METHOD FOR DETECTING AND INFLUENCING THE UPGRADE AND / OR RELEASE OF BIOACTIVE SUBSTANCES USING ELECTRICALLY CONDUCTIVE HYDROGELS

Description

[0001] The invention relates to a method for influencing and detecting the uptake of bioactive substances into a hydrogel material and / or the release of bioactive substances from the hydrogel material.

[0002] Hydrogel-based substance release and uptake systems are promising for applications in biotechnology, particularly in medicine, because hydrogels, compared to many other biomaterials, are similar to human physiological tissue in their water content and mechanical properties, enabling the encapsulation of various substances and their targeted release. By definition, hydrogels are highly hydrated, covalently or physically cross-linked polymers that allow substances to be reversibly bound to affine polymer building blocks of the hydrogel network via various non-covalent interactions. This enables either the targeted removal of substances from a biofluid or living tissue (i.e., sequestration within the hydrogel) or their release from the hydrogel into the biofluid or living tissue.Such substances can be protein-based signaling molecules from the classes of cytokines, chemokines, hormones, neurotransmitters, growth factors, or non-protein-based small molecules, which perform one or more biological functions of the aforementioned protein-based signaling molecules. They can also be non-protein-based chemical / medicinal agents such as antimicrobials, antiseptics, and dyes, which bind reversibly to the affinity-mediating polymer building blocks in the hydrogel via charge interactions and / or hydrophobic interactions or other specific chemical interactions such as hydrogen bonds. Hydrogel systems are known in the prior art in which the physicochemical properties of a hydrogel for the sequestration and / or release of specific substances must be determined during its formation.This is achieved through a specific charge characteristic of the hydrogel network, as described, for example, in WO 2018 / 162009 A2, or through the targeted adjustment of physical properties such as mesh size. It is also known to release covalently coupled substances in a controlled manner using enzymatic triggers, light, or variable pH values; however, a disadvantage of such hydrogel networks is that reversible substance binding is not possible. Controlled sequestration of substances and thus depletion from a biofluid or tissue environment is therefore not possible.

[0003] In known systems that reversibly achieve affinity for substances via non-covalent interactions, the affinity, and thus the control of sequestration or release of substances, is determined by their intrinsic network architecture, i.e., the network architecture imprinted during the formation of the hydrogel network. Subsequent modulation is only possible via the aforementioned external triggers: light, pH changes, or enzymatic cleavage.

[0004] In addition to intrinsically charged hydrogels, electrically conductive hydrogels have been described in biomedical research. They are primarily used as coatings for electrode materials, for example, for neural electrodes, to optimize their biocompatibility and charge injection. Due to their mechanical similarity to tissues and organs, the use of soft, hydrated polymer materials results in fewer rejection reactions compared to the use of metal electrodes. Furthermore, with conventional metal electrodes, charge injection, which is essential for stimulating tissues and organs, only occurs at the electrode surface. Nanostructuring aims to increase the surface area and thus the contact area between the electrode and the tissue fluid (biofluid), resulting in enhanced charge injection.Since the biofluid can penetrate the entire volume of electrically conductive hydrogels, the contact area is significantly larger than that of comparable metal electrodes, even without further structuring. This also leads to a considerably increased charge injection. The term charge storage capacity (in English) is used in the literature to measure the ability to transfer charges into physiological solutions. charge storage capacity ) used.

[0005] To produce such conductive hydrogels, conductive substances such as carbon nanotubes and / or electrically conductive polymers are used. Typically, the conductive substance is dispersed in a hydrogel precursor solution, and the hydrogel is then formed through polymerization and cross-linking in another component that is independent of the conductive substance. A key characteristic of this process is that the electrically conductive component is usually hydrophobic and therefore does not form a hydrogel.

[0006] Due to their diverse applications and simple, cost-effective processing, conductive organic polymers are increasingly used in industrial applications. These polymers function on the principle of semiconductors. Their most important component is a conjugated π-An electron system that extends throughout the entire polymer backbone. The intrinsic electrical conductivity of these polymers is low. To generate free charge carriers, with the aim of increasing the molecular electrical conductivity, charged molecules are needed that remove electrons from the polymer system (p-doping) or introduce electrons into the polymer system (n-doping). This process is called primary doping.

[0007] The most commonly used method is p-doping. The doping and resulting interaction of hydrophobic polymers with highly charged hydrophilic molecules further reduces the hydrophobicity of the polymer-doping complexes, thus allowing the use of these polymers in aqueous solutions or hydrogels.

[0008] Besides primary doping, the arrangement of the conductive polymer chains relative to each other is also crucial. For the transport of charge carriers over longer distances, these must be transferred between the electrically conductive polymer chains. This requires a uniform distribution of the polymer chains within the volume, combined with their close proximity to one another. Aqueous solutions of conductive polymers and the respective doping molecule usually form suspensions. The lack of interaction between the conductive polymer chains results in low electrical conductivity. By structurally reorganizing the conductive polymer chains relative to each other (secondary doping), the necessary close proximity of the polymer chains can be achieved, thereby further increasing the conductivity.This can be achieved, for example, by increasing the ionic strength and the associated shielding effects of the charge (characterized by the Debye length).

[0009] An example using the conductive poly-3,4-ethylenedioxythiophene (PEDOT) is described by Zhenan Bao and other authors (DOI: 10.1038 / s41467-018-05222-4, and US20190390068A1). As described in this publication, a weakly cross-linked hydrogel is initially formed from a mixture of hydrophilic polystyrene sulfonate (PSS) and PEDOT (primary doping) through physical entanglement of the PSS and the use of ionic liquids (secondary doping). This hydrogel is then mechanically stabilized by the formation of a pseudo-interpenetrating network created by a radical cross-linking reaction of acrylate monomers. After polymerization, an electrically conductive hydrogel network results (10.1038 / s41467-018-05222-4, and US20190390068A1), with the secondary polymer network serving only to stabilize and improve the mechanical properties of the material.The electrical properties of the hydrogel result solely from the primary, non-covalent PEDOT:PSS hydrogel.

[0010] Another method for obtaining electrically conductive hydrogels is known from US 9,299,476 B2. According to the method known from US 9,299,476 B2, a biopolymer-based hydrogel network carrying functional anionic groups is formed and deposited on the surface of an electrode. The primary network thus obtained is swollen in or transferred into a solution containing 3,4-ethylenedioxythiophene (EDOT) monomers, and an electrical polymerization of the EDOT monomers to poly-3,4-ethylenedioxythiophene (PEDOT) is triggered by the influence of an electrical voltage. A pseudo-interpenetrating polymer network is formed through the ionic interaction of PEDOT with the anionic functional groups of the primary network. These anionic groups simultaneously act as p-dopers on the PEDOT (primary doping) and, in combination, produce an electrically conductive hydrogel material.Due to the predetermined distribution of the covalently bound anionic groups in the hydrogel and the subsequent polymerization of the 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.

[0011] The hydrogel materials known to date enable sequestration and / or release of substances through their intrinsically imprinted physicochemical characteristics, or, in the case of electrically conductive hydrogel materials, exhibit limited modulatability and specificity for interaction with certain bioactive substances.

[0012] US2020 / 0191757 discloses the determination of the concentration of metal ions in a biofluid by measuring a change in electrical resistance of a hydrogel brought into contact with the biofluid.

[0013] The object of the invention is to propose a method by which concentrations of bioactive substances in a hydrogel material or in an environment of a hydrogel material can be influenced and determined.

[0014] The problem is solved by a method with the features according to claim 1. Further developments are specified in the dependent claims.

[0015] The invention comprises a method for detecting and influencing the uptake of bioactive molecules into a hydrogel material and / or the release of bioactive molecules from the hydrogel material, wherein the hydrogel material is, by definition, a polymer network formed from anionically charged building blocks and uncharged building blocks, the affinity for bioactive molecules of which is configurable based on parameters defining the anionically charged building blocks and which has an electrically conductive component whose electrical resistance and electrical charge storage capacity depend on an interaction with hydrogel building blocks and a binding of bioactive molecules to the hydrogel material, wherein the electrically conductive component is suitable for changing the anionic charge of the hydrogel material and its affinity for bioactive molecules by the influence of an electrical potential.In this method, the predefined hydrogel material is brought into contact with a biofluid, whereby a change in the electrical resistance and / or a change in the charge storage capacity of the hydrogel material is detected, and based on the detected change in electrical resistance and / or the detected change in charge storage capacity, an uptake of bioactive molecules into the hydrogel material or a release of bioactive molecules from the hydrogel material into the biofluid is determined, and / or a concentration of bioactive molecules in the biofluid and / or a concentration of bioactive molecules in the hydrogel material is influenced by an electrical potential acting on the hydrogel material.

[0016] The polymer network formed from anionically charged and uncharged building blocks is an anionically charged polymer network. The anionically charged polymer network can be configured using parameters that define the anionically charged building blocks.

[0017] For the sake of simplicity, the hydrogel material, which is electrically conductive by definition, will be referred to below as hydrogel material.

[0018] For the purposes of this invention, biofluids are understood to be physiological solutions, cell cultures, and living tissue. The instruction to contact the hydrogel material with a biofluid can therefore be interpreted as contact by immersion in a physiological solution or as surface contact of the hydrogel material with living tissue. in vivo and in vitro be understood.

[0019] For the purposes of this invention, bioactive molecules are understood to be protein-based and non-protein-based bioactive substances, active ingredients, and small molecules that exhibit signaling properties of cytokines, chemokines, hormones, neurotransmitters, or growth factors and cause other biological effects. Bioactive molecules can, in particular, be pharmaceutical active ingredients. A common characteristic of all the aforementioned bioactive molecules is a molecular weight of 70 kDa or less.

[0020] The method according to the invention comprises detecting the uptake of bioactive substances into a hydrogel material and / or the release of bioactive molecules from the hydrogel material, as well as influencing the uptake of bioactive molecules into a hydrogel material and / or the release of bioactive molecules from the hydrogel material. Thus, it is possible to detect and influence bioactive molecules, or—according to the alternative—to detect or influence bioactive molecules.

[0021] The fact that changes in the electrical resistance and / or charge capacity of the hydrogel material are detected to monitor the uptake and / or release of bioactive molecules is based on the understanding that these changes are influenced by the binding of bioactive molecules to the hydrogel material. Thus, the electrical resistance of the hydrogel material increases as a result of sequestration and binding to or within the hydrogel material, while the electrical conductivity of the hydrogel material decreases due to the loading of the anionic groups with bioactive molecules.Conversely, when bioactive molecules are dissolved from or out of the hydrogel material, the electrical resistance of the hydrogel material is reduced, while the electrical conductivity of the hydrogel material increases.

[0022] The charge storage capacity is calculated based on cyclic voltammetry measurements. For this, the current flow between the working electrode (hydrogel material) and the counter electrode (carbon electrode) is measured in a three-electrode setup. During five cycles, the applied electrical potential is varied from -0.6 to 0.8 V (potential between the working electrode and the Ag / AgCl reference electrode). The scan rate is 50 mV / s. The negative portion of the area under the curve is then integrated (MultiTrace 4.3, PalmSens 4), and the charge storage capacity is calculated using the following formula: Ladungsspeicherkapazit ä t = Stromst ä rke I ∗ Spannung U Scan − Rate

[0023] The software outputs the integral value I * U in the unit [A] * [V]. Here, [A] = [C / s]. Dividing the scan rate [V / s] by the charge [C], which is transferred through the material, yields the charge. This charge is then expressed as a ratio to the volume, since the interface with the surrounding medium encompasses the entire volume of the hydrogel, making a calculation of the surface area / contact area impossible.

[0024] To detect the uptake of bioactive molecules into and / or the release of bioactive molecules from the hydrogel material, an alternative method is to determine the change in the hydrogel material's electrical conductivity. This change in electrical conductivity then indicates whether bioactive molecules have been absorbed into or released from the hydrogel material. The binding of bioactive molecules in the electrically conductive hydrogel material occurs via non-covalent interactions between the bioactive molecules and the polymer chains of the hydrogel material. Ionic interactions, in particular, play a crucial role in this process.The interaction of anionic and cationic groups of the bound bioactive molecules with the anionic polymer network of the hydrogel material and the cationic, electrically conductive polymer results in a change in the doping of the conductive polymer. The anionic groups in the bound molecules can contribute to the doping and increase the conductivity. Cationic groups, on the other hand, compensate for the negative charges of the anionic polymer component and thus have a negative impact on the doping. Additionally, hydrophobic regions of the bioactive substances can influence the interaction of the PEDOT polymer chains with each other. Depending on the molecular type and concentration of the bound bioactive molecules, a specific change in the electrical properties of the hydrogel material occurs.

[0025] The uptake and / or release of bioactive molecules can be understood as a change in the concentration of bioactive molecules within the hydrogel material. Furthermore, it can be stipulated that a discrete concentration of a bioactive molecule is assigned to a discrete conductivity value, a discrete electrical resistance value, or a discrete value of the electrical charge storage capacity.

[0026] The electrical resistance of the hydrogel material can be determined as DC resistance or as impedance. For impedance measurement, a frequency range of 0.01 Hz to 1 MHz can be specified. It can be stipulated that, to detect the binding of bioactive molecules to or within the hydrogel, a change in the impedance of the hydrogel material is measured at at least one frequency in the range of 0.1 Hz to 1 MHz. Changes in charge storage capacity can also be taken into account.

[0027] To influence the concentration of bioactive molecules in the biofluid and / or the concentration of bioactive molecules in the hydrogel material, the hydrogel material is subjected to an electrical potential. This electrical potential alters the anionic charge of the hydrogel material and its affinity for bioactive molecules, thus affecting the binding of bioactive molecules to or within the hydrogel material.

[0028] The release of bioactive molecules from the hydrogel material requires that the bioactive molecules are bound to or within the hydrogel material before contact with the biofluid. Therefore, it can be arranged that, to release bioactive molecules, the hydrogel material is electrically or chemically loaded with a predetermined concentration of the specified bioactive molecules before contact with the biofluid.

[0029] According to one embodiment of the method according to the invention, the polymer network is configurable in its composition by means of at least three parameters defining the anionically charged building blocks, selected from a group of parameters P0, P1, P2, P3, wherein parameter P0 corresponds to a value from the number of ionized, anionic groups, assuming 30% ionization of all anionic groups, per unit volume of the hydrogel material swollen under physiological conditions, parameter P1 corresponds to a value from the number of strongly anionic groups, with an intrinsic pKa value less than 2.5, per unit volume of the hydrogel material swollen under physiological conditions, and parameter P2 corresponds to a value from the number of strongly anionic groups, with an intrinsic pKa value less than 2.5.The molar mass of each repeating unit corresponds to the repetition unit, and parameter P3 corresponds to a value describing the amphiphilicity of the anionic building blocks, where an electrical resistance and / or an electrical charge storage capacity of the hydrogel material is defined by parameter values ​​of a parameter configuration of the hydrogel material. A detailed definition of the parameter values ​​and their determination can be found in the description below.

[0030] An interaction of bioactive molecules can also be based on the substance-specific value Pp, which is calculated from the ratio of the net charge of a bioactive molecule to the water-accessible surface area of ​​the bioactive molecule. For protein-based substances, any protein structure in the Protein Data Bank (PDB) can be used. http: / / www.rcsb.org / ) available. The net load of the selected protein structure is calculated using the Delphi Web Server. (http: / / compbio.clemson.edu / sapp / delphi webserver / )Calculated with standard settings at pH 7. The protein surface area accessible to the solvent water is determined using the PyMol software. (www.pymol.org)The net charge of water is calculated using a solvent radius of 1.4 Å. 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 the unit 10⁻⁶ < x [1 / A 2 < or A 2 < ]. For non-protein-based substances, the net charge derivable from the chemical structure, which corresponds to the excess of anionic or cationic groups, and the molecular surface area accessible to the solvent water, which was derived analogously to the formula for parameter P3 using ChemDraw 19.0 and ChemAxon MarvinSketch 19.21 software, are calculated. The resulting value is multiplied by a factor of 1,000,000 to obtain the unit 10⁻⁶ < x [1 / A 2 < or A 2 < ]. Loading of the hydrogel material, i.e., the immobilization of a

[0031] The predetermined concentration of bioactive molecules in or on the hydrogel material can be achieved in various ways. According to a first method, which can also be called the first loading method, a predetermined concentration of the bioactive molecules intended for binding is mixed with the anionically charged hydrogel building blocks and thereby integrated into the polymer network. During the subsequent formation of the electrically conductive hydrogel materials through the incorporation of the electrically conductive component, the bioactive molecules are already present in the polymer network at the predetermined concentration. The advantage of this approach is that the entire quantity of bioactive molecules is quantitatively contained in the hydrogel material after polymer network formation. In other words, the loading of the bioactive molecules occurs independently of the parameters P0, P1, P2, and P3, and independently of the substance-specific value Pp.The reaction conditions prevailing during hydrogel material formation can have an adverse influence on the structure of the bioactive molecules, which is why not all bioactive molecules are suitable for immobilization using the first loading method.

[0032] According to a second method, which can also be referred to as the second loading method, electrically conductive hydrogel material is formed with a predetermined parameter configuration and values. This hydrogel material is then brought into contact with an aqueous solution or biofluid as the loading solution, with a predetermined molecular concentration, for a predetermined duration. During this process, the bioactive molecules in solution are absorbed and bound into the hydrogel material from the aqueous solution or biofluid, depending on the parameters P0, P1, P2, and P3. Finally, the loaded hydrogel material is removed from the loading solution. The bioactive molecules immobilized on or within the hydrogel material can then be released into an environment, preferably a biofluid or living tissue, by applying an electrical potential.Furthermore, due to the influence of the electrical potential, there is a possibility that bioactive molecules from the environment of the hydrogel material are sequestered into the hydrogel material.

[0033] According to a third loading method, which can also be referred to as the third loading method, a predefined electrically conductive hydrogel material, possessing a predefined parameter configuration with predefined parameter values, is brought into contact with an aqueous solution or biofluid as the loading solution, with a predefined molecular concentration, for a predefined duration and is subjected to an electrical potential. During this process, bioactive molecules are absorbed (sequestered) from the loading solution into the hydrogel material, depending on the predefined parameter values. It may be necessary to maintain the potential acting on the hydrogel material to keep the binding of the bioactive molecules constant. Otherwise, if the potential or current is changed, the bioactive molecules can be released from the hydrogel material.It is essential that the sequestration or release of bioactive molecules is based not only on the influence of an electrical potential but also on an affinity of the hydrogel material for certain bioactive molecules, as determined by the parameters P0, P1, P2, P3, Pp.

[0034] It has been shown that the structure formation and distribution of the electrically conductive component within the hydrogel material, which influences its electrical conductivity, can be affected by the parameter values ​​of a parameter configuration. Therefore, it is possible to define a hydrogel material that exhibits a specific electrical conductivity based on a given parameter configuration with predefined parameter values.

[0035] The process can further include subjecting the hydrogel material to an electrical potential relative to an Ag / AgCl reference electrode in the range of 1 mV to 1000 mV, preferably in the range of 400 mV to 600 mV, for the uptake of bioactive molecules. For the release of bioactive molecules, the hydrogel material can be subjected to an electrical potential relative to an Ag / AgCl reference electrode in the range of -1 mV to -1000 mV, preferably in the range of -400 mV to -600 mV.

[0036] The hydrogel material can be subjected to a constant electric current greater than 0 mA for the uptake of bioactive molecules, whereby the direction of the electric current flow is changed to release bioactive molecules.

[0037] Further details, features, and advantages of embodiments of the invention will become apparent from the following description of exemplary embodiments, with reference to the accompanying drawings and tables. These show: Figure 1: a schematic representation to illustrate the hydrogel material, Figure 2: a further schematic representation to further illustrate the invention,

[0038] Anionically charged hydrogel building blocks are abbreviated as GB below, with different anionically charged building blocks additionally identified by a number. Uncharged building blocks of the hydrogel material are abbreviated as UGB, with different uncharged building blocks also identified by a number. For the sake of simplicity and brevity, hydrogel materials are referred to as hydrogels in the tables. Hydrogel material types are referred to as hydrogel types in the tables.

[0039] The Figure 1Figure 1 shows a schematic representation to explain the electrically conductive hydrogel material. Figure A of the Figure 1 Figure 1 shows an example of the synthesis of the polymer network 2 from anionically charged building blocks 3 and crosslinking molecules 4 as a template for the formation of the hydrogel material 1. The parameter configuration is defined by the parameters P1, P2, and P3 of the polymer network 2. Figure B shows the structure of the electrically conductive hydrogel material 1 as a pseudo-interpenetrating polymer network (IPN) consisting of the polymer network 2 and an electrically conductive component 5. The polymer network 2 contains anionic groups and is defined by the parameters P1, P2, and P3. The electrically conductive hydrogel material 1 is formed by polymerization or crosslinking of the polymer network 2 with the electrically conductive component 5. Figure C of the Figure 1The doping of the conductive component 5 is shown as an example. In this example, the electrically conductive component 5 is PEDOT: Doping occurs via interaction with sulfate / sulfonate groups in the anionically charged polymer network 2. The integral charge density P1 of the polymer network 2, the local charge density P2, and the hydrophobicity P3 of the anionic group-bearing polymer in the polymer network 2 are crucial for the interaction with PEDOT 5 and the resulting electrical properties of the conductive hydrogel material 1. Figure D illustrates the effect of applying an electrical potential on the electrically conductive hydrogel material. The positive charge of PEDOT can be continuously adjusted by applying an electrical potential. The adjustment ranges from neutral (0) through medium (+1) to strongly positive (+3).

[0040] The Figure 2Figure 1 shows a further schematic representation to further illustrate the invention. It depicts an electrically conductive hydrogel material 1, which is composed of sulfated and sulfonated polymers as charged building blocks 3 and PEG as a crosslinker 4, forming a polymer network 2, and PEDOT as an electrically conductive component 5 incorporated into the polymer network. To the right, reference numeral 6 shows a positively charged signal protein bound to negatively charged PEDOT. The binding properties of the signal protein 6 in the hydrogel material 1 can be influenced by an electrical potential. To electrically influence the hydrogel material 1, it is electrically connected to a voltage source 7. The hydrogel material 1 enables electrodynamic modulation of specific electrostatic interactions between the hydrogel polymers and the signal protein 6. Examples of implementation: Synthesis of the polymer network

[0041] For the synthesis of the hydrogel material 1, three anionically charged polymer network systems 2 were used: (1) one consisting of two hydrogel building blocks, one anionically charged building block (GB1-5) with maleimide and one uncharged building block (UGB2) with thiol groups (hereinafter referred to as the maleimide-thiol two-component system); or (2) one consisting of three hydrogel building blocks (one anionically charged building block (GB1-5) with maleimide groups and one uncharged building block with maleimide groups (UGB1)) and one uncharged building block (UGB2) with thiol groups (hereinafter referred to as the maleimide-thiol three-component system); or (3) a system consisting of two hydrogel building blocks crosslinked via EDC / NHS-based activation of the carboxyl groups of the anionically charged hydrogel building block (GB1) and reaction with the amino groups on the second, uncharged hydrogel building block (UGB3) (hereinafter referred to as the maleimide-thiol three-component system). EDC / NHS system), used.

[0042] The properties of the anionically charged hydrogel building blocks (GB1 to GB5) and the uncharged hydrogel building blocks (UGB1 to UGB3) are Table 1 to be taken. Maleimide-thiol two-component system for the production of an anionically charged polymer network as a precursor to the hydrogel material according to the invention.

[0043] GB1 (heparin maleimide, 15 kDa) and UGB2 (star-shaped thiol-functionalized polyethylene glycol, starPEG, 10 kDa) are dissolved in 0.1 x phosphate-buffered saline ((PBS, pH = 6)) at a concentration of 0.0015 mol / L each. 1 x PBS consists of 137 mM NaCl, 2.7 mM KCl, and 12 mM total phosphate consisting of HPO₄²⁻ and H₂PO₄. The mixing ratios and concentrations are given in Table 2. All subsequent steps up to the mixing of the hydrogel building blocks are carried out on ice (pH adjustment to achieve a gelation time of 30 min). For a molar ratio of 1 of both building blocks, equal volumes of the two solutions are mixed by pipetting and / or in a mixing device.The mixture is then centrifuged to remove air bubbles, and the samples are pipetted onto gold electrodes (11 mm diameter, 100 µl) or a coverslip (8 mm diameter, 67 µl) and covered with an 11 mm or 8 mm hydrophobic (Sigmacote® treated) coverslip. Crosslinking of the hydrogel occurs via the reaction of the thiol and maleimide groups (Michael addition reaction). Polymerization takes place for at least 30 minutes at room temperature in a humidified chamber to prevent the gels from drying out. For cryogelation, the samples are polymerized overnight at -15 °C. The fully polymerized hydrogels are then swollen overnight in 1 x PBS (0.154 mmol / l NaCl, pH 7.4). The solids content of the gels is approximately 3% w / v. Maleimide-thiol three-component system for the production of a polymer network as a precursor to the hydrogel material according to the invention.

[0044] A three-component system is used to precisely adjust the integral negative charge in the hydrogel material. starPEG thiol, starPEG maleimides, and maleimide-functionalized anionically charged hydrogel building blocks are dissolved in 0.01 x PBS at a predetermined concentration and mixed in various ratios depending on the desired integral charge. The mixing ratios are given in Table 2. The gelation time is reduced to less than 5 minutes by adding 0.01 M HCl or 0.01 M NaOH. The solutions are then mixed, and the sample is prepared analogously to the two-component system at room temperature (30 minutes) or 15 °C (overnight). The fully polymerized hydrogels are swollen overnight in 1 x PBS (pH 7). The solids content of the gels is approximately 3% w / v.

[0045] The properties of the uncharged and anionically charged building blocks are shown in Table 1. EDC / NHS system for producing a polymer network as a precursor to the hydroquel material according to the invention.

[0046] For the EDC / NHS system, to achieve a molar ratio of UGB3 sternPEG-amine to non-maleimidated 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) are dissolved in ice-cold MilliQ water. To obtain 1 mL of final gel volume, 85.20 µl of EDC solution and 48.25 µl of NHS solution are pipetted 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 starPEG solution is added, and the mixture is again vortexed. Immediately afterwards, the desired molded parts are produced in the same way as with the two-component system. Polymerization takes place overnight at room temperature or -15 °C in a humid chamber to prevent the hydrogels from drying out.The total solids content of the gels is approximately 10% at a final starPEG concentration of 0.00784 mol / l and a final heparin concentration of 0.00392 mol / l. The fully polymerized hydrogels were swollen overnight in 1 x PBS (pH 7). Synthesis of the electrically conductive hydrogel material as a pseudo-interpenetrating network

[0047] The synthesis of the electrically conductive pseudo-interpenetrating polymer network is possible for all hydrogel materials according to the previously described formation procedures. For the synthesis of the electrically conductive pseudo-interpenetrating poly-3,4-ethylenedioxythiophene (PEDOT) network, with PEDOT as the electrically conductive component, the fully swollen anionically charged polymer networks are incubated in the first step for 3 hours in a 0.4 M ammonium peroxodisulfate (APS) solution in 1 M HCl at room temperature. Subsequently, the hydrogel is incubated for 6 hours in a 0.4 M 3,4-ethylenedioxythiophene (EDOT) solution in mineral oil in a rotary shaker. During this time, the EDOT undergoes oxidative polymerization to form an electrically conductive PEDOT network, which turns black with increasing polymerization of the PEDOT. Figure 3The hydrogel materials produced in this way are then washed overnight in mineral oil to remove unreacted monomers. To remove the mineral oil, the hydrogel materials are subsequently washed in hexane. This washing process can be repeated. Finally, the hydrogel materials are washed in PBS (pH 7.4) for at least 24 hours.

[0048] All hydrogel material types are in Table 2 In summary, the concentrations given are those 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 have not been functionalized with PEDOT. Hydrogel material type UGB1-UGB2 15 is a pure PEG-PEG hydrogel that does not carry anionic charges but is functionalized with PEDOT. Electrical characterization

[0049] All hydrogels were electrically characterized by impedance spectroscopy or cyclic voltammetry in a 3-electrode setup in 100 ml of 1x PBS (pH 7). For both methods, 100 µl of gel samples (unswelled volume) were prepared on a gold mesh electrode, which served 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 No. 6.0726.100) served as the reference electrode. A potentiostat (Metrohm Autolab PGSTAT204 or PalmSens 4) was used as the measuring instrument. Impedance spectroscopy

[0050] For impedance spectroscopy, an effective potential of 10 mV rms is applied, and the impedance and phase angle are measured in a frequency range of 0.01 Hz to 10⁻⁵ Hz with 10 measurement points per decade. The impedance of different hydrogel material types was compared at a frequency of 0.01 Hz. Electrical conductivity is inversely proportional to impedance; as impedance increases, electrical conductivity decreases. Cyclic voltammetry

[0051] The charge storage capacity is calculated based on cyclic voltammetry measurements. Using the aforementioned three-electrode setup, the current flow between the working electrode (hydrogel) and the counter electrode (carbon electrode) is measured while the applied electrical potential is varied from -0.6 to 0.8 V (potential between the working electrode and the Ag / AgCl reference electrode) over five cycles. The scan rate is 50 mV / s. The negative portion of the area under the curve is then integrated (MultiTrace 4.3, PalmSens 4), and the charge storage capacity of the hydrogel is calculated using the following formula: Ladungsspeicherkapazit ä t = I ∗ U Scan − Rate

[0052] The software outputs the integral value I * U in the unit [A] * [V]. Here, [A] = [C / s]. Dividing the scan rate [V / s] by the charge [C], which is transferred through the material, yields this charge. This charge is then expressed as a percentage of the volume, since the interface with the surrounding medium is present throughout the entire volume of the hydrogel, and therefore a surface area cannot be calculated. The values ​​are given in charge per milliliter of hydrogel. Electrical properties of hydrogel materials

[0053] Immediately after oxidative polymerization, PEDOT possesses an inherent positive charge (10.1021 / acs.jpcb.9b01745, 10.1021 / acsapm.8b00061). Through non-covalent interactions between PEDOT and the anionically charged polymer network (hydrogel), as well as non-covalent interactions between the individual PEDOT polymer chains, a pseudo-interpenetrating network forms between the anionically charged polymer network and PEDOT. Simultaneously, the negative charges of the anionically charged polymer network act as p-doping for PEDOT. The degree of doping of PEDOT changes depending on the integral and local charge density (parameters P0 / P1 and P2). This has a direct influence on the electrical properties (impedance and charge storage capacity) of the electrically conductive hydrogel material.With the same P2 and P3, a charge storage capacity of 3040 to 1505 mC / ml and an impedance of 30 to 93 Ω were obtained for the hydrogel material types GB1-UGB2 01 and GB1-UGB2-UGB1 02-04 over a P1 range of 110 to 2 µmol / ml, and in a P1 range of 128 to 2 µmol / ml, a charge storage capacity of 3040 to 1894 mC / ml and an impedance of 36 to 74 Ω were obtained for the hydrogel material types GB2-UGB2 05 and GB2-UGB2-UGB1 06-08 (Table 3-1 and 3-2). Since PEDOT is a hydrophobic polymer, the distribution and crosslinking of the PEDOT chains among themselves is strongly dependent on the hydrophobicity of the environment and thus on the amphiphilicity of the anionically charged polymer network (parameter P3), which also has an influence on the electrical properties of the electrically conductive hydrogel material.The exemplary embodiments show that with a high value of parameter P3 of 6.8 * 10 -3 < 1 / A 2 < (GB2-UGB2-UGB1 08), see Table 2, compared to a low value for P3 of -5.9 * 10 -3 < 1 / A 2 < (GB1-UGB2-UGB1 04), with constant values ​​for P2 (4.5 mmol / (g / mol)) and P1 (2 µmol / ml), the higher hydrophobicity leads to a higher charge storage capacity (1894 vs. 1505 mC / ml) and lower impedance (74 Ω vs. 93 Ω) (Table 3-1 and 3-2). If the distance between the strongly anionically charged groups along the polymer chains is increased (reduced value P2, which represents a lower local charge density), then with similar P3 and approximately the same P1, a significantly lower charge storage capacity is obtained with increased impedance.This difference is particularly evident when comparing low P1 of 9 mmol / ml, P2 of 0.9 mmol / (g / mol) and P3 of 0.7*10 -3< 1 / A 2< (GB4-UGB2-UGB1 12) compared to a P1 of 11 or 4 mmol / ml, P2 of 4.5 mmol / (g / mol) and P3 of -5.9*10 -3< 1 / A 2< (GB1-UGB2-UGB1 02 / 03). Despite the lower value for P3 and an almost identical or lower value for P1, the hydrogels with higher P2 (GB1-UGB2-UGB1 02 / 03) exhibit 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). Despite the moderate values ​​for P1 and P3, GB4-UGB2-UGB1 12 behaves like a hydrogel without anionically charged groups after PEDOT functionalization, see UGB1-UGB2 15 (pure PEG hydrogel material, 921 mC / ml and 167 Ω).Conductive hydrogels with different electrical properties can thus be obtained by changing the degree of doping (P1, P2) and the hydrophobicity (P3). Hydrogels with high conductivity can be obtained by a high integral number of anionic groups (i.e., a high value for P0 or P1) and a small distance between the strongly anionic groups (P2). P0 appears to have a similar influence on the electrical properties as P1, presumably due to the proportion of strongly anionic groups. The weakly anionic groups, for example, carboxyl groups with an intrinsic pKa in the range of 3.5 to 4.5, likely play a subordinate role in the doping of PEDOT. The interaction between the hydrophilic / amphiphilic anionic polymer and the hydrophobic electrically conductive polymer (PEDOT) can also be configured by the parameter P3.Increased hydrophobicity of the anionically charged hydrogel building blocks (high value for parameter P3) allows for the generation of enhanced conductivities (i.e., lower impedances) even at significantly lower P1 values. This is due to the high affinity of the hydrophobic PEDOT units for the hydrophobic groups on the anionically charged hydrogel building blocks, which may positively influence doping. Additionally, the hydrophobic groups facilitate the penetration and distribution of the monomer units (EDOT) within the anionically charged polymer network during PEDOT synthesis. These electrically conductive hydrogel materials, with their very high 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 closely resemble biological tissue, the organism's foreign body reactions to electrodes based on the electrically conductive hydrogel materials according to the invention can be significantly reduced compared to conventional metal electrodes. Additionally, with conventional metal electrodes, charge transfer for electrical stimulation occurs only at the contact surface between the metal and the tissue (physiological solution). Because of the distribution of the conductive polymer within the bulk material and the possibility for biofluids to diffuse into the hydrogel, charge transfer can occur throughout the entire volume. This allows lower voltages to be used for the same charge injection, thus reducing heat generation and the resulting potential tissue damage compared to conventional metal electrodes. Drug sequestration and release

[0054] Sample preparation for drug sequestration and release is identical to that used for characterizing electrical properties. 100 µl of the various anionically charged polymer networks were applied to a gold mesh electrode and made electrically conductive by subsequent penetration and polymerization of EDOT monomers to PEDOT. Drug sequestration

[0055] After thorough washing of the electrically conductive hydrogel materials in PBS, various substances were sequestered at a concentration of 100 ng / ml of substance in 2.5 ml of PBS containing 0.1% BSA (to simulate the physiological situation with a carrier protein). This corresponds to 250 ng per protein and hydrogel material. Sequestration took place in a 5 ml low bindingEppendorf tubes were used to minimize nonspecific binding of proteins to the reaction vessel. Recording took place over 24 hours. Sequestration was performed at 500 mV, 0 mV (passive), and -500 mV. Active sequestration was carried out in a 3-electrode setup similar to that used for electrical characterization. The conductive hydrogel on the gold electrode served as the working electrode. This electrode, along with the reference electrode (an Ag / AgCl wire), was placed in a 5 ml reservoir. low bindingEppendorf tube. The counter electrode, a porous carbon electrode (BioLogic A-010530) with a significantly larger surface area compared to the working electrode, was located in a separate vessel containing 100 ml of PBS. The circuit was closed by a salt bridge consisting of a PVC tube swollen with a 25% polyacrylamide hydrogel (specify according to the manufacturer's instructions) and filled with PBS. The tube was additionally sealed with a 1000 Da dialysis membrane within the Eppendorf tube to prevent the substances from entering. A potentiostat (Metrohm Autolab PGSTAT204 or PalmSens 4) was used to apply a defined potential. 100 µl samples of the solution were taken before and after sequestration. After determining the concentrations of the various proteins according to the manufacturer's instructions using the Multiplex Assay Kit (Luminex Technology, Thermo Fisher), the percentage of protein taken up was calculated.

[0056] The sequestration of substances is primarily influenced by the integral P0 or P1 and the local charge density (P2) at the various applied voltages throughout the entire hydrogel material (anionically charged polymer network – PEDOT pseudo IPN). The highest sequestration was measured without an applied potential, regardless of the charge of the bound molecule (Tables 4-1, 4-2, 4-3, and 4-4). Compared to non-conductive hydrogels (without PEDOT) with similar P1 and P2 values, the electrically conductive hydrogel materials exhibit lower uptake of positively charged substances (GB2-UGB2-UGB1 09; 72.3% SDF-1α; 72.7% FGF-2; 70.8% IL-8 versus GB2-UGB2-UGB1 19; 98.2% SDF-1α; 85.0% FGF-2; 96.7% IL-8) (see Tables 4-1, 4-2, 4-3 and 4-4). The opposite effect is observed for negatively charged molecules.Uptake of 49.9% GM-CSF and 37.1% EGF was measured at GB2-UGB2-UGB1 09 compared to 43.3% GM-CSF and 33.5% EGF at GB2-UGB2-UGB1 19. This effect is even more pronounced at lower P1 levels. For example, 77.8% GM-CSF and 69.2% EGF were bound at GB2-UGB2-UGB1 10 compared to 0.0% GM-CSF and 21.0% EGF at GB2-UGB2-UGB1 20. This is likely due to the positive charge of the PEDOT. Without the application of an electrical potential, PEDOT exhibits a positive charge of approximately 33% of the monomer units, which can interact with negatively charged substances and likely bind them (10.1021 / acsapm.8b00061, 10.1021 / acsami.5b04768). Additionally, it was shown that at a low P1, positively charged substances were also bound more effectively by the electrically conductive hydrogels compared to the non-conductive control.Thus, GB2-UGB2-UGB1 showed the following binding rates: 10 (76.9% SDF-1α, 86.1% FGF-2, 39.9% IL-8), compared to GB2-UGB2-UGB1 (20, 60.5% SDF-1α, 41.6% FGF-2, 17.1% IL-8). This suggests that the conductive polymer PEDOT plays a crucial role in the binding of predominantly positively charged substances, primarily through mutual charge compensation with the anionically charged groups of the hydrogel material. Both the ionic binding of negatively charged domains in the protein by PEDOT and hydrophobic interactions between the protein and PEDOT are important in this process.

[0057] Applying a positive potential of 500 mV reduces the overall sequestration of substances (Tables 4-1, 4-2, 4-3, and 4-4). This is particularly noticeable with positively charged substances. With negatively charged substances, such as GM-CSF and EGF, however, a slight increase or nearly identical sequestration is observed. For example, 49.9% of GM-CSF and 37.1% of EGF were bound before the potential was applied, and 59.2% of GM-CSF and 67.0% of EGF were bound after the potential was applied at GB2-UGB2-UGB1 09. Similarly, 77.8% of GM-CSF and 69.2% of EGF were bound before the potential was applied, and 78.4% of GM-CSF and 64.8% of EGF were bound after the potential was applied at GB2-UGB2-UGB1 09. This may be because applying a positive potential increases the charge of the PEDOT. (10.1021 / acsapm.8b00061, 10.1021 / acsami.5b04768).This leads to a positive effect on the binding of negatively charged proteins such as GM-CSF and EGF due to the increased ionic interaction. However, since the negative charge of the anionic component in the hydrogel is constant and the stronger positive charge of the PEDOT can lead to partial charge compensation, the overall uptake of positively charged proteins is reduced.

[0058] Applying a negative potential can reduce or even completely neutralize the positive charge of the PEDOT (10.1021 / acsapm.8b00061, 10.1021 / acsami.5b04768). At a potential of -500 mV, a reduction in substance sequestration is predominantly observed, independent of the charge of the substances and independent of the P1 and P2 of the hydrogel materials (Tables 5-1, 5-2, 5-3 and 5-4). Thus, the sequestration of SDF-1α decreases from 72.3% to 47.2%, NGF-β from 94.1% to 75.0%, and IL-8 from 70.8% to 35.4% in GB2-UGB2-UGB1 09. A similar trend can be observed for GB2-UGB2-UGB1 10 and GB3-UGB2-UGB1 11 / 12. This confirms the assumption that the positive charge of PEDOT plays an important role in the binding of predominantly positively charged substances. This can be caused, for example, by the binding of negatively charged domains of the substances (proteins) to the positive charges of PEDOT, the phenomenon described previously.The sequestration of negatively charged substances shows a very strong reduction at -500 mV. For example, the sequestration of GM-CSF decreases from 49.9% to 17.9% and EGF from 37.1% to 0.0% in GB2-UGB2-UGB1 09, and of GM-CSF from 77.8% to 44.9% and EGF from 69.2% to 42.8% in GB2-UGB2-UGB1 10. The reason for this strong reduction in the sequestration of negatively charged substances at a negative potential is presumably due to the reduction in the positive charge of the PEDOT. This results in significantly fewer binding sites for negatively charged substances. Drug release

[0059] Following sequestration (loading of the hydrogels according to the previously mentioned third loading method), the bioactive molecules were released under electrical control. Due to the highest average sequestration, samples loaded with substances at a potential of 0 mV (passively) for 24 h were used for this purpose. Controlled release of actively sequestered substances can also be performed according to the protocol described below.

[0060] After loading the hydrogels, a brief washing step was performed to remove weakly bound proteins. For this, the hydrogel was first centrifuged at 3000 rpm for 1 minute to remove any adhering liquid. The hydrogels were then washed in 1 ml of PBS with BSA and centrifuged again at 3000 rpm for 1 minute. Release was also carried out using the 3-electrode setup described in the Drug Sequestration chapter, at applied potentials of 500 mV, 0 mV, and -500 mV. Samples were taken for the initial solution (control), after sequestration, after washing, and for release after 0 min, 10 min, 30 min, 1 h, 6 h, 8 h, and 24 h. Since saturation of the released proteins could already be observed after 8 h, the results of the release lying in the plateau after 8 h are shown (Table 5-1, 5-2, 5-3 and 5-4).The concentration of the various substances was determined according to the manufacturer's instructions using the Multiplex Assay Kit (Luminex Technology, Thermo Fisher).

[0061] The release of bound substances can be adjusted depending on the configured parameters of the anionically charged polymer network in combination with the charge of the PEDOT (Tables 5-1, 5-2, 5-3, and 5-4). At a high P1 (60 µmol / ml, GB2-UGB2-UGB1 09), only a very small release of positively charged substances occurs (0.0% SDF-1α; 0.0% FGF-2; 0.5% IL-8). For positively charged substances, however, a moderately small release can be observed (2.3% GM-CSF; 9.1% EGF). Due to the likely overcompensation of the positive charge of the PEDOT by the anionic groups, the release of negatively charged substances is probably significantly faster than the release of positively charged proteins. At low P1 (2 µmol / ml, GB2-UGB2-UGB1 10) there is an increased release of the positively charged substances (0.8 % SDF-1α; 0.3 % FGF-2; 11.7 % IL-8).Negatively charged substances, on the other hand, are released in lower quantities compared to a higher P1 (0.4% GM-CSF; 2.0% EGF). If P2 is reduced while maintaining similar P1 values ​​(GB3-UGB2-UGB1 11 / 12), an increased release of negatively charged substances (4.3 / 3.7% GM-CSF; 94.1 / 73.5% EGF) can be achieved with a virtually unchanged 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, Table 5). This is also due to the compensation of positive charges in the PEDOT by anionic groups despite the lower charge density.

[0062] Applying a potential of 500 mV results in the retention of bound substances, regardless of their charge. This effect is particularly evident for GB2-UGB2-UGB1 09, where the release of almost all 14 proteins was reduced to 0% (Tables 5-1, 5-2, 5-3, and 5-4). Similarly, at moderate P1 (9 µmol / ml) and very low P2 (0.94 mmol / (g / mol)) in GB3-UGB2-UGB1 12, the release of all substances was reduced to almost 0% (Tables 5-1, 5-2, 5-3, and 5-4). This could be due to the swelling rate of the hydrogels. At a positive potential, the increased positive charge of the PEDOT compensates for the presence of more anionically charged groups. Assuming that there are more anionic groups in the hydrogel material compared to the positive charges of the PEDOT, the overall charge of the hydrogel shifts towards neutral, resulting in a loss of water from the hydrogel and consequently a shrinkage.This leads to a reduced mesh size of the polymer chains, which in turn results in reduced protein release, regardless of their charge. Applying a potential of -500 mV results in increased release for most of the substances (Tables 5-1, 5-2, 5-3, and 5-4). 5) This primarily affects negatively charged bioactive molecules. For example, after applying the potential, the release of TNF-α, GM-CSF, and EGF for the hydrogel material type GB2-UGB2-UGB1 09 increases from 1.4 / 2.3 / 9.2% to 5.3 / 10.4 / 67.2%, and for GB2-UGB2-UGB1 10 from 1.7 / 0.4 / 2.0% to 53.2 / 5.9 / 72.6%. This trend can also be observed for the hydrogel material types GB3-UGB2-UGB1 11 / 12. Interestingly, substances that already exhibit a higher release without an applied potential are released in the greatest quantity when a negative potential is applied. This is most likely due to the reduction / neutralization of the positive charge of the PEDOT.This results in a higher number of free anionic groups, which, through ionic interactions, cause the repulsion of negatively charged proteins. However, increased release can also be observed with positively charged substances. For example, the release of FGF-2, IL-4, and IL-8 for the hydrogel material type GB2-UGB2-UGB1 10 increases from 0.3% to 2.6%, 0.6% to 1.7%, and 11.7% to 26.3%, respectively. This could also be due to the increased number of free anionically charged groups resulting from the lack of charge compensation with PEDOT. The resulting increased net negative charge of the hydrogel leads to swelling. This promotes release regardless of charge, which can also lead to a higher release of positively charged substances. Hydrogel biocompatibility

[0063] For the biocompatibility study, the PC12 cell line and the GB1-UGB2 17 hydrogel material type were used. This cell line consists of pheochromocytoma cells, which can be differentiated into neuron-like cells within a few days upon addition of NGF-β. The PC12 cell line is a widely used model for neuronal differentiation. For PC12 cell culture, the washed and PBS-swollen electrically conductive hydrogels were first rinsed in 70% ethanol for 10 minutes to kill any microorganisms and then washed in sterile PBS. After treatment with collagen type I (50 µg / ml, 1 ml / 100 µl gel, Gibco™< ) in 20 mM sterile acetic acid for 2 hours at room temperature, the gels were washed in PBS for 30 minutes. For NGF-β-loaded gels, an additional incubation with NGF-β (100 ng / ml, Sigma-Aldrich) in PBS with 1% BSA was performed overnight. Subsequently, 50,000 cells per cm² of gel surface were seeded.Cells were seeded and incubated in RPMI1640 (Gibco™<) with the addition of 10% equine serum (Gibco™<), 5% fetal calf serum (Gibco™<), and 1% penicillin and streptamycin (Gibco™<) to the medium. Depending on the cultivation conditions, pure medium, the addition of 100 ng / ml NGF-β to the medium, or a nutrient-reduced medium (reduced) with only 1% equine serum (Gibco™<) and 0.5% fetal calf serum was used. After incubation of 5 or 7 days with medium changes every 2 days, the cells were fixed for 20 minutes at room temperature in 4% PFA in PBS. For imaging, the cells were stained with phalloidin (Abcam) and DAPI (Pierce) according to the manufacturers' instructions. Images were acquired using a fluorescence microscope. Cell count and cell circumference were analyzed using FIJI / ImageJ. watershed and analyze particles (cell count) and the skeletonize Plugins.

[0064] Cell adhesion and cell proliferation were observed on the gels under all cultivation conditions. A significant increase in cell count and a decrease in cell circumference were observed on the hydrogels between 7 and 5 days without the addition of NGF-β when comparing the two. (Table 6).This indicates high cell proliferation. The addition of NGF-β to the medium leads to the differentiation of PC12 cells after only 5 days (Table 6). This was confirmed by an increase in cell circumference due to the growth of axon-like structures. Adding NGF-β to the hydrogel but not to the medium results in only a slight increase in cell circumference and a small increase in cell number (Table 6). Without a stimulus, the amount of released NGF-β is too low 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 leads to increased differentiation. Therefore, the cells possess the ability to proliferate and differentiate on the hydrogels under all cultivation conditions, confirming the high biocompatibility of the hydrogels. Reference symbol list

[0065] 1 Electrically conductive hydrogel material / Hydrogel material 2 Polymer network 3 Anionically charged building blocks 4 Crosslinker molecule 5 Electrically conductive component / PEDOT 6 Bioactive substance / Bioactive substances / Signal protein 7 Voltage source Table 1

[0066] Table 1: Overview of the charged and uncharged hydrogel building blocks of the polymer networks Charged and uncharged building blocks Building blocks abbreviation M [g / mol] Number of maleimide groups [1 / mol] Number of anionic groups [1 / mol] Number of anionic groups (pKa<2.5, [1 / mol]) P2 [mmol / (g / mol)] P3 *10 -3< [ 1 / Å 2< ] Heparin GB1 14000 7 87,6 67,6 4,5 -5,9 Poly(4-styrenesulfonic acid-co-maleic acid) 3:1 GB2 21100 9 142,8 92 4,5 6,8 Poly(acrylic acid-co-acrylamidoethane hydrogen sulfate) 1:1 GB3 26200 12 149 80 3,2 -2,5 Poly(acrylic acid-co-acrylamidoethane hydrogen sulfate) 9:1 GB4 18400 12 149 16 0,9 0,7 6-O,N-desulfated heparin GB5 12300 7 42,5 22,5 1,8 -6,8 4-arm polyethylene glycol, maleimide-terminated UGB1 10000 0 0 0 0 -2,82 4-arm polyethylene glycol, thiol-terminated UGB2 10000 0 0 0 0 -2,82 4-arm polyethylene glycol, amine-terminated, thiol-terminated UGB3 10000 0 0 0 0 -2,82 Table 2

[0067] Table 2: Composition and synthesis of polymer networks as a precursor to a hydrogel material Hydrogel material type charged polymer building block Solvents for charged and uncharged building blocks GBx (mg / ml) UGB1 (mg / ml) UGB2 (mg / ml) GB1-UGB2 01 GB1 0.01 x PBS, pH 4 30,8 0,0 21,8 GB1-UGB2-UGB1 02 GB1 0.01 x PBS, pH 4 3,7 14,3 17,3 GB1-UGB2-UGB1 03 GB1 0.01 x PBS, pH 4 1,3 16,4 16,8 GB1-UGB2-UGB1 04 GB1 0.01 x PBS, pH 4 0,4 17,1 16,8 GB2-UGB2 05 GB2 0.01 x PBS, pH 4 31,8 0,0 32,1 GB2-UGB2-UGB1 06 GB2 0.01 x PBS, pH 4 4,1 14,8 25,7 GB2-UGB2-UGB1 07 GB2 0.01 x PBS, pH 4 1,4 16,6 16,8 GB2-UGB2-UGB1 08 GB2 GB2: 0.01 PBS, pH 4; UGB: 0.01 PBS, pH 5.9 0,4 17,1 16,8 GB2-UGB2-UGB1 09 GB2 0.01 x PBS, pH 4; 21,2 6,9 16,8 GB2-UGB2-UGB1 10 GB2 0.01 x PBS, pH 4; 0,7 17,0 16,8 GB3-UGB2-UGB1 11 GB3 0.01 x PBS, pH 4 19,8 9,5 16,8 GB4-UGB2-UGB1 12 GB4 0.01 x PBS, pH 4 13,9 9,5 16,8 GB1-UGB2 13 GB1 0.1 x PBS, pH 6 44,8 0,0 31,7 GB5-UGB2-UGB1 14 GB5 0.01 x PBS, pH 4 6,2 12,5 16,8 UGB1-UGB2 15 - UGB1: 0.01 PBS, pH 4; UGB2: 0.01 PBS, pH 6.4 0,0 17,3 16,8 GB1-UGB2 16 GB1 0.1 x PBS, pH 6 44,8 0,0 31,7 GB1-UGB2 17 GB1 0.1 x PBS, pH 6 44,8 0,0 31,7 GB1-UGB2 18 GB1 0.1 x PBS, pH 6 44,8 0,0 31,7 GB2-UGB2-UGB1 19 GB2 0.01 x PBS, pH 4 30,8 0,0 14,9 GB2-UGB2-UGB1 20 GB2 0.01 x PBS, pH 4 0,6 14,9 15,2 GB3-UGB2-UGB1 21 GB3 0.01 x PBS, pH 4 29,9 0,0 22,8 GB4-UGB2-UGB1 22 GB4 0.01 x PBS, pH 4 28,9 0,0 19,6

[0068] Tables 3-1 and 3-2:electrical properties of the various electrically conductive hydrogel materials Table 3-1 Hydrogel material type GB1-UGB2 01 GB1-UGB2-UGB1 02 GB1-UGB2-UGB1 03 GB1-UGB2-UGB1 04 GB2-UGB2 05 GB2-UGB2-UGB1 06 GB2-UGB2-UGB1 07 GB2-UGB2-UGB1 08 Relative swelling degree 1,29 1,58 1,46 1,11 1,11 1,47 1,11 1,11 P0 (µmol / ml) after swelling 46 4 2 1 51 5 2 1 P1 (µmol / ml) after swelling 110 11 4 2 128 12 5 2 P2 (mmol / (g / mol)) 4,5 4,5 4,5 4,5 4,5 4,5 4,5 4,5 P3 (1 / A-2)*10^3 -5,9 -5,9 -5,9 -5,9 6,8 6,8 6,8 6,8 charged polymer building block GB 1 GB 1 GB 1 GB 1 GB 2 GB 2 GB 2 GB 2 Charge storage capacity (mC / ml) 3040 2350 1811 1505 2730 3040 2140 1894 ± SD 28,28 14,14 21,21 128,69 14,14 56,57 28,28 82,02 Impedance at 0.01 Hz (Ω) 30 42 81 93 42 36 68 74 ± SD 1,52 2,12 4,06 4,65 2,08 1,82 3,39 3,70 Table 3-2 Cryogel Hydrogel material type GB2-UGB2-UGB1 09 G B2-UGB2-UGB1 10 G B3-UGB2-UGB1 11 G B4-UGB2-UGB1 12 GB1-UGB2 13 GB5-UGB2-UGB1 14 UGB1-UGB2 15 GB1-UGB2 16 GB1-UGB2 17 GB1-UGB2 18 Relative swelling degree 1,50 1,30 1,43 1,39 1,25 1,25 1,25 1,25 1,60 1,60 P0 (µmol / ml) after swelling 24 1 25 26 34 6 0 34 26 26 P1 (µmol / ml) after swelling 60 2 43 9 81 9 0 81 81 81 P2 (mmol / (g / mol)) 4,5 4,5 3,2 0,9 4,5 1,8 0,0 4,5 4,5 4,5 P3 (1 / A-2)*10^3 6,8 6,8 -2,5 0,7 -5,9 -6,8 -2,8 -5,9 -5,9 -5,9 charged polymer building block GB 2 GB 2 GB 3 GB 4 GB 1 GB5 UGB1 GB 1 GB 1 GB 1 Charge storage capacity (mC / ml) 1819 1659 1222 910 2887 2453 921 37 2853 30 ± SD 176,65 37,43 16,97 59,40 122,20 70,24 22,48 2,14 94,52 0,90 Impedance at 0.01 Hz (Ω) 92 103 93 150 53 62 167 8946 53 10636 ± SD 3,36 6,18 25,51 20,39 0,91 1,37 8,46 1143,08 1,26 1112,39

[0069] Tables 4-1 to 4-4: Binding of bioactive substances by electrically conductive hydrogel materials

[0070] Tables 5-1 to 5-4: Release of bioactive substances through electrically conductive hydrogel materials at different electrical potentials

[0071] Table 6:Biocompatibility of electrically conductive hydrogel materials with PC12 cells and neuron-like cells differentiated therefrom

Claims

1. A method for detecting and influencing an absorption of bioactive molecules (6) in a hydrogel material (1) and / or a release of bioactive molecules (6) from the hydrogel material (1), wherein the hydrogel material (1) is a polymer network (2) that is formed of anionically charged building blocks (3) and uncharged building blocks, has an affinity for bioactive molecules (6) that is configurable by means of parameters that define the anionically charged building blocks (3), and has an electrically conductive component (5), the electrical resistance and electrical charge storage capacity of which depend on an interaction with the hydrogel building blocks (3) and on bonding of bioactive molecules (6) to the hydrogel material (1), wherein the electrically conductive component (5) is suitable for changing the anionic charge of the hydrogel material (1) and its affinity for bioactive molecules (6) by the influence of an electrical potential, in which method the hydrogel material (1) is brought into contact with a biofluid, wherein a change in the electrical resistance and / or a change in the charge storage capacity of the hydrogel material (1) is detected, and an absorption of bioactive molecules (6) into the hydrogel material (1) or a release of bioactive molecules (6) from the hydrogel material (1) into the biofluid is determined on the basis of the detected change in the electrical resistance and / or on the basis of the detected change in the charge storage capacity, and / or wherein a concentration of bioactive molecules (6) in the biofluid and / or a concentration of bioactive molecules (6) in the hydrogel material (1) is influenced by an electrical potential acting on the hydrogel material (1).

2. The method according to Claim 1, characterised in that, for the release of bioactive molecules (6), the hydrogel material (1) is loaded with a predefined concentration of a predefined bioactive molecule (6) before contact with the biofluid.

3. The method according to Claim 1 or 2, characterised in that the polymer network (2) is configurable in composition by means of at least three parameters that define the anionically charged building blocks (3), selected from a group of parameters P0, P1, P2, P3, wherein parameter P0 corresponds to a value of the number of the ionised, anionic groups, assuming 30% ionisation of all anionic groups, per unit volume of the hydrogel material (1) swollen under physiological conditions, parameter P1 corresponds to a value of the number of the highly anionic groups, with an intrinsic pKs value of less than 2.5, per unit volume of the hydrogel material (1) swollen under physiological conditions, parameter P2 corresponds to a value of the number of the highly anionic groups, with an intrinsic pKs value of less than 2.5, per repeating unit divided by the molar mass of the repeating unit, and parameter P3 corresponds to a value for describing the amphiphilia of the anionic building blocks (3), wherein an electrical resistance and / or an electrical charge storage capacity of the hydrogel material (1) is predefined by parameter values of a parameter configuration of the hydrogel material (1).

4. The method according to Claim 1 to 3, characterised in that, for the detection of bonding of bioactive molecules (6), a change in the impedance of the hydrogel material (1) is measured at at least one frequency in the range of 0.1 Hz to 1 MHz.

5. The method according to one of Claims 1 to 4, characterised in that, for the absorption of bioactive molecules (6), an electrical potential in the range of 1 mV to 1000 mV, preferably in the range of 400 mV to 600 mV, is applied to the hydrogel material (1), and that, for the release of bioactive molecules, an electrical potential in the range of -1 mV to -1000 mV, preferably in a range of -400 mV to -600 mV, is applied to the hydrogel material (1).

6. The method according to one of Claims 1 to 5, characterised in that, for the absorption of bioactive molecules (6), a constant electrical current of greater than 0 mA is applied to the hydrogel material (1), wherein the direction of the flow of electrical current is changed to release bioactive molecules (6).