NEW HYDROGEN GELS

Description

Field of invention

[0001] The invention relates to hydrogels, methods for their production and their use. State of the art

[0002] Hydrogels are three-dimensional networks of cross-linked hydrophilic polymers with a high water content. These materials are known as matrix materials for biological applications such as drug delivery, wound dressings, tissue engineering, and can also be used in cell culture. Their aqueous and porous structure allows for efficient nutrient transport to the cells.

[0003] Many natural or synthetic polymers have already been used to produce hydrogels, for example collagen, gelatin, and polyethylene glycol (PEG). Various reactions and mechanisms have been investigated for crosslinking these hydrogels, such as photopolymerization, Michael addition, and similar processes.

[0004] Controlling the cross-linking reaction is a major challenge, especially when the hydrogel is intended for cell encapsulation. If the gel polymerizes too quickly, it is often not homogeneously cross-linked. If it polymerizes too slowly, the components to be encapsulated, such as cells, can detach and are not homogeneously enclosed.

[0005] PM Kharkar, et al., HHS Author Manuscripts, 2016, 2, 165-179, describes hydrogels produced by reacting multi-arm poly(ethylene glycol) macromonomers functionalized with alkenyl groups or maleimide groups with multi-arm poly(ethylene glycol) macromonomers functionalized with thiols.

[0006] EA Phelps et al., Advanced Materials, 2011, 24, 64-50, describes hydrogels produced by reacting a 4-arm PEG maleimide macromer, a 4-arm PEG acrylate macromer or a 4-arm PEG vinylsulfone macromer with a thiol-containing peptide.

[0007] PM Kharkar, et al., Polymer Chemistry, 2015, 6, 5565-5574, describes a hydrogel obtained by the reaction of an arylthiol-functionalized four-arm polyethylene glycol (PEG-4-arylSH) with a maleimide-functionalized four-arm polyethylene glycol (PEG-4-PD-MI) via a Michael-type addition reaction.

[0008] N.Toda, et al., Angew. Chem. Int. Ed, 2013, 52, 12592-12596, describes the nucleophilic substitution of sulfonyl groups in heteroaromatic groups by thiol groups, but not this reaction for crosslinking to produce hydrogels. Task

[0009] The object of the invention is to provide a method for producing a hydrogel that allows its use, in particular, for coating cells. It is also an object to provide a corresponding hydrogel and its application. Solution

[0010] This problem is solved by the inventions with the features of the independent claims. Advantageous embodiments of the inventions are characterized in the dependent claims. The wording of all claims is hereby incorporated by reference into the content of this description. The inventions also include all meaningful and, in particular, all mentioned combinations of independent and / or dependent claims. A process for producing a hydrogel comprising the following steps: a) Preparation of a composition comprising a1) at least one macromer comprising at least two thiol groups as functional groups, a2) at least one macromer comprising at least two aromatic or heteroaromatic groups as functional groups, each of which is substituted with at least one sulfonyl group, wherein at least one component a1) or a2) has at least three of the aforementioned functional groups; b) Reaction of the two macromers via the functional groups to form a hydrogel.

[0011] The following section describes individual process steps in more detail. These steps do not necessarily have to be carried out in the order given, and the process described may also include further, unmentioned steps.

[0012] A macromer is defined as a compound having a mean molar mass of less than 500 kDa, preferably less than 100 kDa, and particularly less than 50 kDa. The mean molar mass is determined as the weight-mean molecular weight by gel permeation chromatography (GPC).

[0013] Macromers with a mean molar mass of less than 50 kDa, and especially less than 30 kDa, are particularly preferred.

[0014] In a particular embodiment of the invention, the mean molar mass of a macromer is between 100 Da and 500 kDa, preferably between 200 Da and 200 kDa, and in particular between 800 Da and 100 kDa.

[0015] It is important that the macromer carries the corresponding functional groups and that these are available for reaction.

[0016] Macromers are preferred, which 2, 3, 4, 5, 6, 7, 8, 9 or have 10 functional groups, preferably 2, 3, 4,5, 6, 7, 8 functional groups, especially preferred 2, 3, 4, 5 or 6 functional groups, in particular 2, exhibit 3 or 4 functional groups.

[0017] The formation of a hydrogel means that a hydrogel is formed through cross-linking. Sufficient cross-linking reactions therefore take place. This can be controlled by the type and quantity of the components used.

[0018] In a further preferred embodiment, at least one component a1) or a2) has at least 4 of the aforementioned functional groups.

[0019] In a preferred embodiment, both components a1) and a2) have at least 3, preferably at least 4, of the aforementioned functional groups. Both components a1) and a2) are particularly preferably exhibited. 3, 4, 5, 6, 7, 8, 9 or 10 functional groups, preferably 3, 4, 5, 6, 7, 8 functional groups, especially preferred 3, 4,5 or 6 functional groups, in particular 3 or 4 functional groups.

[0020] Water-soluble macromers are preferred. This means that the macromers must be present in solution to the necessary extent under the reaction conditions.

[0021] Preferred are macromers based on oligomers or polymers. These can be natural or synthetic oligomers or polymers. Examples of synthetic oligomers or polymers include poly(meth)acrylates such as poly(meth)acrylamides, poly(meth)acrylic acid, polyHPMA or polyHEMA, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane (PU), polyvinylpyrrolidone (PVP), polyamides, poly(amidoamine) (PAMAM), polyesters, polylactides, polyglycolic acid (PGA) or poly(lactide-co-glycolide) (PLGA), polyanhydrides, poly(ortho)esters, polyacetals, poloxamers (block copolymers of ethylene oxide (PEG) and propylene oxide (PPG)) such as PEG-co-PPG-co-PEG), poly-2-oxazolines, polyphosphazenes, polyglycerol, polyamines such as polylysine or polyethyleneimine (PEI), polycarbonates, polyglutamic acid, especially polygamma-glutamic acid, polyaspartic acid (PASA), polyphosphonates, or natural oligomers. Oligomers such as DNA, RNA, gelatin, polyhydroxyalkanoates (PHA), poly-gamma-glutamic acid, proteins or peptides such as collagens,VPM, albumin or fibrin, polysaccharides such as agarose, chitin, chitosan, chondroitin, mannan, inulin, dextran, cellulose, alginates or hyaluronic acid. Polyethylene glycol-based oligomers are preferred. The oligomers and polymers are functionalized with the corresponding functional groups.

[0022] In the case of peptide-based oligomers, the thiol groups are preferentially provided by the corresponding amino acids such as cysteine ​​or homocysteine. Peptide-based means that the corresponding oligomer is composed of at least 80% natural or synthetic amino acids by molecular mass. Such oligomers therefore possess at least two thiol groups, in particular at least two cysteine ​​groups.

[0023] The use of natural polymers, at least in part, also allows the introduction of specifically cleavable sites in the hydrogel, for example by enzymes.

[0024] It may be necessary to link the functional groups to the oligomer or polymer via a short linker, for example, via one or more ester, ether, or amide bonds. Linkers with a molar mass of less than 1500 mol are preferred, preferably less than 800 mol, and particularly less than 500 mol or less than 200 mol.

[0025] The thiol groups are preferably present as free thiol groups. It is also possible that they are modified with groups that are cleaved off before the hydrogel is formed.

[0026] Macromer a2) is a macromer comprising at least two aromatic groups, each substituted with at least one sulfonyl group. These are groups of the formula (1): M-Ar-SO₂-R₁< (1) where Ar represents an electron-deficient aryl group or an electron-deficient heteroaryl group. This makes it possible to choose reaction conditions under which the thiol groups of the first macromer can undergo nucleophilic aromatic substitution at the Ar group, with the SO₂-R₁< group serving as the leaving group.

[0027] M represents a covalent bond to the macromer and is preferably a single bond, ether, or carbonyl group. The carbonyl group can be part of an ester or amide bond. Thus, the corresponding esters or amides can be used as the Ar group for coupling to the macromer, such as appropriately substituted benzoic acid esters or benzoic acid amides.

[0028] An aryl group according to this invention contains 6 to 40 carbon atoms; a heteroaryl group according to this invention contains 1 to 40 carbon atoms and at least one heteroatom, provided that the sum of the carbon atoms and heteroatoms is at least 5. The heteroatoms are preferably selected from nitrogen, oxygen, and / or sulfur. An aryl group or heteroaryl group is understood to be either a simple aromatic cycle, i.e., benzene, or a simple heteroaromatic cycle, for example, pyridine, pyrimidine, thiophene, etc., or a fused aryl or heteroaryl group, for example, naphthalene, naphthalimide, anthracene, quinoline, isoquinoline, etc.

[0029] An electron-deficient aryl group or heteroaryl group is defined as an aryl group or heteroaryl group whose n-electron density is reduced by negative induction effects or negative resonance effects (-I effects or -M effects, respectively). A list of substituents or groups that cause these effects can be found in any standard organic chemistry textbook. Examples include, without limitation, for -I substituents: OH, halogens, especially fluorine and chlorine, NO₂, unsaturated groups; for -M substituents: NO₂, CN, aryl groups, or heteroaryl groups. These electron-withdrawing groups must, of course, be conjugated to the leaving group -SO₂-R₁<, i.e., in the case of carbocycles, in the ortho or para position, in order to exert the desired effect. In the case of heteroaryl groups, the heteroatoms contribute to the reduction in electron density according to their position.There can also be several different groups.

[0030] In the case of electron-deficient aryl groups, Ar is selected from the group comprising nitrobenzenes, benzaldehydes, benzonitriles, and benzoic acid esters, which may be further substituted with one or more R2< groups as defined below. An example of such an aryl group is a compound based on nitrobenzoic acid with one or two nitro groups, for example, nitrobenzoic acid esters or nitrobenzoic acid amides, which have a -SO2-R1< group at least at one position. Preferably, this group is arranged in a metaposition to a nitro group. Particularly preferred is a nitro group in the 3-position and the -SO2-R1< group in the 4-position. An example of such a compound is 3-nitro-4-sulfomethylbenzoic acid.

[0031] In the case of electron-deficient heteroaryl groups, Ar is selected from the group comprising mononuclear heteroaromatics such as pyridines, pyrimidines, pyrazines, pyridazines, triazines (such as 1,3,5-triazine, 1,2,4-triazine or 1,2,3-triazine), tetrazines (such as 1,2,4,5-tetrazine, 1,2,3,4-tetrazine or 1,2,3,5-tetrazine), oxazoles, isooxazoles, thiazoles (such as 1,2-thiazole or 1,3-thiazole), isothiazoles, oxadiazoles (such as 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole), and thiadiazoles (such as 1,2,3-thiadiazole). 1,2,4-Thiadiazole, 1,2,5-Thiadiazole or 1,3,4-Thiadiazole, imidazole, pyrazole, triazoles, such as in particular 1,2,4-triazole or 1,2,3-triazole, tetrazole, polynuclear heteroaromatics, such as quinolines, isoquinolines, naphthalimide, benzimidazole, benzoxazole, benzothiazole, benzopyridazine, benzopyrimidine, quinoxaline, benzotriazole, purine, pteridine, indolizine and benzothiadiazole, which may be further substituted with one or more groups R 2< as defined below.

[0032] Preferred heteroaryl groups are oxadiazoles and benzothiazole. In a further particularly preferred embodiment, Ar is an oxadiazole group, in particular a 1,3,4-oxadiazole group, which is preferably substituted with at least one phenyl group, in particular with a phenyl group.

[0033] R 1< stands for N(R 2< ) 2 , a straight-chain alkyl group with 1 to 20 C atoms or a branched or cyclic alkyl group with 3 to 20 C atoms, or an alkenyl or alkynyl group with 2 to 20 C atoms, wherein the alkyl, alkenyl or alkynyl group may each be substituted with one or more R 2< residues and wherein one or more non-adjacent CH 2 groups may be replaced by O, NR 2< , S, R 2< C=CR 2< , C=C, C=O, C(=O)O or C(=O)NR 2< , or an aryl group or heteroaryl group, each of which may be substituted with one or more R 2< residues.

[0034] R 2< is the same or different in each occurrence: H, D, F, Cl, Br, I, N(R 3< ) 2 , CN, NO 2 , OR 3< , SR 3< , C(=O)OR 3< , C(=O)N(R 3< ) 2 , C(=O)R 3< , a straight-chain alkyl group with 1 to 20 C atoms or an alkenyl or alkynyl group with 2 to 20 C atoms or a branched or cyclic alkyl group with 3 to 20 C atoms, wherein the alkyl, alkenyl or alkynyl group may each be substituted with one or more R 3< residues, wherein one or more non-adjacent CH 2 groups are replaced by R 3< C=CR 3< , C=C, C=O, NR 3< , O, S, C(=O)O or C(=O)NR 3< may be replaced, or an aryl group or heteroaryl group, each of which may be substituted with one or more R 3< residues.

[0035] R 3< is the same or different H, D, F, OH, or an aliphatic, aromatic and / or heteroaromatic organic residue, in particular a straight-chain alkyl group with 1 to 20 C atoms, in which one or more H atoms may also be replaced by F.

[0036] In a preferred embodiment, R 1< represents N(R 2< ) 2 , a straight-chain alkyl group with 1 to 10 C atoms or a branched or cyclic alkyl group with 3 to 10 C atoms, wherein the alkyl group may be substituted with one or more R 2< groups and wherein one or more non-adjacent CH 2 groups may be replaced by O, NR 2< , S, C=O, C(=O)O or C(=O)NR 2< , or an aryl group or heteroaryl group, each of which may be substituted with one or more R 2< groups.

[0037] R 2< is the same or different in each occurrence: H, D, F, Cl, Br, I, N(R 3< ) 2 , CN, NO 2 , OR 3< , SR 3< , C(=O)OR 3< , C(=O)N(R 3< ) 2 , C(=O)R 3< , a straight-chain alkyl group with 1 to 10 C atoms or an alkenyl or alkynyl group with 2 to 10 C atoms or a branched or cyclic alkyl group with 3 to 10 C atoms, wherein the alkyl, alkenyl or alkynyl group may each be substituted with one or more R 3< substituents, wherein one or more non-adjacent CH 2 groups are replaced by R 3< C=CR 3< , C=C, C=O, NR 3< , O, S, C(=O)O or C(=O)NR 3< may be replaced, or an aryl group or heteroaryl group, each of which may be substituted with one or more R 3< residues.

[0038] R 3< is the same or different in each occurrence: H, D, F OH or a straight-chain alkyl group with 1 to 5 C atoms, in which one or more H atoms may also be replaced by F or OH.

[0039] In a particularly preferred embodiment, R 1< represents N(R 2< ) 2 , a straight-chain alkyl group with 1 to 6 C atoms or a branched or cyclic alkyl group with 3 to 6 C atoms, wherein the alkyl group may be substituted with one or more R 2< groups and wherein one or more non-adjacent CH 2 groups may be replaced by O, NR 2< , S, C=O, C(=O)O or C(=O)NR 2< , or an aryl group or heteroaryl group with 5 to 10 aromatic ring atoms, each of which may be substituted with one or more R 2< groups.

[0040] R 2< is the same or different in each occurrence H, D, F, OH, C(=O)OH, a straight-chain alkyl group with 1 to 5 C atoms or an aryl group or heteroaryl group with 5 to 10 aromatic ring atoms, in which one or more H atoms bonded to carbon may also be replaced by F or NO 2.

[0041] Particularly preferably, R<1> represents a substituted or unsubstituted methyl group, ethyl group, propyl group, preferably substituted with F or COOH, or N(R<2>)<2, particularly NHR<2>, where R<2> represents an aryl group or heteroaryl group with 5 to 10 ring atoms, in which one or more H atoms bonded to carbon may also be replaced by F, OH, NH2, or NO2. Particularly preferably, R<1> represents methyl, CH2-COOH, or NH-phenyl, wherein the N is bonded to the SO2 group.

[0042] In a preferred embodiment of the invention, at least one macromer is based on poly(meth)acrylates such as poly(meth)acrylamides, poly(meth)acrylic acid, polyHPMA or polyHEMA, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane (PU), polyvinylpyrrolidone (PVP), polyamides, poly(amidoamine) (PAMAM), polyesters such as polylactides, polyglycolic acid (PGA) or poly(lactide-co-glycolide) (PLGA), polyanhydrides, poly(ortho)esters, polyacetals, poloxamers (block copolymers of ethylene oxide (PEG) and propylene oxide (PPG)) such as PEG-co-PPG-co-PEG), poly-2-oxazolines, polyphosphazenes, polyglycerol, polyamines such as polylysine or polyethyleneimine (PEI), polycarbonates, polyglutamic acid, in particular polygamma-glutamic acid, polyaspartic acid (PASA). Polyphosphonates, and the other macromer on DNA, RNA, gelatin, polyhydroxyalkanoates (PHA), poly-gamma-glutamic acid, peptides such as collagens, VPM, albumin or fibrin, polysaccharides such as agarose, chitin, chitosan, chondroitin,Mannan, inulin, dextran, cellulose, alginates, or hyaluronic acid. This makes it possible to integrate biochemical reactivity into the hydrogel, such as cleavage or degradability, for example through ester or carbonate groups in the macromer or through enzymatic reactions. Examples of suitable peptides are enzymatically cleavable dithiol peptides such as VPM (sequence: GCRDVPMSMRGGDRCG).

[0043] Preferably, both macromers are used such that the number of functional groups SH:Ar-SO₂-R₁ of the two macromers contributing to the crosslinking is 2:1 to 1:2, preferably 1.5:1 to 1:1.5, particularly preferably 1.2:1 to 1:1.2, and especially 1:1. If several different compounds with the respective functional group are used, the values ​​refer to the total number of these groups, for example, when using different compounds with thiol groups. For instance, one thiol compound can be used for modification and another for crosslinking.

[0044] Preferably, both macromers are present in solution, preferably in aqueous solution. It may be necessary to adjust the pH, preferably by using a buffer.

[0045] In a preferred embodiment, a first solution containing the first macromer comprising thiol groups and a second solution containing the second macromer comprising the aromatic sulfonyl group are provided. These two solutions are then combined.

[0046] In a preferred embodiment, the pH of the macromer solutions used, particularly those of the given composition, is between 6 and 9 (at 25 °C). Preferably, the pH is adjusted using a buffer, preferably with a buffer concentration between 5 mM and 100 mM. Examples of suitable buffers are PBS or HEPES. A higher buffer concentration stabilizes the pH of the gel when using high macromer concentrations, as the leaving group can act as an acid. A pH of 6 to 9 is preferred, more preferably 6.5 to 8, and particularly preferably 6.6 to 7.5. This allows the gelation time to be adjusted between, for example, 3 seconds (pH 8) and 3.5 minutes (pH 6.6) (measured at 25 °C with a constant macromer concentration).

[0047] It is also possible to prepare the composition at a first pH value and then initiate the crosslinking reaction by changing the pH to a second pH value. Preferably, the crosslinking reaction is at least significantly slowed down at the first pH value; in this case, the first pH value is preferably outside the ranges mentioned above. The second pH value is preferably within one of the ranges mentioned above. The pH change can also be achieved by placing the composition in an environment with the corresponding pH value. Preferably, the second pH value is between 6 and 9, more preferably between 6.5 and 8, and particularly preferably between 6.6 and 7.5.

[0048] Furthermore, the reaction can be started or accelerated by changing the pH value.

[0049] In a further preferred embodiment, the macromer content in the composition is 1 to 30 wt.%, preferably 3 to 15 wt.%, particularly preferably 3 to 10 wt.%, based on all macromers used.

[0050] The temperature during the formation of the hydrogel is preferably between 20 °C and 45 °C, preferably between 20 °C and 40 °C.

[0051] The reaction described here for the formation of hydrogels is characterized by several advantages. Unlike known crosslinking reactions, it is neither particularly fast nor particularly slow under physiological conditions; rather, it can be controlled, among other things, via the pH value. This allows for the encapsulation of cells or other substances such as peptides, enzymes, chemical compounds, or similar materials during gel formation. The composition remains viscous for a longer period during gel formation, allowing it to be mixed with low shear forces for an extended time. This enables a homogeneous distribution of cells within the hydrogel without the need for further steps, such as inverting the gel during curing.

[0052] The proposed reaction is also sufficiently rapid under physiological conditions. This allows its use in cell cultures, preferably in three-dimensional cell culture or even in situ. Furthermore, gelation can be controlled via the pH value, enabling its use for the in situ construction of gels, for example in 3D printing or within an organism when a suitable composition is injected.

[0053] In a preferred embodiment, the conditions are selected such that gelation is achieved within 3 seconds to 5 minutes. The gelation time can preferably be adjusted via the macromer concentration, pH value, and temperature. This also allows for the adjustment of the physical properties of the gels, such as long-term stability, swelling behavior, and mechanical properties.

[0054] The reaction is also orthogonal to OH groups, amino groups, carboxylic acid groups and acrylate groups, which do not react under physiological conditions.

[0055] In a preferred embodiment of the invention, the reaction of the two macromers contributes exclusively to the formation of the hydrogel. No other crosslinking reactions take place.

[0056] The reaction rate can be controlled by selecting the aromatic or heteroaromatic group bearing the sulfonyl group and the pH value. This allows the gelation rate to be tailored to the specific application. Unlike other reactions, no starter or accelerator needs to be added.

[0057] The ratio of the two macromers is preferably chosen such that all functional groups have reacted after the reaction. It may depend on whether further functionalizations are carried out.

[0058] For example, it is possible to modify the second macromer by adding thiol-containing compounds before initiating cross-linking and hydrogel formation by adding the first macromer. This allows the hydrogel to be modified with additional functions, such as fluorophores or bioactive reagents.

[0059] Examples of bioactive reagents include tissue growth promoters, chemotherapeutic agents, proteins (glycoproteins, collagen, lipoproteins), cell-binding mediators (e.g., fibronectin, laminin, collagen, fibrin), integrin-binding sequences (e.g., cyclo(RGDfC)), cadherin-binding sequences, growth factors, differentiation factors, or fragments of the aforementioned reagents. Examples include epidermal growth factor (EGF), endothelial growth factor (VEGF), fibroblast growth factors such as bFGF, insulin-like growth factors (e.g., IGF-I, IGF-II), transforming growth factors (e.g., TGF-α, TGF-β), DNA fragments, RNA fragments, aptamers, or peptidomimetics; cell-binding mediators such as VEGF are preferred.

[0060] The modification can be used, for example, to create appropriate environments in the hydrogel depending on the cells to be cultivated.

[0061] The reagents are preferably used in effective concentrations, for example in the range of 0.01 to 100 mM, preferably 0.1 mM to 50 mM, in particular 0.2 mM to 10 mM, in particular 0.5 to 5 mM based on the swollen gel.

[0062] The invention also relates to a composition for the production of a hydrogel comprising at least two macromers a1) and a2) as described for the process.

[0063] The invention also relates to a hydrogel obtained using the inventive method.

[0064] The process according to the invention yields a hydrogel comprising a first plurality of macromers which is cross-linked with a second plurality of macromers, wherein the cross-linking is carried out via a plurality of Ar-S bonds, where Ar is an aromatic or heteroaromatic group.

[0065] Such a bond is obtained from nucleophilic substitution by thiols on electron-deficient aromatics. Advantageous embodiments of the process are described.

[0066] The hydrogels according to the invention are stable for a long time, preferably up to 6 weeks. They can be easily modified and maintained under physiological conditions. They are particularly suitable for cell encapsulation, three-dimensional cell cultures, organoids, biomaterials, injectable biomaterials, cell therapies, tissue modification, tissue regeneration, tissue transplantation, regenerative medicine, 3D printing, 3D bioprinting, wound dressings or wound treatment, drug delivery systems, in vitro models for investigating or testing diagnostics or therapeutics, or cell transplantation.

[0067] Because the reaction occurs under physiological conditions, it is particularly applicable in the biological field. For example, it is conceivable that the two reacting macromers are combined or mixed in situ. This can be done, for instance, using a multi-component syringe.

[0068] The invention relates to a method for coating cells, wherein the hydrogel is formed in the presence of the cells in order to coat them. This can be used, for example, for cell culture, in particular for three-dimensional cell culture.

[0069] The invention also relates to a kit for the production of a hydrogel comprising the macromers a1) and a2) as described for the process.

[0070] The described reaction is also suitable for the additional crosslinking of existing gels. In such a process, a gel comprising at least two of the functional groups of component a1) or a2), as known, for example, from A. Farrukh, JI Paez, M. Salierno, A. del Campo, Angew. Chem. Int. Ed. 2016, 55, 2092-2096, is provided by polymerizing corresponding monomers into polyacrylamide gels and reacted with a macromer having corresponding functional groups according to macromer a1) or a2), wherein in this case the macromers a1) or a2) have at least two of the functional groups, so that the gel is crosslinked by this reaction.

[0071] The invention therefore also relates to a method for modifying gels, comprising the steps of: a) Providing a gel comprising at least two functional groups according to component a1) or at least two functional groups according to component a2); b) Adding a composition comprising at least one macromer according to the other component, wherein the macromer has at least two functional groups; c) Modifying the gel thereof by reaction of the functional groups, wherein the reaction conditions are chosen such that the thiol groups can undergo nucleophilic aromatic substitution at the Ar group, with the SO2-R1 group serving as the leaving group.

[0072] The method is preferably used for subsequent modification of the gel after its production. This makes it possible to modify the gel under physiological conditions, for example to adjust its mechanical parameters.

[0073] Due to the pH dependence and / or temperature of the reaction, it is possible, for example, that this modification only occurs when the conditions change in a certain way. Examples

[0074] The embodiments are shown in the figures. In the examples, the macromers are referred to as polymers. Fig. 1 Schematic representation of the preparation of a hydrogel according to the invention; Fig. 2a) Photograph of a PEG-thiol-MS hydrogel (5 wt.% polymer concentration in 10 mM HEPES buffer); b) Shear moduli during gelation (5 wt.% polymer, 10 mM HEPES buffer pH 6.6, T = 25°C); Fig. 3a Shear moduli during gelation of the different hydrogels at 25°C (each 5 wt.%; 10 mM HEPES buffer; pH 8); Fig. 3b Shear moduli during gelation of the different hydrogels at 37°C (each 5 wt.%; 10 mM HEPES buffer; pH 8); Fig. 4 Effect of pH (at 5 wt.% polymer content, 25°C) on the crosslinking kinetics and shear moduli (a) Thiol-Mal, Thiol-MS, b) Thiol-VS); Fig. 5 Shear modulus as a function of temperature (conditions: at 30 min, 5 wt.%, pH 7.0); Fig. 6 Influence of polymer content and HEPES buffer concentration on the mechanical properties (columns, left scale) and the pH value (squares, right scale) of the prepared thiol-MS gels.Conditions: pH = 7.5, T = 25°C, at 60 min; Fig. 7 Comparison of the normalized mass of swollen thiol-X gels. Gels were incubated in cell culture medium at 37°C for 6 weeks and 4 weeks, respectively (a) 10 wt.% polymer content, pH 8.0; b) 5 wt.% polymer content at pH 7.0). Thiol-MS gels prepared under these conditions are hydrolysis-stable even after 6 weeks of incubation in cell culture medium; Fig. 8 Fibroblast L929 encapsulated in the various enzymatically cleavable thiol-X hydrogels. Live / dead assay of L929 single fibroblast cells encapsulated in materials (ac) for 1 day: Compared to the other systems, cells cultured in thiol-MS hydrogels showed a more homogeneous distribution throughout the material (a, Z-stack) and similar viability (c); Fig. 9(a) Schemes of enzymatically cleavable gels used for encapsulating cell spheroids. (bc) Migration behavior of cells from encapsulated spheroids.The results of the migration test after 3 days of culture showed that the migration distance in thiol-MS gels was intermediate. Staining: FITC-phalloidin (actin fibers), DAPI (nucleus); Fig. 10 Morphology of individual cells (mouse fibroblast L929) enclosed in the various enzymatically cleavable thiol-X hydrogels after 3 days of cell culture. Compared to the other systems, cells cultured in thiol-MS hydrogels showed a more homogeneous distribution, with fewer clusters or aggregations. Staining: FITC-phalloidin (actin fibers), DAPI (nucleus); Scale bar is 50 µm in each case. Chemical synthesis

[0075] Chemicals and solvents were acquired in µm purity and used directly unless otherwise noted. 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline was acquired from Ark Pharm, USA. 4-arm polyethylene glycol polymers (PEG, 20 kDa, based on pentaerythritol) functionalized with maleimide (PEG-Mal), vinyl sulfone (PEG-VS), thiol (PEG-SH), and N-succinimidyl carboxymethyl ester (NHS-PEG), and linear methoxylated PEG polymer (5 kDa), also functionalized with NHS, SH, Mal, and VS, respectively, were acquired from Jenkem, USA. Buffer solutions were freshly prepared. 10 mM HEPES (pH 8.0, 7.0, and 6.7) and phosphate-buffered saline (PBS, pH 7.4 and 7.0) were used.

[0076] Deuterated solvents were obtained from Deutero GmbH Germany (D-56288 Kastellaun). Deuterated phosphate buffer salt (PBS) was prepared by dissolving the correct amounts of disodium phosphate, monosodium phosphate, sodium chloride, and potassium chloride in D₂O; followed by pD adjustment with 20% DCl solution (Merck) until pD values ​​of 8.0, 7.4, 7.0, 7.0, and 6.0 were achieved. The pH was monitored using a pH meter, and the following correction factor was applied: pD = pH obs + 0.4 (see Bates et al., Anal. Chemie. 1968, 40(4), 700–706).

[0077] Thin-layer chromatography (TLC) plates (ALUGRAM® < SIL G / UV254) and silica gel for column chromatography (60 Å pore size, 63–200 µm particle size) were obtained from Macherey-Nagel, Düren, Germany (52355). TLC plates were observed under 254 or 365 nm light. HPLC analysis and purification of the compounds were performed using a JASCO 4000 HPLC system (Japan) equipped with a diode array, UV-Vis detector, and fraction collector. Reprosil C18 columns were used for semi-preparative (250 × 25 mm) and analytical (250 × 5 mm) runs. Solvent gradients were used with a combination of the following eluents: solvent A (MilliQ-water + 0.1% TFA) and solvent B (95% ACN / 5% MilliQ-water + 0.1% TFA), typically over 40 minutes. Purification of modified polymers was typically performed by dialysis against acetone and water.Spectra / Por 3 dialysis tubing (molecular weight exclusion limit MWCO= 3.5 kDa) from Spectrum Inc. was used.

[0078] The solution spectra < ¹H NMR and < ¹³C NMR were recorded at 25 °C on a Bruker Avance 300 MHz or a Bruker Avance III UltraShield 500 MHz. The latter was equipped with a cooled 5 mm TCI cryoprobe, a proton-optimized triple-resonance inverse NMR probe with external water cooling (CP TCI 500S2, HC / ND-05 Z). Unless otherwise stated, all measurements were performed at 298 K. Tetramethylsilane (TMS) (δ = 0 ppm) was used as an internal reference. Chemical shifts are given in parts per million (ppm), and coupling constants are given in Hertz. The following abbreviations are used: s-singlet, d-doublet, t-triplet, q-quartet, m-multiplet. The degree of substitution of the PEG polymer was calculated by end-group determination.The integral of the signal corresponding to the PEG backbone (3.70–3.40 ppm) was tuned to 440H and compared to the integral of the protons corresponding to the incorporated molecule 2 (the aromatic CHs at 8.10–7.70 ppm and the methylene at 4.20 ppm). Functionalization levels and yields of >91% were achieved in all cases. The data were analyzed in MestReNova.

[0079] Mass spectra were recorded using Agilent Technologies 1260 Infinity Liquid Chromatography / Mass Selective Detector (LC / MSD) and 6545 Accurate-Mass Quadrupole Time-of-Flight-MS (LC / Q-TOF-MS) with electrospray chemical ionization. UV / VIS spectra were acquired using a Varian Cary 4000 UV / VIS spectrometer (Varian Inc., Palo Alto, USA).

[0080] The rheological properties of hydrogels were determined using a Discovery HR-3 rheometer (TA Instruments, USA), equipped with 12 mm parallel plates and a Peltier stage, at 25 and 37°C. Trios v4 software was used. Data were recorded and analyzed in Origin 9.1.

[0081] The following protocols were adopted with some modifications: G. Liang et al Chem. Commun., 2017, 53, 3567-3570; J. Ling et al ChemBioChem 2018, 19, 1060. Synthese von tert-Butyl (2-((4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)amino)-2-oxoethyl)Carbamat (1):

[0082] Boc-Gly-OH (1 eq., 2.28 mmol, 0.394 g) was dissolved in anhydrous THF (3 mL) at 0 °C. Isobutyl chloroformic acid ester (1.2 eq., 2.85 mmol, 0.314 mL) and N-methylmorpholine (2.6 eq., 5.7 mmol, 0.627 mL) were carefully added to the solution by syringe under a nitrogen atmosphere and stirred for 30 minutes. A solution of 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline (0.25 eq., 0.57 mmol, 0.136 g) in THF (3 mL) was added dropwise to the mixture, stirred for a further 2 h at 0 °C, and then overnight at room temperature. Saturated NaHCO3 was added and the reaction mixture was extracted with ethyl acetate (2 x 30 mL). The combined organic phase was dried with sodium sulfate, filtered, evaporated, purified by preparative HPLC (5B to 95B, 280 nm, reaction time = 28 min), and a white solid was obtained after freeze-drying, 165 mg (yield = 73%). Synthese von 2-Amino-N-(4-(5-(5-(Methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acetamid (2):

[0083] Compound 1 (45 mg) was dissolved in 1:1 TFA / DCM (2 mL), stirred at room temperature for 2 h, and evaporated under a nitrogen flow. The final product was obtained after HPLC purification (5B to 95B, 280 nm, reaction time = 18 min), (Y = >99%). The pure compound was immediately coupled to the PEG polymer, as decomposition was otherwise observed within 1 week after storage at -20°C. Synthese from PEG-MS:

[0084]

[0085] Freshly prepared compound 2 (50 µmol, 15 mg) and N-methylmorpholine (18 µmol, 20 µL) were dissolved in dry DMF (2 mL), purged with nitrogen, and stirred for 15 min. 20 kDa, 4-arm PEG-NHS (100 mg, 5 µmol) was dissolved in dry DMF (1 mL) and added under a nitrogen stream. The mixture was stirred overnight at room temperature under an inert atmosphere, then dialyzed in acetone and water and freeze-dried. A white solid polymer was obtained and characterized by <1H NMR in DCM-d 2. A degree of functionalization of >91% and a yield of >90% were calculated.

[0086] The 2-(methylsulfonyl)-5-phenyl-1,3,4-oxadiazole group was selected as an MS substrate for thiol coupling. Under the described heteroaromatic MS rings, this substrate reacts with thiols with high conversion and moderate reaction rate. [N. Toda, S. Asano, CF Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596. X. Chen, H. Wu, C.-M. Park, TH Poole, G. Keceli, NO Devarie-Baez, AW Tsang, WT Lowther, LB Poole, SB King, M. Xian, CM Furdui, ACS Chemical Biology 2017, 12, 2201-2208.] 4-armed PEG-MS macromers (20 kDa) were synthesized on a 500 mg scale in good yield (degree of substitution >91%) within three synthesis steps. Rheological measurements on hydrogels

[0087] The gelation of the 4-arm PEG-MS and 4-arm PEG-thiol mixture was investigated. Crosslinking conditions applied were 5.

[0088] wt.% polymer content in 10 mM HEPES buffer, pH 6.6, at 25°C. An MS:thiol ratio of 1:1 was used for the experiments. Studies showed that thiol-MS gels formed a cross-linked gel within 3–4 min (see Table 1). This corresponds to a favorable cross-linking time, allowing for thorough mixing and homogenization of precursor solutions. Rheological investigations revealed that the cross-linked gels achieve a shear storage modulus of G' ~ 1 kPa ( Figur 2b A swollen PEG-thiol MS hydrogel (5 wt% polymer concentration in 10 mM HEPES buffer) shows Figur 2a .

[0089] Freshly prepared 20 kDa, 4-arm PEG-X polymer solutions were used for these studies. The polymer was dissolved in the appropriate solvent, vortexed, ultrasonic bathed, and centrifuged to remove bubbles. 21 µL of a 5% w / v PEG-X solution was applied to the Peltier bottom plate of the rheometer, followed by 21 µL of a 5% w / v PEG-thiol solution, which was then mixed with the pipette tip directly on the plate. The top plate was brought close to place the sample between the two plates, and the sample was subsequently sealed with paraffin oil to prevent evaporation during measurement. The total time for sample loading, including the start of the measurement, was approximately 2–3 minutes.

[0090] The gel time and final shear modulus of the hydrogel were determined rheometrically. Strain tests (0.1 to 1000% strain at a frequency of 1 Hz) and frequency tests (0.01 to 100 Hz at a strain of 1%) were performed to determine the linear viscoelastic regime. Time tests were conducted within the linear viscoelastic regime with the following parameters: gap 300 µm, axial force (0.0 ± 0.1 N), frequency 1 Hz, strain 1%, temperature 25 or 37°C.

[0091] Hydrogels were tested at 5 wt.% polymer content, HEPES buffer pH 8.0 and T = 25°C ( Figur 3a ) and T-37 °C ( Figur 3b ) manufactured.

[0092] Figur 3a This study compares the crosslinking kinetics of thiol-MS with those of thiol-mal and thiol-VS systems. The experiments were performed under cell culture-typical conditions (5 wt% polymer in 10 mM HEPES buffer, pH 8.0, at 25°C). [EA Phelps, NO Enemchukwu, VF Fiore, JC Sy, N. Murthy, TA Sulchek, TH Barker, AJ Garcia, Advanced Materials 2012, 24, 64-70. A. Farrukh, JI Paez, A. del Campo, Advanced Functional Materials 2019, 29, 1807734.] Under these conditions, the thiol-MS gel formed in 3-4 s (Table 1). This corresponds to a short crosslinking time but is acceptable for mixing and homogenizing gel precursors. In comparison, the thiol-mal gel required 1 s for cross-linking and inhomogeneous gels were obtained, while the thiol-VS system had a gelation time of approximately 10 min and took approximately 2 h to complete the cross-linking.These results show the following trend in the gelation rate: Thiol-Mal > Thiol-MS > Thiol-VS, in agreement with the reported reaction rates for model compounds, [X. Chen, H. Wu, C.-M. Park, TH Poole, G. Keceli, NO Devarie-Baez, AW Tsang, WT Lowther, LB Poole, SB King, M. Xian, CM Furdui, ACS Chemical Biology 2017, 12, 2201-2208. F. Saito, H. Noda, JW Bode, ACS Chemical Biology 2015, 10, 1026-1033. H. Wang, F. Cheng, M. Li, W. Peng, J. Qu, Langmuir 2015, 31, 3413-3421.] as shown in Table 2.

[0093] The shear modulus values ​​of the crosslinked gels after 1 h were G' 25°C = 2000 Pa for Thiol-VS, 1000 Pa for Thiol-MS, and 470 Pa for Thiol-Mal. The higher stiffness of Thiol-MS gels is likely due to greater system homogeneity resulting from the slower gelation kinetics, leading to fewer network defects and a higher degree of crosslinking.

[0094] This result contradicts previous reactivity studies of thiol-mal and thiol-MS couplings on small model molecules, which showed similar reaction conversions in phosphate buffer salt (PBS) at pH 7.4 [N. Toda, S. Asano, CF Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596]. We hypothesized that the hydrolysis of mal groups, which occurs at basic pH, might be the reason for the lower mechanical properties of thiol-mal. To test this hypothesis, the stability of a 4 wt% PEG-mal solution in deuterated PBS at pD 8.0 was investigated by 1H NMR.

[0095] Hydrolysis of Mal groups was detected after 2 hours. Therefore, hydrolysis of Mal groups is not expected to significantly affect the mechanical properties of thiol-Mal within the range of tested conditions. Thiol-VS achieved the highest shear modulus; this is related to a higher conversion rate or the slowest curing, ensuring a network with far fewer defects. Overall, these results show that thiol-MS crosslinking represents an intermediate kinetic between the very fast-curing thiol-Mal and the slow thiol-VS-based materials. The observed crosslinking time, on the order of a few seconds, allows for convenient mixing and pipetting of the components at low shear forces and should be suitable for cell encapsulation. dependence on pH value

[0096] The gelation of the 4-arm PEG-MS and 4-arm PEG-thiol mixture was investigated ( Figur 1 The applied crosslinking conditions were 5 wt% polymer content in 10 mM HEPES buffer, pH 6.6, at 25°C. An MS:thiol ratio of 1:1 was used for the experiments. The bulk gelation time for the thiol-X hydrogels was determined at different pH values. The experiments were performed at 5 wt% polymer solution in 10 mM HEPES buffer, T = 25°C. The gelation time was estimated as the time between mixing the components and the point at which pipetting to the mixture was no longer possible. Studies showed that thiol-MS gels formed a crosslinked gel within 3–4 min (see Table 1). This corresponds to a favorable crosslinking time, allowing for thorough mixing and homogenization of precursor solutions.

[0097] The reaction rate of polar thiol-X coupling is pH-dependent in the pH range between 6 and 9. This is due to the deprotonation of the thiol group (pKa ~ 8) to the thiolate anion, which acts as a nucleophile in these reactions [MH Stenzel, ACS Macro Letters 2013, 2, 14-18]. This feature offers an interesting possibility for pH-controlled curing kinetics under physiologically relevant conditions. Thiol-MS crosslinking was analyzed in the pH range of 8.0-6.6. A decrease in the crosslinking rate was observed with decreasing pH ( Figuren 4a and 4band Table 1). It is noteworthy that changing the pH from 8.0 to 6.6 allowed the gel time to be adjusted from a few seconds to a few minutes (Table 1), providing an ideal experimental time window for 3D cell encapsulation applications. In contrast, the gel time of Thiol-Mal varied only within a few seconds, while Thiol-VS ranged from a few minutes to a few hours. These results highlight the advantages of Thiol-MS gels over Thiol-Mal and Thiol-VS in terms of handling and adaptability to application requirements.

[0098] The shear modulus of thiol-MS crosslinked hydrogels was slightly influenced by pH: at pH 8.0, they showed a lower G', probably due to the occurrence of very rapid crosslinking, which led to some inhomogeneities and defects in the network. This was not the case for thiol-MS gels formed at pH 7.5–6.6, where similar final G's were obtained. Therefore, this appears to be the optimal interval in which the crosslinking rate can be adjusted without compromising the quality and mechanical stability of the gel. In comparison, the thiol-Mal system showed a decrease in mechanical properties at pH ≥ 7.5, but a similar final shear modulus at pH 7.0–6.6, while thiol-VS showed a clear trend toward slower gelation kinetics and a slightly reduced shear modulus with decreasing pH. Measurements were taken in 10 mM HEPES buffer at pH values ​​of 8.0, 7.5, and 6.6. 7.0 and 6.6 at 5 weights.% polymer content and carried out at 25 °C. It should be noted that the fastest curing systems (Mal at pH ≥ 7.0 and MS at pH ≥ 7.5) cure immediately when the rheometer is loaded. Figuren 4a and 4b ) Influence of temperature on gelling time

[0099] Freshly prepared 20 kDa, 4-arm PEG-X polymer solutions were used for these studies. The polymer was dissolved in the appropriate solvent, vortexed, ultrasonic bathed, and centrifuged to remove bubbles. 30 µL of a 5% w / v PEG-X solution was added to a plastic Eppendorf tube, followed by 30 µL of a 5% w / v PEG-thiol solution while continuously pipetting. The bulk gel time was recorded as the time at which the hardness mixture no longer flowed and continuous pipetting was no longer possible. The temperature was controlled using a temperature-controlled water bath.

[0100] Temperature can also be used to adjust the thiol-MS gel properties. Lowering the temperature from 45°C to 25°C allowed for a decrease in the shear modulus ( Figur 5 ) and an extension of the gelling time (see Table 3). Influence of polymer content and HEPES buffer concentration on thiol-MS hydrogels

[0101] Hydrogels with increasing polymer content of 1.3, 2.5, 5.0, 7.5, and 10.0 wt% were prepared at a constant pH of 7.5 and T = 25°C, using either 10 mM or 50 mM HEPES buffer concentrations. The pH of the prepared hydrogels was measured using a pH meter with a flat surface electrode (PH100 ​​Waterproof ExStik®, Extech Instruments, USA).

[0102] Finally, the influence of the polymer content on the thiol-MS hydrogel crosslinking kinetics and the shear modulus was investigated ( Figur 6a, 6b The gelling time was shorter with increasing polymer content (in the range of 18–2 s, see Table 4). Furthermore, G'The homogenization rate increased with a polymer concentration from 1.3 to 7.5 wt% and decreased at polymer concentrations above 10 wt%. This result was surprising, as precursor solutions could already be homogenized correctly at 10 wt%; therefore, poor mixing was not expected to be responsible for this behavior.

[0103] To investigate the reaction mechanism, the pH value of the resulting gels was measured (see Figur 6a, 6b It was found that the pH of freshly prepared thiol-MS gels decreased with increasing polymer concentration. Gels with 1.3–7.5 wt% polymer concentrations showed a pH between 7.5 and 6.5, while gels at 10 wt% had a pH close to 5.1. This can be explained by the release of methanesulfinic acid as a leaving group during thiol-MS coupling. At high polymer concentrations, the leaving group is produced in higher concentrations, leading to a decrease in the pH of the crosslinking medium and thus a decrease in the achieved final shear modulus. This effect can be controlled by increasing the buffering capacity of the crosslinking medium, which is achieved by increasing the HEPES buffer concentration from 10 mM to 50 mM (see [reference]). Figur 6 The latter is known to be cytocompatible. These results show that the polymer content can also be used to control the mechanical properties of the gel. At higher concentrations (10 wt%), the pH should be controlled by increasing the buffering capacity. Swelling measurements on thiol-X hydrogels

[0104] For these studies, 5% w / v precursor solutions were used, prepared in 10 mM HEPES buffer pH 7.0 and previously cooled in an ice bath. 50 µL of a 5% w / v PEG-X solution was transferred to a flexible PDMS cylindrical mold (0.75 cm diameter), rapidly mixed with 50 µL of a 5% w / v PEG-thiol solution, and crosslinked in a humid chamber at 37°C for 4 h. The resulting hydrogels were carefully removed, swollen in Milli-Q water for 24 hours, and the mass of the swollen gel was then determined (M s ).

[0105] The gel was dried in an oven at 37°C for 48 hours and the mass of the dry hydrogel was determined (M d ). The degree of swelling (SR) was calculated using the following formula: SR = M s − M d M d

[0106] The experiments were performed three times. Mean values ​​and standard deviations were reported.

[0107] The degree of swelling (SR) of 5% thiol-MS gels was measured in water at pH 7.0. A swelling of 33.6 mg water / mg polymer was obtained (see Table 5). Thiol-VS gels showed similar SR values, while thiol-Mal increased approximately 1.5-fold. These results suggest a similar degree of crosslinking for thiol-MS and thiol-VS networks and a lower degree of crosslinking for thiol-Mal gels.

[0108] Hydrolytic stability is a relevant material property for hydrogels used in 3D cell culture.

[0109] Therefore, the hydrolytic stability of 5 wt% thiol-MS gels was determined by gravimetric analysis of the swollen gel after incubation in cell culture medium at 37°C for different time points over 4 weeks ( Figur 7a The mass of the swollen thiol-MS gels reached 1.2 times the initial mass within the first two weeks, indicating low gel erosion and high hydrolytic stability. It is worth noting that the long-term stability of the gels is advantageous for long-term cell culture and allows for fine-tuning of the degradation properties through copolymerization with specific degradable sequences. [EA Phelps, NO Enemchukwu, VF Fiore, JC Sy, N. Murthy, TA Sulchek, TH Barker, AJ Garcia, Advanced Materials 2012, 24, 64-70.] The stability of the thiol-MS system was similar to that of thiol-VS, which is typically used for long-term cultures, [MP Lutolf, GP Raeber, AH Zisch, N. Tirelli, JA Hubbell, Advanced Materials 2003, 15, 888-892.] and much higher than that of thiol-Mal gels (1.2-fold in 2 days and hydrogel disintegration on day 18). [N. Boehnke, C. Cam, E. Bat, T. Segura, HD Maynard, Biomacromolecules 2015, 16, 2101-2108.]The hydrolysis of thiol-mal gels is attributed to the low stability of the thioether-succinimide bond, which can undergo retro-Michael and exchange reactions in the presence of other soluble thiols in cell culture media. These results are consistent with studies using model MS compounds, which demonstrate superior stability of thio-heteroaromatic conjugates resulting from thiol-MS coupling compared to thiol-mal compounds under therapeutically relevant conditions. [N. Toda, S. Asano, CF Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596.] Finally, experiments performed with 10 wt% thiol gels showed that thiol-MS gels remained hydrolytically stable for more than 6 weeks (see [reference]). Figur 7b ). Use for cell encapsulation PEG hydrogel preparation for 3D cell culture

[0110] 3D-PEG hydrogels were prepared by adapting the described protocol (Phelps et al., Advanced Materials 2012, 24, 64-70; and Farrukh et al., Adv. Funct. Mater. 2018). The precursor solution of 20 kDa 4-arm PEG Mal / VS / MS (100 mg mL⁻¹, 10% w / v) was prepared by dissolving it in HEPES buffer (10 mM, pH 8.0) under sterile laminar flow. Solutions of Cyclo(RGDfC) (3.45 mg mL⁻¹, 5 mM) and VPM peptide were used. (GCRDVPMSMRGDRCG, 26.6 mg mL⁻¹ (15.68 mM) were also prepared in sterile HEPES buffer (pH 8.0). These concentrations were kept constant throughout all cell experiments.

[0111] A 4-arm PEG Mal / MS / VS stock solution (10% w / v) was mixed with 5 mM cyclo(RGDfC) at a volume ratio of 2:1 and incubated for 30 min at 37°C. The cell suspension (10 x 10⁻⁶ cells / mL) in RPMI medium (2 µL) was added to the above solution, and 8 µL drops of the resulting mixture were dispensed into two Ibidi 15 µL angiogenesis slide plates. Immediately, the VPM peptide solution (2 µL, 15.8 mM) was added to each µL plate, carefully mixed with the pipette tip, and cross-linked. Polymerization of the Mal and MS 3D hydrogels was carried out for 15 min, while VS hydrogels were polymerized for 45 min at 37°C and 5% CO₂. After gelation, the RPMI medium was added and the culture was maintained for 1-3 days. Alternatively, for spheroid culture, the RPMI medium (2 µL) was mixed with the cyclo(RGDfC)-modified PEG precursor solution (6 µL, as described above) and each (8 µL) was added to the µ-titer plate.A fibrin clot was added to each titer plate, followed by the addition of 15.8 mM VPM peptide (2 µL), which gelled at 37°C for 15–45 min. The medium was added to each titer plate and replaced with fresh medium every 24 hours during cell culture.

[0112] In this procedure, the PEG-MS component is first functionalized with the cyclo(RGDfC) peptide, then mixed with L929 fibroblasts, and finally crosslinked with an enzymatically cleavable dithiol peptide (VPM). A composition of 4 wt% PEG-MS, 1 mM RGD peptide, and 3.14 mM VPM was used. [EA Phelps, NO Enemchukwu, VF Fiore, JC Sy, N. Murthy, TA Sulchek, TH Barker, AJ Garcia, Advanced Materials 2012, 24, 64-70. A. Farrukh, JI Paez, A. del Campo, Advanced Functional Materials 2019, 29, 1807734.] After mixing, the solution remained low-viscosity, allowing homogenization of the mixture by pipetting at low shear forces. A stable gel formed within 15 min, visible to the naked eye. The distribution of cells within the hydrogel was analyzed using Z-stacking imaging with a confocal microscope. A uniform distribution of cells across the thickness of the hydrogel was observed ( Figur 8a ). Cell culture conditions

[0113] The Fibroblast L929 cell line (ATCC) was cultured at 37 °C and 5% CO₂ in RPMI 1640 medium (Gibco, 61870-010), supplemented with 10% FBS (Gibco, 10270) and 1% P / S (Invitrogen). For suspended cell cultures, L929 cells (10 × 10⁶ cells < 1 mL⁻¹) were suspended directly in the PEG precursor solution during polymerization.

[0114] For spheroid culture, a fibrin clot of the Fibroblast L929 cell line was prepared using the following literature reports: (JL West, Biomaterials 2008, 29, 2962-2968; CA DeForest, KS Anseth, Nature Chemistry 2011, 3, 925-931).

[0115] In summary, a pellet of 10 × 10⁶ cells (mL⁻¹) was dissociated in fibrinogen (10 mg mL⁻¹ in PBS), and 2 µL drops were applied to a hydrophobic, Sigmacote-coated glass slide. 1 µL of thrombin solution (5 UN mL⁻¹ in PBS) was added to each drop of fibrinogen, and the cells were placed in an incubator for 15 min to obtain a fibrin clot. Fixation and staining

[0116] 3D PEG hydrogel samples were fixed with 4% PFA solution for 2 h at room temperature and washed with PBS. The samples were blocked with 1% BSA solution for 1 h, followed by permeabilization with 0.5% Triton X-100 for 1 h. FITC-phalloidin (1:200 in water, Thermo Fisher Scientific) was used to stain actin fibers and DAPI (1:500 in water, Life Technology) to stain nuclei. The samples were incubated with antibodies for 5 h at room temperature and then washed with PBS. Live-dead assay

[0117] The cell culture medium was removed and the samples were incubated for 5 min with fluorescein diacetate (40 µg mL⁻¹) and propidium iodide (30 µg mL⁻¹) in PBS. The samples were washed twice with PBS and imaged using a Zeiss LSM 880 confocal microscope.

[0118] Live / dead assays on cells encapsulated in thiol-MS gels for 1 day demonstrate the cytocompatibility of the material according to the invention (>90% viability, Figur 8a, 8b , 8c These results suggest that the system's crosslinking kinetics are ideal for obtaining homogeneous constructs under comfortable and cytocompatible experimental conditions.

[0119] Conversely, thiol-mal hydrogels resulted in immediate curing upon mixing of precursor solutions, which made proper homogenization difficult and led to cell agglomeration in the upper part of the gel. On the other hand, the thiol-vs system allowed for good mixing, but the slow gelation kinetics led to cell sedimentation at the bottom of the gel. These results are consistent with previous reports by Peyton et al. on the effect of the crosslinking rate on the distribution of fluorescent beads encapsulated in thiol-mal hydrogels [LE Jansen, LJ Negrón-Piñeiro, S. Galarza, SR Peyton, Acta Biomaterialia 2018, 70, 120-128] and by Shikanov et al., who pointed out the need to invert thiol-vs gels during curing to avoid cell deposition. [J. Kim, YP Kong, SM Niedzielski, RK Singh, AJ Putnam, A Shikanov, Soft Matter 2016, 12, 2076-2085.In this context, thiol-MS hydrogels exhibit more suitable kinetics and overcome these inconveniences. Migration assay

[0120] To demonstrate that cells cultured in thiol-MS hydrogels remain functional, a migration assay was performed. L929 fibroblast spheroids were encapsulated in the degradable thiol-MS hydrogels, [A. Farrukh, JI Paez, A. del Campo, Advanced Functional Materials 2019, 29, 1807734.] cultured for 3 days, fixed, and stained. The cell migration distance from the spheroid was quantified as an indicator of gel degradation and the cell's ability to move within the gel. Figur 9a-c The cells migrated a distance of d ~425 µm. The results were compared with those obtained for thiol-Mal and thiol-VS as materials for 3D cell encapsulation. The migration distance was ~470 µm for Mal and ~360 µm for VS systems ( Figur 9c This result is due to the differences in the degree of cross-linking (G' 37°C = VS > MS > Mal, see Figur 3b )) and in hydrolytic stability (MS = VS >> times, Figur 7 ). Reduced connectivity or faster degradation creates space for the cells, i.e., leads to longer migration routes.

[0121] Furthermore, after 3 days of incubation, the cells cultured in thiol-MS hydrogels were more homogeneously distributed in the gel and showed less clustering than in the other two systems (see Figur 10 ).

[0122] The thiol-MS reaction is suitable for crosslinking hydrogels in cell encapsulation. This reaction achieves kinetics between thiol-mal and thiol-VS systems and attains high conversion. The resulting crosslinked units exhibit good hydrolytic stability and cytocompatibility. Under aqueous mild conditions, the MS-thiol reaction is orthogonal to alcohols, amines, carboxylic acids, and acrylate functional groups [D. Zhang, NO Devarie-Baez, Q. Li, JR Lancaster, M. Xian, Organic Letters 2012, 14, 3396-3399. A. Farrukh, JI Paez, M. Salierno, A. del Campo, Angew. Chem. Int. Ed. 2016, 55, 2092-2096. A. Farrukh, JI Paez, M. Salierno, W. Fan, B. Berninger, A. del Campo, Biomacromolecules 2017, 18, 906-913.] which enables the application of this crosslinking mechanism to almost any natural polymer scaffold of interest in the biomedical field.The reactivity of the thiol-MS pair can be regulated by the pH used and the selection of different MS-aromatic substrates. [N. Toda, S. Asano, CF Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596.] The combination of all these properties makes thiol-MS a superior alternative to other reactive chemicals for 3D cell encapsulation. Table 1: Gelation times of various gels measured in 10mM HEPES buffer, T= 25°C, pH= 8-6.6 Gel pH 8,0 pH 7,5 pH 7,0 pH 6,6 Thiol-Mal < 1 s 1-2 s 2-3 s 5-6 s Thiol-MS 3 s 6 s 12 s 3,5 Min Thiol-VS 8 Min 22 Min 88 Min 190 Min Table 2: Reported second-order reaction rates for selected nucleophilic thiol-X couplings under mild aqueous conditions. X Reaktionsrate k 2 (M -1< S -1< ) Quelle Mal 734, 0 F. Saito, H. Noda, J. W. Bode, ACS Chemical Biology 2015, 10, 1026-1033. MS 0,4-16,0 X. Chen, H. Wu, C.-M. Park, T. H. Poole, G. Keceli, N. O. Devarie-Baez, A. W. Tsang, W. T. Lowther, L. B. Poole, S. B. King, M. Xian, C. M. Furdui, ACS Chemical Biology 2017, 12, 2201-2208. VS 0,08-1,0 H. Wang, F. Cheng, M. Li, W. Peng, J. Qu, Langmuir 2015, 31, 3413-3421. Table 3: Determined bulk gelation time for thiol-MS hydrogels at different temperatures (5 wt.% in 10 mM HEPES buffer). Gel T: 45 °C 25 °C 15 °C 5 °C Thiol-MS (pH 7, 0) 7 s 12 s 22 s 30 s Table 4: Determined bulk gelation time for thiol-MS hydrogels at different polymer contents (10 mM HEPES buffer, pH 7.5, T = 25 °C) Gel 10, 0 Gew.% 7,5 Gew.% 5,0 Gew.% 2,5 Gew.% 1, 3 Gew.% Thiol-MS 2-3 s 4 s 6 s 10 s 18 s Table 5: Swelling ratio in water of thiol-X hydrogels (5 wt.% polymer content; n= 3). Quellverhältnis Gel [mg Wasser / mg Gel] Relativer Wert Thiol-Mal 48,8 ± 4,3 1,45 Thiol-MS 33,6 ± 1,0 1,00 Thiol-VS 35,3 ± 2,8 1,05

Claims

1. A process for preparing a hydrogel, comprising the following steps: a) preparing a composition comprising a1) at least one macromer comprising as functional groups at least two thiol groups, a2) at least one macromer comprising as functional groups at least two aromatic or heteroaromatic groups each substituted by at least one sulfonyl group, where at least one component a1) or a2) contains at least three of the stated functional groups; b) reacting the two macromers via the functional groups to form a hydrogel, wherein reaction conditions are se- lected, under which the thiol groups of the first macromer are able to perform a nucleophilic aromatic substitution on the group Ar, with the group SO2-R1 serving as leaving group, wherein the functional groups of the macromer a2) are func- tional groups of the formula (1):         M-Ar-SO2-R1     (1) where: Ar is an electron-deficient aryl group with 6 to 40 C atoms or electron-deficient heteroaryl group with 1 to 40 C atoms and at least one heteroatom, with the proviso that the sum total of C atoms and heteroatoms makes at least 5, wherein the electron-withdrawing groups and / or hetero atoms are in conjugation with the leaving group -SO2-R1, and wherein Ar is selected from the group encompassing nitrobenzenes, benzaldehydes, benzonitriles, benzoic esters, pyridines, pyrimidines, pyrazines, pyridazines, triazines, tetrazines, oxazoles, isoxazole, thiazoles, isothiazole, oxadiazoles, thiadiazoles, such as 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole or 1,3,4-thiadiazole, imidazole, pyrazole, triazoles, tetrazole, quinolines, isoquinolines, benzimidazole, benzoxazole, benzothiazole, benzopyridazine, benzopyrimidine, quinoxaline, benzotriazole, naphthalimide, purine, pteridine, indolizine and benzothiadiazole, where Ar may be substituted in each case additionally by one or more groups R2, M is a covalent connection to the macromer; R1 is N(R2)2, a straight-chain alkyl group having 1 to 20 C atoms or a branched or cyclic alkyl group having 3 to 20 C atoms, or an alkenyl or alkynyl group having 2 to 20 C atoms, where the alkyl, alkenyl or alkynyl group may be substituted in each case by one or more radicals R2 and where one or more nonadjacent CH2 groups may be replaced by O, NR2, S, R2C=CR2, C≡C, C=O, C(=O)O or C(=O)NR2, or is an aryl group or heteroaryl group which may be substituted in each case by one or more radicals R2. R2, identical or different at each occurrence, is H, D, F, Cl, Br, I, N(R3)2, CN, NO2, OR3, SR3, C(=O)OR3 C(=O)N(R3)2, C(=O)R3, a straight-chain alkyl group having 1 to 20 C atoms or an alkenyl or alkynyl group having 2 to 20 C atoms or a branched or cyclic alkyl group having 3 to 20 C atoms, where the alkyl, alkenyl or alkynyl group may be substituted in each case by one or more radicals R3, where one or more nonadjacent CH2 groups may be replaced by R3C=CR3, C≡C, C=O, NR3, O, S, C(=O)O or C(=O)NR3, or is an aryl group or heteroaryl group which may be substituted in each case by one or more radicals R3. R3, identical or different at each occurrence, is H, D, F, OH, or an aliphatic, aromatic and / or heteroaromatic organic radical, more particularly a straight-chain alkyl group having 1 to 20 C atoms, in which one or more H atoms may also be replaced by F.

2. The process as claimed in claim 1, characterized in that the macromer has an average molar mass of less than 500 kDa, wherein the average molar mass is determined as weight-average molecular weight by gel permeation chromatography (GPC).

3. The process as claimed in either of claims 1 and 2, characterized in that the macromers contain 2, 3, 4, 5, 6, 7, 8, 9 or 10 functional groups.

4. The process as claimed in any of claims 1 to 3, characterized in that the macromers are based on oligomers or polymers, such as poly(meth)acrylates such as poly(meth)acrylamides, poly(meth)acrylic acid, polyHPMA or polyHEMA, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane (PU), poly¬vinylpyrrolidone (PVP), polyamides, poly(amido¬amines) (PAMAM), polyesters, polylactides, poly¬glycolic acid (PGA) or poly(lactide-co-glycolide) (PLGA), polyanhydrides, poly(ortho)esters, poly¬acetals, poloxamers (block copolymers of ethylene oxide (PEG) and propylene oxide (PPG)) such as PEG-Co-PPG-Co-PEG), poly-2 oxazolines, polyphospha-zenes, polyglycerol, polyamines such as polylysine or polyethylenimine (PEI), polycarbonates, poly¬glutamic acid, more particularly poly-gamma-glutamic acid, polyaspartic acid (PASA), polyphos¬phonates, DNA, RNA, gelatin, polyhydroxyalkanoates (PHA), proteins or pep¬tides such as collagens, VPM, albumin or fibrin, polysaccharides such as agarose, chitin, chitosan, chondroitin, mannan, inulin, dextran, cellulose, alginates or hyaluronic acid.

5. The process as claimes in any of the claims 1 to 4, characterized in that Ar is a 1,3,4-oxadiazole group, which is substituted by a phenyl group.

6. The process as claimed in any of claims 1 to 6, characterized in that the macromer content of the composition is 1 to 30 wt%.

7. The process as claimed in any of claims 1 to 7, characterized in that the gelling takes place under physiological conditions.

8. A hydrogel obtained as claimed in any of claims 1 to 7.

9. A composition for preparing a hydrogel, comprising components a1) and a2) as claimed in any of claims 1 to 7.

10. A kit for preparing a hydrogel, comprising compo¬nents a1) and a2) as claimed in any of claims 1 to 6.

11. The use of a hydrogel as claimed in claim 8 for encapsu¬lating cells, for three-dimensional cell cultures, organoids, biomaterials, injectable biomaterials, cell therapies, tissue modification, tissue regeneration, tissue transplantation, regenerative medicine, 3D printing, 3D bioprinting, wound dressings or wound treatment, transport agents for active ingredients, invitro models for studying or testing diagnostic or therapeutic agents, or cell trans¬plantations.

12. A process for modifying gels, comprising the steps of: a) providing a gel thereof, comprising at least two functional groups as per component a1) or at least two functional groups as per component a2); b) adding a composition comprising at least one macromer as claimed in any of claims 1 to 6 as per the respectively other component, where the macromer contains at least two functional groups; c) modifying the gel or the precursor thereof by reacting the functional groups of the macromer with the gel, wherein the reaction conditions are selected according to claim 1.