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

Abstract

The present invention relates to a method for separating and isolating different substances of substance groups A and B in a sulfated and / or sulfonated hydrogel, depleting the substances of substance group A from a biofluid while simultaneously otherwise releasing substances of substance group A or B from the sulfated and / or sulfonated hydrogel into the biofluid, and / or weakening the binding of substances of substance group B in the sulfated and / or sulfonated hydrogel, wherein the sulfated and / or sulfonated hydrogel is selected from the group of Type 1, Type 2, Type 3, or Type 4 hydrogels, and the hydrogel is composed of an uncharged component and a charged component. In the present invention, the charged components are characterized by a parameter calculated by dividing the number of sulfate and / or sulfonate groups per repeat unit by the molar mass of the repeat unit: 0.0040-0.0060 mol / g for Type 1, 0.0025-0.0040 mol / g for Type 2, 0.0005-0.0025 mol / g for Type 3, and 0.0040-0.0100 mol / g for Type 4. The swollen hydrogels have a storage modulus of less than 20 kPa, and the swollen hydrogels have a sulfate or sulfonate group concentration, in mmole / mL, of 0.09-0.20 for Type 1, 0.05-0.18 for Type 2, 0.01-0.12 for Type 3, and 0.16-0.08 for Type 4. The concentration of material group A and material group B in the biofluid is influenced by the choice of hydrogel type.

Description

[Technical field]

[0001] The invention relates to a method for the differentiated sequestration of substances of different substance groups A and B using hydrogels comprising sulfated and / or sulfonated components and for the differential and simultaneous release of substances of substance group A or B from hydrogels comprising sulfated and / or sulfonated components into a biofluid in order to deplete substances of substance group A and / or reduce the binding of substances of substance group B in hydrogels comprising sulfated or sulfonated components.The invention also relates to a hydrogel which is synthetically obtainable on the basis of poly(4-styrenesulfonic acid-co-maleic acid) as a network component and also of amine- or thiol-containing crosslinking molecules as another network component and which has the characteristics of a material for the differentiated sequestration of substances of different substance groups and which is used in the above-mentioned method. Substance groups A and B are not chemically defined substance groups, but may contain different substances that, depending on the type of hydrogel, give rise to the differentiated segregation according to the invention in corresponding constellations and compositions 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 field 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. In a broader sense, it is therefore 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, for example for culturing mammalian cells or for antibacterial finishes of surfaces.

[0003] Hydrogels containing sulfated components, such as star PEG-glycosaminocyclo-hydrogels, are also known from WO 2010 / 060485 A1 and are 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, where the former include, for example, proteases, lipolases, amylases, and the latter include, for example, hormones, neurotransmitters, cytokines, growth factors, and chemokines.

[0005] The interactions that are crucial for their affinity to 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 proteins, which are positively charged.

[0006] This principle has already been widely used in the art in the development of hydrogels, including for 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). In addition, the release of FGF-2 and VEGF can be progressively greater the lower the degree of sulfatedness of the underlying 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 for the preparation of hydrogels intended for use in, for example, contact lenses.

[0007] However, covalently crosslinked, water-containing polymer networks bearing sulfonic acid groups, i.e., hydrogels, are not currently known, in which 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 the relevant biological context, biofluids containing a variety of different proteins are present at various concentrations, but the signaling molecules are present in the biofluids at very low levels, ranging from only 100 pg / mL to 2000 ng / mL, whereas other proteins, e.g., albumin, are present at high concentrations of about 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 to estimate. However, in addition to the net charge under physiological conditions, which can be estimated based on the IEP, the charge distribution, such as the presence of positive charge clusters, as well as secondary interactions (which are influenced by the protein size and protein structure, as well as weaker non-ionic intermolecular forces, such as hydrophobic interactions, hydrogen bonds, or dipole interactions) also play a role, making it extremely difficult to predict the absolute or relative binding of proteins to hydrogels. In addition, there are further competing processes, which have often been ignored, such as the interaction of the target protein with albumin, which is responsible for regulating osmotic pressure and controlling transport processes, and also between albumin and hydrogel. Furthermore, the binding constants used in the literature to predict molecular interactions between charged polymers and proteins are only valid in describing the interactions of individual molecule-protein complexes in solution, whereas in hydrogels, adjacent polymer chains, three-dimensional charge distribution, or affinity centers within the polymer network have a strong influence on the resulting interactions and thus on the binding of proteins into the hydrogel.

[0010] Thus, the selectivity of binding between hydrogels (building blocks) containing sulfated and / or sulfonated components and molecular components of complex biofluids could not be elucidated so far, and therefore the demonstrated methods and processes have only allowed to a very limited extent to selectively modulate the levels of signaling molecules 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 invention is to propose a method which makes it possible to consciously influence the concentration of certain substances in a biofluid. [Means for solving the problem]

[0012] The object is achieved by a method and material having the features of the independent claims. As a material suitable for carrying out the process, a fully synthetic hydrogel system based on poly(4-styrenesulfonic acid-co-maleic acid) was used as a synthetic component, which is negatively charged under physiological conditions and therefore in close association with partially positively charged biomolecules, thus isolating the material from various substance groups. 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 the hydrogelation. The risk of adverse immunogenic reactions can therefore be eliminated. [Brief description 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 PREFERRED EMBODIMENTS

[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, as hydrogels containing sulfate and / or sulfonate groups, hydrogels with 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 WO2014040591A2. The first building block or component of the hydrogel is a molecule that is uncharged under physiological conditions (also called uncharged building block = UGB), preferably with a molar mass between 20 g / mole and 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, UGBs have at least two functional groups, preferably between 4 and 8 functional groups, which are particularly favorable for crosslinking. Suitable functional groups for crosslinking (at GB or UGB) include: amine, thiol, carboxyl, anhydride, maleimide, vinylsulfone, 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 is therefore negatively charged under physiological conditions (charged building block = GB) (optionally with a molar mass of 2,000 to 250,000 g / mole) and is able to bind proteins in the hydrogel network mainly via electrostatic (ionic) interactions and, to a lesser extent, via weaker bonds such as van der Waals forces, hydrogen bonds or hydrophobic interactions. In the present invention, the affinity centers that predominantly 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 also 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) (as well as in copolymers having units containing the above-mentioned crosslinking groups).

[0016] The affinity center, negatively charged sulfate or sulfonate groups, are intentionally distributed only along the GB, whereas the UGB is the component that minimizes protein adsorption. Any carboxyl groups present on the GB that are not available for cross-linking reactions are believed to be irrelevant for protein binding in the hydrogel, since the GB sulfate or sulfonate groups are significantly more acidic and therefore preferably deprotonated.

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

[0018] Biofluids in the present invention are aqueous solutions having various salt contents, preferably physiological salinities, 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 difference in 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 determined significantly on the one hand by 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 by the network parameters of the hydrogels, which were determined from: (1) the concentration of sulfate or sulfonate in the swollen hydrogel (mmoles of sulfate or sulfonate / mL) in the hydrogels swollen under physiological conditions; and (2) the number of sulfate or sulfonate groups per repeat unit of the GB divided by the molar mass of the repeat unit of the GB (number of groups / (g / mole)) as the characteristic size of the charge of the GB. 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 of adjacent polymer chains) between the GBs and the proteins (signaling molecules, enzymes). On the other hand, parameter (2) indicates the density and distribution of interaction centers (corresponding to negatively charged sulfate or sulfonate groups) on the different GBs and will therefore determine the interaction of the protein with each individual GB and therefore includes specific interactions via spatial modification of the charge centers of the polymer chains, which are 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 hydrogels 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 therefore can be quantitatively described by identifying 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 in order to bind and sequester proteins, the steric accessibility of the hydrogel network must be guaranteed, 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 a biofluid 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 for the treatment of neurodegenerative and autoimmune and cancer diseases, diabetes, in skin wound healing and bone regeneration, for the inhibition of tumor growth, as well as for disinfection and antibacterial treatments in 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, as well as cardiac, muscular, renal, liver and nervous tissues, as well as enrichment or depletion and separation of protein mixtures, particularly signaling molecule or enzyme mixtures.

[0023] Another aspect of the 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 mentioned above, 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. The 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 crosslinking directly with a polymer containing amine groups or with a crosslinker molecule having at least two amino groups, respectively, to form amides, or by functionalization of the activated carboxyl groups by means of a bifunctional crosslinker molecule, respectively, having an amino group and a group capable of Michael-type addition, followed by crosslinking via Michael-type addition of a polymer containing thiol groups or with a crosslinker molecule having at least two thiol groups, respectively. The groups capable of Michael-type addition are preferably selected from maleimide groups, vinylsulfone groups and acrylate groups. The polymers containing amine or thiol groups as uncharged building blocks are preferably selected from the classes of polyethylene glycol (PEG), poly(2-oxazoline) (POX), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) and / or polyacrylamide (PAM). The short-chain crosslinker molecules containing amine or thiol groups used instead are preferably non-polymeric bifunctional crosslinker molecules. Poly(4-styrenesulfonic acid-co-maleic acid) as charged building blocks are advantageously selected with a molar ratio of 4-styrenesulfonic acid to maleic acid ranging from 6:1 to 1:6 and a molar mass varying from 5,000 to 100,000 g / mole.In a particularly preferred embodiment of the 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 reactive amino acid in the peptide sequence. The enzymatically cleavable peptides can advantageously be cleaved using human or bacterial proteases, such as matrix metalloproteinase (MMP) responsive, e.g., PQGIWGQ, IPVSLRSG or VPMSMRGG, cathepsin responsive, e.g., VPMSMRGG, elastase responsive, e.g., AAPV or APEEIMDRQ, blood coagulation enzyme responsive, e.g., thrombin responsive, GGF-pipecolic acid RYSWGCG or GG-cyclohexylalanine ARSWGCG, FXa responsive, e.g., GGIEGRMGGWCG, kallikrein responsive, e.g., CGGGPFRIGGWCG, or bacterial-protease responsive, e.g., aureolysin responsive, ADVFEA or AAEAA, elastase responsive, AAPV, or protease IV responsive sequence, MKATKLVLGAVILGSTLLAG. Hydrogels constructed in this way allow for self-regulating release and degradation mechanisms. In another embodiment of the invention, bioactive and / or antiadhesive molecules bearing amino or carboxyl groups and / or cell-engineering peptides, in particular those selected from KCWG-RGDSP, KCWG-EIDGIELT, KCWG-IKLLI, KGCWGGRNIAEIIKDI, KGCWGGSDPGYIGSRSDDSA, KGCWGGPQVTRGDVFTMP, KGCWGGKGGNGEPRGDTYRAY, are attached by forming a covalent bond via a lysine or cysteine ​​in the sequence to the charged building block poly(4-styrenesulfonic acid-co-maleic acid) or a derivative thereof bearing a group allowing Michael type addition to the hydrogel network.These bioactive molecules may be antimicrobial substances, such as antibiotics or disinfectants, or pharmaceuticals. The anti-adhesive molecules are preferably polyethylene glycol (PEG) or poly(2-oxazoline) (POX). The cell-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 cell-engineering peptides are covalently linked to the hydrogel network via an enzymatically cleavable peptide sequence. The enzymatically cleavable peptides are, as already mentioned, preferably sensitive to human or bacterial proteases, such as MMPs, cathepsins, elastases, blood clotting enzymes. This type of hydrogel allows for self-regulating release and degradation mechanisms while retaining 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 (in g / mole) can be varied independently of each other 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 through the sulfonate groups of poly(4-styrenesulfonic acid-co-maleic acid) as a charged building block (GB), and has purposefully gradable 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 hydrogel material described above. This material offers the possibility to map all the important functions of the natural extracellular matrix (ECM) in a modular manner, i.e. almost independently of each other. More specifically, these functions are, firstly, the framework, support and protection for growing cells, secondly, the regulation of cell adhesion, thirdly, the graded sequestration and reversible release of therapeutically relevant signaling molecules, and fourthly, the possibility of remodeling on demand by ingrowing cells. The physical properties, such as the stiffness and the degree of hydration of the material, are tunable within a wide range. In a preferred embodiment of the present invention, for this purpose, covalently crosslinked hydrogels 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 uncharged building blocks via a stable amide group. In an alternative embodiment, preferred for 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 6-10 molecules of N-(2-aminoethyl)maleimide per molecule of poly(4-styrenesulfonic acid-co-maleic acid), then purified and isolated.These derivatives of poly(4-styrenesulfonic acid-co-maleic acid) with N-(2-aminoethyl)maleimide are then subjected to spontaneous, bio-orthogonal reactions when mixed with living cells and biomolecules in aqueous solutions or complex biofluids, but do not result in any change in their functionality within the context of the aforementioned hydrogel structures via 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 that have in their sequence the amino acid cysteine, which has a thiol group in the side chain, and that have been previously conjugated to one or more arms of a four-arm polyethylene glycol (PEG) (as described in 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 fast, resulting in a direct reaction without undesired side reactions with other biomolecules or with proteins on the cell surface, so that the Michael-type addition reaction can be used as a bioorthogonal crosslinking reaction for polymerizing cells. It is also possible to functionalize the hydrogels by means of cell-engineering peptides, for example the amino acid sequence CWGRGDSP, via the maleimide groups of poly(4-styrenesulfonic acid-co-maleic acid) derivatives. Furthermore, the hydrogel material may be functionalized with antimicrobial substances, for example positively charged antibiotics.

[0025] To carry out the above-mentioned method, as an alternative to covalently crosslinked hydrogel materials, physically crosslinked hydrogel materials may be applied. 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 bonded onto 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 with lysine and alanine or with 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 the cell culture after culturing human endothelial cells on the hydrogel for 24 hours.

[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 as the property of the charged unit [mole / g] in column A. The properties of the swollen hydrogels are given as the concentration of sulfate or sulfonate groups in mmole / mL in column B and the storage modulus in kPa in column C. Column D lists the properties of the uncharged building blocks.

[0037] Column E shows the molar concentration of sulfate or sulfonate groups per mole of polymer of the charged building block (units: mole / mole), Column F shows the hydrogel properties as a function of 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 facilitate steric access within the hydrogel for all of the structurally similar signaling molecules discussed here.

[0039] [Table 9]

[0040] Table 7 lists substances from Groups A and B that are present at relevant concentrations in biofluids.

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

[0042] For example, the hydrogel from UGB1-GB1 01 in Table 1 (AB1) was found to be particularly advantageous for modulating the concentration 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). Moreover, chemokines, characterized by a strong positive net charge 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 in the hydrogel by this hydrogel type and are therefore depleted from the adjacent biofluid (MIP1-alpha and MIP1-beta bound at almost 60%, while the other chemokines, eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, RANTES and SDF-1alpha, are bound at more than 95% (Table 3)), while, surprisingly, the following chemokines, FGF family (corresponding to PROSITE ID: PS00247) and TGFb1 family (PROSITE ID: PS00248) are strongly bound in the hydrogel by this hydrogel type and are therefore depleted from the adjacent biofluid (MIP1-alpha and MIP1-beta bound at almost 60%, while the other chemokines, eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, RANTES and SDF-1alpha, are bound at more than 95% (Table 3)), are not bound at all (Table 3). The strongly positively charged signaling molecules FGF-2 (IEP, 9.58) and TGFb1 (IEP, 8.59) related to the class of growth factors and structurally similar proteins (corresponding to PROSITE ID: PS00250) were bound very weakly with 31% and 18%, and PLGF (IEP, 8.37) was bound even weaker with less than 10% (Table 3), as well as 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), the IL-10 protein ... 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) were only weakly bound to less than 30% of the complex biofluids (TNF-alpha binding, approximately 38%) (Table 3) and therefore present in the biofluids at largely uncharged concentrations.Furthermore, the slight negative net charge of EGF (IEP, 4.8) and growth factors with less than 10% binding are poorly bound, resulting in signaling factors essential for cell survival, such as EGF, FGF-2, TGFβ, or PLGF, remaining unaffected by the sequestration achieved by the hydrogel approach 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), as well as 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 to more than 59.9% as expected (Table 3) and therefore depleted from the biofluid.

[0045] By contacting a biofluid with a hydrogel having specific charge and network properties of type 1 as described in Table 5.1, growth factors having proteins structurally similar to DKK1, bNGF, PDGF-BB, VEGF, as well as chemokines (e.g., eotaxin, Gro-alpha, IL-8, IP-10, MCP-1alpha, MIP1beta, Rantes, SDF1alpha) and cytokines having proteins structurally similar to IFN-gamma, IL4 and sclerostin according to the sequence motifs noted, protein families can be depleted from the biofluid or their concentration in the biofluid can be increased by pre-charging the hydrogel, while advantageously keeping the concentration of the above-mentioned signaling molecules EGF, FGF-2, TGFb1, PLGF, IL-10, IL1beta, IL6 and TNFalpha almost unchanged. Furthermore, this hydrogel has an interaction between GB1 and thrombin and antithrombin, which are important enzymes 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] Yet 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, as well as sclerostin (approximately 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 clotting 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 2). 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 the WE [mole / g] of 0.0038, however, both parameters are within the range given for Type 2 hydrogels in Table 5.1. However, that hydrogel was formed from another charged synthetic gel building block (GB5) using a different crosslinking reaction (crosslink type 2, Table 1) and has nearly identical binding and sequestration patterns to AB2 (see Table 3), but differs by having 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 displays particularly advantageous sequestration patterns.

[0047] Yet another advantageous exemplary embodiment (AB3) is represented by hydrogel type UGB1-GB3 03 (see Table 2), which has a 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, compare with 0.12 mmole / mL for AB1 or AB2) and also the lower number of sulfate or sulfonate groups per WE divided by the molar mass of the WE of 0.0019 compared to 0.005 or 0.0035 for AB1 or AB2 (see Table 5.2, column A). In this type of hydrogel, bNGF, PDGF-BB and VEGF A, eotaxin, GRO-alpha, IP-10, Rantes, SDF-1alpha, IFN-gamma, and IL-4 are bound by the hydrogel at more than 50% and are thereby depleted from the biofluid. In contrast, IL-8, MCP-1 and IL-12p40 are bound by the hydrogel at only about 45% (see Table 3), HGF, MIP-1alpha, MIP-1beta, IL-1beta, IL-10, IL-6, TNFalpha, and DKK1 are bound even weaker at 16-36% (see Table 3), and FGF-2, TGFb1, EGF, PLGF, GM-CSF, and sclerostin are hardly bound (less than 12%) (see Table 3), so the majority of signaling molecules are significantly less bound than AB1 and AB2. With this type of gel it is possible to separate with high selectivity, 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 the WE of 0.0045 mole / g, binds all the factors studied 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 studied 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), with a high structural similarity of the tertiary structure resulting from the interaction of four cysteines by disulfide bridges, and characterized by a strong positive net charge, IEP>9, or positively charged domains as in MIP1 alpha and MIP1 beta, and a molecular weight of less than 10 kDa (see table of protein properties), FGF-2 (IEP, 9.58), TGFb1 (IEP, 8.59), and structurally similar FGF family proteins (corresponding to PROSITE IDs: PS00247), TGF family (see ... ID: PS00250) and a class of signaling molecules related to cytokines with a slightly basic EP, e.g., IL-10 (IEP, 7.65), as well as signaling molecules with an IEP 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 (corresponding to PROSITE ID: PS00253) and the IL-10 protein family (corresponding to PROSITE ID: PS00520). Another advantageous exemplary embodiment, UGB2-GB4 20 (see Table 1), has a charge characteristic that can belong to 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 belongs to type 4, but is formed using a different crosslinking reaction (VN: 2 in Table 1), and has an almost identical binding or segregation pattern as UGB1-GB4 04 (see Table 3). Therefore, this result also proves the effective predictive performance of the charge characteristics of types 1 to 5 described in Table 5.1 for the separate segregation of substances of a group of substances.

[0050] Yet another exemplary embodiment is the uncharged hydrogel (UGB2-UGB3 26) formed from UGB1 and UGB3, which, as expected, shows little to no binding of signaling molecules from the biofluid as a negative control. With the exception of a small degree of sequestration of PDGF-BB by 19.7±23.1% and IP 10 by 16.1±16.4% (which cannot be classified as significant due to the large standard deviations), no sequestration occurs in this hydrogel (less than 1.3% for 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 equivalent 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, especially with the lack of interaction of the uncharged hydrogel (UGB2-UGB3 26) with signaling molecules (i.e., absence of binding and sequestration effect).

[0051] At the same time, individual factors can be released in the biofluid in parallel and independently of the sequestration and thus 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 tuned by targeted precharging of the hydrogel or the signaling molecule 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 of the number of sulfate or sulfonate groups per WE divided by the molar mass of the WE result in high binding of the signaling molecules. Corresponding to these parameters, the following order of binding to the hydrogel is obtained, 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 interactions known in the literature as "specific interactions", that is, between the protein and negatively charged polyelectrolytes (for example, glucose-amino glycans). 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.

[0053] Further possibilities for adjusting the levels of soluble signaling molecules are based on the use of different hydrogel types, either in sequence or in combination, to precisely adjust 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 multi-phase hydrogel materials (i.e., by mixing different types of hydrogel types or precharging with signaling molecules or proteins), as well as other advantageous concentration ratios can also be set in adjacent biofluids.

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

[0055] In order to be able to effectively regulate the complex biological functions of signaling molecules, for example for therapeutic concepts, it is necessary to actively manage signaling molecules that are distinct from the immediate biological environment of the cells or from the biological application, in addition to the slow release of individual signaling molecules from hydrogels precharged with signaling molecules. Therefore, interactions with a large number of related signaling molecules must be taken into account. If hydrogels containing sulfated or sulfonated components are charged with signaling molecules for slow release, other therapeutically important molecules from the biological environment may be simultaneously sequestered and therefore inactivated with the therapeutically desirable release of the signaling molecules, which may result 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 carrying sulfated or sulfonated groups. Overall, very complex release and sequestration scenarios are possible, which overall determine the biological effect, and in particular the molecular separation performance of the material.

[0056] Further advantageous embodiments of the present invention include the use of hydrogels according to types 1 to 4 of Table 5 for modulating the concentration or level of biologically active proteins at low total levels.

[0057] The essential advantage of the method is its broad range of applications: 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] In wound healing, primarily pro-inflammatory chemokines (e.g., eotaxin, GRO-a, 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 field of application of the present invention is the regulation of the concentration of substances in biofluids, which play a role in determining the fate of cells in vitro and in vivo. Dysregulation of proinflammatory 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 aim of regulating the concentration of these inflammatory factors from biofluids by applying various hydrogels containing sulfated or sulfonated components according to the present invention represents a possible application scenario.

[0061] In a broader context, the application of hydrogels containing sulfated or sulfonated components according to the invention allows the targeted purification of under-stood or structurally similar signaling molecules from complex protein mixtures. The described gel system can therefore be used biotechnologically for the targeted purification of proteins from cell lysates of microbial or eukaryotic origin. In this case, the cell lysates are prompted by the hydrogel according to the invention to bind to the signaling molecules. In a second step, the bound material can be removed again from the hydrogel for further use by washing with a high concentration of saline solution or a positively charged polyelectrolyte (e.g. chitosan) and the resulting separation. Furthermore, there is also a negative selection to separate signaling molecules weakly bound to the hydrogels of the types 1 to 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 a variety of 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 as well as at low levels of signaling molecules such as 100 pg / mL to 2000 ng / mL in physiological electrolytes such as biofluids, and thus it is possible to exploit the selective differences in binding of individual signaling molecules to different hydrogel types.

[0063] Exemplary embodiments of UGB1-GB1 01 to UGB1-GB4 011 shown in Table 1 are covalently crosslinked hydrogels according to crosslinking reaction 1, where 6 to 24 carboxyl groups (depending on the molar ratio of 1.5 to 6 in the 4-arm star PEG) of GB4 (poly(4-styrenesulfonate-co-malate), styrenesulfonate:maleate molar ratio=3:1, Table 1) are activated with EDC / sulfo-NHS to react directly with the 4-arm, 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, where 6 to 24 carboxyl groups (depending on the molar ratio of 1.5 to 6 in the tetrafunctional star PEG) of a molecule of poly(4-styrenesulfonic acid-co-maleic acid) (GB5) with a molar ratio of styrenesulfonic acid to maleic acid=1:1 are activated by EDC / SN HS and directly converted into hydrogels by means of a four-armed, amine-terminated UGB1 (see Table 1) to obtain hydrogels with sulfonate concentrations variable from 0.13 to 0.05 mmole / mL and storage moduli variable in phase 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-styrenesulfonate-co-maleic acid) activated with EDC / sulfo-NHS in a 3:1 molar ratio of styrenesulfonate:maleic acid are functionalized in a first step with the short-chain bifunctional crosslinker molecule N-(2-aminoethyl)maleimide, followed by purification and isolation. These GB4 derivatives were converted into hydrogels by mixing saline (or serum) with UGB-2 (thiol-terminated 4-arm PEG), resulting in 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 eight carboxyl groups of EDC / sulfo-NHS-activated GB5 (poly(4-styrenesulfonate-co-maleic acid) with a 1:1 molar ratio of styrenesulfonate:maleic acid are functionalized with a short-chain bifunctional crosslinker molecule N-(2-aminoethyl)maleimide in a first step, followed by purification and isolation. These GB5 derivatives were then converted into hydrogels by mixing saline solution (or serum) with UGB-2 (thiol-terminated 4-arm PEG), resulting in hydrogels with sulfonate group concentrations ranging from 0.14 to 0.04 mmole / mL and storage moduli grading from 4.6 to 0.3 kPa.

[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 the sequestration experiments.

[0068] Surprisingly, fully synthetic hydrogels with a wide range of advantageous properties and combinations of properties and without adverse immunogenic reactions can be prepared based on the exemplary embodiments 04-5. Thus, the wide range of sulfonate group concentrations from 0.04 to 0.28 mmole / mL and the equally large variation of storage modulus from 0.2 to 21.8 kPa of the swollen hydrogels can be widely adjusted independently of each other. 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 hydrogels, but at the same time the storage modulus increases gradually from 1.1, 5.5, 12.5, and 19.6 kPa, i.e. the sulfonate concentration and stiffness of the hydrogels can be modulated independently of each other over a wide range. Similarly, the selection of UGB1-GB5 19 with a sulfonate concentration of 0.05, UGB1-GB4 11 with a sulfonate concentration of 0.06, and UGB1-GB4 10 with a sulfonate concentration of 0.09, all have a nearly constant storage modulus of 8.0-8.5 kPa, but the sulfonate concentration can be varied independently of the hydrogel stiffness (see Table 1). In addition, with further variation in the UGB:GB molar ratio ranging from 1.5-3 and 3-6 for VN1 and 0.5-0.75 and 1-1.5 and 1.5-2 for VN2, and further variation in the concentrations of GB and UGB, many other performance combinations can be made.

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

[0070] In yet another exemplary embodiment, hydrogels of types UGB1-GB1 01, UGB1-GB4 20, UGB1-GB5 23, and UGB1-GB3 26 were functionalized with the adhesion peptide CWRGDSP, precharged with VEGF-A and FGF-2, and used on the gel surface of a serum-free cell culture medium for the culture of human endothelial cells. The morphology of adherent cells was analyzed for UGB1-GB1 01, UGB2-GB4 20, UGB2-GB5 23, and UGB2-UGB3 26 after a 24-hour culture time, as shown in Figure 1. Endothelial cells were expected to have different morphologies on different hydrogels and could be represented by two parameters, the aspect ratio and circularity. A high aspect ratio and a low circularity corresponded to the desired elongated endothelial cell morphology, which was 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, while the uncharged reference gel UGB2-UGB3 26 had a significantly lower aspect ratio of 2.1 ± 1.2. The circularities of endothelial cells on hydrogels UGB1-GB1 01, UGB1-GB4 20, and UGB1-GB5 23, and UGB1-GB3 26 were, correspondingly, 0.5 ± 0.1, 0.4 ± 0.1, 0.3 ± 0.1, and 0.7 ± 0.3. These differences were highly significant statistically. Thus, the endothelial cells described in Table 5.1 showed significantly different behaviors on hydrogels of different charges and sequestration properties 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 desired morphology increased in the following order: UGB1-GB1 01 < UGB1-GB4 20 < UGB2-GB5 23. The UGB2-GB5 23 of type 2 hydrogel showed the best results in this experiment.This ordering therefore demonstrates a direct effect of charge properties and segregation patterns on 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) as well as uncharged type 5 (UGB1-UGB3 26) hydrogels were used for polymerization of human mesenchymal stromal cells. The hydrogels were cell-responsive, i.e., in addition to functionalization of the hydrogels with the adhesion-mediating peptide CWGRGDSP, the cleavable peptide sequence GCGGPQGIWGQGGCG, enzymatically cleavable by matrix metalloproteases secreted by these cells, was pre-conjugated onto each uncharged building block and used for cross-linking with VN2. The metabolic activity of the embedded cells was characterized after 24 hours with 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 showed the highest metabolic activity when embedded in type 2 gels and the lowest metabolic activity when embedded in the uncharged reference gel. Thus, hydrogels, especially type 2, proved to be particularly advantageous for the culture of live human mesenchymal cells in 3D.

[0072] In yet 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 carrying a positively charged group. The 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). The antibacterial activity was measured by the size of the inhibition zone (distance from the hydrogel on the culture plate, see Materials and Methods). For hydrogels of the types UGB1-GB1 01, UGB2-GB4 20, UGB2-GB5 23, and UGB2-UGB3 26, no inhibition zone was detectable without prior gentamicin sequestration, i.e., those hydrogels had no antibacterial effect. In the case of pre-segregation of gentamicin by hydrogels, 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, as well as 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 very small amounts of gentamicin were nonspecifically sequestered and then released again in the uncharged reference gel UGB2-UGB3 26.Among other hydrogels, antibacterial effects were found in the following order: UGB2-GB4 20 < UGB2-GB5 23 < UGB1-GB1 01. This result demonstrates the compartmentalized isolation of positively charged drug molecules. In addition, by varying the sulfation concentration of gentamicin, it was also demonstrated that the antibacterial effect changes.

Example

[0073] Materials and Methods: (Region-selective desulfation of heparin (produced from GB2 from GB1 and GB3)) For the synthesis of region-selectively desulfated heparin (GB2 and GB3), heparin (MW: 14,000, Merck Millipore, producer number: 375095, Germany, GB1) was first dissolved in deionized ultrapure water and desalted using an Amberlite IR-120H+ ion exchange column (Sigma-Aldrich, Germany). To the heparin solution, pyridine (Sigma-Aldrich, Germany) was added until pH 6 was reached to form a heparin-pyridine salt, which was concentrated by solvent evaporation in a B-490 rotary evaporator (Buechi, 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 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 solution thus obtained 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 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, 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 heparin-pyridine was dissolved in a mixture of DMSO and deionized ultrapure water (95:5) and incubated at 90°C for 24 h. The so obtained 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 6O-N-desulfurized heparin was concentrated by solvent evaporation with B-490 (Büchi, Germany) and then freeze-dried (GEA Lyovac GT2, Germany).

[0076] GB and UGB characterization (see Table 2): The molecular weights of GB1–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 molecular weights of GB1, GB2, and GB3 were 0.1351 mL g -1 An increment of RI, dn / dc, of 100 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 heparin derivatives (GB1-GB3) was determined based on the S:N molar ratio by elemental analysis (Elementar, Vario MICRO cube, Germany). Since 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 therefrom. 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, amino-, thiol- or maleimide-terminated four-arm PEG are based on information from the manufacturer (Jenkem Technology, USA). The molar mass and structure of UGB4 (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 number: 375095, Germany), a desulfurized heparin derivative based on GB1, was dissolved in deionized ultrapure water at 4° C. for 30 s at 500 rpm using a vortex mixer (IKA, Germany) by the method described above according to the type of hydrogel as the charged component (GB) for GB2 and GB3 (see Table 2). In addition, 4-arm amine-terminated PEG (MW, 10,000, Jenkem Technology, USA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma-Aldrich, Germany), and N-hydroxysulfosuccinimide (sulfo-NHS, Sigma-Aldrich, Germany) were dissolved in the same manner for all types of hydrogels as UGB1. To ensure complete dissolution of the hydrogel building blocks, these solutions were further treated with an ultrasonic bath (RK 100H, Bandelin, Germany) at 4° C. for 5 min.

[0081] To activate GB1-GB3, EDC and sulfo-NHS (2:1 ratio of EDC:sulfo-NHS) were added to the dissolved GB (4 moles of sulfo-NHS, 8 moles of EDC per mole of UGB1 in the final reaction mixture) and incubated at 4°C for the times listed in Table 4. After the activation period of GB1, GB2, and GB3, dissolved amino-terminated 4-arm PEG (UGB1) was further 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): Poly(4-styrenesulfonic acid-co-maleic acid) with a molar ratio of 4-styrenesulfonic acid:maleic acid = 3:1 (Sigma-Aldrich, Manufacturer No.: 434566, Germany, GB4, see Table 2) and with a molar ratio of 4-styrenesulfonic acid:maleic acid = 1:1 (Sigma-Aldrich, Manufacturer No.: 434558, Germany, GB5, see Table 2), and UGB1 (tetravalent PEG, amine-terminated, MW = 10,000, Jenkem Technology, USA) were used to prepare covalently crosslinked hydrogels in the following manner: GB4 or GB5 and UGB1 were dissolved in deionized ultrapure water at 1 / 3 of the total volume of the hydrogel mixture, at a concentration three times that shown in Table 1, and further treated in an ultrasonic bath (RK 100H, Bandelin, Germany) at 4 °C for 5 min. Afterwards, the whole solution was 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 whole reaction mixture and tempered at 4° C. The molar ratio of UGB1:EDC:sulfo-NHS is 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 waiting time of 30 seconds, the reaction mixture was mixed on a vortex shaker (VWR, Germany) for 10 seconds, followed by another waiting time of 20 seconds. After this procedure, UGB1 was pipetted into the activated GB4 or GB5 on a vortex shaker with a total activation time of 1 min, then mixed with the vortex shaker for another 10 s. The final reaction mixture (where the concentrations of UGB1 and GB4 or GB5 correspond to those listed in Table 1) can now be poured into any form by pipetting, 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 about 100 μm were formed by pipetting 11 μL of the final reaction mixture per cover slip. 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 of Pompe et.al., Biomolecules, 2003, 4, 1072-1079. For modification with RGD peptide, the cast hydrogels swollen in phosphate buffered saline containing EDC / sulfo-NHS were dissolved in 1 / 15M phosphate buffered saline at 4°C at a concentration of 50 mM EDC and 25 mM sulfo-NHS, and the remaining carboxyl groups of GB4 or GB5 were activated for 20 min and then washed out with borate buffer (100 mM, pH 8.0, 4°C). The activated hydrogel was then treated with a peptide sequence [ka] The mixture was reacted with a solution having a concentration of 50 mg / mL (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 (HUVEC, Lonza, Germany) were subcultured on fibronectin-coated cell culture flasks supplemented with Promocell C-22010 (PromoCell GmbH, Germany) at 37°C and 5% CO2 to achieve 80% confluency. Cells from passages 2 to 6 were used in subsequent experiments.

[0085] The RGD-functionalized, surface-bound hydrogels were then applied to the hydrogel surface along with VEGF-A and FGF-2 at 1 cm 2 The cells were incubated at a concentration of 0.565 μg per well for 18 h at RT 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 h of incubation, cells were washed, fixed and characterized by light microscopy (Olympus IX73 inverted microscope, Hamburg, Germany). Aspect ratios were 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 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 hydrogel based on poly(4-styrenesulfonic acid-co-maleic acid) 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, respectively, are dissolved in 2.7 mL of deionized ultrapure water and stirred on ice for 10 min. The sample container is a 25 mL snap-cover glass. Then, 65.14 mg of NHS (0.30 mmole) dissolved in 400 μL and 115.02 mg (0.60 mmole) of EDC dissolved in 200 μL of ice-cold deionized ultrapure water are added to the GB4 or GB5 solution and waited for 5 min. 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 for activating GB4 or GB5 was completed. The reaction mixture was stirred on ice for another 10 minutes. Finally, the ice bath was removed and the mixture was stirred overnight at room temperature. The product was placed in a dialysis tube with an exclusion size of MWCO=5 kDa. Dialysis was carried out for 2 days; on the first day, dialysis was carried out for 6 hours against 2.5 liters of 1 molar sodium chloride solution, with the solution being replaced every 2 hours. After 6 hours, dialysis was carried out overnight against deionized ultrapure water. On the second day, dialysis was carried out against deionized ultrapure water, with the water being replaced every 2 hours. In the last step, the solution was freeze-dried.

[0088] Exclusion volume chromatography for characterization of GB4 / GB5-maleimide derivatives: Volume exclusion chromatography (SEC) is used to determine the maleimide groups per GB4 or GB5 molecule. For this purpose, the peptide RGD-SP (M = 990 g / mole) is coupled to the GB4 / GB5-maleimide derivatives in various excesses. The molar excesses of RGD-SP examined are in this case 6.8, 10 or 12 with respect to GB4 or GB5. Calibration is performed by first mixing 35 μL of the respective 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) is used, into which is inserted an HPLC system Agilent 1100 (Germany). The eluent is phosphate buffered saline solution, the flow rate is 0.5 mL / min. The injection volume is 50 μL.

[0090] The maleimide groups are also measured under the same elution conditions. In each case, 35 μL of the RGD concentrate are mixed with 35 μL of the GB4 / GB5-maleimide derivative solution in a sample vessel. The 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-armed, thiol-terminated PEG (UGB2, Mw=10,000 Da, Jenchem, USA, Table 2) are dissolved in phosphate buffered saline at twice the concentrations listed in Table 1. Equal volumes of both components are then pipetted into a microreaction vessel and shaken on a vortex shaker for 10 seconds. A volume of the reaction mixture is then withdrawn and placed on a 9 mm diameter cover slip coated with Sigma Cote®. Another cover slip coated with Sigma Cote® is placed on top of the drop. After 30 minutes, the cover slip is removed and the gel slice is allowed to swell in phosphate buffered saline for 12 hours. At high UGB2 concentrations, the pH of UGB2 can be adjusted to a pH of 5-7 using 1 M HCl for optimal gelation.

[0092] Fabrication of hydrogels in 3D cell culture with human mesenchymal cells: 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, hydrogels were formed by mixing according to the method described above, and immediately after a gelation period of 5 min, they were swollen in phosphate-buffered saline solution and cell culture medium.

[0094] Cell culture: Mesenchymal stem cells (MSCs, ATCC, isolated from adipose tissue, Germany) were cultured in DMEM with 10% fetal simian serum and 1% penicillin / streptomycin under standard culture conditions (5% CO2, 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 concentrations of GB4 or GB5 derivatives were three times higher than those shown in Table 1). After mixing, the solution was incubated at 37°C for 10-15 minutes, and then the concentrated cell suspension was extrapolated to 6 × 10 6 The hydrogels were mixed into 1 / 3 of the final gel volume with cells / mL. 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 min of gelation, the cell culture medium described above was added and the hydrogels were incubated at 37°C for 24 h as described above.

[0096] PrestoBlue® test: The PrestoBlue® test is carried out after a 24-hour incubation period following the seeding of the cells into 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 at 37° C. for 1 hour. The fluorescence is then measured using a microplate photometer (Tecan Spark®, Germany). The excitation wavelength was 560 nm and the fluorescence was measured at a wavelength of 590 nm. The metabolic activity of the cells was expressed as relative fluorescence values ​​(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 h and then washed twice with phosphate-buffered saline solution. The antibacterial activity of the gentamicin-charged hydrogels was measured by 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 distributed 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 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 a digital caliper. Three subject definitions were evaluated in ten independent experiments (n=30), and significant differences were found in all conditions (one-way anova).

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

number

[0099] Each reported data is the average (MW) from at least four independent samples, in addition the standard deviation (SD) is given.

[0100] In addition, the storage and loss moduli of the hydrogels were measured by oscillatory rheometry (units: kilobascals) using a shear rheometer of type Ares from TA Instruments (United Kingdom). For this purpose, 8 mm slices were punched out 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)) and measured at room temperature with a 9 mm plate-plate instrument set at increasing frequencies from 1 to 100 rad / s at low deformation (2%) and averaged over the full frequency range (one measurement per sample). The reported values ​​are the average of four independently prepared hydrogel discs, with ± standard deviation. The storage moduli of the four hydrogel types are also shown 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 hydrogel mesh size 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 polymerized between two 5 mm glass slides treated with Sigmacote (Sigma-Aldrich, Germany) (Menzel Glasses) for 16 h at room temperature, and then swollen in phosphate-buffered saline (PBS) (Sigma-Aldrich, Germany) buffered to pH 7.4 with 0.9% NaCl for 24 h. The total volume was adjusted to provide the same molar amount for 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 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, whose respective concentrations are shown in Table 7 (corresponding to solution 1 before incubation).

[0104] Until the protein concentration was measured, the solutions 1 and 2 were stored at -80°C. To measure the protein concentration 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 (before incubation)) x 100

[0106] Table 4 shows the activation times (units, minutes) of the carboxyl groups of GB1 to GB4 and the gel volumes used for the binding studies.

[0107] The sulfated or sulfonated hydrogels were characterized by the charge distribution of the charged building blocks (GB) (units, number of 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 performed based on the molar concentration of the hydrogel building blocks upon hydrogelation (see Table 1) and the volumetric swelling (Table 1), 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 (concentration of GB in the unswollen hydrogel) × (number of repeating units) × (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] Extraction experiments did not result in any measurable dissolution of the gel components, therefore the hypothesis that the gel components are fully incorporated appears to be plausible.

[0110] Table 3 shows the percentage of the amount of signaling molecule bound in the hydrogel, normalized by the concentration of soluble factors prior to incubation, as discussed above. The percentages are by weight.

[0111] The molecular sizes of substances (also called signal substances or factors) are expressed in units of kilodaltons (kDa).

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

[0113] The isoelectric points (IEP) and molar masses were determined based on the fully biologically processed amino acid sequences 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 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 method for separating and isolating a substance from an aqueous solution of physiological salt concentration containing a protein, said aqueous solution containing substances of substance group A and substances of substance group B which have a lower binding affinity to said sulfated and / or sulfonated hydrogel than the substances of substance group A, comprising: The method further comprising: (a) contacting an aqueous solution of physiological salt concentration containing said protein with said sulfated and / or sulfonated hydrogel in order to remove substances of substance group A from said aqueous solution and sequester said substances in said sulfated and / or sulfonated hydrogel; Or, (b) a method for releasing a substance of substance group B from the sulfated and / or sulfonated hydrogel precharged with the substance of substance group B simultaneously with the method of (a), into an aqueous solution of physiological salt concentration containing the protein, Where: the sulfated and / or sulfonated hydrogel is selected from the group of Type 1, Type 2, Type 3 or Type 4 hydrogels, and the hydrogel is composed of uncharged and charged building blocks; The charged building blocks have a parameter, calculated by dividing the number of sulfate and / or sulfonate groups per repeat unit of the charged building blocks by the molar mass of the repeat unit, of 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; and the swollen hydrogel has a storage modulus of less than 20 kPa; and the swollen hydrogel has a molar concentration of sulfate or sulfonate groups per mL of hydrogel of 0.09-0.20 mmole / mL for Type 1, 0.05-0.18 mmole / mL for Type 2, 0.01-0.12 mmole / mL for Type 3, and 0.16-0.8 mmole / mL for Type 4; and the charged building blocks have a molar concentration of sulfate or sulfonate groups per mole of polymer of 60-80 moles / mole for type 1, 30-75 moles / mole for type 2, 10-30 moles / mole for type 3, and 80-120 moles / mole for type 4; and the charged building blocks have a molar concentration of the charged building blocks per mL of hydrogel of 0.0015 to 0.0025 mmole / mL for type 1, 0.0015 to 0.0030 mmole / mL for type 2, 0.0010 to 0.0040 mmole / mL for type 3, and 0.0018 to 0.0050 mmole / mL for type 4; and the effect on the concentration of the substance of substance group A and the concentration of the substance of substance group B in the aqueous solution is determined by the selection of the type of hydrogel; When using a type 1 hydrogel, at least one of the following substances is selected as the substance group A: bNGF, PDGF-BB, VEGF-A, eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1 alpha, MIP-1 beta, Rantes, SDF1-alpha, IFN-gamma, IL-12p40, IL-4, sclerostin and / or DKK1; and at least one of the following substances is selected as the substance group B: FGF-2, TGFb1, EGF, HGF, PLGF, GM-CSF, IL-1 beta, IL-10, IL-6 and / or TNF-alpha; When using a type 2 hydrogel, at least one of the following substances is selected as the substance group A: bNGF, PDGF-BB, VEGF-A, eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, Rantes, SDF1-alpha, IFN-gamma, IL-12p40, IL-4, and / or DKK1; and at least one of the following substances is selected as the substance group B: FGF-2, TGFb1, EGF, HGF, PLGF, GM-CSF, IL-1 beta, IL-10, IL-6, TNF-alpha, and / or sclerostin; When using type 3 hydrogels, at least one of the following substances is selected as the substance group A: bNGF, PDGF-BB, VEGF-A, eotaxin, GRO-alpha, IP-10, Rantes, SDF1-alpha, IFN-gamma, and / or IL-4; and at least one of the following substances is selected as the substance group B: FGF-2, TGFb1, EGF, HGF, PLGF, IL-8, MCP-1, MIP-1 alpha, MIP-1 beta, GM-CSF, IL-1 beta, IL-10, IL-12p40, IL-6, TNF-alpha, sclerostin, and / or DKK1; when using type 4 hydrogels, at least one of the following substances is selected as the substance group A: bNGF, PDGF-BB, VEGF-A, Eotaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1 alpha, MIP-1 beta, Rantes, SDF1-alpha, IFN-gamma, IL-1 beta, IL-10, IL-12p40, IL-4, IL-6, and / or TNF-alpha; and at least one of the following substances is selected as the substance group B: EGF, PLGF, and / or GM-CSF; the charged building blocks are selected from the group consisting of heparin, N-desulfurized heparin, 6O-N-desulfurized heparin, and poly(4-styrenesulfonic acid-co-maleic acid); the uncharged building block is selected from the group consisting of an amine-terminated four-arm polyethylene glycol (PEG), a thiol-terminated four-arm PEG, a maleimide-terminated four-arm PEG, and a four-arm PEG end-capped with an enzymatically cleavable peptide sequence; The method of claim 1, wherein the hydrogel is formed by covalently crosslinking the charged and uncharged building blocks.

2. The charged building blocks of the hydrogel have a parameter, the number of sulfate and / or sulfonate groups per repeating unit divided by the molar mass of the repeating unit, of 0.0050 mole / g for type 1, 0.0035 mole / g or 0.0038 mole / g for type 2, 0.0019 mole / g for type 3, and 0.0045 mole / g for type 4; and 2. The method of claim 1, wherein the swollen hydrogel has a molar concentration of sulfate or sulfonate groups per mL of hydrogel of 0.12 mmole / mL for type 1, 0.12 or 0.14 mmole / mL for type 2, 0.06 mmole / mL for type 3, and 0.28 or 0.16 mmole / mL for type 4.

3. the charged building blocks have a molar concentration of sulfate or sulfonate groups per mole of polymer of 70.2 mole / mole for type 1, 48.4 or 75.0 mole / mole for type 2, 23.4 mole / mole for type 3, and 90.0 mole / mole for type 4; and The charged building blocks have a molar concentration of the charged building blocks per mL of hydrogel of 0.0018 mmole / mL for type 1, 0.0025 or 0.0018 mmole / mL for type 2, 0.0027 mmole / mL for type 3, and 0.0031 or 0.0018 mmole / mL for type 4; and 2. The method of claim 1, wherein the swollen hydrogel has a molar concentration of sulfate or sulfonate groups per mL of hydrogel of 0.12 mmole / mL for type 1, 0.12 or 0.14 mmole / mL for type 2, 0.06 mmole / mL for type 3, and 0.28 or 0.16 mmole / mL for type 4.

4. 4. The method according to claim 1, wherein the hydrogel of type 1, type 2, type 3 or type 4 is precharged with substances of substance group B, which are then released from the hydrogel and change their concentration in the aqueous solution by contacting the aqueous solution with the hydrogel.

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