of parameters defining the anionic building blocks, and the hydrogel material has a conductive component whose resistance and charge storage capacity depend on its interaction with the hydrogel building blocks and the bonding of the bioactive substance to the hydrogel material, wherein the conductive component is adapted to alter the anionic charge of the hydrogel material and its affinity for the bioactive substance by influencing the potential. In the method, the hydrogel material defined above is brought into contact with a biofluid, wherein changes in the resistance and / or charge storage capacity of the hydrogel material are detected, and the absorption of bioactive substances into the hydrogel material or the release of bioactive substances from the hydrogel material into the biofluid is determined by means of the detected changes in resistance and / or charge storage capacity, and / or wherein the concentration of bioactive substances in the biofluid and / or the concentration of bioactive substances in the hydrogel material is affected by the potential acting on the hydrogel material.

[0014] A polymer network formed by anionic and uncharged building units is an anionic polymer network. Anionic polymer networks can be configured by defining anionic building units.

[0015] For simplicity, the term "hydrogel material" will be used below to refer to conductive hydrogel materials according to the definition.

[0016] In the context of this invention, physiological solutions, cell cultures, and living tissues are understood as biofluids. Therefore, the treatment instructions for contacting hydrogel materials with biofluids can be understood as contact through immersion in physiological solutions or as contact between the hydrogel material and the surface of living tissues both in vivo and in vitro.

[0017] In the context of this invention, bioactive substances are understood to be protein-based or non-protein-based bioactive materials, active ingredients, and small molecules that possess signaling properties such as cytokines, chemokines, hormones, neurotransmitters, and growth factors, and produce other biological effects. Bioactive substances can particularly be pharmaceutically active ingredients. For the aforementioned bioactive substances, a molecular weight of 70 kDa or less is a suitable characteristic.

[0018] The method of the present invention includes detecting the absorption of bioactive substances in a hydrogel material and / or the release of bioactive substances from the hydrogel material, and influencing the absorption of bioactive substances in the hydrogel material and / or the release of bioactive substances from the hydrogel material. This allows for the detection and influence of bioactive substances, or—according to an alternative—the detection or influence of bioactive substances.

[0019] To detect changes in the resistance and / or capacitance of hydrogel materials by measuring the absorption and / or release of bioactive substances from them, this is based on the understanding that changes in the resistance and / or capacitance of hydrogel materials are influenced by the bonding of bioactive substances to the hydrogel material. Thus, the resistance of the hydrogel material increases due to encapsulation and bonding to or within the hydrogel material, while the conductivity decreases due to the loading of anionic groups with the bioactive substances. Conversely, when the bioactive substances detach from or from the hydrogel material, the resistance decreases, while the conductivity increases.

[0020] The charge storage capacity was calculated using cyclic voltammetry. For this purpose, the current between the working electrode (hydrogel material) and the counter electrode (carbon electrode) was measured in a three-electrode configuration, while the applied potential was varied from -0.6 V to 0.8 V (the potential between the working electrode and the Ag / AgCl reference electrode) over five cycles. The scan rate was 50 mV / s. The negative portion of the area under the curve was then integrated (MultiTrace 4.3, PalmSens 4), and the charge storage capacity was calculated from this integral using the following formula:

[0021]

[0022] The software outputs the integral as a numerical value I*U in units of [A]*[V]. Here, [A] = [C / s]. The charge transferred through the material [C] is obtained by dividing the scan rate [V / s]. This is then divided by the volume, because the interface with the surrounding medium involves the entire volume of the hydrogel and therefore the surface area / contact area cannot be calculated.

[0023] To detect the absorption and / or release of bioactive substances from hydrogel materials, alternatively, changes in the conductivity of the hydrogel material can be determined, whereby changes in conductivity reveal the absorption and / or release of bioactive substances. The bonding of bioactive substances in conductive hydrogel materials occurs through non-covalent interactions between the bioactive substances and the polymer chains of the hydrogel material. Ionic interactions play a particularly important role here. The doping of the conductive polymer changes through the interactions of the bonded anionic and cationic groups of the bioactive substances with the anionic polymer network of the hydrogel material and with cationic conductive polymers. Anionic groups in the bonded material can contribute to doping and improve conductivity. Conversely, cationic groups compensate for the negative charge of the anionic polymer components and thus have a detrimental effect on doping. Furthermore, the hydrophobic regions of the bioactive substances may affect the interactions between the PEDOT polymer chains. Therefore, the electrical properties of the hydrogel material vary specifically depending on the molecular type and concentration of the bonded bioactive substances.

[0024] The absorption and / or release of bioactive substances can be understood as changes in the concentration of bioactive substances in hydrogel materials. Furthermore, it can be proposed to correlate discrete conductivity values, discrete resistance values, or discrete charge storage capacity values ​​with discrete concentrations of bioactive substances.

[0025] The resistance of a hydrogel material can be determined as either a DC resistance or an impedance. For impedance detection, a frequency range of 0.01 Hz to 1 MHz can be preset. It can be proposed that, in order to detect the bonding of bioactive substances on or within the hydrogel, the impedance change of the hydrogel material be measured at least within a frequency range of 0.1 Hz to 1 MHz. Changes in charge storage capacity can also be taken into account here.

[0026] To influence the concentration of bioactive substances in biofluids and / or hydrogel materials, an electric potential is applied to the hydrogel material. By affecting the electric potential on the hydrogel material, the anionic charge of the hydrogel material and its affinity for bioactive substances are altered, thus affecting the bonding of bioactive substances in or on the hydrogel material due to the change in potential.

[0027] The prerequisite for releasing bioactive substances from hydrogel materials is that the bioactive substances have been bonded to or contained within the hydrogel materials before contact with biofluids. Therefore, it can be proposed that, in order to release the bioactive substances, the hydrogel materials be electrically or chemically loaded with a predetermined concentration of a predetermined bioactive substance before contact with biofluids.

[0028] According to one embodiment of the method of the present invention, the polymer network can be configured in terms of its composition by means of at least three parameters defining the anionicly charged building blocks, the parameters being selected from the group consisting of parameters P0, P1, P2, and P3, wherein parameter P0 is a value corresponding to the number of ionized anionic groups per volume unit of a hydrogel material swollen under physiological conditions, assuming that 30% of all anionic groups are ionized, parameter P1 is a value corresponding to the number of ionized anionic groups per volume unit of a hydrogel material swollen under physiological conditions having an intrinsic pK of less than 2.5. s The value is derived from the number of strong anionic groups, with parameter P2 corresponding to an intrinsic pK of less than 2.5 for each repeating unit. s The value is obtained by dividing the number of strongly anionic groups by the molecular weight of the repeating unit, and parameter P3 corresponds to the amphiphilic value used to describe the anionic building block, wherein the electrical resistance and / or charge storage capacity of the hydrogel material are predetermined by the parameter values ​​configured for the hydrogel material. Specific definitions and determinations of these parameter values ​​can be found in the specification below.

[0029] The interactions of bioactive substances can also be based on a substance-specific value Pp, calculated as the ratio of the net charge of the bioactive substance to its water-accessible surface area. For protein-based substances, this can be found in the Protein Database (PDB). http: / / www.rcsb.org / Any protein structure can be obtained from Delphi Web Server. The net charge of the selected protein structure is calculated using Delphi Web Server. http: / / compbio.clemson.edu / sapp / delphi_ webserver / The calculations are performed using a standard setting at pH 7. The protein surface area accessible to water is used in [the following context]. Using PyMol software at the aqueous solvent radius ( www.pymol.org The calculation is as follows: Then, Pp is calculated by dividing the net charge by the protein surface area accessible to the solvent water and multiplying by a factor of 1,000,000 to obtain a unit of 10. -6 x[1 / A 2 bzw.A -2 For non-protein-based substances, the net charge corresponding to the excess of anionic or cationic groups and the molecular surface area accessible to the solvent water, which can be derived from the chemical structure, are calculated. This molecular surface area is derived using ChemDraw 19.0 and ChemAxon MarvinSketch 19.21 software, similar to the formation process of parameter P3. The obtained value is multiplied by a factor of 1,000,000 to obtain the unit 10. -6 x[1 / A 2 bzw.A -2 ].

[0030] Loading hydrogel materials, i.e., immobilizing a predetermined concentration of bioactive substances in or on the hydrogel material, can be carried out in different ways. According to a first method, also referred to as a first loading method, one or more bioactive substances set for bonding at a predetermined concentration are mixed with anionicly charged hydrogel building units and simultaneously integrated into a polymer network. Then, in the subsequent formation of a conductive hydrogel material by incorporating conductive components, the bioactive substances have already been contained in the polymer network at a predetermined concentration. Advantageously, in this manner, the total amount of this or these bioactive substances is quantitatively contained in the hydrogel material after the formation of the polymer network. In other words, the loading of this or these bioactive substances is carried out independently of parameters P0, P1, P2, P3 and the substance-specific value Pp. The dominant reaction conditions during the formation of the hydrogel material may have an adverse effect on the structure of the bioactive substances; therefore, not all bioactive substances are suitable for immobilization according to the first loading method.

[0031] According to the second method, also known as the second loading method, a conductive hydrogel material is formed with predetermined parameter configurations and values, and then the conductive hydrogel material is contacted with an aqueous solution or biofluid having a predetermined substance concentration as a loading solution for a predetermined time period. Here, depending on parameters P0, P1, P2, and P3, the bioactive substance in solution is absorbed from the aqueous solution or biofluid and bonded to the hydrogel material. The loaded hydrogel material is then removed from the loading solution. Then, by influencing the potential, the bioactive substance immobilized on or therein in the hydrogel material can be released into the surrounding environment, preferably the biofluid or living tissue. Here, by influencing the potential, it is further possible to encapsulate the bioactive substance from the surrounding environment of the hydrogel material into the hydrogel material.

[0032] According to a third method for loading, also known as the third loading method, a predetermined conductive hydrogel material with predetermined parameter configurations having predetermined parameter values ​​is contacted with an aqueous solution or biofluid having a predetermined substance concentration as a loading solution for a predetermined time period and simultaneously exposed to the effect of potential. Here, depending on the predetermined parameter values, the bioactive substance is absorbed (encapsulated) from the loading solution into the hydrogel material. It may be necessary to maintain the potential acting on the hydrogel material so that the bonding of the bioactive substance remains constant. Otherwise, when the potential or current changes, the bioactive substance may be released from the hydrogel material. Importantly, the encapsulation or release of the bioactive substance is based not only on the potential but also on the affinity of the hydrogel material for certain bioactive substances, predetermined by parameters P0, P1, P2, P3, and Pp.

[0033] It has been shown that the electrical conductivity of hydrogel materials, the structural formation and distribution of conductive components within the hydrogel material, can be influenced by the parameter values ​​configured in the parameter settings. Therefore, it is proposed to define a hydrogel material having a predetermined electrical conductivity based on a predetermined parameter configuration with predetermined parameter values.

[0034] This method can be further proposed to apply a potential in the range of 1 mV to 1000 mV, preferably 400 mV to 600 mV (for the Ag / AgCl reference electrode), to the hydrogel material in order to absorb the bioactive substance. To release the bioactive substance, a potential in the range of -1 mV to -1000 mV, preferably -400 mV to -600 mV (for the Ag / AgCl reference electrode), can be applied to the hydrogel material.

[0035] To absorb bioactive substances, a constant current greater than 0 mA can be applied to the hydrogel material, wherein the direction of current flow is changed to release the bioactive substances.

[0036] The present invention further includes a conductive hydrogel suitable for performing the above-described methods. The hydrogel material of the present invention has a polymer network formed by anionic and uncharged building units, or is composed of such a polymer network, and the polymer network can be configured in terms of its composition by means of at least three parameters defining the anionic building units, the parameters being selected from the group consisting of parameters P0, P1, P2, and P3, wherein parameter P0 corresponds to a value derived from the number of ionized anionic groups per volume unit of the hydrogel material swollen under physiological conditions, assuming that 30% of all anionic groups are ionized, and parameter P1 corresponds to a value derived from the number of ionized anionic groups per volume unit of the hydrogel material swollen under physiological conditions having an intrinsic pK of less than 2.5. s The value is derived from the number of strong anionic groups, with parameter P2 corresponding to an intrinsic pK of less than 2.5 for each repeating unit. s The value is obtained by dividing the number of strongly anionic groups by the molecular weight of the repeating unit, and parameter P3 corresponds to the amphiphilic value used to describe the anionic building block. The hydrogel material also has a conductive component incorporated into the polymer network, wherein the conductivity, resistance, and / or charge storage capacity of the hydrogel material can be predetermined by parameter values ​​configured in the parameters of the hydrogel material.

[0037] The parameters P0 to P3 are defined in detail using the following process:

[0038] Parameter P0: The value of P0, given in μmol / ml, corresponds to 30% of the total number of anionic groups relative to the volume of the hydrogel swollen under physiological conditions (0.154 mmol / L NaCl, pH buffered to 7.4). It can be calculated, for example, from the polymer concentration of the hydrogel building blocks under swollen conditions in physiological solution.

[0039] Parameter P1: The value of P1, which can be given in μmol / ml, corresponds to the number of strong anionic groups with a pKs value of less than 2.5 relative to the volume of the hydrogel that swells under physiological conditions (0.154 mmol / l NaCl, pH buffered to 7.4).

[0040] Parameter P2: The value of P2, given in mmol / (g / mol), corresponds to the number of strong anionic groups with a pKs value of less than 2.5 in each anionic building block, divided by the corresponding molecular weight of the anionic building block.

[0041] Parameter P3: The parameter P3, describing the amphiphilicity of the anionic building block, is calculated by dividing the octanol / water partition coefficient (LogP value) of the anionic building block by the surface area accessible to water in the solvent of the anionic building block. This can be performed using the software ChemDraw 19.0 and ChemAxon MarvinSketch 19.21 as follows: Using ChemDraw 19.0, each anionic building block with a polymer backbone length of 22 carbon atoms, or, in the case of a sugar-based structure, each anionic building block with a total of 2 disaccharide units, is drawn as a complete chemical structure. Subsequently, using the software ChemAxon MarvinSketch 19.21, the octane / water partition coefficient (LogP value) is calculated by reading the structural formula drawn by ChemDraw 19.0, and... The solvent radius is used to calculate the accessible surface area of ​​the solvent water. The obtained value is multiplied by a factor of 1000 to obtain the unit 10. -3 x[1 / A 2 bzw.A -2 ].

[0042] The hydrogel material of this invention is an important component for carrying out the method of this invention. The features involved in the hydrogel of this invention can therefore be used to explain the method of this invention in detail, and in particular to define the hydrogel material, and vice versa. The hydrogel material of this invention is based on a polymer network with anionic groups, the polymer network being defined in terms of its properties by parameters P0, P1, P2, and P3. The electrical properties of the conductive hydrogel material formed by the (pseudo) interpenetrating network created by the conductive components are controlled by these parameters P0 to P3. Advantageously, the charge properties of the conductive hydrogel material can be modulated in a continuously varying manner by applying a potential or by influencing the potential, wherein the modulation of the charge properties in relation to parameters P0, P1, P2, and P3 controls the affinity of the conductive hydrogel material for bioactive substances, and thereby allows for the continuous, reversible, and real-time regulation of the reduction (encapsulation) of substances from the biofluid in contact with the conductive hydrogel or the release of bioactive substances from the hydrogel material into the biofluid, directly dependent on the applied potential.

[0043] By using various charged building blocks with anionic groups and varying their concentrations in the hydrogel material, as well as the number and density of strong anionic groups along the polymer chains of uncharged building blocks, hydrogel materials with different configurations having the same degree of crosslinking and solids content can be synthesized according to parameters P0-P3. To produce conductive hydrogel materials, PEDOT polymer is preferably chemically polymerized around the primary hydrogel network as a pseudo-interpenetrating network, serving as the conductive component. Depending on the configured parameters or parameter values ​​and their configurations, the resulting hydrogel materials differ in their electrical properties and in the encapsulation and release of bioactive substances.

[0044] Compared to parameter P1, parameter P0, which describes all actually ionized anionic groups in the swollen hydrogel regardless of the intrinsic pKa, exerts a slightly lower influence on the electrical properties of the resulting hydrogel material. The weak anionic groups included in parameter P0 are not effective dopant for PEDOT, thus affecting conductivity or resistance compared to parameter P1, which describes strongly acidic groups with pKa < 2.5. Conversely, the overall number of strongly negatively charged anionic groups (P1) directly affects conductivity or resistance. Minimal conductivity is achieved in completely undoped hydrogel materials (P1 = 0) or in the absence of conductive components. The greater the number of strongly charged anionic groups in the hydrogel material, the higher the conductivity and the lower the resistance. This is because p-doping of PEDOT generates ionized charge carriers. The greater the number of dopant cells, the more ionized charge carriers can be formed. A similar direct influence on the electrical properties can be expected for the local charge density (P2) of the strongly anionic groups. Reducing the local charge density while keeping P0 and P1 constant leads to a decrease in the conductivity of the hydrogel material. This is due to the local electrostatic interaction between the negatively charged polymer and the positively charged PEDOT polymer chains. Too low a local negative charge results in a weak interaction between the positively charged PEDOT polymer chains and the anionic polymer or anionic building blocks. This makes doping more difficult, leading to lower conductivity. Therefore, even with moderate P1 values ​​and very small P2 values, an increase in conductivity relative to undoped hydrogel materials may not be observed. Similarly, the interaction between the amphiphilic anionic polymer and the hydrophobic conductive polymer (PEDOT) can be configured using parameter P3. Increased conductivity can be achieved even at significantly small P1 values ​​through the increased hydrophobicity of the anionic building blocks. This is because the hydrophobic PEDOT units have a high affinity for the hydrophobic groups on the anionic building blocks, which advantageously influences doping. In addition, the hydrophobic groups during PEDOT synthesis facilitate better penetration of the monomer unit (EDOT) into the polymer network and thus make better insertion of the PEDOT chain easier.

[0045] For the encapsulation and release of substances that depend on voltage or current, the overall (P0 or P1) and local charge density (P2) play a primary role throughout the hydrogel material under different applied voltages / currents. In the absence of applied voltage, the positive charge of PEDOT compensates for a portion of the negative charge of the anionic building blocks. Thus, negatively charged molecules can be bonded to a greater extent compared to hydrogel materials composed of polymer networks without conductive components. Bonding of positively charged bioactive substances is also possible depending on the affinity of the bioactive substance for anionic polymers (i.e., for anionic building blocks). The ratio of the positive charge of PEDOT to the anionic groups of the charged building blocks can be set under constant PEDOT polymerization conditions by configuring parameters P0, P1, P2, and P3. This allows for the encapsulation (absorption) of either positively or negatively charged bioactive substances. Furthermore, by applying voltage, the charge of PEDOT can be set from neutral to 66% positive charge per monomer unit. This allows for continuous variation in the setting of the bonding. The release of the bonded bioactive substances can also be determined by the configuration parameters P0, P1, P2, and P3 of the polymer network, which are associated with the charge of PEDOT (i.e., the conductive component). Furthermore, the charge of PEDOT can be continuously varied by applying voltage or current. This allows for the continuous variation of the release of various charged bioactive substances, either decreasing or increasing.

[0046] The anionic building blocks may be selected from the group consisting of: poly(acrylic acid-co-4-acrylamidomethylbenzenesulfonic acid), poly(acrylic acid-co-acrylamidoethanesulfonic acid), poly(acrylic acid-co-acrylamidoethane hydrogen sulfate), poly(4-styrenesulfonic acid-co-maleic acid), sulfated glycosaminoglycans, especially heparin, selectively desulfated heparin derivatives, heparan sulfate, chondroitin sulfate, keratin sulfate, and dermatan sulfate. The uncharged building units can be polymers containing amino or thiol groups or crosslinking agent molecules with at least two amino or thiol groups, wherein the anionic and uncharged building units are crosslinked to the polymer network, which can be achieved by activating poly(acrylic acid-co-4-acrylamidomethylbenzenesulfonic acid) and / or poly(acrylic acid-co-acrylamidoethanesulfonic acid) and / or poly(acrylic acid-co-acrylamidoethane hydrogen sulfate) and / or poly(4-styrenesulfonic acid-co-maleic acid) and / or sulfated glycosaminoglycans, especially heparin, with EDC / sulfonated NHS. And / or selectively desulfated heparin derivatives and / or heparan sulfate and / or chondroitin sulfate and / or keratin sulfate and / or dermatan sulfate carboxyl groups, and directly crosslinked with the polymer containing amino groups or the crosslinking agent molecule having at least two amino groups in the case of forming an amide, or functionalized with activated carboxyl groups by means of a bifunctional crosslinking agent molecule, the bifunctional crosslinking agent molecule having an amino group and a group capable of Michael addition, and subsequently crosslinked with the polymer containing thiol groups or the crosslinking agent molecule having at least two thiol groups by Michael addition, respectively.

[0047] The electrical impedance of the hydrogel material of the present invention, measured at a frequency of 0.01 Hz, can vary from 150 Ω to 10 Ω. Preferably, the parameter values ​​of the parameter configuration are selected to achieve an impedance of 30 Ω. A preferred impedance value of 30 Ω is achieved when the parameter configuration has the following parameters P1, P2, and P3: P1 = 110 μmol / ml, P2 = 4.5 mmol / (g / mol), and P3 = -5.9 A. -2 An additional advantageous impedance of 63Ω was achieved when the parameters P1, P2, and P3 were configured as follows: P1 = 12 μmol / ml, P2 = 4.5 mmol / (g / mol), and P3 = 6.8 A. -2 .

[0048] The charge storage capacity of the hydrogel material of the present invention can vary in the range of 900 mC / ml to 4000 mC / ml. Preferably, the parameter values ​​of the parameter configuration are selected to achieve a charge storage capacity of 3040 mC / ml. A preferred charge storage capacity of 2480 mC / ml is achieved when the parameter configuration has the following parameters P1, P2, and P3: P1 = 110 μmol / ml, P2 = 4.5 mmol / (g / mol), and P3 = -5.9 A. -2 When the parameters P1, P2, and P3 are configured as follows, an additional advantageous charge storage capacity of 3040 mC / ml is achieved: P1 = 12 μmol / ml, P2 = 4.5 mmol / (g / mol), and P3 = 6.8 A. -2 .

[0049] The conductive component may be a π-conjugated conductive polymer or a polymer composed of polypyrrole, polyaniline, polythiophene and / or poly(3,4-ethylenedioxythiophene) (PEDOT).

[0050] An improved embodiment of the conductive hydrogel according to the invention may be proposed in which the polymers comprising amino and thiol groups as uncharged building units are selected from the following categories: polyethylene glycol (PEG), poly(2-oxazoline) (POX), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) and / or polyarylamide (PAM), and the crosslinking agent molecules comprising amino or thiol groups are non-polymeric bifunctional crosslinking agent molecules.

[0051] Alternatively, it can be proposed to use polymers with conjugated, enzymatically cleavable peptides as uncharged building blocks for forming polymer networks, wherein the peptides have lysine or cysteine ​​as reactive amino acids in the peptide sequence. Enzymatically cleavable peptides can be cleaved by human or bacterial proteases, particularly MMPs, cathepsins, elastases, Staphylococcus aureus metalloproteinases (Aureolysin), and / or thrombin.

[0052] Another advantageous improvement to the conductive hydrogel according to the invention may propose that bioactive and / or anti-adhesion molecules having amino or carboxyl groups and / or cellularly beneficial peptides are covalently bonded to the hydrogel network via lysine or cysteine ​​residues in the sequence of the anionicly charged building units poly(acryloyl-co-4-acrylamidomethylbenzenesulfonic acid) and / or poly(acryloyl-co-acrylamidoethanesulfonic acid) and / or poly(acryloyl-co-acrylamidoethane hydrogen sulfate) and / or poly(4-styrenesulfonate-co-maleic acid) and / or sulfated glycosaminoglycans such as heparin and / or selectively desulfated heparin derivatives and / or heparan sulfate and / or chondroitin sulfate and / or keratin sulfate and / or dermatan sulfate or their derivatives. The bioactive molecules may be antimicrobial substances such as antibacterial agents or preservatives, or pharmaceutical active ingredients.

[0053] Preferably, the conductive hydrogel material is configured with parameters P0, P2, P3 or parameters P1, P2, P3. These parameter values ​​can vary within a predetermined range. The value of parameter P0 can be preset to the range of 0 to 80 μmol / ml, the value of parameter P1 can be preset to the range of 0 to 150 μmol / ml, the value of parameter P2 can be preset to the range of 0 to 10 mmol / (g / mol), and the value of parameter P3 can be preset to -7 x 10⁻⁷ mmol / (g / mol). -3 Up to 7x 10 -3 A -2 Within the range.

[0054] The hydrogel material of the present invention can preferably have a storage modulus of 0.2 kPa to 22 kPa.

[0055] The conductive hydrogel material of the present invention can be proposed for use in vivo for element management to control angiogenesis in the treatment of immune diseases, cancer, diabetes, neurodegenerative diseases, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, skin wounds, and bone regeneration. Another use or application of the conductive hydrogel material is in the electrical stimulation of cells or tissues.

[0056] In addition, the hydrogel material of the present invention can be used to specifically purify proteins from cell lysis products of microbial or eukaryotic origin.

[0057] Furthermore, the hydrogel material of the present invention can be used for in vitro cell and organ culture of induced pluripotent stem cells (iPS) and other stem cells and precursor cells that are not iPS, primary cells obtained from patients, immortalized cell lines, and heart tissue, muscle tissue, kidney tissue, liver tissue and nerve tissue.

[0058] The core of this invention is to provide a hydrogel of the invention, which, as an important feature, carries anionic (i.e., negatively charged) groups under physiological conditions (ionic strength and pH). The physicochemical properties of the hydrogel material of this invention are described here by the parameters P0, P1, P2, and P3, particularly the (anionic) charge characteristics that determine the inherent affinity for substances and the chemical environment of the anionic group that determines nonionic interactions. In addition to the properties already described, the physical properties of the hydrogel material of this invention, such as swelling, stiffness, and mesh width, can also be altered or affected by influencing the potential. Here, the presence of covalent bonding signals can be graded over a wide range and largely independent of parameters P0-P3.

[0059] To prepare the hydrogel material of the present invention, a predetermined native polymer network having a predetermined parameter configuration with predetermined parameter values ​​is swollen in a solution containing a conductive component, and subsequently polymerized or cross-linked to form a conductive polymer network. The native polymer network and its anionic groups act on the conductive component via electrostatic interactions in a doping manner. According to the present invention, the prepared hydrogel material consists of one or more conductive polymer systems that interact with the predetermined native polymer network via physical interactions. Surprisingly, it has been shown that the structure and distribution of the conductive component can be influenced by parameters P0-P3 and, consequently, by electrical properties such as resistance, conductivity, and charge storage capacity of the hydrogel material, as well as mechanical and physicochemical properties. Furthermore, by influencing the potential (i.e., by applying a voltage to the conductive component), the charge characteristics of the polymer network and thereby the affinity for bioactive substances can be modulated continuously, reversibly, and in real time.

[0060] Other advantageous properties are derived from the unexpected effects of combining the electrical stimulation of cells with the release or encapsulation of bioactive substances using the hydrogel material of the present invention. Furthermore, the mechanical properties of the material can be altered in real time depending on parameters P0 to P3 by modulating the current.

[0061] The preparation of the hydrogel material of the present invention will be explained in detail below with the aid of examples:

[0062] To electrically functionalize the hydrogel material, a conductive polymer, poly-3,4-ethylenedioxythiophene (PEDOT), was polymerized as the conductive component around a predetermined native polymer network. For this purpose, the entire swollen polymer network was first cultured for 3 hours at room temperature in a solution consisting of 0.4 M ammonium persulfate (APS) dissolved in 1 M HCl. Subsequently, it was cultured for 6 hours at room temperature in 0.4 M 3,4-ethylenedioxythiophene (EDOT) in mineral oil. During this second culture step, oxidative polymerization of PEDOT was performed through the native polymer network. SPH-PEDOT was obtained and subsequently washed in mineral oil, hexane, and PBS. Compared to PEDOT electrode sites, electrodes are not required due to the method used. Furthermore, it allows for the production of solids of any volume. Conductive.

[0063] Following oxidative polymerization, PEDOT inherently possesses a positive charge (see 10.1021 / acs.jpcb.9b01745, 10.1021 / acsapm.8b00061). A pseudo-interpenetrating network is formed between the anionic polymer network and PEDOT through non-covalent interactions between PEDOT and the anionic building blocks of the hydrogel, as well as non-covalent interactions (most likely hydrophobic interactions) between individual PEDOT chains. Simultaneously, the negative charge of the anionic polymer network serves as dopant for PEDOT. The doping degree of PEDOT varies depending on the overall and local charge density defined by parameters P0 or P1 and P2. This directly affects the electrical properties of the conductive hydrogel material. Since PEDOT is a hydrophobic polymer, the distribution and cross-linking of PEDOT chains are heavily dependent on the hydrophobicity of the surrounding environment (defined by parameter P3), which also influences the electrical properties of the hydrogel material.

[0064] In addition to releasing charged molecules, conductive hydrogels with different electrical properties can be obtained by varying the doping degree according to predetermined values ​​of P1, P2, and hydrophobicity P3. High doping yields hydrogel materials with high conductivity. These materials can be used as biocompatible electrodes. Due to the mechanical properties of hydrogel materials that are very similar to biological tissue, the rejection response of organisms to SPH-PEDOT electrodes can be reduced compared to classic metal electrodes. Furthermore, in classic metal electrodes, charge transfer for electrical stimulation occurs only at the interface between the metal and the tissue (physiological solution). Due to the distribution of conductive components within the hydrogel material and the possibility of physiological fluid diffusing into the hydrogel material, charge transfer can occur within the volume of the hydrogel material. Therefore, advantageously, a smaller voltage is required for the same charge injection, resulting in less heat generation and thus tissue protection.

[0065] The hydrogel material of the present invention can be combined with the method of the present invention to achieve sustained release of active ingredients into biological fluids or living tissues.

[0066] Further details, features, and advantages of the present invention will become apparent from the subsequent description of embodiments with reference to the accompanying drawings and tables. In the drawings:

[0067] Figure 1 : A schematic diagram illustrating the hydrogel material of the present invention.

[0068] Figure 2 : Another schematic diagram used to further illustrate the invention.

[0069] Figure 3 Table 1: A summary of the charged and uncharged hydrogel building blocks of the polymer network.

[0070] Figure 4 Table 2 shows the composition and synthesis of the polymer network used as a precursor to the hydrogel material.

[0071] Figure 5 Images showing the visual appearance of conductive porous and non-porous hydrogel materials in cross-section.

[0072] Figure 6 Table 3 shows the electrical properties of different conductive hydrogel materials.

[0073] Figure 7 Table 4 shows the bonding of bioactive substances through conductive hydrogel materials.

[0074] Figure 8 Table 5 shows the release of bioactive substances through conductive hydrogel materials at different potentials, and...

[0075] Figure 9 Table 6 shows the biocompatibility of the conductive hydrogel material with respect to PC12 cells and different neural cell types.

[0076] Anionicly charged hydrogel building blocks are abbreviated as GB in the following text, where building blocks carrying different anions are additionally characterized by numbers. Uncharged building blocks of hydrogel materials are abbreviated as UGB, where different uncharged building blocks are characterized by numbers. For simplicity and space saving, hydrogel materials are referred to as hydrogels in the table. Hydrogel material types are referred to as hydrogel types in the table.

[0077] Figure 1 A schematic diagram of the conductive hydrogel material 1 used to illustrate the present invention is shown. Figure 1Image A exemplarily illustrates a polymer network 2 synthesized from anionic building blocks 3 and crosslinking agent molecules 4 as a template for forming the hydrogel material 1 of the present invention. A parameter configuration having parameters P1, P2, and P3 is predetermined for the polymer network 2. Image B shows the structure of the conductive hydrogel material 1 as a pseudo-interpenetrating polymer network (IPN) formed from the polymer network 2 and a conductive component 5. The polymer network 2 carries anionic groups and is determined by parameters P1, P2, and P3. The conductive hydrogel material 1 is produced by polymerization or crosslinking of the polymer network 2 with the conductive component 5. Figure 1 Image C exemplarily illustrates the doping of conductive component 5. In this embodiment, conductive component 5 is PEDOT: doped through interaction with sulfate / sulfonic acid groups in the anionic polymer network 2. The overall charge density P1, local charge density P2, and hydrophobicity P3 of the polymer carrying anionic groups in the polymer network 2 play a decisive role in the interaction with PEDOT 5 and the resulting electrical properties of the conductive hydrogel material 1. Image D exemplarily illustrates the effect of applied potential on the conductive hydrogel material. The positive charge of PEDOT can be continuously varied by influencing the potential. Here, it is varied from neutral (0) through moderate (+1) positive charge to strong (+3) positive charge.

[0078] Figure 2 Another schematic diagram is shown to further illustrate the invention. A conductive hydrogel material 1 is shown, which is formed from sulfated and sulfonated polymers as charged building units 3, PEG as a crosslinking agent 4 (forming a polymer network 2), and PEDOT as a conductive component 5 bound within the polymer network. A positively charged signal protein bonded to the negatively charged PEDOT is shown on the right side, indicated by reference numeral 6. The bonding characteristics of the signal protein 6 in the hydrogel material 1 can be influenced by affecting the electrical potential. To electrically influence the hydrogel material 1, the hydrogel material 1 is electrically contacted with a voltage source 7. The hydrogel material 1 of the present invention can achieve electrodynamic modulation of specific electrostatic interactions between the hydrogel polymer and the signal protein 6. Example:

[0079] Synthetic polymer networks

[0080] To synthesize the hydrogel material 1, three anionic polymer network systems 2 were used: (1) a system formed by two hydrogel building units, namely an anionic building unit (GB1-5) with maleimide groups and an uncharged building unit (UGB2) with thiol groups (hereinafter referred to as the maleimide-thiol two-component system) or (2) a system formed by three hydrogel building units (an anionic building unit (GB1-5) with maleimide groups and an uncharged building unit (UGB1) with maleimide groups and an uncharged building unit (UGB2) with thiol groups (hereinafter referred to as the maleimide-thiol three-component system) or (3) a system formed by two hydrogel building units, which was formed by EDC / NHS-based activation of the carboxyl group of the anionic hydrogel building unit (GB1) and crosslinking with the amino group on the uncharged second building unit (UGB3) (hereinafter referred to as the EDC-NHS system).

[0081] The properties of anionic hydrogel building units (GB1 to GB5) and uncharged hydrogel building units (UGB1 to UGB3) can be derived from... Figure 3 As can be seen from Table 1.

[0082] A maleimide-thiol two-component system was used to synthesize anionicly charged electrolytes that serve as precursors for the hydrogel material of this invention. Sexual polymer network

[0083] GB1 (heparin-maleimide, 15 kDa) and UGB2 (star-shaped thiol-functionalized polyethylene glycol, starPEG, 10 kDa) were dissolved at a concentration of 0.0015 mol / L each in 0.1x phosphate-buffered saline (PBS, pH 6). 1x PBS consisted of 137 nm NaCl, 2.7 mM KCl, and 12 mM total phosphate (from HPO4). 2- It consists of (and H2PO4). The mixing ratio and concentration can be determined from... Figure 4 As shown in Table 2, all subsequent steps up to the mixing of the hydrogel building blocks were performed on ice. (The pH was set to achieve a gelation time of 30 minutes.) For a molar ratio of 1 for the two building blocks, equal volumes of the two solutions were mixed by means of a dropper and / or in a mixer. The mixture was then centrifuged to remove air bubbles, and the sample was dropped onto a gold electrode (11 mm diameter, 100 μl) or a coverslip (8 mm diameter, 67 μl) and covered with a hydrophobic coverslip of 11 mm or 8 mm in size. (For Sale) Coverage. Crosslinking of the hydrogel occurs via the reaction of thiol groups with maleimide groups (Michael addition reaction). Polymerization is carried out in a humid chamber at room temperature for at least 30 minutes to avoid gel drying. For cryogelation, the sample is polymerized overnight at -15°C. The fully polymerized hydrogel is then swollen overnight in 1x PBS (0.154 mmol / L NaCl, pH 7.4). The solids content of the gel is approximately 3% (m / v).

[0084] A maleimide-thiol three-component system was used to synthesize a polymer network that serves as a precursor for the hydrogel material of this invention.

[0085] To accurately set the overall negative charge in the hydrogel material, a three-component system was used. Star-shaped PEG-thiol, star-shaped PEG-maleimide, and maleimide-functionalized anionic hydrogel building block polymers were dissolved in 0.01x PBS at predetermined concentrations and mixed in different ratios depending on the desired overall charge. The mixing ratio can be determined from... Figure 4 As shown in Table 2, the gelation time was set to less than 5 minutes by adding 0.01M HCl or 0.01M NaOH. The solutions were then mixed and samples were generated at room temperature (30 minutes) or 15°C (overnight), similar to the two-component system. The fully polymerized hydrogel was swollen overnight in 1x PBS (pH 7). The solids content of the gel was approximately 3% (m / v).

[0086] The characteristics of uncharged and anionic building blocks in Figure 3 As shown in Table 1.

[0087] The EDC / NHS system was used to synthesize the polymer network that serves as a precursor for the hydrogel material of this invention.

[0088] For the EDC / NHS system, with a molar ratio of UGB3 star-shaped PEG-amine to unmaleiminated GB1 of 2, 0.167 mg / μl of UGB3, 0.145 mg / μl of GB1, 0.1 mg / μl of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), and 0.1 mg / μl of NHS (N-hydroxysulfosuccinimide) were dissolved in ice-cooled MilliQ water. To obtain a final gel volume of 1 ml, 85.20 μl of EDC solution and 48.25 μl of NHS solution were first added dropwise to 533.22 μl of heparin solution, mixed in a laboratory mixer (vortexed), and incubated on ice for 15 min. Then, 333.33 μl of star-shaped PEG solution was added and remixed by vortexing. The desired gel was then produced directly, exactly as with the two-component system. Polymerization was carried out overnight in a humid chamber at room temperature or -15°C to prevent the hydrogel from drying out. With a final star-shaped PEG concentration of 0.00784 mol / L and a final heparin concentration of 0.00392 mol / L, the total solids content of the gel was approximately 10%. The fully polymerized hydrogel was swollen overnight in 1x PBS (pH 7).

[0089] Synthesis of conductive hydrogel materials as pseudo-interpenetrating networks

[0090] The synthesis of conductive pseudo-interpenetrating polymer networks is possible for all hydrogel materials with the aforementioned formation process. To synthesize a conductive pseudo-interpenetrating poly-3,4-ethylenedioxythiophene (PEDOT) network with PEDOT as the conductive component, in the first step, the fully swollen, anionicly charged polymer network was cultured for 3 hours at room temperature in a 0.4 M ammonium persulfate (APS) solution in 1 M HCl. Subsequently, the hydrogel was cultured for 6 hours in a 0.4 M 3,4-ethylenedioxythiophene (EDOT) solution in mineral oil in a rotary shaker. During this time, EDOT oxidatively polymerized into a conductive PEDOT network, and the network became increasingly black as the polymerization of PEDOT progressed. Figure 5 The prepared hydrogel was then washed overnight in mineral oil to remove unreacted monomers. To remove the mineral oil, the hydrogel was then washed in hexane. This washing process can be repeated. The hydrogel was then washed in PBS (pH 7.4) for at least 24 hours.

[0091] All hydrogel material types are summarized in Figure 4Table 2 shows the concentrations given in the finished gel mixture. Hydrogel material types GB1-UGB2 16, GB1-UGB2 18, GB2-UGB2-UGB1 19, GB2-UGB2-UGB1 20, GB3-UGB2-UGB1 21, and GB4-UGB2-UGB1 22 are hydrogels that are not functionalized with PEDOT. Hydrogel material type UGB1-UGB2 15 is a pure PEG-PEG hydrogel without anionic charge, but it has been functionalized with PEDOT.

[0092] Electrical characterization

[0093] Electrical characterization of all hydrogels was performed in a three-electrode setup using impedance spectroscopy or cyclic voltammetry in 100 ml of 1xPBS (pH 7). For both assays, 100 μl gel samples (unswollen volume) were prepared on a gold mesh electrode used as the working electrode. A porous carbon electrode (BioLogic A-010530) with a significantly larger surface area compared to the working electrode was used as the counter electrode. An Ag / AgCl electrode (Metrohm, part number 6.0726.100) was used as the reference electrode. A potentiometer (Metrohm Autolab PGSTAT204 or PalmSens 4) was used as the measuring instrument.

[0094] Impedance spectrum

[0095] To obtain the impedance spectrum, a 10 mV voltage is applied. rms The effective potential is in the range of 0.01 Hz to 10 Hz. 5 Impedance and phase angle were measured at 10 measurement points every ten decathos (Hz) within the frequency range. Impedance of different hydrogel material types was compared at a frequency of 0.01 Hz. Conductivity is inversely proportional to impedance; increased impedance corresponds to decreased conductivity.

[0096] Cyclic voltammetry

[0097] The charge storage capacity was calculated using cyclic voltammetry. For this purpose, the current between the working electrode (hydrogel) and the counter electrode (carbon electrode) was measured using the aforementioned three-electrode structure, with the applied potential varying from -0.6 V to 0.8 V (the potential between the working electrode and the Ag / AgCl reference electrode) over five cycles. The scan rate was 50 mV / s. The negative portion of the area under the curve was then integrated (MultiTrace 4.3, PalmSens 4), and the charge storage capacity of the hydrogel was calculated from this integral using the following formula:

[0098]

[0099] The software outputs the integral as a numerical value I*U in units of [A]*[V]. Here, [A] = [C / s]. The charge transferred through the material [C] is obtained by dividing by the scan rate [V / s]. This is then divided by the volume, since the interface with the surrounding medium exists throughout the entire volume of the hydrogel and therefore the surface area cannot be calculated. The value is given as charge per milliliter of hydrogel.

[0100] Electrical properties of hydrogel materials

[0101] Following oxidative polymerization, PEDOT inherently possesses a positive charge (10.1021 / acs.jpcb.9b01745, 10.1021 / acsapm.8b00061). Through non-covalent interactions between PEDOT and the anionic polymer network (hydrogel), as well as non-covalent interactions between individual PEDOT polymer chains, a pseudo-interpenetrating network is formed between the anionic polymer network and PEDOT. Simultaneously, the negative charge of the anionic polymer network serves as p-doping for PEDOT. The doping degree of PEDOT varies depending on the overall and local charge density (parameters P0 / P1 and P2). This directly affects the electrical properties (impedance and charge storage capacity) of the conductive hydrogel material. Under the same conditions of P2 and P3, charge storage capacities of 3040 to 1505 mC / ml and impedances of 30 to 93 Ω were obtained for hydrogel material types GB1-UGB2 01 and GB1-UGB2-UGB1 02-04 in the P1 range of 110 to 2 μmol / ml, and for hydrogel material types GB2-UGB2 05 and GB2-UGB2-UGB1 06-08 in the P1 range of 128 to 2 μmol / ml. Figure 6 Table 3 shows charge storage capacities ranging from 3040 to 1894 mC / ml and impedances ranging from 36 to 74 Ω. Since PEDOT is a hydrophobic polymer, the distribution and crosslinking of PEDOT chains are heavily dependent on the hydrophobicity of the surrounding environment and thus on the amphiphilicity of the anionic polymer network (parameter P3), which also affects the electrical properties of the hydrogel materials. These examples show that at 6.8 × 10⁻⁶ mC / ml… -3 1 / A 2 For (GB2-UGB2-UGB1 08) with a higher parameter P3 value, see Table 2 and -5.9*10 -3 1 / A 2Compared to the lower parameter P3 value of (GB1-UGB2-UGB1 04), the higher hydrophobicity resulted in a higher charge storage capacity (1894 vs. 1505 mC / ml) and lower impedance (74 Ω vs. 93 Ω) while the values ​​of P2 (4.5 mmol / (g / mol)) and P1 (2 μmol / ml) remained constant. Figure 6 (See Table 3). Increasing the distance along the polymer chain of the strongly anionic groups (resulting in a decreased P2 value, representing a smaller local charge density) yields a significantly reduced charge storage capacity and increased impedance at similar P3 and almost identical P1. This difference is particularly evident at a lower P1 of 9 mmol / ml, a P2 of 0.9 mmol / (g / mol), and a P2 of 0.7*10. -3 1 / A 2 P3 (GB4-UGB2-UGB1 12) with 11 or 4 mmol / ml P1, 4.5 mmol / (g / mol) P2 and -5.9*10 -3 1 / A 2In the comparison of P3 (GB1-UGB2-UGB1 02 / 03), although the P3 value is small and the P1 value is almost the same or small, the hydrogel with higher P2 (GB1-UGB2-UGB1 02 / 03) has a significantly higher charge storage capacity (2350 and 1811 mC / ml) and lower impedance (42 and 81 Ω) compared to GB4-UGB2-UGB1 12 (910 mC / ml and 150 Ω). Although it has moderate P1 and P3 values, GB4-UGB2-UGB1 12 behaves like a hydrogel without anionic groups after PEDOT functionalization, see UGB1-UGB2 15 (pure PEG hydrogel material, 921 mC / ml and 167 Ω). Thus, conductive hydrogels with different electrical properties can be obtained by changing the doping degree (P1, P2) and hydrophobicity (P3). High-conductivity hydrogels can be obtained by using a larger overall number of anionic groups (i.e., higher P0 or P1 values) and a smaller distance between groups carrying strong anions (P2). P0 exhibits a similar effect on electrical properties as P1, which may be attributed to the proportion of strong anionic groups. Weak anionic groups, such as carboxyl groups with an intrinsic pKa in the range of 3.5 to 4.5, may play a minor role in PEDOT doping. The interaction between the hydrophilic / amphiphilic anionic polymer and the hydrophobic conductive polymer (PEDOT) can also be configured by the parameter P3. Due to the increased hydrophobicity of the anionic hydrogel building blocks (higher parameter P3 value), increased conductivity (i.e., lower impedance) can already be achieved at a significantly lower P1. This is because the hydrophobic PEDOT units have a high affinity for the hydrophobic groups on the anionic hydrogel building blocks, which can advantageously influence doping. In addition, the hydrophobic groups during PEDOT synthesis have already facilitated the penetration and distribution of monomer units (EDOT) in the anionic polymer network. Conductive hydrogel materials with very high electrical conductivity (low impedance) and high charge storage capacity can be used as biocompatible electrode materials for stimulating cells or tissues. Due to the high hydration and softness of these hydrogels, which are very similar to biological tissues, rejection responses in organisms to electrodes based on the conductive hydrogel materials of this invention can be significantly reduced compared to classic metal electrodes. Furthermore, in classic metal electrodes, charge transfer for electrical stimulation occurs only at the interface between the metal and the tissue (physiological solution). Due to the distribution of the conductive polymer within the bulk material and the possibility of biofluids diffusing into the hydrogel, charge transfer can occur throughout the entire volume. This allows a smaller voltage to be used for the same charge injection, reducing heat generation and the resulting potential tissue damage compared to classic metal electrodes.

[0102] Encapsulation and release of active ingredients

[0103] Sample preparation for the encapsulation and release of the active ingredient was performed in exactly the same manner as the characterization of electrical properties. 100 μl of different anionic polymer networks were applied to the gold mesh electrode and made conductive by subsequent permeation and polymerization of EDOT monomers into PEDOT.

[0104] Seal-up of active ingredients

[0105] After basic washing of the conductive hydrogel material in PBS, different substances were encapsulated at a concentration of 100 ng / ml in 2.5 ml PBS with 0.1% BSA (to simulate the physiological state with carrier proteins). This corresponded to 250 ng for each protein and hydrogel material. Encapsulation was performed in 5 ml low-bonding Eppendorf tubes to minimize non-specific bonding of proteins to the reaction vessel. Uptake was carried out over 24 hours. Encapsulation was performed at 500 mV, 0 mV (passive), and -500 mV. Active encapsulation was performed in a 3-electrode setup, similar to electrical characterization. The conductive hydrogel on the gold electrode was used as the working electrode. The working electrode was placed in a 5 ml low-bonding Eppendorf tube along with the reference electrode (Ag / AgCl wire). The counter electrode (porous carbon electrode, BioLogic A-010530), with a significantly larger surface area compared to the working electrode, was placed in a separate container with 100 ml PBS. The circuit was closed by a salt bridge formed from a PVC tube filled with 25% polyacrylamide hydrogel swollen in PBS (as per manufacturer's instructions). The tube was further sealed in an Eppendorf container with a 1000 Da dialysis membrane to prevent material ingress. A potentiometer (Metrohm Autolab PGSTAT204 or PalmSens 4) was used as the measuring instrument to apply the defined potential. 100 μl of solution was taken as samples before and after sealing. The amount of protein absorbed was calculated as a percentage after determining the concentrations of different proteins using a Multiplex Assays Kit (Luminex Technology, ThermoFisher) according to the manufacturer's instructions.

[0106] For the encapsulated material, under different applied voltages, the overall charge density P0 or P1 and the local charge density (P2) in the hydrogel material (anionic polymer network - PEDOT pseudo-IPN) play a major role. Here, regardless of the charge of the bonded molecules, the highest encapsulation capacity can be measured without applying a potential. Figure 7(See Table 4). Compared to non-conductive hydrogels (without PEDOT) with similar P1 and P2, conductive hydrogel materials showed a smaller absorption for positively charged substances (GB2-UGB2-UGB1 09; 72.3% SDF-1α; 72.7% FGF-2; 70.8% IL-8 vs. GB2-UGB2-UGB1 19; 98.2% SDF-1α; 85.0% FGF-2; 96.7% IL-8) (see Table 4). The opposite effect was observed in the case of negatively charged molecules. Absorption of 49.9% GM-CSF and 37.1% EGF could be measured in the case of GB2-UGB2-UGB1 09, compared to 43.3% GM-CSF and 33.5% EGF in the case of GB2-UGB2-UGB1 19. This effect was more pronounced at lower P1. Thus, in the case of GB2-UGB2-UGB1 10, 77.8% GM-CSF and 69.2% EGF can be bonded, compared to 0.0% GM-CSF and 21.0% EGF in the case of GB2-UGB2-UGB1 20. This is likely due to the positive charge of PEDOT. PEDOT has approximately 33% positive charge in its monomer units without an applied potential, which can generate interactions with negatively charged materials and potentially bond them (10.1021 / acsapm.8b00061, 10.1021 / acsami.5b04768). Furthermore, it was shown that even positively charged materials can bond better with conductive hydrogels at lower P1 compared to the non-conductive control group. Thus, in the case of GB2-UGB2-UGB1 10, 76.9% SDF-1α, 86.1% FGF-2, and 39.9% IL-8 can be bonded, compared to 60.5% SDF-1α, 41.6% FGF-2, and 17.1% IL-8 in the case of GB2-UGB2-UGB1 20. This likely means that the conductive polymer PEDOT plays an important role even for bonding to generally positively charged substances, mainly due to the mutual charge compensation with the anionic groups of the hydrogel material. Negatively charged domains in proteins play an important role in the ionic bonding of PEDOT and the hydrophobic interactions between proteins and PEDOT.

[0107] If a positive potential of 500mV is applied, the overall amount of material sequestration decreases ( Figure 7(See Table 4). This phenomenon is primarily observed in the case of positively charged materials. In the case of negatively charged materials, such as GM-CSF and EGF, a smaller increase or almost the same encapsulation is observed. Thus, in the case of GB2-UGB2-UGB109, 49.9% GM-CSF and 37.1% EGF can be bonded before the potential is applied, while 59.2% GM-CSF and 67.0% EGF can be bonded after the potential is applied; or in the case of GB2-UGB2-UGB109, 77.8% GM-CSF and 69.2% EGF can be bonded before the potential is applied, while 78.4% GM-CSF and 64.8% EGF can be bonded after the potential is applied. The reason for this is likely that the charge of PEDOT is increased by applying a positive potential. (10.1021 / acsapm.8b00061, 10.1021 / acsami.5b04768). This enhanced ionic interaction has a favorable effect on the binding of negatively charged proteins such as GM-CSF and EGF. Conversely, the overall uptake of positively charged proteins is reduced because the negative charge of the anionic component is constant in the hydrogel and the stronger positive charge of PEDOT may lead to partial charge compensation.

[0108] If a negative potential is applied, the positive charge of PEDOT may decrease or even be completely neutralized (10.1021 / acsapm.8b00061, 10.1021 / acsami.5b04768). At a potential of -500 mV, a reduction in material encapsulation is generally observed, independent of the charge of the material and independent of P1 and P2 of the hydrogel material. Figure 8(See Table 4). Thus, in GB2-UGB2-UGB1 09, the sequestration of SDF-1α decreased from 72.3% to 47.2%, NGF-β from 94.1% to 75.0%, and IL-8 from 70.8% to 35.4%. The same trend was observed for GB2-UGB2-UGB1 10 and GB3-UGB2-UGB1 11 / 12. This confirms the hypothesis that the positive charge of PEDOT plays a crucial role in the bonding of positively charged substances. This can, for example, result from the bonding of negatively charged domains of the substance (protein) to the positive charge of PEDOT (i.e., the previously described phenomenon). A very significant reduction in sequestration was observed at -500 mV when sequestering negatively charged substances. Therefore, in GB2-UGB2-UGB1 09, the GM-CSF encapsulation percentage decreased from 49.9% to 17.9% and the EGF percentage decreased from 37.1% to 0.0%, or in GB2-UGB2-UGB1 10, the GM-CSF encapsulation percentage decreased from 77.8% to 44.9% and the EGF percentage decreased from 69.2% to 42.8%. This significant reduction in the encapsulation of negatively charged materials at negative potentials is likely due to the reduction in the positive charge of PEDOT. Consequently, there are significantly fewer bonding sites available for negatively charged materials.

[0109] Release of active ingredients

[0110] Following encapsulation (loading the hydrogel according to the third loading method described above), the bioactive molecules were released electrically. For this purpose, samples loaded with the substance at a potential of 0 mV (passively) for 24 hours were used due to the highest average encapsulation rate. Controlled release of actively encapsulated substances can also be performed according to the experimental protocol described below.

[0111] After loading the hydrogel, a short washing step was performed first to remove weakly bonded proteins. For this purpose, the hydrogel was first centrifuged at 3000 rpm for 1 minute to remove adhering liquid. The hydrogel was then washed with BSA in 1 ml PBS and centrifuged again at 3000 rpm for 1 minute. Release was also performed in the three-electrode setup described in the "Active Ingredient Immobilization" section at applied potentials of 500 mV, 0 mV, and -500 mV. Samples were taken at 0 min, 10 min, 30 min, 1 h, 6 h, 8 h, and 24 h after the initial solution (control group), after immobilization, after washing, and after release. Since protein saturation was observed after 8 h, a plateau in the release results was observed after 8 h. Figure 8(See Table 5). Different concentrations of substances were determined using Multiplex Assay Kits (Luminex Technology, ThermoFisher) according to the manufacturer's instructions.

[0112] The release of the bonded substance can be determined by the configuration parameters of the anionic polymer network combined with the charge of PEDOT. Figure 8 (See Table 5). At high P1 (60 μmol / ml, GB2-UGB2-UGB1 09), only very little release of positively charged substances occurred (0.0% SDF-1α; 0.0% FGF-2; 0.5% IL-8). Conversely, a slightly smaller release was observed for positively charged substances (2.3% GM-CSF; 9.1% EGF). Since the positive charge of PEDOT is likely to be overcompensated by anionic groups, the release of negatively charged substances is likely to occur significantly faster than the release of positively charged proteins. At low P1 (2 μmol / ml, GB2-UGB2-UGB1 10), an increased release of positively charged substances occurred (0.8% SDF-1α; 0.3% FGF-2; 11.7% IL-8). Conversely, less release of negatively charged substances was observed compared to higher P1 (0.4% GM-CSF; 2.0% EGF). If P2 is reduced (GB3-UGB2-UGB1 11 / 12) while maintaining a similar P1 value, an increased release of negatively charged substances (4.3 / 3.7% GM-CSF; 94.1 / 73.5% EGF) can be achieved while maintaining almost the same release of positively charged substances (1.6 / 0.2% SDF-1α; 0.0 / 0.0% FGF-2; 1.4 / 1.2% IL-8). Figure 8 (See Table 5). The reason is the same: although the charge density is low, the positive charge in PEDOT is compensated by anionic groups.

[0113] When a potential of 500 mV is applied, the bonded substances remain unchanged, independent of their charge. This effect is particularly pronounced for GB2-UGB2-UGB1 09. Here, the release of almost all 14 proteins is reduced to 0% ( Figure 8 (Table 5). Even with moderate P1 (9 μmol / ml) and very small P2 (0.94 mmol / (g / mol)) in GB3-UGB2-UGB1 12, the release of all substances can be reduced to almost 0%. Figure 8(See Table 5). The reason may lie in the swelling rate of the hydrogel. Under positive potential, the increased positive charge of PEDOT more than compensates for the anionic groups. If we assume that there are more anionic groups in the hydrogel material compared to the positive charge of PEDOT, the total charge of the hydrogel shifts towards neutral, resulting in water loss and shrinkage. This leads to a reduced mesh width of the polymer chains, which in turn results in reduced protein release, regardless of its charge.

[0114] When a potential of -500mV is applied, increased release is observed in most of these substances. Figure 8 (See Table 5). This primarily involves negatively charged bioactive molecules. Thus, after applying a potential, the release of TNF-α, GM-CSF, and EGF increased from 1.4 / 2.3 / 9.2% to 5.3 / 10.4 / 67.2% for hydrogel material type GB2-UGB2-UGB1 09 and from 1.7 / 0.4 / 2.0% to 53.2 / 5.9 / 72.6% for GB2-UGB2-UGB1 11 / 12. This trend was also observed for hydrogel material types GB3-UGB2-UGB1 11 / 12. Significantly, substances that already exhibited significant release without an applied potential were released to a maximum extent upon applying a negative potential. The most likely reason is the reduction / neutralization of the positive charge of PEDOT. This results in multiple free anionic groups that, through ionic interactions, repel negatively charged proteins. However, increased release was also observed with positively charged substances. Therefore, for GB2-UGB2-UGB1 10, the release of FGF-2, IL4, and IL8 increased from 0.3% to 2.6%, from 0.6% to 1.7%, and from 11.7% to 26.3%, respectively. This is likely due to the increased number of free anionic groups resulting from the lack of charge compensation with PEDOT. The resulting increase in the negative net charge of the hydrogel leads to swelling. This facilitates charge-independent release, allowing for the release of positively charged substances in even greater quantities.

[0115] Biocompatibility of hydrogels

[0116] To investigate biocompatibility, the PC12 cell line and hydrogel material type GB1-UGB2 17 were used. This cell line is a pheochromocytoma cell line that can differentiate into neural cells within days by adding NGF-β. The PC-12 cell line is a widely used model for neuronal differentiation. For culturing with PC12 cells, the washed and swollen conductive hydrogel in PBS was first rinsed in 70% ethanol for 10 minutes to kill any potential microorganisms, and then washed again in sterile PBS. The hydrogel was then treated with type I collagen (50 μg / ml, 1 ml / 100 μl gel, Gibco). TM After treatment with 20 mM sterile acetic acid at room temperature for 2 hours, the gel was washed in PBS for 30 minutes. For gels loaded with NGF-β, they were additionally incubated overnight in PBS with 1% BSA using NGF-β (100 ng / ml, Sigma-Aldrich). Subsequently, each cm 2 50,000 cells were seeded onto the gel surface. Cell seeding and culture were performed on an RPMI 1640 (Gibco) gel. TM In this process, 10% horse serum (Gibco) is added to the medium. TM 5% fetal bovine serum (Gibco) TM ) and 1% penicillin and streptomycin (Gibco) TM This is done using [a specific method / method]. Depending on the culture conditions, pure medium, medium with 100 ng / ml NGF-β added, or medium containing only 1% horse serum (Gibco) may be used. TM Cells were cultured in a reduced-nutrient medium (reduced) containing 0.5% fetal bovine serum and 0.5% fetal bovine serum. After culturing for 5 or 7 days with the medium changed every 2 days, cells were fixed for 20 minutes in 4% PFA in PBS at room temperature. For imaging, cells were stained with phalloidin (Abcam) and DAPI (Pierce) according to the manufacturer's instructions. Images were taken using a fluorescence microscope. Cell number and cell extent were analyzed using FIJI / ImageJ with the watershed and analyze particles (cell number) and skeletonize plugins.

[0117] Cell adhesion and cell proliferation were observed on the gel under all culture conditions. In a 7-day vs. 5-day comparison, without NGF-β addition, the cell number increased significantly despite a smaller cell extent on the hydrogel. Figure 9 (See Table 6). This corresponds to greater cell proliferation. Adding NGF-β to the medium resulted in PC12 cells differentiating after 5 days (…). Figure 9Table 6). This can be confirmed by the increase in cell extent due to the growth of axonal structures. Adding NGF-β to the hydrogel, rather than to the medium, resulted in only a slight increase in cell extent with only a small increase in cell number. Figure 9 (See Table 6). Without stimulation, the amount of NGF-β released was too small to achieve complete cell differentiation. This is due to the high affinity of NGF-β for the hydrogel. Reducing the nutrient content in the medium could potentially lead to increased differentiation. These cells then demonstrated the ability to proliferate and differentiate on the hydrogel under all culture conditions, confirming the hydrogel's high biocompatibility.

[0118] List of reference numerals

[0119] 1. Conductive hydrogel materials / hydrogel materials

[0120] 2 Polymer Network

[0121] 3. Anionic structural units

[0122] 4. Crosslinking agent molecules

[0123] 5 Conductive Components / PEDOT

[0124] 6 Bioactive substances / Multiple bioactive substances / Signaling proteins

[0125] 7. Voltage source

Claims

1. A method for detecting and influencing the absorption of a bioactive molecule (6) in a hydrogel material (1) and / or the release of the bioactive molecule (6) from the hydrogel material (1), wherein The hydrogel material (1) is a polymer network (2) formed by anionic building blocks (3) and uncharged building blocks. The affinity of the polymer network for bioactive molecules (6) can be configured by defining the parameters of the anionic building blocks (3). The hydrogel material has a conductive component (5), the resistance and charge storage capacity of which depend on the interaction with the hydrogel building blocks and the bonding of bioactive molecules (6) to the hydrogel material (1). The conductive component (5) is suitable for altering the anionic charge of the hydrogel material (1) and its affinity for bioactive molecules (6) by means of electrical potential, wherein, The hydrogel material (1) is brought into contact with the biofluid. The changes in resistance and / or charge storage capacity of the hydrogel material (1) are detected, and based on the detected changes in resistance and / or charge storage capacity, the absorption of bioactive molecules (6) into the hydrogel material (1) or the release of bioactive molecules (6) from the hydrogel material (1) into the biofluid is determined, and the concentration of bioactive molecules (6) in the biofluid and / or the concentration of bioactive molecules (6) in the hydrogel material (1) is changed by applying an electric potential to the hydrogel material (1).

2. The method according to claim 1, characterized in that, In order to release the bioactive molecules (6), the hydrogel material (1) is loaded with a predetermined concentration of predetermined bioactive molecules (6) before contact with the biofluid.

3. The method according to claim 1, characterized in that, The polymer network (2) can be configured in terms of its composition using at least three parameters that define the anionicly charged structural units (3), wherein the parameters are selected from the group consisting of parameters P0, P1, P2, and P3. The parameter P0 corresponds to the number of ionized anionic groups per volume unit of the hydrogel material (1) that swells under physiological conditions, assuming that 30% of all anionic groups are ionized. Parameter P1 is the intrinsic pK of less than 2.5 per volume unit of hydrogel material (1) that swells under physiological conditions. s The value is derived from the number of strong anionic groups. Parameter P2 corresponds to an intrinsic pK of less than 2.5 for each repeating unit. s The value is obtained by dividing the number of strong anionic groups by the molecular weight of the repeating unit, and Parameter P3 corresponds to the amphiphilic value used to describe the anionic building block (3), wherein the resistance and / or charge storage capacity of the hydrogel material (1) are predetermined by the parameter values ​​configured by the parameters of the hydrogel material (1).

4. The method according to claim 1, characterized in that, In order to detect the bonding of bioactive molecules (6), the impedance change of the hydrogel material (1) is measured at a frequency in the range of at least 0.1 Hz to 1 MHz.

5. The method according to claim 1, characterized in that, In order to absorb bioactive molecules (6), a potential in the range of 1 mV to 1000 mV is applied to the hydrogel material (1), and in order to release bioactive molecules, a potential in the range of -1 mV to -1000 mV is applied to the hydrogel material (1).

6. The method according to any one of claims 1 to 5, characterized in that, In order to absorb bioactive molecules (6), a constant current greater than 0 mA is applied to the hydrogel material (1), wherein the direction of the current flow is changed to release the bioactive molecules (6).

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