Novel hydrogels

Abstract

The present invention relates to hydrogels, methods for making them, and their uses. The hydrogels are based on the reaction of thiols with electron-deficient heteroaromatic compounds. This reaction can be carried out under physiological conditions, making them suitable for cell encapsulation.

Description

[Technical Field]

[0001] The present invention relates to hydrogels, methods for making them and their uses. [Background technology]

[0002] Hydrogels are three-dimensional networks of cross-linked hydrophilic polymers containing a high percentage of water. Known uses of such materials include matrix materials for biological applications such as active ingredient delivery, wound care, and tissue engineering, and they can also be used in cell culture. Due to their aqueous and porous structure, hydrogels can effectively transport nutrients to cells.

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

[0004] In particular, controlling the crosslinking reaction is a major challenge. This is especially true when hydrogels are made for the purpose of enveloping cells. If the gel polymerizes too quickly, it often does not crosslink uniformly. If the gel polymerizes too slowly, the components to be encapsulated, such as cells, may settle and are not encapsulated uniformly. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide a method for making a hydrogel that can be used, inter alia, for coating cells. A further object is to provide such a hydrogel and its uses. [Means for solving the problem]

[0006] This object is achieved by the invention having the features of the independent claims. Advantageous developments of the invention are characterized in the dependent claims, the text of all claims of which is hereby incorporated by reference. The invention also covers all reasonable combinations of the independent and / or dependent claims, in particular all specified combinations.

[0007] a) a1) at least one macromer having at least two thiol groups as functional groups; a2) at least one macromer having as functional groups at least two aromatic or heteroaromatic groups, each substituted with at least one sulfonyl group; wherein at least one component a1) or a2) has at least three of the functional groups described; b) reacting the two macromers via functional groups to form a hydrogel; A method of making a hydrogel, comprising:

[0008] The individual method steps are described in more detail below. The steps do not necessarily have to be performed in the order specified, and the methods outlined may include additional steps not described.

[0009] Macromers are understood to be compounds having an average molar mass of less than 500 kDa, preferably less than 100 kDa, in particular less than 50 kDa, the average molar mass being determined as weight-average molecular weight by gel permeation chromatography (GPC).

[0010] Particularly preferred macromers are those having an average molar mass of less than 50 kDa, especially less than 30 kDa.

[0011] In one particular embodiment of the invention, the average molar mass of the macromers is between 100 Da and 500 kDa, preferably between 200 Da and 200 kDa, in particular between 800 Da and 100 kDa.

[0012] Here, it is important that the macromer has the corresponding functional groups and that these groups are available for reaction.

[0013] Preferred macromers in this regard are those having 2, 3, 4, 5, 6, 7, 8, 9 or 10 functional groups, preferably 2, 3, 4, 5, 6, 7 or 8 functional groups, more preferably 2, 3, 4, 5 or 6 functional groups, especially 2, 3 or 4 functional groups.

[0014] "Forming a hydrogel" means that a hydrogel is formed as a result of crosslinking. Thus, the crosslinking reaction is carried out to a sufficient extent, which can be controlled by the nature and amounts of the components used.

[0015] In another preferred embodiment, at least one component a1) or a2) has at least four of the mentioned functional groups.

[0016] In one preferred embodiment, components a1) and a2) both have at least 3, preferably at least 4 of the mentioned functional groups. More preferably, components a1) and a2) both have 3, 4, 5, 6, 7, 8, 9 or 10 functional groups, preferably 3, 4, 5, 6, 7 or 8 functional groups, more preferably 3, 4, 5 or 6 functional groups, especially 3 or 4 functional groups.

[0017] Water-soluble macromers are preferred, meaning that the macromer will be in solution to the extent required under the conditions of the reaction.

[0018] Preferred macromers are those based on oligomers or polymers. These can be natural or synthetic oligomers or polymers. Examples of synthetic oligomers or polymers are poly(meth)acrylates such as poly(meth)acrylamide, poly(meth)acrylic acid, polyHPMA, or polyHEMA, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane (PU), polyvinylpyrrolidone (PVP), polyamides, poly(amidoamine)s (PAMAM), polyesters, polylactides, polyglycolic acid (PGA), or poly(lactide-co-glycolide) (PLGA), polyanhydrides, poly(ortho)esters, polyacetals, poloxamers (polyethylene oxide (PEG) and propylene oxide (PPG) copolymers such as PEG-Co-PPG-Co-PEG), and the like. Examples of suitable oligomers include polyamines such as ethylene glycol copolymers, poly-2-oxazolines, polyphosphazenes, polyglycerols, polylysines, and polyethyleneimines (PEIs), polycarbonates, polyglutamic acids, particularly poly-gamma-glutamic acids, polyaspartic acids (PASAs), and polyphosphonates. Examples of suitable oligomers include proteins or peptides such as DNA, RNA, gelatin, polyhydroxyalkanoates (PHAs), poly-gamma-glutamic acids, collagen, VPM, albumin, and fibrin, and polysaccharides such as agarose, chitin, chitosan, chondroitin, mannan, inulin, dextran, cellulose, alginates, and hyaluronic acid. Preferred oligomers are those based on polyethylene glycol. Oligomers and polymers are functionalized with the appropriate functional groups.

[0019] In the case of peptide-based oligomers, the thiol groups are preferably provided by corresponding amino acids such as cysteine ​​or homocysteine. "Peptide-based" here means that the oligomer in question is composed of natural or unnatural amino acids to the extent of at least 80% of its molecular mass. Such oligomers therefore have at least two thiol groups, in particular at least two cysteines.

[0020] The at least partial use of natural polymers also allows for the introduction of sites into the hydrogel that are specifically cleavable, for example by enzymes.

[0021] It may be necessary for the functional group to be linked to the oligomer or polymer via a short linker, for example one or more ester, ether or amide bonds. Preferred linkers are those having a molar mass of less than 1500 mol, preferably less than 800 mol, in particular less than 500 mol or less than 200 mol.

[0022] The thiol groups are preferably in the form of free thiol groups, although it is also possible to have groups that are removed before the hydrogel is formed.

[0023] Macromer a2) is a macromer having at least two aromatic groups, each substituted with at least one sulfonyl group. Preferred groups are of formula (1): M-Ar-SO2-R 1 (1) where Ar is an electron-deficient aryl or heteroaryl group. As a result, the thiol group of the first macromer can undergo aromatic nucleophilic substitution on the Ar group, forming SO2-R 1 It is possible to choose reaction conditions under which the group acts as a leaving group.

[0024] M is preferably the covalent bond to the macromer and is preferably a single bond, an ether, or a carbonyl group. The carbonyl group may also be part of an ester or amide bond. Thus, as the Ar group, a corresponding ester or amide, such as a correspondingly substituted benzoic acid ester or benzoic acid amide, can be used for coupling to the macromer.

[0025] An aryl group in the sense of the present invention has 6 to 40 C atoms, and a heteroaryl group in the sense of the present invention has 1 to 40 C atoms and at least one heteroatom, provided that the sum of C atoms and heteroatoms is at least 5. The heteroatoms are preferably selected from N, O and / or S. An aryl or heteroaryl group here refers either to a single aromatic ring, i.e. benzene, or to a single heteroaromatic ring, such as pyridine, pyrimidine, thiophene, etc., or to fused aryl or heteroaryl groups, such as naphthalene, naphthalimide, anthracene, quinoline, isoquinoline, etc.

[0026] Electron-deficient aryl or heteroaryl groups refer to aryl or heteroaryl groups that have reduced π-electron density as a result of a negative inductive or mesomeric effect (-I or -M effect). Lists of substituents or groups that produce these effects can be found in any standard textbook on organic chemistry. Non-limiting examples that may be mentioned are OH, halogens, especially fluorine and chlorine, NO, unsaturated groups for the -I substituent, and NO, CN, aryl or heteroaryl groups for the -M substituent. These electron-withdrawing groups (EWGs) must, of course, be joined to a leaving group -SO-R to exert the desired effect. 1 In the case of carbocyclic ring systems, conjugation must occur in the ortho or para positions. In the case of heteroaryl groups, the heteroatoms contribute correspondingly to the electron density reduction depending on their position. Two or more different groups may be present.

[0027] Examples of electron-deficient aryl groups are nitrobenzene, benzaldehyde, benzonitrile, benzoesters, which may additionally have one or more R 2 Examples of such aryl groups are nitrobenzoic acid-based compounds having one or two nitro groups, e.g., -SO2-R 1The nitrobenzoic acid ester or nitrobenzoic acid amide has a group. This group is preferably located in the meta position relative to the nitro group. The nitro group at the 3-position and the —SO—R group at the 4-position are preferably present. 1 The group is particularly preferred. An example of such a compound is 3-nitro-4-sulfomethylbenzoic acid.

[0028] Examples of electron-deficient heteroaryl groups are, for example, pyridine, pyrimidine, pyrazine, pyridazine, 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, oxazole, isoxazole, thiazoles such as 1,2-thiazole or 1,3-thiazole, isothiazole, 1,2,3-oxadiazole, 1,2,4, monocyclic heteroaromatic compounds such as oxadiazoles, such as 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazoles, such as 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole or 1,3,4-thiadiazole, imidazoles, pyrazoles, in particular triazoles, such as 1,2,4-triazole or 1,2,3-triazole, tetrazoles, and additionally one or more R as defined below; 2 and polycyclic heteroaromatic compounds such as quinoline, isoquinoline, naphthalimide, benzimidazole, benzoxazole, benzothiazole, benzopyridazine, benzopyrimidine, quinoxaline, benzotriazole, purine, pteridine, indolizine, and benzothiadiazole, optionally substituted by groups.

[0029] Preferred heteroaryl groups are oxadiazole and benzothiazole.

[0030] In one preferred embodiment of the invention, Ar is a polycyclic or monocyclic heteroaryl group substituted by at least one further aryl or heteroaryl group, preferably phenyl.

[0031] In another particularly preferred embodiment, Ar is an oxadiazole group, especially a 1,3,4-oxadiazole group, preferably substituted by at least one phenyl group, especially one phenyl group.

[0032] In another embodiment, Ar is an aryl group having at least one, preferably one or two, -I or -M substituents, preferably F or NO2, more preferably NO2.

[0033] R 1 is N(R 2 )2, a linear 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, or a branched or cyclic alkyl group having 3 to 20 C atoms, wherein the alkyl, alkenyl or alkynyl group each comprises one or more radicals R 2 and one or more non-adjacent CH groups are optionally substituted by O, NR 2 , S, R 2 C=CR 2 , C≡C, C═O, C(═O)O or C(═O)NR 2 or each may be replaced by one or more radicals R 2 is an aryl or heteroaryl group optionally substituted by

[0034] R 2 are the same or different, and are H, D, F, Cl, Br, I, N(R 3 )2, CN, NO2, OR 3 , S.R. 3 , C(=O)OR 3 , C(=O)N(R 3 )2, C(=O)R 3 , a linear 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, wherein the alkyl, alkenyl or alkynyl group each comprises one or more radicals R 3and one or more non-adjacent CH groups are optionally substituted by R 3 C=CR 3 , C≡C, C=O, NR 3 , O, S, C(═O)O or C(═O)NR 3 or each may be replaced by one or more radicals R 3 is an aryl or heteroaryl group optionally substituted by

[0035] R 3 are each identical or different and are H, D, F, OH, or an aliphatic, aromatic and / or heteroaromatic organic radical, in particular a linear alkyl group having 1 to 20 C atoms, in which one or more H atoms may be replaced by F.

[0036] In a preferred embodiment, R 1 is N(R 2 ) 2, a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms, where the alkyl group is each one or more radicals R 2 and one or more non-adjacent CH groups are optionally substituted by O, NR 2 , S, C=O, C(=O)O or C(=O)NR 2 or each may be replaced by one or more radicals R 2 is an aryl or heteroaryl group optionally substituted by

[0037] R 2 are the same or different, and are H, D, F, Cl, Br, I, N(R 3 )2, CN, NO2, OR 3 , S.R. 3 , C(=O)OR 3 , C(=O)N(R 3 )2, C(=O)R 3, a linear alkyl group having 1 to 10 C atoms, or an alkenyl or alkynyl group having 2 to 10 C atoms, or a branched or cyclic alkyl group having 3 to 10 C atoms, wherein the alkyl, alkenyl or alkynyl group each contains one or more radicals R 3 and one or more non-adjacent CH groups are optionally substituted by R 3 C=CR 3 , C≡C, C=O, NR 3 , O, S, C(═O)O or C(═O)NR 3 or each may be replaced by one or more radicals R 3 is an aryl or heteroaryl group optionally substituted by

[0038] R 3 are each the same or different and are H, D, F, OH, or a straight-chain alkyl group having 1 to 5 C atoms, in which one or more H atoms may be replaced by F or OH.

[0039] In one particularly preferred embodiment, R 1 is N(R 2 ) 2, a linear alkyl group having 1 to 6 C atoms or a branched or cyclic alkyl group having 3 to 6 C atoms, where the alkyl group is each one or more radicals R 2 and one or more non-adjacent CH groups are optionally substituted by O, NR 2 , S, C=O, C(=O)O or C(=O)NR 2 or each may be replaced by one or more radicals R 2 and n is an integer of 1 to 10. The aryl group or heteroaryl group has 5 to 10 aromatic ring atoms and may be substituted by:

[0040] R 2are the same or different and are H, D, F, OH, C(=O)OH, a linear alkyl group having 1 to 5 C atoms, or an aryl or heteroaryl group having 5 to 10 aromatic ring atoms, and one or more H atoms bonded to carbon may be replaced by F or NO2.

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

[0042] In a preferred embodiment of the present invention, the at least one macromer is selected from the group consisting of poly(meth)acrylates such as poly(meth)acrylamides, poly(meth)acrylic acids, poly(HPMA), or poly(HEMA); polyesters such as polyethylene glycols (PEG), polyvinyl alcohols (PVA), polyurethanes (PU), polyvinylpyrrolidone (PVP), polyamides, poly(amidoamines) (PAMAM), polylactides, polyglycolic acids (PGA), or poly(lactide-co-glycolide) (PLGA); polyanhydrides, poly(ortho)esters, polyacetals, polyolefins containing ethylene oxide (PEG) and propylene oxide (PG) such as poloxamers (PEG-Co-PPG-Co-PEG). Some macromers are based on polymers such as block copolymers with PEG (PPG), poly-2-oxazolines, polyphosphazenes, polyglycerols, polyamines such as polylysine or polyethyleneimine (PEI), polycarbonates, polyglutamic acid, particularly poly-gamma-glutamic acid, polyaspartic acid (PASA), and polyphosphonates. Other macromers are based on DNA, RNA, peptides such as gelatin, polyhydroxyalkanoates (PHAs), poly-gamma-glutamic acid, collagen, VPM, albumin, or fibrin, and polysaccharides such as agarose, chitin, chitosan, chondroitin, mannan, inulin, dextran, cellulose, alginate, or hyaluronic acid. As a result, biochemical reactivity, e.g., cleavage or decomposition by ester or carbonate groups in the macromers, or by enzymatic reactions, can be incorporated into the hydrogel. An example of a suitable peptide is an enzymatically cleavable dithiol peptide such as VPM (sequence: GCRDVPMSMRGGDRCG).

[0043] Preferably, the functional groups SH in the two macromers that contribute to crosslinking are: Ar—SO—R 1Both macromers are used so that the ratio of the functional groups is 2:1 to 1:2, preferably 1.5:1 to 1:1.5, more preferably 1.2:1 to 1:1.2, and particularly 1:1. When two or more different compounds having different functional groups are used, for example, when different compounds having thiol groups are used, the numerical values ​​are based on the total number of these groups. For example, one thiol compound can be used for modification and another compound can be used for crosslinking.

[0044] Both macromers are preferably in solution, preferably aqueous. The pH may need to be adjusted, preferably with a buffer.

[0045] In one preferred embodiment, a first solution containing a first macromer having a thiol group and a second solution containing a second macromer having an aromatic sulfonyl group are prepared, and the two solutions are then combined.

[0046] In a preferred embodiment, the pH of the macromer solution, particularly the composition, used is between 6 and 9 (at 25°C). The pH is preferably adjusted with a buffer, preferably at a buffer concentration of 5 mM to 100 mM. Examples of buffers are PBS or HEPES. Using a relatively high buffer concentration allows the pH of the gel to be stabilized when a high macromer concentration is used, since the leaving group can act as an acid. A pH of 6 to 9, preferably 6.5 to 8, more preferably 6.6 to 7.5, is preferred. As a result, it is possible to establish gelation times (measured at 25°C with a constant macromer concentration) of, for example, 3 seconds (pH 8) to 3.5 minutes (pH 6.6).

[0047] The crosslinking reaction can also be initiated by forming the composition at a first pH and then changing the pH to a second pH. The crosslinking reaction is preferably at least significantly slowed at the first pH, so the first pH is preferably outside the ranges described above. The second pH is preferably within one of the ranges described above. The pH change can also be achieved by placing the composition in a medium of the corresponding pH. Preferably, the second pH is between 6 and 9, preferably between 6.5 and 8, more preferably between 6.6 and 7.5.

[0048] The reaction can also be initiated or accelerated by changing the pH.

[0049] In another preferred embodiment, the macromer content of the composition is from 1 wt% to 30 wt%, preferably from 3 wt% to 15 wt%, more preferably from 3 wt% to 10 wt%, based on all macromers used.

[0050] The temperature at which the hydrogel is formed is preferably 20°C to 45°C, more preferably 20°C to 40°C.

[0051] The reaction for forming hydrogels described herein features numerous advantages. In contrast to known crosslinking reactions, this reaction is neither particularly fast nor particularly slow under physiological conditions, but rather can be controlled by factors including pH. This allows for the encapsulation of cells or other substances, such as peptides, enzymes, and chemical compounds, during gel formation. During gel formation, the composition remains viscous for a longer period, allowing for longer mixing times with lower shear forces. This allows for uniform distribution of cells throughout the hydrogel without the need for additional steps, such as rotating the gel during hardening.

[0052] Furthermore, the proposed reaction is sufficiently fast under physiological conditions, allowing its use in cell culture, preferably in three-dimensional cell culture, or even in situ. The gelation can also be controlled by pH, allowing for its use in situ injection of the corresponding composition, 3D printing, or the construction of gels in living organisms.

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

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

[0055] In a preferred embodiment of the present invention, the reaction of the two macromers contributes only to the formation of a hydrogel; no other cross-linking reactions occur.

[0056] The reaction rate can be controlled by selecting the aromatic or heteroaromatic group bearing the sulfonyl group and by selecting the pH. The gelation rate can be adapted to the specific application. In contrast to other reactions, no initiators or accelerators need to be added.

[0057] The ratio of the two macromers is preferably selected so that after the reaction all functional groups are reacted, although the ratio may vary depending on whether further functionalization is carried out.

[0058] Thus, for example, it is possible to pre-modify the second macromer by adding a thiol-containing compound before the addition of the first macromer initiates crosslinking and formation of the hydrogel. In this way, the hydrogel can be modified with additional functional groups, such as fluorophores or bioactive reagents.

[0059] Examples of bioactive agents are tissue growth promoters, chemotherapeutic agents, proteins (glycoproteins, collagen, lipoproteins), cell binding mediators such as fibronectin, laminin, collagen, fibrin, or integrin-binding sequences (e.g., cyclo(RGDfC)) or cadherin-binding sequences, growth factors, differentiation factors, or fragments of the above-mentioned agents. Examples are 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, with cell binding mediators such as VEGF being preferred.

[0060] The modifications can be used, for example, to create an appropriate environment within the hydrogel depending on the cells to be cultured.

[0061] The reagent is preferably used at an effective concentration, which may be, for example, in the range of 0.01 mM to 100 mM, preferably 0.1 mM to 50 mM, particularly 0.2 mM to 10 mM, and especially 0.5 mM to 5 mM, based on the swollen gel.

[0062] The present invention also relates to a composition for preparing a hybrid gel comprising at least two macromers a1) and a2) as described in the method.

[0063] The present invention also relates to a hydrogel obtainable by the method of the present invention.

[0064] The present invention also relates to a hydrogel comprising a first plurality of macromers crosslinked with a second plurality of macromers, wherein the crosslinking is via a plurality of Ar-S bonds, where Ar is an aromatic or heteroaromatic group.

[0065] This type of bond can be obtained from nucleophilic substitution of electron-deficient aromatic compounds with thiols as described above. Advantageous embodiments describe methods.

[0066] The hydrogels of the present invention have a long-term stability, preferably up to 6 weeks. They can be obtained and modified under physiological conditions in a simple manner.

[0067] The hydrogels of the present invention are particularly suitable for cell encapsulation, three-dimensional cell culture, organoids, biomaterials, injectable biomaterials, cell therapy, tissue engineering, tissue regeneration, tissue transplantation, regenerative medicine, 3D printing, 3D bioprinting, wound dressings or wound treatment, delivery agents for active ingredients, in vitro models for researching or testing diagnostic or therapeutic agents, or cell transplantation.

[0068] The reactions described can be used in particular in the field of biology, since they occur under physiological conditions. For example, it is conceivable to mix or combine two macromers that react with each other only in situ. This can be achieved, for example, using a multi-component syringe.

[0069] The present invention relates to a method for coating cells, in which a hydrogel is formed in the presence of cells to coat the cells, which can be used, for example, for cell culture, particularly three-dimensional cell culture.

[0070] The present invention also relates to a kit for preparing a hydrogel comprising macromers a1) and a2) as described in the method.

[0071] The described reaction is also suitable for achieving additional crosslinking of existing gels. In this type of process, as known, for example, from A. Farrukh, J.I. Paez, M. Salierno, A. del Campo, Angew. Chem. Int. Ed. 2016, 55, 2092-2096, a gel containing at least two functional groups of component a1) or a2) is prepared by copolymerization of such a monomer into a polyacrylamide gel and reacted with a macromer having a corresponding functional group according to macromer a1) or a2), where the macromer a1) or a2) has at least two functional groups, and as a result of this reaction the gel is crosslinked.

[0072] Therefore, the present invention also provides a method for producing a pharmaceutical composition comprising: a) providing a gel or a precursor thereof having 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 having at least two functional groups, depending on each of the other ingredients; c) modifying the gel or its precursor by reacting it with a functional group; The present invention relates to a method for modifying a gel, comprising:

[0073] This method is preferably used for post-modification of the gel after its preparation, as a result of which it is possible to modify the gel under physiological conditions, for example to adapt its mechanical parameters.

[0074] Due to the pH dependence and / or temperature of the reaction, for example, this modification can only occur if a defined change in conditions occurs.

[0075] Further details and features are evident from the following description of preferred embodiments in conjunction with the dependent claims. In this connection, each feature can be realized alone or in combination with one another in any combination. The possibilities for achieving the objective are not limited to the examples. For example, the specification of ranges always includes all unmentioned intermediate values ​​and all possible subintervals. [Brief explanation of the drawings]

[0076] [Figure 1] FIG. 1 is a schematic diagram of the preparation of a hydrogel according to the present invention. [Figure 2] a) Photograph of PEG-thiol-MS hydrogel (polymer concentration 5 wt% in 10 mM HEPES buffer); b) Shear modulus during gelation (5 wt% polymer, 10 mM HEPES buffer (pH 6.6), T=25°C). [Figure 3a]FIG. 1 shows the shear modulus during gelation of various hydrogels at 25° C. (5 wt % each; 10 mM HEPES buffer; pH 8). [Figure 3b] FIG. 1 shows the shear modulus during gelation of various hydrogels at 37° C. (5 wt % each; 10 mM HEPES buffer; pH 8). [Figure 4] Figure 1 shows the effect of pH (at 5 wt% polymer content and 25°C) on crosslinking kinetics and shear modulus (a) Thiol-Mal, Thiol-MS, b) Thiol-VS). [Figure 5] FIG. 1 shows shear modulus as a function of temperature (conditions: 30 min, 5 wt %, pH 7.0). [Figure 6] Effect of polymer content and HEPES buffer concentration on the mechanical properties (bars, left scale) and pH (squares, right scale) of prepared thiol-MS gels. Conditions: pH=7.5, T=25°C, 60 min. [Figure 7] Figure 1 shows a comparison of the normalized mass of swollen Thiol-X gels. The gels were incubated in cell culture medium at 37 °C for 6 or 4 weeks (a) 10 wt% polymer fraction, pH 8.0; b) 5 wt% polymer content at pH 7.0. Thiol-MS gels prepared under these conditions are stable to hydrolysis, even after 6 weeks of incubation in cell culture medium. [Figure 8] Figure 1 shows fibroblast L929 cells encapsulated in various enzymatically cleavable Thiol-X hydrogels. Single-cell live / dead assays of L929 fibroblasts encapsulated in the materials for 1 day (a-c): Compared to the other systems, cells cultured in Thiol-MS hydrogels showed a more uniform distribution throughout the material (a, Z-stack) and similar viability (c). [Figure 9](a) Scheme of the enzymatically cleavable gel used to encapsulate cell spheroids; (b and c) migration behavior of cells from encapsulated spheroids. Results of migration studies after 3 days of culture showed intermediate migration distances in the thiol-MS gel. Staining: FITC-phalloidin (actin filaments), DAPI (nuclei). [Figure 10] Morphology of individual cells (mouse fibroblast L929) encapsulated in various enzymatically cleavable Thiol-X hydrogels after 3 days of cell culture. Compared to other systems, cells cultured in Thiol-MS hydrogels showed a more uniform distribution and less clustering or aggregation. Staining: FITC-phalloidin (actin filaments), DAPI (nuclei); scale: 50 μm. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0077] Examples are shown in the drawings, in which the macromers are referred to as polymers.

[0078] chemical synthesis Chemicals and solvents were obtained at purity and used directly unless otherwise noted. 4-(5-Methyl-sulfonyl)-1,3,4-oxadiazol-2-yl)aniline was obtained from Ark Pharm (USA). Four-arm polyethylene glycol polymer (PEG, pentaerythritol-based, 20 kDa) functionalized with maleimide (PEG-Mal), vinyl sulfone (PEG-VS), thiol (PEG-SH), and N-succinimidyl carboxymethyl ester (NHS-PEG), as well as linear methoxylated PEG polymers (5 kDa) similarly functionalized with NHS, SH, Mal, and VS, respectively, were obtained from Jenkem (USA). Buffer solutions were freshly prepared. 10 mM HEPES (pH 8.0, pH 7.0, and pH 6.7) and phosphate-buffered saline (PBS, pH 7.4 and pH 7.0) were used.

[0079] Deuterated solvents were obtained from Deutero GmbH, Germany (D-56288 Kastellaun). Deuterated phosphate buffered saline solution (PBS) was prepared by dissolving the correct amounts of disodium phosphate, monosodium phosphate, sodium chloride, and potassium chloride in DO, followed by pD adjustment with 20% DCl solution (Merck) to reach pD values ​​of 8.0, 7.4, 7.0, 7.0, and 6.0. 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).

[0080] Thin-layer chromatography (TLC) plates (ALUGRAM™ SIL G / UV254) and silica gel for column chromatography (60 Å pore size, 63 μm–200 μm particle size) were obtained from Macherey-Nagel (52355 Düren, Germany). TLC plates were observed under 254 nm or 365 nm light. HPLC analysis and purification of compounds were performed using a JASCO 4000 HPLC (Japan) equipped with a diode array, UV-Vis detector, and fraction collector. A Reprosil C18 column was used for semi-preparative (250 mm × 25 mm) and analytical (250 mm × 5 mm) runs. A solvent gradient was typically used over 40 min with the following eluent combination: solvent A (MilliQ water + 0.1% TFA) and solvent B (95% ACN / 5% MilliQ water + 0.1% TFA). The modified polymers were typically purified by dialysis against acetone and water. Spectra / Por 3 dialysis tubing (molecular weight cut-off limit MWCO=3.5 kDa) from Spectrum Inc. was used.

[0081] 1 H-NMR and 13C-NMR solution spectra 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 He-cooled 5 mm TCI CryoProbe (CP TCI 500S2, HC / ND-05 Z), a proton-optimized triple-resonance NMR inverse probe with external water cooling. All measurements were performed at 298 K unless otherwise noted. Tetramethylsilane (TMS) (δ = 0 ppm) was used as the internal reference. Chemical shifts are reported in ppm, and coupling constants are reported in Hertz. The following abbreviations are used: s-singlet, d-doublet, t-triplet, q-quartet, and m-multiplet. The degree of substitution of the PEG polymer was calculated by end-group quantification. The integrals of the signals corresponding to the PEG backbone (3.70 ppm to 3.40 ppm) were set to 440 H and compared with the integrals of the protons corresponding to the incorporated molecule 2 (aromatic -CH from 8.10 ppm to 7.70 ppm and methylene at 4.20 ppm). In all cases, degrees of functionalization greater than 91% and yields greater than 91% were achieved. Data were analyzed with MestReNova.

[0082] Mass spectra were recorded using an Agilent Technologies 1260 Infinity liquid chromatography / mass selective detector (LC / MSD) and a 6545 accurate-mass quadrupole time of flight (LC / Q-TOF-MS) with electrospray chemical ionization. UV / VIS spectra were recorded using a Varian Cary 4000 UV / VIS spectrometer (Varian Inc., Palo Alto, USA).

[0083] The rheological properties of the hydrogels were determined at 25°C and 37°C on a discovery HR-3 rheometer (TA Instruments, USA) equipped with 12 mm parallel plates and a Peltier platform. The software was Trios v4. Data were recorded and analyzed in Origin 9.1. [ka]

[0084] 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.

[0085] Synthesis of tert-butyl (2-((4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)amino)-2-oxoethyl)carbamate (1): Boc-Gly-OH (1 equiv., 2.28 mmol, 0.394 g) was dissolved in anhydrous THF (3 ml) at 0° C. Isobutyl chloroformate (1.2 equiv., 2.85 mmol, 0.314 ml) and N-methylmorpholine (2.6 equiv., 5.7 mmol, 0.627 ml) were carefully added to the solution using a 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 equiv., 0.57 mmol, 0.136 g) in THF (3 ml) was added dropwise to the mixture, which was then stirred at 0° C. for an additional 2 hours and then at room temperature overnight. Saturated NaHCO was added, and the reaction mixture was extracted with ethyl acetate (2 × 30 ml). The combined organic phases were dried over sodium sulfate, filtered, evaporated and purified by preparative HPLC (5B→95B 280 nm, reaction time=28 min) to give 165 mg of a white solid after lyophilization (yield=73%).

[0086] Synthesis of 2-amino-N-(4-(5-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acetamide (2): Compound 1 (45 mg) was dissolved in 1:1 TFA / DCM (2 ml), the solution was stirred at room temperature for 2 hours, and evaporated under a stream of nitrogen. After HPLC purification (5B → 95B 280 nm, reaction time = 18 min), the final product was obtained (Y = > 99%). The pure compound was readily coupled to the PEG polymer, otherwise decomposition was observed within 1 week after storage at -20 °C.

[0087] Synthesis of PEG-MS: [ka] Freshly prepared compound 2 (50 μmol, 15 mg) and N-methylmorpholine (18 μmol, 20 μL) were dissolved in dry DMF (2 ml), flushed 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 stream of nitrogen. The mixture was stirred overnight at room temperature under an inert atmosphere, then dialyzed in acetone and water and lyophilized. A white solid polymer was obtained, which was purified by filtration in DCM d2. 1 Characterization was performed by H-NMR. A degree of functionalization of over 91% and a yield of over 90% were calculated.

[0088] The 2-(methylsulfonyl)-5-phenyl-1,3,4-oxadiazole group was selected as the MS substrate for thiol coupling. Among the heteroaromatic MS rings described, 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, CM 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). A four-arm PEG-MS macromer (20 kDa) was synthesized in good yield (>91% substitution) on a 500 mg scale over three synthetic steps.

[0089] Rheological measurements on hydrogels The gelation of 4-arm PEG-MS and 4-arm PEG-thiol mixtures was studied. The crosslinking conditions used were 5 wt% polymer content in 10 mM HEPES buffer (pH 6.6) at 25 °C. A 1:1 MS:thiol ratio was used in the experiments. Studies showed that the thiol-MS gel formed a crosslinked gel within 3 to 4 min (see Table 1). This corresponds to a convenient crosslinking time, allowing thorough mixing and homogenization of the precursor solutions. Rheological studies revealed that the crosslinked gel reached a shear storage modulus G' of approximately 1 kPa (Figure 2b). The swollen PEG-thiol-MS hydrogel (5 wt% polymer concentration in 10 mM HEPES buffer) is shown in Figure 2a.

[0090] A freshly prepared solution of 20 kDa 4-arm PEG-X polymer was used in these studies. The polymer was dissolved in the corresponding solvent, mixed using a vortexer, placed in an ultrasonic bath, and centrifuged to remove bubbles. 21 μL of 5% (w / v) PEG-X solution, followed by 21 μL of 5% (w / v) PEG-thiol solution, was applied to the lower Peltier plate of the rheometer, and mixing was performed directly on the plate using a pipette tip. The upper plate was brought close together to sandwich the sample between the two plates, and the sample was subsequently sealed with liquid paraffin to prevent evaporation during the measurement. The total sample loading time, including the start of the measurement, was approximately 2–3 min.

[0091] The gelation time and final shear modulus of the hydrogels were determined using a rheometer. Strain runs (0.1% to 1000% strain at 1 Hz) and frequency runs (0.01 Hz to 100 Hz at 1% strain) were performed to determine the linear viscoelastic region. Time runs were performed within the linear viscoelastic region using the following parameters: 300 μm slot, 0.0 ± 0.1 N axial force, 1 Hz frequency, 1% strain, and 25°C or 37°C temperature.

[0092] Hydrogels were prepared with a polymer content of 5 wt %, HEPES buffer (pH 8.0), and T = 25 °C (Figure 3a) and T = 37 °C (Figure 3b).

[0093] Figure 3a compares the crosslinking kinetics of thiol-MS with those of thiol-Mal and thiol-VS systems. Experiments were performed under conditions typical of cell culture (5 wt% polymer in 10 mM HEPES buffer (pH 8.0) at 25°C) (EA Phelps, NO Enemchuwu, 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, thiol-MS gels formed in 3–4 s (Table 1). This corresponds to a short crosslinking time, but is acceptable for mixing and homogenization of the gel precursors. In comparison, the thiol-Mal gel took 1 second to crosslink and the resulting gel was heterogeneous, while the thiol-VS system produced a gelation time of approximately 10 minutes and required approximately 2 hours for complete crosslinking. These results, as shown in Table 2, show the following trend in gelation rate: thiol-Mal > thiol-MS > thiol-VS, consistent with the reaction rates identified for model compounds (X. Chen, H. Wu., C. M. Park, T. H. Poole, G. Keceli, N. O. Devarie-Baez, A. W. Tsang, W. W. Lowther, L. B. Poole, S. B. King, M. Xian, C. M. Furdui, ACS Chemical Biology 2017, 12, 2201-2208; F. Saito, H. Noda, J. W. Bode, ACS Chemical Biology 2015, 10, 1026-1033; H. Wang, F. Cheng, M. Li, W. Peng, J. Qu, Langmuir 2015, 31, 3413-3421).

[0094] The shear modulus values ​​of the crosslinked gel after 1 hour were G' 25℃was 2000 Pa for thiol-VS, 1000 Pa for thiol-MS, and 470 Pa for thiol-Mal. The higher stiffness of the thiol-MS gel is likely due to slower gelation kinetics resulting in a more homogeneous system, resulting in fewer defects in the network and a higher degree of cross-linking.

[0095] This result contradicts previous reactivity studies of thiol-Mal and thiol-MS couplings on small model molecules, which showed similar conversion rates in phosphate-buffered saline (PBS) (pH 7.4) (N. Toda, S. Asano, C.F. Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596). We hypothesized that hydrolysis of the Mal group, which occurs at basic pH, may be the reason for the lower mechanical properties of thiol-Mal. To test this hypothesis, we investigated the stability of a 4 wt% PEG-Mal solution in deuterated PBS at pD 8.0. 1 It was studied by H-NMR.

[0096] Hydrolysis of Mal groups was detected after 2 h. Therefore, hydrolysis of Mal groups is not expected to significantly affect the mechanical properties of thiol-Mal within the range of conditions tested. Thiol-VS achieved the highest shear modulus, which is consistent with a higher conversion rate or the slowest cure, ensuring a much less defect-prone network. Overall, these results indicate that thiol-MS crosslinking exhibits intermediate kinetics between the very rapidly crosslinking thiol-Mal and the slower thiol-VS-based materials. The observed crosslinking times in the range of a few seconds allow for convenient mixing and pipetting of the components at low shear forces, making it suitable for cell encapsulation.

[0097] pH dependence The gelation of 4-arm PEG-MS and 4-arm PEG-thiol mixtures was studied (Figure 1). The crosslinking conditions used were 5 wt% polymer content in 10 mM HEPES buffer (pH 6.6) at 25 °C. A 1:1 MS:thiol ratio was used in the experiments. The gelation time of the bulk thiol-X hydrogel was determined at different pH values. Experiments were performed with a 5 wt% polymer solution in 10 mM HEPES buffer at T = 25 °C. The gelation time was estimated as the interval between mixing of the components and the point at which the mixture could no longer be pipetted. Studies showed that the thiol-MS gel formed a crosslinked gel within 3 to 4 minutes (see Table 1). This corresponds to a convenient crosslinking time that allows thorough mixing and homogenization of the precursor solutions.

[0098] The reaction rate of polar thiol-X coupling is pH-dependent in the pH range 6-9. This is due to the deprotonation of the thiol group (pKa approximately 8) to a thiolate anion, which acts as a nucleophile in these reactions (M.H. Stenzel, ACS Macro Letters 2013, 2, 14-18). This feature provides an intriguing opportunity for pH-controlled curing kinetics under physiologically relevant conditions. Thiol-MS crosslinking was analyzed over a pH range of 8.0-6.6. A decrease in crosslinking rate was observed with decreasing pH (Figures 4a and 4b and Table 1). It is noteworthy that by varying the pH from 8.0 to 6.6, the gelation time could be adjusted from a few seconds to several minutes (Table 1), providing an ideal experimental timeframe for 3D cell encapsulation applications. In contrast, the gelation time of thiol-Mal only varied within a few seconds, while that of thiol-VS ranged from a few minutes to several hours. These results demonstrate the advantages of the thiol-MS gel compared to thiol-Mal and thiol-VS in terms of handling and suitability for application requirements.

[0099] The shear modulus of thiol-MS crosslinked hydrogels was slightly affected by pH. At pH 8.0, they exhibited a relatively low G', likely due to very rapid crosslinking, resulting in numerous inhomogeneities and defects within the network. This was not the case for thiol-MS gels formed at pH 7.5–6.6, which yielded similar final G' values. This therefore appears to be the optimal interval in which the crosslinking rate can be adjusted without compromising gel quality and mechanical stability. In comparison, the thiol-Mal system exhibited a decrease in mechanical properties at pH ≥ 7.5 but similar final shear moduli at pH 7.0–6.6, whereas thiol-VS showed a clear trend toward slower gelation kinetics and a slightly decreased shear modulus with decreasing pH. To this end, measurements were performed at pH values ​​of 8.0, 7.5, 7.0, and 6.6, with a polymer content of 5 wt%, and in 10 mM HEPES buffer at 25°C. It is noteworthy here that the fastest curing systems (Mal at pH ≥ 7.0 and MS at pH ≥ 7.5) cure instantly with rheometer loading (Figures 4a and 4b).

[0100] Effect of temperature on gelation time Freshly prepared solutions of 20 kDa 4-arm PEG-X polymer were used in these studies. The polymer was dissolved in the corresponding solvent, mixed using a vortexer, placed in an ultrasonic bath, and centrifuged to remove bubbles. 30 μL of 5% (w / v) PEG-X solution, followed by 30 μL of 5% (w / v) PEG-thiol solution, were added to a plastic Eppendorf tube with continuous mixing using a pipette. The "bulk" gelation time was recorded as the time at which the hardened mixture became non-flowable and continuous pipetting was no longer possible. Temperature was controlled using a thermoregulated water bath.

[0101] Temperature can also be used to tune the properties of thiol-MS gels. Decreasing the temperature between 45°C and 25°C decreased the shear modulus (Figure 5) and extended the gelation time (see Table 3).

[0102] Effect of polymer content and HEPES buffer concentration on thiol-MS hydrogels Hydrogels were prepared using either 10 mM or 50 mM HEPES buffer with increasing polymer content values ​​of 1.3 wt%, 2.5 wt%, 5.0 wt%, 7.5 wt%, and 10.0 wt% at a constant pH of 7.5 and T of 25° C. The pH of the prepared hydrogels was measured using a pH meter with a flat electrode (PH100 ​​Waterproof ExStik™, Extech Instruments, USA).

[0103] Finally, we investigated the effect of polymer content on the crosslinking kinetics and shear modulus of thiol-MS hydrogels (Figures 6a and 6b). The gelation time decreased with increasing polymer content (ranging from 18 s to 2 s; see Table 4). Furthermore, G' increased with increasing polymer concentration from 1.3 wt% to 7.5 wt% and decreased at polymer concentrations above 10 wt%. This result was surprising, since the precursor solution could be precisely homogenized even at 10 wt%. Therefore, insufficient mixing effects were not expected to be responsible for this behavior.

[0104] To study the reaction mechanism, we measured the pH of the resulting gels (see Figures 6a and 6b). We found that the pH of freshly prepared thiol-MS gels decreased with increasing polymer concentration. Gels from 1.3 wt% to 7.5 wt% exhibited pH values ​​between 7.5 and 6.5, whereas 10 wt% gels had a pH close to 5.1. This can be explained by the liberation of methanesulfinic acid as a leaving group during thiol-MS coupling. At higher polymer fractions, leaving groups are generated at higher concentrations, which reduces the pH of the crosslinking medium and thus the achieved final shear modulus. This effect can be controlled by increasing the buffer capacity of the crosslinking medium, which is achieved by increasing the HEPES buffer concentration from 10 mM to 50 mM (see Figure 6). The latter concentration is known to remain cytocompatible. These results indicate that the polymer content can also be used to control the mechanical properties of the gels. At relatively high concentrations (10 wt%), pH should be controlled by increasing the buffer capacity.

[0105] Swelling measurements on Thiol-X hydrogels These studies were performed using a 5% (w / v) precursor solution prepared in 10 mM HEPES buffer (pH 7.0) and pre-cooled in an ice bath. 50 μL of 5% (w / v) PEG-X solution was placed in a flexible PDMS cylindrical mold (0.75 cm diameter) and rapidly mixed with 50 μL of 5% (w / v) PEG-thiol solution. The hydrogel was then crosslinked in a humidity chamber at 37 °C for 4 h. The resulting hydrogel was carefully removed and allowed to swell in MilliQ water for 24 h. The mass of the swollen gel was then determined (M s ).

[0106] The gel was dried in an oven at 37 °C for 48 h, and the mass of the dried hydrogel was determined (M d The swelling ratio (SR; swelling degree) was calculated according to the following formula: SR=(M s -M d ) / M d

[0107] The experiment was performed in triplicate, and the mean and standard deviation were reported.

[0108] The swelling ratio (SR) of 5% thiol-MS gels was measured in water at pH 7.0. A swelling of 33.6 mg of water per mg of polymer was obtained (see Table 5). The thiol-VS gels showed similar SR values, while the thiol-Mal gels showed an approximately 1.5-fold increase. These results indicate a similar degree of cross-linking in the thiol-MS and thiol-VS networks, and less cross-linking in the thiol-Mal gels.

[0109] Hydrolytic stability is an important material property for hydrogels used in 3D cell culture.

[0110] Therefore, the hydrolytic stability of 5 wt% thiol-MS gels was determined by gravimetric analysis of the swollen gels after incubation in cell culture medium at 37°C for 4 weeks at different time points (Figure 7a). Within the first 2 weeks, the mass of the swollen thiol-MS gel reached 1.2 times its original mass, suggesting low gel erosion and high hydrolytic stability of the thiol-MS gel. It should be noted that the long-term stability of the gels is advantageous for long-term cell culture, and the degradation characteristics can be finely tuned by 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 commonly used for long-term culture (MP Lutolf, GP Raeber, AH Zisch, N. Tirelli, JA Hubbell, Advanced Materials 2003, 15, 888-892), but much higher than that of the thiol-Mal gel (1.2-fold increase over 2 days and hydrogel collapse on day 18) (N. Boehnke, C. Cam, E. Bat, T. Segura, HD Maynard, Biomacromolecules 2015, 16, 2101-2108). Hydrolysis of the thiol-Mal gel 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 the cell culture medium. This result is consistent with studies using model MS compounds showing superior stability of thioheteroaromatic conjugates obtained from thiol-MS coupling relative to thiol-Mal compounds under therapeutically relevant conditions (N. Toda, S. Asano, C.F. Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596).Finally, experiments performed with 10 wt % gels showed that the thiol-MS gels remained hydrolytically stable for more than 6 weeks (see Figure 7b).

[0111] Use in cell encapsulation PEG hydrogel preparation for 3D cell culture 3D PEG hydrogels were prepared by adapting the protocol described (Phelps et al., Advanced Materials 2012, 24, 64-70 and Farrukh et al., Adv. Funct. Mater. 2018). A precursor solution of 20 kDa 4-arm PEG Mal / VS / MS (100 mg / mL, 10% (w / v)) was prepared by dissolving it in HEPES buffer (10 mM, pH 8.0) under sterile laminar flow. cyclo(RGDfC) (3.45 mg / mL, 5 mM) and VPM peptide (GCRD) were added. VPM A solution of SMRGDRCG (26.6 mg / mL, 15.68 mM) was similarly prepared in sterile HEPES buffer (pH 8.0). These concentrations were kept constant during all cell experiments.

[0112] A 4-arm PEG Mal / MS / VS stock solution (10% (w / v)) was mixed with 5 mM cyclo(RGDfC) in a 2:1 volume ratio and incubated at 37 °C for 30 min. A cell suspension (10 × 10 6Cells (1000 cells / mL) were added to the above solution, and 8 μL drops of the resulting mixture were placed into each Ibidi 15 microtiter plate angiogenesis slide. A solution of VPM peptide (2 μL, 15.8 mM) was immediately added to each microtiter plate and carefully mixed and crosslinked using a pipette tip. Polymerization of 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, RPMI medium was added and the cultures were maintained for 1 to 3 days. Alternatively, for spheroid culture, RPMI medium (2 μL) was mixed with cyclo(RGDfC)-modified PEG precursor solution (6 μL, as above) and each solution (8 μL) was introduced into the microtiter plate. After the fibrin clot was added to each plate, 15.8 mM VPM peptide (2 μL) was added and allowed to gel for 15 to 45 min at 37 °C. Culture medium was added to each titer plate and replaced with fresh medium once every 24 hours during cell culture.

[0113] Using this process, PEG-MS components were first functionalized with cyclo(RGDfC) peptide, then mixed with L929 fibroblasts, and finally crosslinked with enzymatically cleavable dithiol peptide (VPM). The composition used was 4 wt% PEG-MS, 1 mM RGD peptide, and 3.14 mM VPM (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 highly fluid, and the mixture could be homogenized by pipetting with low shear. A stable gel formed within 15 minutes and was visible to the naked eye. The distribution of cells within the hydrogel was analyzed by Z-stack imaging with a confocal microscope. A uniform distribution of cells throughout the thickness of the hydrogel was observed ( Figure 8 a).

[0114] Cell culture conditions Fibroblast L929 cell line (ATCC) was cultured in RPMI 1640 medium (Gibco, 61870-010) supplemented with 10% FBS (Gibco, 10270) and 1% P / S (Invitrogen) at 37°C and 5% CO. For suspension cell culture, L929 cells (10 × 10 6 cells / mL) were directly suspended in the PEG precursor solution during polymerization.

[0115] For spheroid culture, fibrin clots of the fibroblast L929 cell line were prepared using the following literature reports (JL West, Biomaterials 2008, 29, 2962-2968; CA DeForest, KS Anseth, Nature Chemistry 2011, 3, 925-931).

[0116] In summary, 10 x 10 6 The cells / mL pellet was dissociated in fibrinogen (10 mg / mL in PBS) and 2 μL drops were applied to Sigmacote-coated hydrophobic slides. 1 μL of thrombin solution (5 μl / mL in PBS) was added to each fibrinogen drop, and the cells were placed in an incubator for 15 min to obtain fibrin clots.

[0117] Fixation and staining 3D PEG hydrogel samples were fixed with 4% PFA solution for 2 hours at room temperature and washed with PBS. Samples were blocked with 1% BSA solution for 1 hour and then permeabilized with 0.5% Triton X-100 for 1 hour. FITC-phalloidin (1:200 in water, Thermo Fisher Scientific) was used to stain actin fibers, and DAPI (1:500 in water, Life Technology) was used to stain nuclei. Samples were incubated with antibodies for 5 hours at room temperature and then washed with PBS.

[0118] Live-dead assay The cell culture medium was removed, and the samples were incubated with fluorescein diacetate (40 μg / mL) and propidium iodide (30 μg / mL) in PBS for 5 min. Samples were washed twice with PBS and documented using a Zeiss LSM 880 confocal microscope.

[0119] 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, Figures 8a, 8b, and 8c). These results suggest that the crosslinking kinetics of the system are ideal for obtaining homogeneous constructs under convenient and cytocompatible experimental conditions.

[0120] Conversely, the thiol-Mal hydrogels hardened immediately upon mixing with the precursor solution, making proper homogenization more difficult and resulting in cell aggregation at the top of the gel. The thiol-VS system, on the other hand, allowed for effective mixing, but its slow gelation kinetics resulted in cell sedimentation at the bottom of the gel. These results are consistent with a previous report by Peyton et al. (LE Jansen, LJ Negron-Pineiro, S. Galarza, SR Peyton, Acta Biomaterialia 2018, 70, 120-128) on the influence of crosslinking rate on the distribution of fluorescent beads encapsulated in thiol-Mal hydrogels, and a previous report by Shikanov et al. (J. Kim, YP Kong, SM Niedzielski, RK Singh, AJ Putnam, A. Shikanov, Soft Matter 2016, 12, 2076-2085) that noted the need to rotate thiol-VS gels during hardening to prevent cell sedimentation. In this regard, thiol-MS hydrogels exhibit more favorable kinetics and overcome these disadvantages.

[0121] Migration assay To demonstrate that cells cultured in thiol-MS hydrogels remain functional, we performed a migration assay. L929 fibroblast spheroids were encapsulated in degradable thiol-MS hydrogels (A. Farrukh, J.I. Paez, A. del Campo, Advanced Functional Materials 2019, 29, 1807734), cultured for 3 days, fixed, and stained. The cell migration distance of the spheroids was quantified as an indicator of gel degradation and the cell's ability to migrate within the gel (Figures 9a-c). Cells migrated a distance of approximately 425 μm. This result was compared with that obtained using thiol-Mal and thiol-VS as 3D cell encapsulation materials. The migration distance was approximately 470 μm for Mal and approximately 360 μm for VS systems (Figure 9c). This result was due to the difference in crosslinking degree (G'). 37℃ This is due to differences in cross-linking (MS = VS>MS>Mal; see Figure 3b) and hydrolytic stability (MS = VS>>Mal; Figure 7). Lower cross-linking or more rapid degradation allows more space for cells, i.e., longer migration paths.

[0122] Furthermore, after 3 days of incubation, cells cultured in thiol-MS hydrogels were more evenly distributed within the gel and showed fewer instances of clustering than the other two systems (see Figure 10).

[0123] The thiol-MS reaction is suitable for crosslinking hydrogels in the context of cell encapsulation. This reaction achieves kinetics between the thiol-Mal and thiol-VS systems, reaching high conversion rates. The resulting crosslinked units exhibit good hydrolytic stability and cytocompatibility. Under mild aqueous conditions, the MS-thiol reaction is orthogonal to alcohol, amine, carboxylic acid, and acrylate functional groups (D. Zhang, N.O. Devarie-Baez, Q. Li, K.R. Lancaster, M. Xian, Organic Letters 2012, 14, 3396-3399; A. Farrukh, J.I. Paez, M. Salierno, A. del Campo, Angew. Chem. Int. Ed. 2016, 55, 2092-2096; A. Farrukh, J.I. Paez, M. Salierno, W. Fan, B. Berninger, A. del Campo, Biomacromolecules 2017, 18, 906-913), making this crosslinking mechanism applicable to most natural polymer scaffolds of biomedical interest. The reactivity of the thiol-MS couple can be tuned by the pH and the choice of different MS-aromatic substrates used (N. Toda, S. Asano, C.F. Barbas, Angew. Chem., Int. Ed. 2013, 52, 12592-12596). The combination of all these properties makes thiol-MS an excellent alternative to other reactive chemistries for 3D cell encapsulation.

[0124] [Table 1]

[0125] [Table 2]

[0126] [Table 3]

[0127] Table 4

[0128] Table 5

Claims

1. a) a1) at least one macromer having at least two thiol groups as functional groups; a2) at least one macromer having at least two 2-(methylsulfonyl)-1,3,4-oxadiazole groups as functional groups; wherein at least one component a1) or a2) has at least three of said functional groups; b) reacting the two macromers via the functional groups to form a hydrogel; A method of making a hydrogel, comprising:

2. 2. The method according to claim 1, characterized in that the at least one component a1) or a2) has an average molar mass of less than 500 kDa.

3. 3. The method according to claim 1 or 2, characterized in that the at least one component a1) or a2) has 3, 4, 5, 6, 7, 8, 9 or 10 functional groups.

4. 4. The method according to claim 1, wherein the at least one component a1) or a2) is based on an oligomer or a polymer.

5. 5. The method of claim 4, wherein the oligomer or polymer is poly(meth)acrylamide, poly(meth)acrylic acid, poly(meth)acrylate, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane (PU), polyvinylpyrrolidone (PVP), polyamide, poly(amidoamine) (PAMAM), polyester, polylactide, polyglycolic acid (PGA), or poly(lactide-co-glycolide) (PLGA), polyanhydride, poly(ortho)ester, polyacetal, poloxamer (block copolymer of ethylene oxide (PEG) and propylene oxide (PPG)), poly-2-oxazoline, polyphosphazene, polyglycerol, polyamine, polycarbonate, polyglutamic acid, polyaspartic acid (PASA), polyphosphonate, DNA, RNA, gelatin, polyhydroxyalkanoate (PHA), poly-gamma-glutamic acid, protein or peptide, or polysaccharide.

6. The poly(meth)acrylate is polyHPMA or polyHEMA; the poloxamer is PEG-Co-PPG-Co-PEG; The polyamine is polylysine or polyethyleneimine (PEI); the protein or peptide is collagen, a dithiol peptide with the sequence GCRDVPMSMRGGDRCG, albumin, or fibrin, or The polysaccharide is agarose, chitin, chitosan, chondroitin, mannan, inulin, dextran, cellulose, alginate, or hyaluronic acid.

6. The method according to claim 5.

7. 7. The method according to any one of claims 1 to 6, characterized in that the macromer content of the composition is between 1 wt% and 30 wt%.

8. The method according to any one of claims 1 to 7, characterized in that the gelation is carried out under physiological conditions.

9. A hydrogel obtainable by the method according to any one of claims 1 to 8.

10. 1. A hydrogel comprising a first plurality of macromers having at least two thiol groups as functional groups crosslinked with a second plurality of macromers having at least two 2-(methylsulfonyl)-1,3,4-oxadiazole groups as functional groups, wherein at least one of the first or second macromers has at least three of said functional groups, and the crosslinking is via a plurality of Ar—S bonds, where Ar is a 2-(methylsulfonyl)-1,3,4-oxadiazole group.

11. A composition for making a hydrogel comprising components a1) and a2) according to any one of claims 1 to 7.

12. A kit for making a hydrogel comprising components a1) and a2) according to any one of claims 1 to 7.

13. 11. The hydrogel of claim 9 or 10 for cell encapsulation, three-dimensional cell culture, organoids, biomaterials, injectable biomaterials, cell therapy, tissue engineering, tissue regeneration, tissue transplantation, regenerative medicine, 3D printing, 3D bioprinting, wound dressing or wound treatment, delivery agent for active ingredients, in vitro model for researching or testing diagnostic or therapeutic agents, or cell transplantation.

14. a) providing a gel or a precursor thereof having 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 any one of claims 1 to 7, which has at least two functional groups, depending on the other components; c) modifying the gel or its precursor by reacting the functional groups of the macromer with the gel or its precursor; A method for modifying a gel, comprising:

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