Methods and materials for separating and isolating different classes of substances using hydrogels containing sulfated or sulfonated components

Abstract

A method is provided that allows the concentration of certain substances in biofluids to be consciously influenced. [Solution] This method involves separating and isolating different substances from substance groups A and B within a sulfated and / or sulfonated hydrogel, depleting the substances from substance group A from the biofluid, and simultaneously releasing the substances from substance group A or B from the sulfated and / or sulfonated hydrogel into the biofluid by another method, and / or weakening the bonds of the substances from substance group B within the sulfated and / or sulfonated hydrogel.

Description

[Technical Field]

[0001] The present invention relates to a method for the differentiated sequestration of substances of different substance groups A and B using a hydrogel containing sulfated and / or sulfonated components, and for the differential and simultaneous release of substances of substance group A or B into a biofluid from the hydrogel containing sulfated and / or sulfonated components, thereby depleting substances of substance group A and / or reducing the binding of substances of substance group B in the hydrogel containing sulfated or sulfonated components. The present invention also relates to a synthetically obtainable hydrogel based on poly(4-styrenesulfonic acid-co-maleic acid) as a network component and amine- or thiol-containing crosslinking molecules as another network component, characterized as a material for the differentiated sequestration of substances of different substance groups, and used in the above-mentioned method. Substance groups A and B are not chemically defined groups, but may contain different substances that, depending on the type of hydrogel, result in a differentiated segregation according to the invention in a corresponding constellation and composition within the overall system. Substance groups A and B are therefore defined by their behavior within the overall system. [Background technology]

[0002] The field of application of the present invention is in the fields of biotechnology and medicine, where certain substances are selectively removed at the molecular level from a biofluid and sequestered in a hydrogel, while other substances are not specifically sequestered but are selectively released from the hydrogel into the biofluid. Therefore, in a broader sense, it is a molecular-level separation process. In a broader sense, the field of application of the present invention is the use of graded negatively charged hydrogels for technical, biomedical, and biological applications, such as for culturing mammalian cells or for antibacterial finishes on surfaces.

[0003] Hydrogels containing sulfated components, such as starPEG-glycosaminocyclo-hydrogels, are also known from WO 2010 / 060485 A1 and are being considered for use in biotechnological applications or for use in implants or regenerative therapy or as tissue replacement materials.

[0004] A key feature of these materials is the interaction that occurs between sulfate groups on the polymer chains of the hydrogel components and soluble proteins, such as enzymes and signaling molecules that govern metabolic, transport, and signaling functions, including proteases, lipases, and amylases, and hormones, neurotransmitters, cytokines, growth factors, and chemokines.

[0005] The interactions that determine their affinity for proteins are primarily based on electrostatic forces between the sulfate or sulfonate groups of the hydrogel, which are negatively charged under the applied conditions, and the amino acid side chains of the protein, which are positively charged.

[0006] This principle has been applied in the development of hydrogels in the art by incorporating sulfated sugars (glycosaminoglycans = GAGs) for the sustained release of signaling molecules, such as the growth factors VEGF (Vascular Endothelial Growth Factor) and FGF-2 (Fibroblast Growth Factor 2). It has already been widely used, including for the release of FGF-2 and VEGF. Additionally, the release of FGF-2 and VEGF may be progressively greater with a lower degree of sulfated star PEG-GAG hydrogel. Synthetic polymers containing sulfonic acid groups, such as polystyrene sulfonate (PSS) (J. Phys. Chem., B, 2007, 111(13), pp. 3391-3397, DOI: 10.1021 / jp067707d9) or 2-acrylamido-2-methylpropanesulfonic acid (U.S. Pat. Nos. 5,451,617 and 5,011,275, and U.S. Patent Application Publication No. 2008 / 011412), have been used as sulfonated components to prepare hydrogels intended for use in contact lenses, for example.

[0007] However, no covalently crosslinked, water-containing polymer networks bearing sulfonic acid groups, i.e., hydrogels, are currently known, although the carboxyl groups of poly(4-styrenesulfonic acid-co-maleic acid) have been used as functional groups for crosslinking with amine-group-containing short-chain crosslinkers or amine-group-containing polymers.

[0008] Another drawback is that interactions between hydrogels (i.e., building blocks) containing sulfated or sulfonated components and proteins have mostly been studied only from solutions containing one or two signaling molecules, often not under physiologically relevant conditions. However, in relevant biological contexts, biofluids containing a variety of different proteins are present at various concentrations, but the signaling molecules are present in biofluids at extremely low levels, ranging from only 100 pg / mL to 2000 ng / mL, whereas other proteins, such as albumin, are present at high concentrations of approximately 60 mg / mL.

[0009] In this case, the primary electrostatic attraction between the positively charged regions of the signaling molecule and the negatively charged sulfate or sulfonate groups in the hydrogel plays an important role, for which the isoelectric point (IEP) of the protein is often used. However, in addition to the net charge under physiological conditions, which can be inferred based on the IEP, the charge distribution, e.g., the distribution of positively charged charge clusters, can also be estimated. The presence of clusters of proteins, as well as secondary interactions (which are influenced by protein size and structure, as well as weaker non-ionic intermolecular forces such as hydrophobic interactions, hydrogen bonding, or dipole-dipole interactions), play a role, making it extremely difficult to predict the absolute or relative binding of proteins to hydrogels. Additionally, there are often-ignored competing processes, such as the interaction of the target protein with albumin, which is responsible for osmotic pressure regulation and transport processes, and the interaction between albumin and the hydrogel. Furthermore, binding constants used in the literature to predict molecular interactions between charged polymers and proteins are only valid for describing the interaction of individual molecule-protein complexes in solution; in contrast, in hydrogels, neighboring polymer chains, three-dimensional charge distribution, or affinity centers within the polymer network have a strong influence on the resulting interactions and, therefore, on the binding of proteins to the hydrogel.

[0010] Therefore, the selectivity of binding between hydrogels (building blocks) containing sulfated and / or sulfonated components and molecular components of complex biofluids has not been elucidated until now, and the demonstrated methods and processes have therefore only allowed, to a very limited extent, the selective modulation of signaling molecule levels in application-relevant biofluids by hydrogels (building blocks) containing sulfated or sulfonated components. Summary of the Invention [Problem to be solved by the invention]

[0011] The aim of the present invention is to propose a method that makes it possible to consciously influence the concentration of certain substances in a biofluid. [Means for solving the problem]

[0012] This object is achieved by a method and material having the features of the independent claims. Suitable materials for carrying out the process include a fully synthetic hydrogel system based on poly(4-styrenesulfonic acid-co-maleic acid), which is negatively charged under physiological conditions and therefore enters into close association with partially positively charged biomolecules, thereby isolating the substance from a diverse group of substances. Further developments of the method and material are described in the dependent claims. The term "fully synthetic" means that no components of biological origin are required for hydrogelation, thus eliminating the risk of adverse immunogenic reactions. [Brief explanation of the drawings]

[0013] [Figure 1] Representative light microscope images of human endothelial cells cultured on hydrogels for 24 hours are shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] In the context of the present invention, "sequestration of substances from the substance groups A and B" refers to binding of these substances, such as signaling molecules, factors, or enzymes, to affinity centers in the hydrogel material, thereby reducing their concentration or completely removing them from the biofluid upon direct contact with the hydrogel. Hydrogels are composed of charged or uncharged building blocks.

[0015] The present invention includes a hydrogel containing sulfate and / or sulfonate groups having the following properties and composition: a polymer network formed by covalent (chemical) or physical crosslinking between two hydrogel components or hydrogel building blocks, as described, for example, in WO 2014040591 A2. The first building block or component of the hydrogel is a molecule that is uncharged under physiological conditions (also called an uncharged building block, or UGB), preferably having a molar mass of 20 g / mole to 100,000 g / mole. The uncharged molecule is advantageously selected or derived from the class of polyethylene glycol, poly(2-oxazoline), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and / or polyacrylamide (PAM), or short-chain bifunctional crosslinker molecules. Furthermore, the UGB has at least two functional groups, preferably 4 to 8 functional groups, which are particularly suitable for crosslinking. Suitable functional groups for cross-linking (GB or UGB) include: amine, thiol, carboxyl, anhydride, maleimide, vinyl sulfone, acrylate, hydroxyl, isocyanate, epoxide, and aldehyde groups, or groups capable of forming non-covalent bonds based on electrostatic forces, hydrophobic interactions, hydrogen bonds, dipole interactions. The second building block (component) consists of a polymer with (sulfur-based) sulfate or sulfonate groups, which are therefore negatively charged under physiological conditions (charged building block = GB) (optionally with a molar mass of 2,000 to 250,000 g / mole) and are primarily electrostatically Proteins can be bound within the hydrogel network through electrophilic (ionic) interactions, and to a lesser extent through weaker bonds such as van der Waals forces, hydrogen bonds, or hydrophobic interactions. In the present invention, the affinity centers that significantly define protein binding are sulfate or sulfonate groups, which are highly negatively charged under physiological conditions. Sulfated or sulfonated polymers are sulfated glycosaminoglycans obtained from natural sources, such as heparin and selectively desulfurized heparin, chondroitin sulfate, heparan sulfate, keratan sulfate, sulfated hyaluronic acid, as well as sulfated glycopolymers based on mannose, lactose, dextran, and polysulfonated compounds (which may be included as sulfur-containing monomers), such as styrenesulfonic acid (SS), vinylsulfonic acid (VS), 2-acrylamido-2-methylpropanesulfonic acid (AMPS), aminopropanesulfonic acid (APS), or anetholesulfonic acid (AS), also in copolymers with units containing the above-mentioned crosslinking groups.

[0016] The negatively charged sulfate or sulfonate groups of the affinity centers are intentionally distributed only along the GBs, whereas the UGBs are components that minimize protein adsorption. Any carboxyl groups present on the GBs that are not available for cross-linking reactions are believed to be irrelevant for protein binding within the hydrogel because the sulfate or sulfonate groups of the GBs are significantly more acidic and therefore preferably deprotonated.

[0017] Physiological conditions are aqueous solutions with a pH and ionic strength in the tissue that closely corresponds to the physiological salt concentration in human tissue, and therefore are adjusted to pH 7.4 and approximately 0.9% NaCl using a phosphate buffer.

[0018] Biofluids in the present invention are aqueous solutions with various salt contents, preferably physiological salt concentrations, and a mixture of proteins.

[0019] Its protein content is variable, consisting mostly of low levels of water-soluble globular proteins, such as signaling molecules, enzymes, or factors, ranging from 100 pg / mL to 2000 ng / mL, as well as albumin, which regulates osmotic and transport processes in the body and is present in higher concentrations, e.g., about 60 mg / mL.

[0020] According to the concept of the present invention, the binding of proteins regulating metabolic, transport, and signaling functions (e.g., signaling molecules and enzymes) to hydrogels containing sulfated or sulfonated components (GBs) is significantly determined by, on the one hand, the electrostatic interactions between the GBs and the proteins, as well as the size and structure of the proteins (e.g., the presence of specific protein domains), and, on the other hand, the network parameters of the hydrogels, which were measured by: (1) the concentration of sulfate or sulfonate in the swollen hydrogel (mmoles of sulfate or sulfonate / mL) in the hydrogel swollen under physiological conditions; and (2) the number of sulfate or sulfonate groups per GB repeat unit, as the characteristic size of the GB charge, divided by the molar mass of the GB repeat unit (number of groups / (g / mole)). Parameter (1) is related to the number of interaction centers (corresponding to negatively charged sulfate or sulfonate groups in the three-dimensionally swollen hydrogel network) and will therefore significantly affect the binding of proteins (signaling molecules, enzymes) into the hydrogel. This parameter includes the intermolecular interactions (i.e., interactions between adjacent polymer chains) between the GBs and proteins (signaling molecules, enzymes). Meanwhile, parameter (2) indicates the density and distribution of interaction centers (corresponding to negatively charged sulfate or sulfonate groups) on different GBs. This will determine the interaction of the protein with each individual GB, and thus include specific interactions via spatial modification of the charge centers of the polymer chains, known for example as protein-glycosaminoglycan interactions (see Capila, I. Linhardt, RJ Angew Chem Int Ed Engl, 2002, 41(3), 391-412). The binding and release behavior between the hydrogel and proteins (signaling molecules) is determined in the present invention by superimposing the results of the hydrogel network properties obtained from both parameters (1) and (2), and can therefore be quantitatively described by specifying these parameters (see also the results of exemplary embodiments UGB1-GB1 01 to UGB1-GB4 04).

[0021] Furthermore, steric effects must be taken into account, since the steric accessibility of the hydrogel network must be guaranteed in order to bind and sequester proteins, i.e., the size of the protein must not exceed the mesh size of the hydrogel (e.g., estimated from the storage modulus of the hydrogel by methods of rubber elasticity theory, relevant to the materials and methods).

[0022] By contacting biofluids with hydrogels that can be selectively graded for their network structure and their degree of sulfate or sulfonation, the concentration of relevant signaling molecules in the biofluid can be regulated through the specific selectivity of binding of the signaling molecules to the hydrogel, and thus for biological or biotechnological applications, which in a medical context can relate to the modification of soluble signaling molecules for angiogenesis, vascular sprouting, control of immune responses, especially neurodegenerative and autoimmune and cancer diseases, for the treatment of diabetes, in skin wound healing and bone regeneration, for the inhibition of tumor growth, and for disinfection and antibacterial treatments within and on the body. Biotechnological applications include in vitro cell and organ culture of embryonic stem cells (ES), induced pluripotent stem cells (iPS), other non-ES and iPS-related stem and progenitor cells, primary, patient-derived cells, immortalized cell lines, and cardiac, muscular, renal, liver, and nervous tissue, as well as enrichment or depletion and isolation of protein mixtures, particularly signaling molecule or enzyme mixtures.

[0023] Another aspect of the present invention relates to a covalently crosslinked hydrogel material for carrying out the above-mentioned method, based on charged building blocks in the form of poly(4-styrenesulfonic acid-co-maleic acid), as previously mentioned, and uncharged building blocks in the form of a polymer containing amine or thiol groups, or a crosslinker molecule having at least two amino or thiol groups. These charged and uncharged building blocks are crosslinked to form a polymer network, which can be obtained by activating the carboxyl groups of poly(4-styrenesulfonic acid-co-maleic acid) with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) / N-hydroxysulfosuccinimide (sulfo-NHS) and directly crosslinking with a polymer containing amine groups or a crosslinker molecule having at least two amino groups, respectively, to form amides, or by functionalizing the activated carboxyl groups by means of a bifunctional crosslinker molecule having an amino group and a group capable of Michael-type addition, respectively, followed by crosslinking via Michael-type addition with a polymer containing thiol groups or a crosslinker molecule having at least two thiol groups, respectively. The group capable of Michael-type addition is preferably selected from maleimide groups, vinyl sulfone groups, and acrylate groups. Polymers containing amine or thiol groups as uncharged building blocks include polyethylene glycol (PEG), poly(2-oxazoline) (POX), and PEG-100 (PEG-100). Preferably, the crosslinking agent is selected from the class of polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and / or polyacrylamide (PAM). The short-chain crosslinking molecules containing amine or thiol groups used instead are preferably non-polymeric bifunctional crosslinking molecules. Poly(4-styrenesulfonic acid-co-maleic acid) as the charged building block is advantageously selected with a molar ratio of 4-styrenesulfonic acid to maleic acid ranging from 6:1 to 1:6 and a molar mass ranging from 5,000 to 100,000 g / mole. In a particularly preferred embodiment of the present invention, polymers containing enzymatically cleavable peptides are used as uncharged building blocks for polymer network formation. These enzymatically cleavable peptides preferably have either lysine (with an amino group in the side chain) or cysteine ​​(with a thiol group in the side chain) as the reactive amino acid in the peptide sequence. The enzymatically cleavable peptides can be advantageously cleaved using human or bacterial proteases, such as matrix metalloproteinase (MMP)-responsive peptides (e.g., PQGIWGQ, IPVSLRSG, or VPMSMRGG), cathepsin-responsive peptides (e.g., VPMSMRGG), elastase-responsive peptides (e.g., AAPV or APEEIMDRQ), coagulation enzyme-responsive peptides (e.g., thrombin-responsive peptides GGF-pipecolic acid RYSWGCG or GG-cyclohexylalanine ARSWGCG), FXa-responsive peptides (e.g., GGIEGRMGGWCG), kallikrein-responsive peptides (e.g., CGGGPFRIGGWCG), or bacterial protease-responsive peptides (e.g., aureolysin-responsive peptides ADVFEA or AAEAA, elastase-responsive peptides AAPV, or protease IV-responsive peptides (e.g., MKATKLVLGAVILGSTLLAG). The hydrogels constructed in this manner enable a self-regulating release and degradation mechanism.In another embodiment of the present invention, bioactive and / or antiadhesive molecules and / or cell-engineering peptides containing amino or carboxyl groups, particularly those selected from KCWG-RGDSP, KCWG-EIDGIELT, KCWG-IKLLI, KGCWGGRNIAEIIKDI, KGCWGGSDPGYIGSRSDDSA, KGCWGGPQVTRGDVFTMP, and KGCWGGKGGNGEPRGDTYRAY, are covalently bonded via lysines or cysteines in the sequence to the charged building block poly(4-styrenesulfonic acid-co-maleic acid) or its derivatives containing groups capable of Michael-type addition to the hydrogel network. These bioactive molecules may be antimicrobial agents, such as antibiotics or disinfectants, or pharmaceuticals. The antiadhesive molecules are preferably polyethylene glycol (PEG) or poly(2-oxazoline) (POX). The tissue-engineering peptides are preferably peptides derived from structural and functional proteins of the extracellular matrix, such as collagen, laminin, tenascin, fibronectin, and vitronectin. In an advantageous embodiment, the bioactive and / or anti-adhesive and / or tissue-engineering peptides are covalently attached to the hydrogel network via an enzymatically cleavable peptide sequence. As previously mentioned, the enzymatically cleavable peptides are preferably sensitive to human or bacterial proteases, such as MMPs, cathepsins, elastases, and blood clotting enzymes. This type of hydrogel allows for a self-regulating release and degradation mechanism while maintaining the hydrogel network. The hydrogel material preferably has a storage modulus of 0.2 to 22 kPa. The sulfonate concentration in the swollen network and the number of sulfonate groups per repeat unit (WE) divided by the molar mass (MW) of the repeat unit (units: g / mole) can be varied independently according to the ranges defined in Table 5.1 (see below).

[0024] Thus, the present invention provides a fully synthetic, highly hydrated hydrogel, which carries affinity centers for groups of substances via the sulfonate groups of poly(4-styrenesulfonic acid-co-maleic acid) as charged building blocks (GBs), and is purposefully rated. These materials possess adaptable physical and biochemical properties. In this case, poly(4-styrenesulfonic acid-co-maleic acid) is used as the charged building block (GB) in the context of the previously described hydrogel materials. This material offers the possibility of mapping all the important functions of the natural extracellular matrix (ECM) in a modular manner, i.e., largely independent of one another. More specifically, these functions are, first, framework, support, and protection for growing cells; second, regulation of cell adhesion; third, graded sequestration and reversible release of therapeutically relevant signaling molecules; and fourth, the possibility of on-demand remodeling by ingrowing cells. The physical properties, such as the stiffness and hydration of the material, can be tuned over a wide range. In a preferred embodiment of the present invention, covalently crosslinked hydrogels for this purpose were prepared by reacting the carboxyl groups of poly(4-styrenesulfonic acid-co-maleic acid) activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) / N-hydroxysulfosuccinimide (sulfo-NHS) with the terminal amino groups of linear or star-branched polyethylene glycol as an uncharged building block via a stable amide group. In an alternative embodiment, which is preferred when embedding living cells, in a first step the carboxyl groups of poly(4-styrenesulfonic acid-co-maleic acid) activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) / N-hydroxysulfosuccinimide (sulfo-NHS) are graded by means of a short-chain bifunctional crosslinker molecule, N-(2-aminoethyl)maleimide, i.e., functionalized with 6 to 10 molecules of N-(2-aminoethyl)maleimide per molecule of poly(4-styrenesulfonic acid-co-maleic acid), followed by purification and isolation.These derivatives of poly(4-styrenesulfonic acid-co-maleic acid) with N-(2-aminoethyl)maleimide then undergo spontaneous, bioorthogonal reactions when mixed with living cells and biomolecules in aqueous solutions or complex biofluids, but do not result in a change in their functionality within the context of the aforementioned hydrogel structures via a Michael-type addition between the maleimide groups of the functionalized poly(4-styrenesulfonic acid-co-maleic acid) derivatives and the thiol groups of the thiol-containing bifunctional crosslinker molecules or thiol-containing polymers as uncharged building blocks. These building blocks can be, for example, enzymatically cleavable peptides containing the amino acid cysteine ​​(which has a thiol group in its side chain) in their sequence and pre-conjugated to one or more arms of a four-arm polyethylene glycol (PEG) (Tsurkan et al., 2013 (Adv. Mater., 2013, 25, 2606-2610)). This type of crosslinking has the advantage that the reaction between maleimide and free thiol groups is rapid, resulting in a direct reaction without undesired side reactions with other biomolecules or cell surface proteins. Therefore, Michael-type addition reactions can be used as a bioorthogonal crosslinking reaction for polymerizing cells. It is also possible to functionalize hydrogels via the maleimide groups of poly(4-styrenesulfonic acid-co-maleic acid) derivatives by means of cell-engineering peptides, such as the amino acid sequence CWGRGDSP. Furthermore, hydrogel materials can be functionalized with antibacterial agents, such as positively charged antibiotics.

[0025] To implement the above-mentioned method, a physically crosslinked hydrogel material may be used instead of a covalently crosslinked hydrogel material. This, also fully synthetic, material is based on the physical interaction between charged building blocks in the form of poly(4-styrenesulfonic acid-co-maleic acid) and uncharged building blocks in the form of a polymer, where a highly positively charged peptide sequence is covalently attached to the polymer. The highly positively charged peptide sequence preferably contains at least 10 repeats of lysine or arginine, or at least 5 repeats of a dipeptide motif using lysine and alanine or arginine and alanine.

[0026] Further details, features, and advantages of embodiments of the present invention will become apparent from the following description of exemplary embodiments, which are also described and demonstrated by the accompanying tables and figures: Figure 1 shows a representative light microscope image of a cell culture of human endothelial cells on a hydrogel after 24 hours of culture.

[0027] Other favorable concentration ratios can also be established by carefully varying the properties of the network, and the proposed method also makes it possible to modulate the concentrations of these species in any biofluid by extending the analysis to additional signaling molecules.

[0028] [Table 1]

[0029] [Table 2]

[0030] [Table 3]

[0031] [Table 4]

[0032] [Table 5]

[0033] [Table 6]

[0034] [Table 7]

[0035] [Table 8]

[0036] In Tables 5.1, 5.2, 6.1, and 6.2, the number of sulfate or sulfonate groups per repeat unit (WE) divided by the molar mass (MW) of the WE is given in column A as the property of the charged unit [mole / g]. The properties of the swollen hydrogels are given in column B as the concentration of sulfate or sulfonate groups in mmole / mL and in column C as the storage modulus in kPa. Column D lists the properties of the uncharged building blocks.

[0037] Column E shows the molar concentration of sulfate or sulfonate groups of the charged building block per mole of polymer (units: mole / mole). Column F shows the hydrogel properties as GB concentration in mmole / mL, column G shows the concentration of sulfate or sulfonate groups in mmole / mL, and column C is the storage modulus.

[0038] A storage modulus of 20 kPa corresponds to a mesh size of approximately 6 nm, according to the assumptions made in this method, and should therefore provide steric accessibility within the hydrogel for all of the structurally similar signaling molecules discussed here.

[0039] [Table 9]

[0040] Table 7 lists substances from substance groups A and B at relevant concentrations in biofluids.

[0041] The present invention will now be described with reference to exemplary embodiments (AB) in the table shown above. do.

[0042] For example, the hydrogel from UGB1-GB1 01 (AB1) in Table 1 was found to be particularly advantageous for modulating the concentrations of different signaling molecules (see Table 3).

[0043] The class of chemokines has a high structural similarity with a tertiary structure that is stabilized by the interaction of four cysteines through disulfide bridges (corresponding to PROSITE ID: PS00471 and PS00472). Furthermore, chemokines, characterized by a strong positive net charge of IEP>9 or positively charged domains, such as MIP1-alpha and MIP1-beta, and a molar mass of less than 10 kDa (see Table 3), are strongly bound within the hydrogel by this hydrogel type and are thereby depleted from the adjacent biofluid (MIP1-alpha and MIP1-beta binding is approximately 60%, while other chemokines, eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, RANTES and SDF-1alpha, are bound by more than 95% (Table 3)), while, surprisingly, the following chemokines, the FGF family (corresponding to PROSITE ID: PS00247) and the TGFb1 family (PROSITE ID: PS00248) are strongly bound within the hydrogel by this hydrogel type and are therefore depleted from the adjacent biofluid (MIP1-alpha and MIP1-beta binding is approximately 60%, while other chemokines, eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, RANTES and SDF-1alpha, are bound by more than 95% (Table 3)). Strongly positively charged signaling molecules related to the class of growth factors and structurally similar proteins, FGF-2 (IEP, 9.58) and TGFb1 (IEP, 8.59), were bound very weakly, at 31% and 18%, respectively, and PLGF (IEP, 8.37) was bound even weaker, at less than 10% (Table 3). Signaling molecules related to the class of cytokines with slightly basic IEPs, such as IL-10 (IEP, 7.65), as well as signaling molecules with IEPs in the neutral range (pH, 5.5-7), such as IL1-beta, IL6, and TNF-alpha, as well as the IL-1-beta protein family (corresponding to PROSITE ID: PS00253), the IL-10 protein family (corresponding to PROSITE ID: PS00520), the interleukin-6 / GM-CSF / MGF family (PROSITE ID: PS00520), and the IL-10 protein family (PROSITE ID: PS00520) were also bound. Structurally similar proteins of the TNF-alpha protein family (corresponding to PROSITE ID: PS00254) and the TNF-alpha protein family (corresponding to PROSITE ID: PS00251) are only weakly bound to the complex biofluid (TNF-alpha binding, approximately 38%), at less than 30% (Table 3), and are therefore present in the biofluid at concentrations that are largely uncharged.Furthermore, EGF (IEP, 4.8) with its slight net negative charge and growth factors with less than 10% binding affinity are poorly bound. As a result, signaling factors essential for cell survival, such as EGF, FGF-2, TGFβ, or PLGF, remain untouched by the sequestration achieved by the hydrogel method described above.

[0044] The highly basic cytokines IFN-gamma (IEP, 9.52), IL-4 (IEP, 9.25), the WNT and BMP antagonists sclerostin (9.57) and DKK1 (8.72), as well as the structurally similar proteins IL-4 / IL-13 family (corresponding to PROSITE ID: PS00838) and sclerostin-like family (corresponding to InterPro ID: IPR008835), the highly basic growth factors bNGF (IEP, 9.00), PDGF (IEP, 9.4), and VEGF (IEP, 9.2), as well as the structurally similar proteins NGF family (corresponding to PROSITE ID: PS00248) and PDGF family (corresponding to PROSITE ID: PS00250) and IL-12p40, were strongly bound (>59.9%) and therefore depleted from the biofluid, as expected (Table 3).

[0045] By contacting the biofluid with hydrogels having specific charge and network properties of Type 1 as described in Table 5.1, DKK1, bNGF, PDGF-BB, VEGF, as well as chemokines (e.g., eotaxin, Gro-alpha, IL-8, IP-10, MCP-1alpha, MIP1beta, Ranthambolic Acid, etc.) can be produced according to the annotated sequence motifs. By selecting growth factors with proteins structurally similar to the IL-10, IL-1β, IL-6, and IL-1β (e.g., SDF1 alpha), and cytokines with proteins structurally similar to IFN-gamma, IL-4, and sclerostin, protein families can be depleted from the biofluid or their concentrations in the biofluid can be increased by pre-charging the hydrogel, while advantageously keeping the concentrations of the above-mentioned signaling molecules EGF, FGF-2, TGFb1, PLGF, IL-10, IL1 beta, IL-6, and TNF alpha largely unchanged. Furthermore, this hydrogel interacts with GB1 and thrombin and antithrombin, enzymes important in blood clotting (Uwe Freudenberg et al., Journal of Controlled Release (Journal of Controlled Release: Official Journal of the Controlled Release Society), 220, no. Part A, (2015 / 12 / 28): 79-88, doi:10.1016 / j.jconrel,2015.10.028).

[0046] Another particularly advantageous exemplary embodiment (AB2) consists of hydrogel type GB1-GB2 02 (see Table 2), which has approximately the same binding and sequestration pattern as hydrogel type UGB1:GB1 01 (see Table 3), except for weaker binding to two chemokines, MIP-1 alpha and MIP-1 beta, and sclerostin (about 40% each, Table 3), and even weaker binding to IL-10 (15%), IL-6 (8%), TNF-alpha (25%) (see Table 3), and more advantageously, almost non-existent binding to FGF-2 (5%) and TGFb1 (0%), and the lack of interaction of GB2 with the blood coagulation enzymes thrombin and antithrombin (see Uwe Freudenberg et al., Journal of Controlled Release (Journal of Controlled Release: Official Journal of the Controlled Release Society), 220, no. Part 1). A, (2015 / 12 / 28):79-88, doi:10.1016 / j.jconrel.2015.10.028). Another advantageous exemplary embodiment is UGB2-GB5 23 (AB23, see Table 1), which, like AB2, has charge characteristics that can be assigned to Type 2 according to Table 5.1, but has a slightly different sulfonate content of 0.14 mmole / mL (Table 1 or Table 5.2, column B) and a slightly different number of sulfonate groups per repeat unit (WE) divided by the molar weight (MW) of that WE [mole / g] of 0.0038, both of which are within the ranges given for Type 2 hydrogels in Table 5.1. However, that hydrogel, formed from another charged synthetic gel building block (GB5) using a different crosslinking reaction (crosslink type 2, Table 1), has nearly identical binding and sequestration patterns to AB2 (see Table 3), but differs in that it has somewhat stronger binding to two chemokines, MIP-1 alpha and MIP-1 beta. This supports the classification of hydrogels with characteristic charge patterns as described in Table 5.1, and also exhibits particularly advantageous sequestration patterns.

[0047] Another advantageous exemplary embodiment (AB3), represented by the hydrogel type UGB1-GB3 03 (see Table 2), has significantly weaker binding to many proteins (signaling molecules) due to the significantly lower concentration of sulfate or sulfonate groups in the hydrogel compared to AB1 and AB2 (Table 5.2, column B, 0.06, compared to 0.12 mmole / mL for AB1 or AB2), and also due to the lower number of sulfate or sulfonate groups per WE divided by the molar mass of WE (0.0019, compared to 0.005 or 0.0035 for AB1 or AB2) (see Table 5.2, column A). This type of hydrogel exhibits significantly weaker binding to many proteins (signaling molecules), including bNGF, PDGF-BB and VEGF A, eotaxin, GRO-alpha, IP-10, Rantes, S. DF-1 alpha, IFN-gamma, and IL-4 were bound by the hydrogel at over 50% and thus depleted from the biofluid. In contrast, IL-8, MCP-1, and IL-12p40 were bound by the hydrogel at only about 45% (see Table 3), HGF, MIP-1 alpha, MIP-1 beta, IL-1 beta, IL-10, IL-6, TNF alpha, and DKK1 were bound even less at 16-36% (see Table 3), and FGF-2, TGFb1, EGF, PLGF, GM-CSF, and sclerostin were barely bound (less than 12%) (see Table 3), so the majority of signaling molecules were bound significantly less than AB1 and AB2. This type of gel allows for the highly selective separation of, for example, bNGF, PDGF-BB, and VEGF A, eotaxin, GRO-alpha, IP-10, Rantes, SDF-1 alpha, IFN-gamma, and IL-4 from any biofluid.

[0048] Another advantageous embodiment is the hydrogel UGB1-GB4 04 (see Table 1), which, due to the high concentration of sulfate or sulfonate groups in the hydrogel of 0.28 mmole / mL (Table 5.2, column B) as well as the high number of sulfonate groups per WE divided by the molar mass of WE of 0.0045 mole / g, binds all the factors investigated in the hydrogel with high efficiency (72-100%, see Table 3), with the exception of HGF, PLGF, GM-GSF, and EGF, which are therefore depleted from the biofluid (see Table 3).

[0049] In particular, the use of this hydrogel results in the depletion of all investigated structurally similar factors (IEPs and the presence of structural similarities) from the adjacent biofluid, in particular the class of chemokines (corresponding to PROSITE IDs: PS00471 and PS00472) (see Table of Protein Properties), which have a high structural similarity in tertiary structure resulting from the interaction of four cysteines by disulfide bridges and are characterized by a strong positive net charge, IEP > 9, or positively charged domains such as in MIP1 alpha and MIP1 beta, and a molecular weight of less than 10 kDa, FGF-2 (IEP, 9.58), TGFb1 (IEP, 8.59), and structurally similar FGF family proteins (corresponding to PROSITE IDs: PS00247), TGF family proteins (PROSITE IDs: PS00248), and the like. ID: PS00250) and a class of signaling molecules related to cytokines with slightly basic EPs, e.g., IL-10 (IEP, 7.65), as well as signaling molecules with IEPs in the neutral range (pH, 5.5-7), e.g., IL1β, IL6, and TNF, as well as structurally similar proteins of the IL-1β protein family (equivalent to PROSITE ID: PS00253) and the IL-10 protein family (equivalent to PROSITE ID: PS00520). Another advantageous exemplary embodiment, UGB2-GB4 20 (see Table 1), has charge characteristics that can be classified as Type 4 according to Table 5.1, similar to AB4 with the same GB 5 (see Table 5.1, column A: 0.0045), but with a lower sulfonate concentration of 0.16 mmole / mL (see Tables 1 and 5.1), still belonging to Type 4, but formed using a different cross-linking reaction (VN:2 in Table 1), has a nearly identical binding or segregation pattern as UGB1-GB4 04 (see Table 3). Therefore, this result also demonstrates the effective predictive ability of the charge characteristics of Types 1 to 5 listed in Table 5.1 for the differentiated segregation of substances of a group of substances.

[0050] Another exemplary embodiment is an uncharged hydrogel (UGB2-UGB3 26) formed from UGB1 and UGB3, which, as expected, serves as a negative control and shows little to no binding of signaling molecules from the biofluid. With the exception of a small sequestering of PDGF-BB at 19.7±23.1% and IP 10 at 16.1±16.4% (which cannot be classified as significant due to the large standard deviations), no sequestration occurs in this hydrogel (all signaling molecules tested). (See Table 3). This result can clearly be attributed to the absence of charged affinity centers and, therefore, the absence of the charge interactions described above. However, the storage modulus of 4.4 kPa, and therefore the mesh size of the network (Table 1), is nearly identical to that of UGB1-GB2 02, UGB2-GB4 20, and UGB2-GB5 23, i.e., the steric interactions of the charged exemplary embodiments with signaling molecules are comparable. These results substantiate the compartmentalized sequestration claims made in Table 5.1, particularly due to the lack of interaction of the uncharged hydrogel (UGB2-UGB3 26) with signaling molecules (i.e., the absence of binding and sequestration effects).

[0051] At the same time, individual factors can be released into a biofluid, parallel to and independent of the sequestration, and therefore depletion, of other factors, by selectively precharging the hydrogels of Types 1-3 in Table 5. Thus, using various types of hydrogels, the levels of almost any individual signaling molecule in a combined biofluid can be tailored by targeting the precharge of the hydrogel, or signaling molecules can be sequestrated (depleted) almost quantitatively (e.g., applied to the isolation of signaling molecules from various protein-containing solutions).

[0052] Based on the exemplary embodiments of UGB1-GB1 01 to UGB2-UGB3 26, the following relationships among the properties of the protein, the properties of the hydrogel, and the binding of the protein in the hydrogel network, which are important in the description of the present invention, are clear: The strong binding of the signaling molecules described in Table 3 is related, on the protein side, to a positive effective charge (here, as the corresponding parameter, a high basic IEP), and further to the reciprocal of the molecular weight (a high IEP and a small molecular weight improve the binding, see Table 3). Highly positively charged and relatively small chemokines (<9 kDa) bind most strongly. On the hydrogel network side, (1) a high concentration of sulfate or sulfonate groups in the hydrogel (Table 1 and 5.2, column B), and (2) a large value obtained by dividing the number of sulfate or sulfonate groups per WE by the molar mass of WE result in a high binding of the signaling molecule. Corresponding to these parameters, the following order is obtained for the binding of the hydrogel, from the strongest to the weakest: UGB1-GB4 04≒UGB2-GB4 20<UGB1-GB1 01<UGB1-GB2 02≒UGB2-GB5 23<UGB1-GB3 03<<UGB2-UGB3 26. This result supports the experimentally found binding values (see Table 3). In addition, by changing the combination of the above hydrogel network parameters (1) and (2), it is possible to adjust the spatial matching of different interaction centers of the strong interaction known in the literature as "specific interactions", that is, between the protein and a negatively charged polyvalent electrolyte (for example, glucose-amino-glycan). Therefore, in addition to the molecular weight and the effective charge, other structural properties can be used on the protein side to adjust the binding to the hydrogel. These correlations can also explain the low binding of the strongly basic and relatively small signaling molecules FGF-2, TGFb1, and PLGF to the hydrogel types UGB1-GB1 01, UGB1 GB2-02, GB3 UGB1-03, and UGB1-GB4 04. [[ID= 2]]

[0053] Further possibilities for modulating the levels of soluble signaling molecules are based on the use of different hydrogel types, either sequentially or in combination, to precisely tailor a wide variety of specific sequestration and release properties. Targeted variations in network properties (especially the aforementioned parameters (1) and (2)) and further combinations of hydrogel microparticles of different compositions (e.g., charged with individual signaling molecules) in so-called multiphase hydrogel materials (i.e., by mixing different types of hydrogel types or precharging with signaling molecules or proteins), as well as other advantageous possibilities. It is also possible to establish suitable concentration ratios within adjacent biofluids.

[0054] By extending the analysis to additional signaling molecules, the proposed method allows for the modulation of the concentrations of these substances in various types of biofluids.

[0055] To effectively regulate the complex biological functions of signaling molecules, for example, for therapeutic purposes, it is necessary to actively manage signaling molecules that are distinct from the immediate biological environment of cells or biological applications, in addition to the sustained release of individual signaling molecules from hydrogels precharged with signaling molecules. Therefore, interactions with multiple related signaling molecules must be taken into account. When hydrogels containing sulfated or sulfonated components are charged with signaling molecules for sustained release, other therapeutically important molecules may be simultaneously sequestered from the biological environment and thus inactivated with the therapeutically desired release of the signaling molecules, potentially resulting in undesirable side effects. However, on the other hand, it is also possible to specifically sequester and thereby inactivate therapeutically undesirable signaling molecules in hydrogels bearing sulfated or sulfonated groups. Overall, extremely complex release and sequestration scenarios are possible, which ultimately determine the biological effect, particularly the molecular isolation performance, of the material.

[0056] A further advantageous embodiment of the present invention comprises the use of hydrogels according to Types 1 to 4 of Table 5 to regulate the concentration or level of biologically active proteins at low total levels.

[0057] The essential advantage of the method is its wide range of applicability: it can be advantageously used both for the biotechnological purification of or from biofluids and for the separation of protein mixtures by using hydrogels containing sulfated or sulfonated components.

[0058] In an exemplary narrow context, the methods of manipulating factors can be used in vivo to regulate angiogenesis, immune diseases, diabetes, neurodegenerative diseases, and wound healing.

[0059] During wound healing, primarily pro-inflammatory chemokines (e.g., eotaxin, GRO-α, IL-8, IP-10, MCP-1, MCP-3, MCD, Rantes, and SDF-1) are sequestered inside the hydrogel, whereas pro-regenerative factors (e.g., EGF, FGF-2, TGFb1, IL-10, HGF, and PLGF) remain largely unaffected.

[0060] A particularly important application area of ​​the present invention is the modulation of the concentrations of substances in biofluids that play a role in determining cell fate in vitro and in vivo. Dysregulation of pro-inflammatory chemokines and the associated chronic inflammation are responsible for the development of various diseases, such as Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, or rheumatoid arthritis. The application of various hydrogels containing sulfated or sulfonated components according to the present invention aims to modulate the concentrations of these inflammatory factors from biofluids, representing a possible application scenario.

[0061] In a broader context, the application of hydrogels containing sulfated or sulfonated moieties according to the present invention makes it possible to target the purification of signaling molecules of interest or structurally similar molecules from complex protein mixtures. Thus, the described gels The system can be used biotechnologically for the targeted purification of proteins from cell lysates derived from microorganisms or eukaryotes. In this case, the cell lysates are stimulated by the hydrogel of the present invention to bind signaling molecules. In a second step, the bound substances can be removed from the hydrogel for further use by washing with a high concentration of saline or a positively charged polyelectrolyte (e.g., chitosan) and subsequent separation. Furthermore, negative selection is also possible for the separation of signaling molecules weakly bound to the hydrogels of the Type 1-3 binding examples. EGF, FGF-2, TGF-β, IL-10, HGF, and PLGF are possible by using the supernatant after 24 hours of binding.

[0062] A related practical advantage of the present invention is that the binding of various biologically relevant signaling molecules to sulfated or sulfonated hydrogels with targeted graded charge profiles and network structures can be achieved at albumin concentrations as high as 1 mg / mL to 45 mg / mL in physiological electrolytes such as biofluids, as well as at signaling molecule levels as low as 100 pg / mL to 2000 ng / mL, thereby making it possible to exploit the differential selectivity of individual signaling molecules in binding to different hydrogel types.

[0063] The exemplary embodiments UGB1-GB1 01 through UGB1-GB4 011 shown in Table 1 are covalently crosslinked hydrogels according to crosslinking reaction 1, in which 6 to 24 carboxyl groups (depending on the molar ratio of 1.5 to 6 in the four-armed star PEG) of GB4 (poly(4-styrenesulfonate-co-malic acid) molecule, styrenesulfonate:maleic acid molar ratio = 3:1, Table 1) are activated with EDC / sulfo-NHS and react directly with the four-armed, amine-terminated UGB1 to produce hydrogels (see Table 1), with sulfonate concentrations variable from 0.28 to 0.06 mmole / mL and storage moduli variable from 4.8 to 19.6 kPa (see Table 1).

[0064] Exemplary embodiments UGB1-GB5 12 to UGB1-GB5 19 shown in Table 1 are covalently crosslinked hydrogels according to crosslinking principle 1, in which 6 to 24 carboxyl groups (depending on the molar ratio of 1.5 to 6 in the tetrafunctional star PEG) of a poly(4-styrenesulfonic acid-co-maleic acid) molecule (GB5) with a 1:1 molar ratio of styrenesulfonic acid to maleic acid are activated by EDC / SN HS and directly converted into hydrogels by means of the four-armed, amine-terminated UGB1 (see Table 1), resulting in hydrogels with sulfonate concentrations variable from 0.13 to 0.05 mmole / mL and storage moduli variable from 3 to 21.8 kPa (see Table 1).

[0065] Exemplary embodiments UGB2-GB4 20 to UGB2-GB4 22 are covalently crosslinked hydrogels according to crosslinking principle 2, in which the eight carboxyl groups of GB4 (poly(4-styrenesulfonic acid-co-maleic acid)) activated with EDC / sulfo-NHS in a 3:1 molar ratio of styrenesulfonic acid to maleic acid are functionalized with the short-chain bifunctional crosslinker molecule N-(2-aminoethyl)maleimide in a first step, followed by purification and isolation. These GB4 derivatives were converted into hydrogels by mixing saline (or serum) with UGB-2 (a thiol-terminated four-arm PEG), yielding hydrogels with sulfonate group concentrations ranging from 0.16 to 0.05 mmole / mL and storage moduli ranging from 3.7 to 0.2 kPa.

[0066] Exemplary embodiments UGB2-GB5 23 to UGB2-GB5 25 are covalently crosslinked hydrogels according to crosslinking principle 2 (VN2, Table 1), in which the molar ratio of styrene sulfonic acid:maleic acid is 1:1 and the hydrogel is activated with EDC / sulfo-NHS. In a first step, the eight carboxyl groups of the prepared GB5 (poly(4-styrenesulfonic acid-co-maleic acid)) were functionalized with a short-chain bifunctional crosslinker molecule, N-(2-aminoethyl)maleimide, followed by purification and isolation. These GB5 derivatives were then converted into hydrogels by mixing saline solution (or serum) with UGB-2 (a thiol-terminated four-arm PEG). Hydrogels with sulfonate group concentrations ranging from 0.14 to 0.04 mmole / mL and storage moduli ranging from 4.6 to 0.3 kPa were obtained.

[0067] The exemplary embodiment UGB2-UGB3 26 is an uncharged PEG hydrogel in which a thiol-terminated four-armed PEG (UGB-2) was reacted with a maleimide-terminated four-armed PEG (UGB-3) according to crosslinking principle 2. This hydrogel was used as an uncharged negative control in sequestration experiments.

[0068] Surprisingly, fully synthetic hydrogels possessing a wide range of advantageous properties and property combinations without adverse immunogenic reactions can be prepared based on exemplary embodiments 04-5. Thus, the sulfonate group concentration of the swollen hydrogels, ranging from 0.04 to 0.28 mmole / mL, and the equally large variation in storage modulus, ranging from 0.2 to 21.8 kPa, can be adjusted independently of one another. For example, hydrogels UGB2-GB4 21, UGB1-GB5 14, UGB1-GB5 15, and UGB1-GB4 08 have a constant sulfonate group concentration of 0.08 mmole / mL in the swollen hydrogel, while simultaneously exhibiting gradually increasing storage moduli of 1.1, 5.5, 12.5, and 19.6 kPa. That is, the sulfonate concentration and stiffness of the hydrogels can be modulated independently of one another over a wide range. Similarly, the selection of UGB1-GB5 19, UGB1-GB4 11, and UGB1-GB4 10, all with a nearly constant storage modulus of 8.0-8.5 kPa but with a sulfonate concentration of 0.05, 0.06, and 0.09, respectively, allows the sulfonate concentration to be varied independently of the hydrogel stiffness (see Table 1). Additionally, with further variation in the UGB:GB molar ratio, ranging from 1.5-3 and 3-6 for VN1 and from 0.5-0.75, 1-1.5, and 1.5-2 for VN2, and with further variation in the GB and UGB concentrations, many other performance combinations can be created.

[0069] Surprisingly, by crosslinking poly(4-styrenesulfonic acid-co-maleic acid) starting with the activation of one of the closely adjacent acid groups in maleic acid with EDC / sulfoNHS (whose reactivity is affected in unpredictable ways not only by adjacent carboxyl groups but also, in particular, by sulfonate groups and hydrophobic styrene units), it was possible to synthesize hydrogel materials defined by uncharged building blocks with the broadly scalable properties described above. In this case, precise network formation (in the case of VN1) or derivatization (in the case of VN2) was surprisingly achieved using an extremely short activation time of 1 min (i.e., the time during which EDC / sulfoNHS was added to poly(4-styrenesulfonic acid-co-maleic acid) (see Table 4), after which an amino-bearing molecule was subsequently mixed with the activated poly(4-styrenesulfonic acid-co-maleic acid), and the reactants were allowed to react with very vigorous stirring).

[0070] In another exemplary embodiment, hydrogels of type UGB1-GB1 01, UGB1-GB4 20, UGB1-GB5 23, and UGB1-GB3 26 were functionalized with the adhesion peptide CWRGDSP and precharged with VEGF-A and FGF-2, and used on the gel surface in serum-free cell culture medium for the culture of human endothelial cells. The morphology of the adherent cells was observed after 24 hours of culture, as shown in Figure 1. Analysis was performed on UGB1-GB1 01, UGB2-GB4 20, UGB2-GB5 23, and UGB2-UGB3 26. Endothelial cells are expected to have different morphologies on different hydrogels, which can be represented by two parameters: aspect ratio and circularity. A high aspect ratio and a low circularity correspond to the desirable elongated endothelial cell morphology, which is the first step in the formation of a biologically desirable tubular structure. The aspect ratios of endothelial cells cultured on UGB1-GB1 01, UGB2-GB4 20, and UGB2-GB5 23 were 3.4 ± 1.0, 4.1 ± 1.1, and 4.9 ± 1.6, respectively. In contrast, UGB2-UGB3 26 of the uncharged reference gel had a significantly lower aspect ratio of 2.1 ± 1.2. The circularities of endothelial cells on the hydrogels UGB1-GB1 01, UGB1-GB4 20, and UGB1-GB5 23, and UGB1-GB3 26 were, correspondingly, the reciprocals, 0.5 ± 0.1, 0.4 ± 0.1, 0.3 ± 0.1, and 0.7 ± 0.3. These differences were highly significant statistically. Therefore, the endothelial cells described in Table 5.1 showed significantly different behaviors on hydrogels with different charges and isolation performances of type 1 (UGB1-GB1 01), type 4 (UGB2-GB4 20), type 2 (UGB2-GB5 23), and uncharged type 5 (UGB1-GB3 26). On the uncharged hydrogel, the cells showed an undesirable short aspect ratio and high circularity, while the desirable morphology increased in the following order: UGB1-GB1 01 < UGB1-GB4 20 < UGB2-GB5 23. UGB2-GB5 23 of type 2 hydrogel showed the best results in this experiment. Therefore, this ranking demonstrates that the nature of the charge and the isolation pattern directly affect the culture of human endothelial cells.

[0071] In another exemplary embodiment, type 1 (UGB1-GB1 01), type 4 (UGB1-GB4 20), type 2 (UGB1-GB5 23), and uncharged type 5 (UGB1-UGB3 26) hydrogels were used for polymerization of human mesenchymal stromal cells. The hydrogels were cell-responsive; in addition to functionalization of the hydrogels with the adhesion-mediating peptide CWGRGDSP, a cleavable peptide sequence, GCGGPQGIWGQGGCG, enzymatically cleavable by matrix metalloproteinases secreted by these cells, was preconjugated onto each uncharged building block and used for crosslinking with VN2. The metabolic activity of the embedded cells was characterized after 24 hours using the PrestoBlue® test as evidence of viability. The metabolic activity of human mesenchymal cells polymerized in hydrogels, measured as relative fluorescence units, was 7112 ± 3924 for Type 1, 3704 ± 2945 for Type 2, 3160 ± 6023 for Type 4, and 2316 ± 446 for uncharged Type 5, respectively. Thus, human mesenchymal cells exhibited the highest metabolic activity when embedded in Type 2 gels and the lowest metabolic activity when embedded in the uncharged reference gel. Thus, hydrogels, particularly Type 2, were found to be particularly advantageous for culturing live human mesenchymal cells in 3D.

[0072] In another advantageous exemplary embodiment, UGB1-GB1 01, UGB2-GB4 20, UGB2-GB5 23, and UGB2-UGB3 26 were functionalized by compartmentalized sequestration with gentamicin, an antibiotic bearing a positively charged group. Their inhibitory properties were then measured by releasing gentamicin from different hydrogels using two related pathogenic bacterial species, Escherichia coli (E. coli) and Staphylococcus epidermidis (Staph. epidermidis). Antibacterial activity was measured by the size of the inhibition zone (distance from the hydrogel on the culture plate; see Materials and Methods). In the case of hydrogels of the types B1-GB1 01, UGB2-GB4 20, UGB2-GB5 23, and UGB2-UGB3 26, the inhibition zones could not be detected without prior gentamicin sequestration, that is, those hydrogels did not have an antibacterial effect. When gentamicin was pre-fractionated and sequestered by the hydrogel, the following grades of inhibition zones were measured: UGB1-GB1 01: 3.08 ± 0.33 mm, UGB2-GB4 20, E. coli: 3.08 ± 0.33 mm, S. Epidermidis: 2.94 ± 0.35 mm, UGB2-GB4 20: E. coli: 1.78 ± 0.27 mm, S. Epidermidis: 1.76 ± 0.45 mm; UGB2-GB5 23, E. coli: 2.18 ± 0.38 mm, S. Epidermidis: 1.81 ± 0.51 mm, and the uncharged reference gel UGB2 UGB3-26, E. coli: 0.48 ± 0.36 mm, S. Epidermidis: 0.90 ± 0.28 mm. Thus, as expected, only a very small amount of gentamicin was non-specifically sequestered and then released again in the uncharged reference gel UGB2-UGB3 26. In the other hydrogels, antibacterial effects were found in the following order: UGB2-GB4 20 < UGB2-GB5 23 < UGB1-GB1 01. This result demonstrates the fractionated sequestration of positively charged drug molecules. In addition, it was also demonstrated that the antibacterial effect changed by varying the sulfidation concentration of gentamicin.

Example

[0073] Materials and methods: (Region-selective desulfation of heparin (manufactured from GB2 from GB1 and GB3)) For the synthesis of regioselectively desulfurized heparins (GB2 and GB3), heparin (MW: 14,000, Merck Millipore, manufacturer number: 375095, Germany, GB1) was first dissolved in deionized ultrapure water and desalted using an Amberlite IR-120H+ ion exchange column (Sigma-Aldrich, Germany). Pyridine (Sigma-Aldrich, Germany) was added to the heparin solution until a pH of 6 was reached, forming a heparin-pyridine salt, which was concentrated by solvent evaporation in a B-490 rotary evaporator (Büchi, Germany), then lyophilized at -80°C (GEA Lyovac GT2, Germany), and stored at -20°C until further processing.

[0074] To prepare N-desulfurized heparin (N-DSH, GB2), 1 g / L of heparin-pyridine was dissolved in a mixture of DMSO and deionized ultrapure water (95:5) and incubated at 50°C for 1.5 h. The resulting solution was diluted 1:1 with deionized ultrapure water and adjusted to pH 9 with 1 M sodium hydroxide solution (Sigma-Aldrich, Germany). After dialysis (Spectrum Labs, Germany, MWCO = 8 kDa) of the solution against deionized ultrapure water for 3 days, N-acetylation of the desulfurized heparin was performed. For this purpose, 10% (v / v) methanol (Sigma-Aldrich, Germany) and 50 mM sodium carbonate (Sigma-Aldrich, Germany) were added to the solution. The acetylation reaction was carried out for 3 h by cooling to 4 °C and adding 400 μL of acetic anhydride (Sigma-Aldrich, Germany) per mg of heparin every half hour with constant stirring at 400 rpm, and then adjusting the pH to 7.5 by adding 2 M sodium carbonate solution.

[0075] To prepare 6O-N-desulfurized heparin (6ON-DSH, GB3), 1 g / L of heparin was used. Heparin-pyridine was dissolved in a mixture of DMSO and deionized ultrapure water (95:5) and incubated at 90°C for 24 hours. The resulting solution was diluted 1:1 with deionized ultrapure water and adjusted to pH 9 with 1 M sodium hydroxide solution (Sigma-Aldrich, Germany). After dialysis with deionized ultrapure water for 3 days in a dialysis tube (Spectrum Labs, Germany, MWCO = 8 kDa), the 60-N-desulfurized heparin was concentrated by solvent evaporation on a B-490 tube (Büchi, Germany) and subsequently lyophilized (GEA Lyovac GT2, Germany).

[0076] GB and UGB characterization (see Table 2): The molecular weights of GB1 to GB3 were determined by multi-angle light scattering at 690 nm on a Dawn HELEOS II (Wyatt Technology Europe, Germany). The dn / dc values ​​required for molecular weight determination were measured at a wavelength of λ = 690 nm using an RI detector (Optilab T-rEX, Wyatt, Germany). The average molecular weights of GB1, GB2, and GB3 were 0.1351 mL g -1 An RI increment, dn / dc, of 0.001 was required for evaluation of the light scattering experiments. The light scattering results for determining the molar mass were performed using Astra software, version 6.1 (Wyatt Technology, USA).

[0077] The sulfation degree of the heparin derivatives (GB1-GB3) was determined by elemental analysis (Elementar, Vario MICRO cube, Germany) based on the S:N molar ratio. Because each disaccharide unit contains exactly one N atom, the sulfation degree (number of sulfate groups / repeating unit) of GB1, GB2, and GB3 was determined by the S:N molar ratio. Furthermore, the molar mass of the repeating unit was also determined from this. The number of sulfate groups per repeating unit or per mole of polymer was calculated accordingly.

[0078] The molecular weights of GB4 and GB5 (poly(4-styrenesulfonic acid-co-maleic acid)) and the number of sulfate groups per repeat unit or per mole of polymer are based on information from the manufacturer (Sigma-Aldrich, manufacturer number: 434566, Germany).

[0079] The molecular weights and structures of UGB1, UGB2, UGB3, and amino-, thiol-, or maleimide-terminated four-arm PEGs were based on information from the manufacturer (Jenkem Technology, USA). The molar mass and structure of UGB4 (an enzymatically cleavable peptide-terminated four-arm PEG) was produced and used according to the process described in Tsurkan et al., 2013 (Adv. Mater., 2013, 25, 2606-2610).

[0080] Preparation of hydrogels based on heparin or heparin derivatives (see also Table 1): To prepare the hydrogels, heparin (GB1) (Merck-Millipore, manufacturer no. 375095, Germany), a desulfurized heparin derivative based on GB1, was dissolved in deionized ultrapure water using a vortex mixer (IKA, Germany) at 500 rpm for 30 seconds at 4°C according to the hydrogel type (see Table 2) as the charged component (GB) for GB2 and GB3. In addition, four-arm amine-terminated PEG (MW 10,000, Jenke) was added. Technology, USA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma-Aldrich, Germany), and N-hydroxysulfosuccinimide (sulfo-NHS, Sigma-Aldrich, Germany) were used in the same manner as UGB1 for all types of hydrogels. To ensure complete dissolution of the hydrogel building blocks, these solutions were further treated in an ultrasonic bath (RK 100H, Bandelin, Germany) at 4° C. for 5 min.

[0081] To activate GB1-GB3, EDC and sulfo-NHS (EDC:sulfo-NHS ratio = 2:1) were added to the dissolved GB (4 moles of sulfo-NHS and 8 moles of EDC per mole of UGB1 in the final reaction mixture) and incubated at 4°C for the time indicated in Table 4. After the activation period for GB1, GB2, and GB3, dissolved amino-terminated 4-arm PEG (UGB1) was added and mixed for 30 seconds at 500 rpm using a vortex mixer (IKA, Germany).

[0082] Preparation of hydrogel based on poly(4-styrenesulfonic acid-co-maleic acid) after crosslinking reaction 1 (VN1, see Table 1): Covalently crosslinked hydrogels were prepared using poly(4-styrenesulfonic acid-co-maleic acid) (Sigma-Aldrich, Manufacturer No. 434566, Germany, GB4, see Table 2) with a 4-styrenesulfonic acid:maleic acid molar ratio of 3:1 and that of 1:1 (Sigma-Aldrich, Manufacturer No. 434558, Germany, GB5, see Table 2), and UGB1 (tetravalent PEG, amine-terminated, MW = 10,000, Jenkem Technology, USA) as follows: GB4 or GB5 and UGB1 were each dissolved in deionized ultrapure water at a concentration three times that of the hydrogel mixture in Table 1, each in one-third of the total volume, and then sonicated for 5 min at 4 °C using an ultrasonic bath (RK 100H, Bandelin, Germany). The entire solution was then tempered at 4 °C for the next step: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Sigma-Aldrich, Germany) and N-hydroxysulfosuccinimide (sulfo-NHS; Sigma-Aldrich, Germany) were also dissolved in 1 / 6 of the total reaction mixture and tempered at 4 °C. The molar ratio of UGB1:EDC:sulfo-NHS was 1:8:4 (1:2:1 for each amino group of UGB1). In the next step, EDC and sulfo-NHS were added to GB4 or GB5, mixed by pipetting, and after a 30-second waiting period, the reaction mixture was mixed on a vortex shaker (VWR, Germany) for 10 seconds, followed by another 20-second waiting period. After this procedure, UGB1 was pipetted onto the activated GB4 or GB5 on a vortex shaker for a total activation time of 1 min, followed by another 10 s of vortex mixing. The final reaction mixture (where the concentrations of UGB1 and GB4 or GB5 correspond to those listed in Table 1) can now be pipetted into any mold, and gelation is allowed to occur by polymerization for 12 h.The hydrogels were then fully swollen in a solution of phosphate buffered saline by repeated solution exchange for several hours, after which the hydrogels were either used or characterized.

[0083] Preparation of hydrogels for cell culture with human endothelial cells: To culture human endothelial cells, 1 cm 2 Surface-bound gels with a final thickness of approximately 100 μm were formed by pipetting 11 μL of the final reaction mixture per cover glass. The coverslips were pre-coated with a thin film of poly(ethylene-old-maleic anhydride) to ensure covalent attachment of the hydrogel, according to the procedure described in Pompe et al., Biomolecules, 2003, 4, 1072-1079. For modification with RGD peptide, extruded hydrogels swollen in phosphate-buffered saline containing EDC / sulfo-NHS were incubated at 4°C in 1 / 15 M phosphate-buffered saline. The remaining carboxyl groups of GB4 or GB5 were activated for 20 minutes by dissolving 50 mM EDC and 25 mM sulfo-NHS in a solution, followed by washing with borate buffer (100 mM, pH 8.0, 4°C). The activated hydrogel was then treated with a peptide sequence (GlcNAc) dissolved in borate buffer (100 mM, pH 8.0). [ka] The resulting mixture was reacted with a 50 mg / mL solution of Peptides International (Louisville, KY, USA) at room temperature for 2 hours, and then washed extensively with phosphate buffered saline.

[0084] Cell culture: Human umbilical vein endothelial cells (HUVECs, Lonza, Germany) were subcultured on fibronectin-coated cell culture flasks supplemented with Promocell C-22010 (PromoCell GmbH, Germany) at 37°C and 5% CO2 until 80% confluency was achieved. Cells from passages 2–6 were used in subsequent experiments.

[0085] The RGD-functionalized, surface-bound hydrogel was applied to the hydrogel surface along with VEGF-A and FGF-2. 2 The cells were incubated at a concentration of 0.565 μg per well for 18 hours at room temperature and then washed twice with phosphate-buffered saline.

[0086] 50,000 cells / cm on RGD- and VEGF-165- and FGF-2-functionalized hydrogels in Promocell medium without supplemental mixing. 2 After 24 hours of culture, cells were washed, fixed, and characterized by light microscopy (Olympus IX73 inverted microscope, Hamburg, Germany). The aspect ratio was determined by measuring individual cells using Fuji image processing software and then dividing the cell length by the cell width. Circularity was determined by means of Fiji image processing software according to the following formula: circularity = 4 * π * (cell area / perimeter). 2 In each case, 20 cells were measured per image for n = 10 independent samples. Statistical evaluation using GraphPad Prism software and one-way anova tests revealed significant differences under all conditions studied.

[0087] Preparation of poly(4-styrenesulfonic acid-co-maleic acid) based hydrogel after crosslinking reaction 2 (VN2, see Table 1): Derivatization of poly(4-styrenesulfonic acid-co-maleic acid) with maleimide groups: To form hydrogels according to crosslinking reaction 2, GB4 or GB5 must first be functionalized with N-(2-aminoethyl)maleimide trifluoroacetate (Sigma, Germany). For this purpose, 500 mg (25 μmol) of GB4 or GB5 was dissolved in 2.7 mL of deionized ultrapure water and stirred on ice for 10 min. The sample container was a 25 mL snap-cover glass. Next, 65.14 mg of NHS (0.30 mmol) was dissolved in 400 μL, and 115.02 mg (0.60 mmol) of EDC dissolved in 200 μL of ice-cold deionized ultrapure water was added to the GB4 or GB5 solution, followed by a 5-minute wait. In the next step, 76.25 mg (0.30 mmole) of N-(-2-aminoethyl)maleimide trifluoroacetate dissolved in 200 μL of MilliQ water was added dropwise over 40 seconds after the activation time was completed to activate GB4 or GB5. The reaction mixture was then further incubated on ice for 1 hour. The mixture is stirred for another 10 minutes. Finally, the ice bath is removed and the mixture is stirred overnight at room temperature. The product is placed in a dialysis tube with an exclusion size of MWCO = 5 kDa. Dialysis is carried out over two days; on the first day, dialysis is carried out for 6 hours against 2.5 liters of 1 molar sodium chloride solution, with the solution being changed every 2 hours. After 6 hours, dialysis is carried out overnight against deionized ultrapure water. On the second day, dialysis is carried out against deionized ultrapure water, with the water being changed every 2 hours. In the final step, the solution is freeze-dried.

[0088] Exclusion volume chromatography for characterization of GB4 / GB5-maleimide derivatives: Volume exclusion chromatography (SEC) was used to determine the maleimide groups per GB4 or GB5 molecule. For this purpose, the peptide RGD-SP (M = 990 g / mole) was coupled to the GB4 / GB5-maleimide derivative at various excesses. The molar excesses of RGD-SP tested were, in this case, 6.8, 10, or 12 relative to GB4 or GB5. Calibration was performed by first mixing 35 μL of each RGD-SP concentration with 35 μL of phosphate-buffered saline solution in a sample vessel.

[0089] For this study, a BioSEP-SEC S2000 column from Phenomnex (Germany) was used, into which an Agilent 1100 HPLC system (Germany) was inserted. The eluent was phosphate-buffered saline solution, and the flow rate was 0.5 mL / min. The injection volume was 50 μL.

[0090] The maleimide groups were also measured under the same elution conditions. In each case, 35 μL of the RGD concentrate was mixed with 35 μL of the GB4 / GB5-maleimide derivative solution in a sample vessel. This method allows accurate quantification of the conversion of maleimide to GB4 or GB5.

[0091] Preparation of hydrogels based on poly(4-styrenesulfonic acid-co-maleic acid) derivatives after crosslinking reaction 2 (VN2, see Table 1): The GB4 / GB5 maleimide derivative and four-arm, thiol-terminated PEG (UGB2, Mw = 10,000 Da, Jenchem, USA, Table 2) were dissolved in phosphate-buffered saline at twice the concentration listed in Table 1. Equal volumes of both components were then pipetted into a microreaction vessel and shaken on a vortex shaker for 10 seconds. A predetermined volume of the reaction mixture was then withdrawn and placed on a 9 mm diameter Sigma Cote®-coated coverslip. Another Sigma Cote®-coated coverslip was placed on top of the drop. After 30 minutes, the coverslip was removed, and the gel slice was allowed to swell in phosphate-buffered saline for 12 hours. At high UGB2 concentrations, the pH of UGB2 can be adjusted to 5-7 using 1 M HCl for optimal gelation.

[0092] Fabrication of cell culture hydrogels using human mesenchymal cells in 3D: Following the method described above, matrix metalloproteinase (MMP)-cleavable hydrogels were prepared using UGB4 (Table 4, synthesized according to the procedure described by Tsurkan et al., Adv. Mater., 2013, 25(18), 2606-2610) instead of UGB2.

[0093] For this purpose, cells were suspended in GB4 or GB5 derivatives, mixed according to the method described above to form hydrogels, and immediately after a 5-minute gelation period, swelled in phosphate-buffered saline solution and cell culture medium.

[0094] Cell culture: Mesenchymal stem cells (MSCs, ATCC, Germany) isolated from adipose tissue were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin under standard culture conditions (5% CO, 37°C). MSCs from passages 2 to 4 were used for the experiments.

[0095] GB-4 or GB-5 derivatives and UGB were mixed with 1 mole of CGWGGRGDSP per mole of GB and dissolved in phosphate-buffered saline (the concentration of GB4 or GB5 derivatives was three times that shown in Table 1). After mixing, the solution was incubated at 37°C for 10-15 minutes, and then the concentrated cell suspension was added to the final reaction mixture, extrapolating to 6 x 10 cells. 6 The cells / mL were mixed into 1 / 3 of the final gel volume. UGB4 (3x concentration in Table 1) was dissolved in another 1 / 3 of the total gel volume in phosphate-buffered saline and added to the GB4 / 5 derivative cell mixture and mixed by pipetting. After 5 minutes of gelation, the above cell culture medium was added, and the hydrogel was incubated at 37°C for 24 hours as described above.

[0096] PrestoBlue® test: The PrestoBlue® test is performed after 24 hours of incubation following cell seeding in the hydrogel. For this purpose, a 10% solution of PrestoBlue® dye in cell culture medium is added to each hydrogel sample and incubated in a microtiter plate for 1 hour at 37°C. Fluorescence is then measured using a microplate photometer (Tecan Spark®, Germany). The excitation wavelength is 560 nm, and fluorescence is measured at a wavelength of 590 nm. The metabolic activity of the cells is expressed as relative fluorescence units (RFU). Four subject determinations were evaluated in 10 independent experiments (n = 40), and significant differences were found in all conditions (one-way anova).

[0097] Antimicrobial Zone of Inhibition Assay: Hydrogel discs (60 μL) were incubated in 1 mL of 50 μg / mL gentamicin (Sigma Aldrich, Germany) for 18 hours and then washed twice with phosphate-buffered saline. The antibacterial activity of the gentamicin-charged hydrogels was measured by a zone of inhibition assay. For this purpose, Luria broth (LB) agar (Sigma Aldrich, Munich, Germany) plates were prepared according to the manufacturer's instructions. 12-h-grown cultures of Escherichia coli K12 DH5 (DSMZ, Germany) and Staphylococcus epidermidis PCI 1200 (ATCC, USA) were diluted to 0.1, and the optical density (OD) was measured at 600 nm. 250 μL of these bacterial suspensions were spread onto each plate. Bacteria were evenly dispersed using a sterile cotton cloth. Hydrogels UGB1-GB1 01, UGB2-GB4 20, and UGB2-GB5 23, as well as UGB2-UGB3 26, each with or without gentamicin loading, were applied to the four corners of agar plates, and the plates were incubated overnight at 37°C. Zones of inhibition were then measured using digital calipers. Three subject definitions were evaluated in 10 independent experiments (n = 30), and significant differences were found across all conditions (one-way anova).

[0098] Measurement of the physical-chemical properties of the hydrogel: To measure the physical properties of the various types of hydrogels, 67 μL of unpolymerized hydrogel solution was applied in each case to two 9 mm glass slides (Menzel Gla) treated with Sigmacote (Sigma-Aldrich, Germany). The gel slices were polymerized for 16 hours at room temperature in a glass slide (Messeler, Germany), and then removed from the glass slide. The diameters of the gel slices were optically measured using a scanner of type FLA-3100 (Fujitsu, Japan) (diameter in the unswollen state). The gel slices were then swollen for 24 hours in phosphate-buffered saline (PBS) (Sigma-Aldrich, Germany) buffered to pH 7.4 with 0.9% NaCl (under physiological conditions) and measured again using a scanner of type FLA-3100 (Fujitsu, Japan) (diameter in the swollen state). The swelling degree of the hydrogels was calculated from the measured diameters according to the following equation: Swelling degree = diameter of swollen hydrogel 3 / (diameter of unswollen hydrogel) 3 .

number

[0099] Each reported data is the mean (MW) of at least four independent samples, with the standard deviation (SD) also shown.

[0100] Furthermore, the storage and loss moduli of the hydrogels were measured by oscillatory rheometry (units: kilobascals) using a type Ares shear rheometer manufactured by TA Instruments (United Kingdom). For this purpose, 8 mm thin sections were punched from hydrogels swollen for 24 h under physiological conditions (phosphate-buffered saline solution (PBS, buffered to pH 7.4 with 0.9% NaCl (Sigma-Aldrich, Germany)). These were measured at room temperature under low deformation (2%) using a 9 mm plate-plate instrument set to increasing frequencies from 1 to 100 rad / s. The average values ​​were calculated over the entire frequency range (one measurement per sample). The reported values ​​are the average values ​​of four independently prepared hydrogel discs, with ± standard deviations. The storage moduli of the four hydrogel types are also listed in Table 1, whereas the loss moduli were several orders of magnitude lower (data not shown).

[0101] Table 1 shows the physical-chemical properties of the hydrogels.

[0102] The mesh size of a hydrogel can be derived from the experimentally determined storage modulus based on rubber elasticity theory using the following equation (Polymer Physics, Michael Rubinstein and Ralph H. Colby, 2006, Oxford University Press, Oxford):

number

[0103] To determine the binding properties of various types of hydrogels, solutions of unpolymerized hydrogels were placed between two 5 mm glass slides treated with Sigmacote (Sigma-Aldrich, Germany) (Menzel Glasses) at room temperature for 16 hours. The hydrogels were polymerized and then swollen for 24 h in phosphate-buffered saline (PBS) (Sigma-Aldrich, Germany) buffered to pH 7.4 with 0.9% NaCl. The total volume was adjusted to ensure the same molar content among the four hydrogel types: GB1, GB2, GB3, and GB4 (see Table 4). For binding (sequestration) studies, discs of each hydrogel were incubated for 24 h in 0.5 mL Protein LoBind reaction vessels (Eppendorf Tubes, Germany) with a mixture of proteins corresponding to Table 3 dissolved in 400 μL of PBS containing 1% (m / v) bovine serum albumin (Sigma-Aldrich, Germany) and 0.05% (m / v) Proclin 300 (Sigma-Aldrich, Germany) (corresponding to Solution 2 after incubation). To prepare the protein mixture, ProcartaPlex Standards A, B, and C (ebioscience, Germany) were dissolved according to the manufacturer's instructions, and the proteins DKK1 (manufacturer number: 120-30, Peprotech, Germany), sclerostin (manufacturer number: 1406-ST, Peprotech, Germany), and TGFb1 (manufacturer: 100-21, Peprotech, Germany) were added, at their respective concentrations shown in Table 7 (corresponding to solution 1 before incubation).

[0104] Solutions 1 and 2 were stored at -80°C until the protein concentrations were measured. To measure the protein concentrations in solutions 1 and 2, the samples were measured using the ProcartaPlex Human chemokine Panel 1 (ebioscience, Germany) according to the manufacturer's instructions in combination with the corresponding ProcartaPlex Simplex kit on a Bioplex 200 type (Biorad, Germany) instrument.

[0105] The binding (sequestration) of signaling molecules was calculated from the measured concentrations of solutions 1 and 2 according to the following formula: Binding (%) = (concentration of 1 in solution 2 (after incubation) / concentration of 1 in solution 1 (before incubation)) x 100

[0106] Table 4 shows the activation time (unit: minutes) of the carboxyl groups of GB1 to GB4 and the gel volume used for the binding study.

[0107] The sulfated or sulfonated hydrogels were characterized by the charge distribution of the charged building blocks (GBs) (units, moles of sulfate or sulfonate groups per mole of polymer), the concentration of sulfate or sulfonate groups in the hydrogel volume swollen under physiological conditions (units, mmoles of sulfate or sulfonate / mL of hydrogel), and the number of sulfate or sulfonate groups per WE in a specific area divided by the molar mass of the WE. The calculations were based on the molar concentration of the hydrogel building blocks (see Table 1) and the volumetric swelling (Table 1) upon hydrogel formation, assuming that the hydrogel building blocks are quantitatively incorporated into the network.

[0108] The concentration of sulfate or sulfonate groups in the unswollen hydrogel was calculated by multiplying the concentration of GB in the unswollen hydrogel by the number of repeating units and the number of sulfate or sulfonate groups per repeating unit. The concentration of sulfate or sulfonate groups in the swollen gel (Table 1) was calculated by dividing the concentration of sulfate or sulfonate groups in the unswollen hydrogel by the degree of swelling (see Table 1).

[0109] In the extraction experiment, no measurable elution of gel components occurred, and therefore , the hypothesis that the gel components are fully incorporated seems reasonable.

[0110] Table 3 shows the percent amount of signaling molecule bound in the hydrogel, normalized by the concentration of soluble factor before incubation, as discussed above. The percentages are by mass.

[0111] The molecular size of substances (also called signal substances or factors) is expressed in kilodaltons (kDa).

[0112] These proteins (signaling molecules) have been uniquely assigned by abbreviation (Table 3) and UniProt Identification Numbers (Uniprot ID) from the Universal Protein Resource database (UniProt; http: / / www.uniprot.org / ).

[0113] The isoelectric point (IEP) and molar mass were determined based on the fully biologically processed amino acid sequence using the program ExPASy ProtParm (http: / / web.expasy.org / protparam / (Reference: Gasteiger, E., et al., The Proteomics Protocols Handbook, 571-607 (2005))).

[0114] Protein structural parameters were determined using the ExPASy PROSITE database, which is based on UniProt Identification Numbers (http: / / prosite.expasy.org / ; Sigrist, CJA, et al., New and continuing development at ExPASy PROSITE; References: (1) Nucleic Acids Res., 41, (2013); (2) Sigrist, CJA, et al., PROSITE: a documented database using patterns and profiles as motif descriptors., Brief. Bioinform., 3, 265-274 (2002)).

Claims

1. 1. A hydrogel material comprising a covalently crosslinked hydrogel, said hydrogel comprising: a charged building block in the form of poly(4-styrenesulfonic acid-co-maleic acid); and uncharged building blocks in the form of polymers containing amine or thiol groups or crosslinker molecules having at least two amino or thiol groups; Based on the charged and uncharged units are crosslinked to form a polymer network; The polymer network comprises: or by activating the carboxyl groups of the poly(4-styrenesulfonic acid-co-maleic acid) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to directly crosslink the amine group-containing polymer or the crosslinker molecule having at least two amino groups by forming amides, respectively; or functionalization of the activated carboxyl groups with bifunctional crosslinker molecules containing an amino group and a group capable of Michael-type addition, respectively, followed by crosslinking the thiol-containing polymer or the crosslinker molecule having at least two thiol groups, respectively, via a Michael-type addition reaction; the hydrogel is selected from the group of Type 1, Type 2, Type 3 or Type 4 hydrogels; The charged building blocks are the parameter calculated from the number of sulfate and / or sulfonate groups per repeat unit divided by the molar mass of said repeat unit is 0.0040 to 0.0060 mole / g for Type 1, 0.0025 to 0.0040 mole / g for Type 2, 0.0005 to 0.0025 mole / g for Type 3, and 0.0040 to 0.0100 mole / g for Type 4; or the concentration of moles of sulfate or sulfonate groups per mole of polymer is 60 to 80 for Type 1, 30 to 75 for Type 2, 10 to 30 for Type 3, and 80 to 120 for Type 4, and the charged units in the hydrogel, when swollen, have a concentration in mmole / mL of 0.0015 to 0.0025 for Type 1, 0.0015 to 0.0030 for Type 2, 0.0010 to 0.0040 for Type 3, and 0.0018 to 0.0050 for Type 4; The uncharged building blocks are In the case of the polymer containing amine or thiol groups, the polymer containing amine and thiol groups is selected from polyethylene glycol (PEG), poly(2-oxazoline) (POX), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and / or polyacrylamide (PAM) as uncharged building blocks, or When the crosslinker molecule has at least two amino or thiol groups, it is a non-polymeric, bifunctional crosslinker molecule; the swollen hydrogel has a storage modulus of less than 20 kPa; The swollen hydrogel has a concentration of sulfate or sulfonate groups in mmole / mL of 0.09 to 0.20 for Type 1, 0.05 to 0.18 for Type 2, 0.01 to 0.12 for Type 3, and 0.16 to 0.80 for Type 4.

1. A covalently crosslinked hydrogel material comprising:

2. 2. The covalently crosslinked hydrogel material of claim 1, wherein the group allowing Michael-type addition is selected from maleimide groups, vinyl sulfone groups, or acrylate groups.

3. 3. The covalently crosslinked hydrogel material according to claim 1, wherein poly(4-styrenesulfonic acid-co-maleic acid) having a variable molar ratio of 4-styrenesulfonic acid to maleic acid ranging from 6:1 to 1:6 and a molar mass ranging from 5,000 to 100,000 g / mole is selected as the charged building block.

4. 4. The covalently crosslinked hydrogel material according to any one of claims 1 to 3, characterized in that a polymer comprising covalently bound enzymatically cleavable peptides having either lysine or cysteine ​​as the reactive amino acid in the peptide sequence is used as uncharged building blocks for forming the polymer network.

5. 5. The covalently crosslinked hydrogel material of claim 4, wherein the enzymatically cleavable peptide is cleavable by a human or bacterial protease.

6. 6. The covalently crosslinked hydrogel material according to claim 4 or 5, characterized in that bioactive and / or anti-adhesive molecules having amino or carboxyl groups and / or cell-instructing peptides are attached to the polymer network by forming covalent bonds via lysines or cysteines in the sequence to the charged building blocks of poly(4-styrenesulfonic acid-co-maleic acid) or its derivatives having groups allowing Michael-type addition.

7. 7. The covalently crosslinked hydrogel material of claim 6, wherein the bioactive molecule is an antibacterial agent or a pharmaceutical active ingredient.

8. The covalently crosslinked hydrogel material according to claim 6 or 7, characterized in that the anti-adhesive molecule is polyethylene glycol (PEG) or poly(2-oxazoline) (POX).

9. 9. The covalently crosslinked hydrogel material according to any one of claims 6 to 8, characterized in that the cell-instructing peptide is a peptide derived from a structural and functional protein of the extracellular matrix.

10. 10. The covalently crosslinked hydrogel material according to any one of claims 4 to 9, characterized in that the bioactive and / or anti-adhesive and / or cell-attracting peptides are covalently bound to the polymer network via enzymatically cleavable peptide sequences.

11. The covalently crosslinked hydrogel material of any one of claims 1 to 10, wherein the hydrogel material has a storage modulus of 0.2 to 22 kPa.

12. A hydrogel material consisting of a physically crosslinked hydrogel, said hydrogel being based on physical interactions between charged building blocks in the form of poly(4-styrenesulfonic acid-co-maleic acid) and uncharged building blocks in the form of a polymer, with a strongly positively charged peptide sequence covalently attached onto said polymer; the hydrogel is selected from the group of Type 1, Type 2, Type 3 or Type 4 hydrogels; The charged building blocks are the parameter calculated from the number of sulfate and / or sulfonate groups per repeat unit divided by the molar mass of said repeat unit is 0.0040 to 0.0060 mole / g for Type 1, 0.0025 to 0.0040 mole / g for Type 2, 0.0005 to 0.0025 mole / g for Type 3, and 0.0040 to 0.0100 mole / g for Type 4; or the concentration of moles of sulfate or sulfonate groups per mole of polymer is 60 to 80 for Type 1, 30 to 75 for Type 2, 10 to 30 for Type 3, and 80 to 120 for Type 4, and the charged units in the hydrogel, when swollen, have a concentration in mmole / mL of 0.0015 to 0.0025 for Type 1, 0.0015 to 0.0030 for Type 2, 0.0010 to 0.0040 for Type 3, and 0.0018 to 0.0050 for Type 4; As the uncharged building blocks, the polymer is selected from polyethylene glycol (PEG), poly(2-oxazoline) (POX), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and / or polyacrylamide (PAM); the swollen hydrogel has a storage modulus of less than 20 kPa; The swollen hydrogel has a concentration of sulfate or sulfonate groups in mmole / mL of 0.09 to 0.20 for Type 1, 0.05 to 0.18 for Type 2, 0.01 to 0.12 for Type 3, and 0.16 to 0.80 for Type 4. A physically crosslinked hydrogel material, characterized in that:

13. 13. The physically crosslinked hydrogel material of claim 12, wherein the strongly positively charged peptide sequence comprises at least 10 repeats of lysine or arginine, or at least 5 repeats of a dipeptide motif using lysine and alanine or using arginine and alanine.

14. A hydrogel material according to any one of claims 1 to 13, for use in manipulating factors for regulating angiogenesis, immune diseases, cancer, diabetes, neurodegenerative diseases, Crohn's disease, colon ulcers, multiple sclerosis, asthma, rheumatoid arthritis, or skin wound healing, and bone regeneration in vivo.

15. Use of the hydrogel material according to any one of claims 1 to 13 for purifying a target protein from a cell lysate of microbial or eukaryotic origin.

16. 14. Use of the hydrogel material of any one of claims 1 to 13 for negatively selecting and separating EGF, FGF-2, TGF-β, IL-10, HGF, and PLGF signaling molecules from a biofluid, said signaling molecules being weakly bound to said hydrogel.

17. 14. Use of the hydrogel material according to any one of claims 1 to 13 for in vitro cell and organ culture from embryonic stem cells (ES), induced pluripotent stem cells (iPS-) and other stem and progenitor cells without ES and iPS-, primary cells taken from patients, immortalized cell lines, as well as cardiac, muscular, renal, liver and nervous tissue.

Images