SE2250471A1 - Formation of cross-linked hydrogels - Google Patents

Abstract

A method of forming a cross-linked hydrogel, comprising to provide a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thiol groups of a thiol-containing moiety are protected with enzyme-responsive thiol protection groups, and to add an enzyme to the polymer composition, the enzyme being configured for enzymatic deprotection of the protected thiol protection group, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalent crosslinked hydrogel.

Description

FORMATION OF CROSS-LINKED HYDROGELS TECHNICAL FIELD id="p-1" id="p-1" id="p-1"

[001] The present document relates to a method for providing cross-linked hydrogels, in which polymer molecules provided with protected thiol groups are deprotected using enzymatic deprotection, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalent cross-linked hydrogel.

BACKGROUND ART id="p-2" id="p-2" id="p-2"

[002] Engineered hydrogels are used extensively in a wide range of biomedical applications and are key components for tissue engineering of soft tissues and in development of advanced tissue and disease models. Hydrogels can be tailored to mimic specific aspects of the native extracellular matrix (ECM), such as stiffness, porosity, degradability and cell adhesion.[1] The physicochemical properties ofthe hydrogels influence cell viability, proliferation, cell behavior, and differentiation.[2,3] ln addition to facilitate 3D cell culture, hydrogels are also important components in bioinks for 3D bioprinting. I\/lost ECI\/I-mimicking hydrogels are polymer based. [003] Cross-linking of the polymers is central for maintaining the structural integrity of the materials and for controlling their mechanical properties. Numerous different polymer cross- linking methods have been developed and explored for generating hydrogels, including both covalent and non-covalent chemistries. Whereas the former result in hydrogels with long-term structural robustness, the latter allows for dynamic remodulation ofthe materials by e.g., cells. Non-covalently cross-linked hydrogels are also often shear thinning, which is an attractive feature for 3D bioprinting and other applications requiring syringe extrusion of the materials, such as cell-injection therapy and soft tissue filler treatments. id="p-4" id="p-4" id="p-4"

[004] Chemical reactions between thiols and thiol-reactive species comprised of e.g., maleimides, methacrylates, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, alkyne have been extensively used for cross-linking of hydrogels for cell-encapsulation, 3D cell culture and 3D bioprinting. Notably, the commercially available cell culture hydrogel system HyStem® is based on thiolated hyaluronic acid cross-linked using thiol-reactive cross-linkers. ln addition, possibilities to cross-link hydrogels using disulfide bonds have been widely explored for a wide range of different polymers, including alginate, hyaluronan, gelatin, poly(ethylene glycol) (PEG), and poly(carboxybetaine).[4-8] 2 [005] Disulfide bonds are present in many proteins and are crucial for maintaining their structure and function. ln tissues, disulfide formation is controlled by the redox potential in the extracellular space mediated by cell secretion of reducing agents, such as glutathione.[9] Disulfides thus represent a dynamic cytocompatible covalent cross-linking chemistry, resulting in cross-links that can form and break at time scales of relevance for cell-matrix remodeling.[10] [006] Unfortunately, the rate of oxidation of thiols into disulfides is difficult to control and to a large extent dictated by environmental factors (such as presence of oxygen), making this strategy for fabrication of hydrogels unreliable and difficult to use in most applications. ln addition, free thiols can react with thioesters through transesterification to form a new thioester.[11] This reaction is often referred to as native chemical ligation (NCL) and has been widely used in chemical synthesis of large peptides and proteins, and has also been explored for covalent cross-linking of hydrogels from soluble macromolecular precursors.[12] Similar to when using thiol-reactive cross-linkers or when relying on disulfide-mediated cross-linking, NCL-triggered hydrogel cross-linking require free thiols. Since thiols are prone to oxidation, these strategies are complicated to implement for 3D cell culture and bioprinting. Oxidation of thiols is spontaneous in aqueous solutions in the presence of oxygen and at physiological pH. Spontaneous oxidation ofthiolated hydrogel precursors can result in uncontrolled hydrogel formation due to formation of disulfides or prevent formation of hydrogels when thiol- reactive cross-linkers or NCL strategies are used. [007] Different strategies to overcome the obstacles with spontaneous thiol oxidation have been explored and that does not require that the thiol containing hydrogel precursors are prevented from contact with oxygen. [008] ln one study, S-protected thiolated hyaluronic acid was synthesized and the thiols protected by N-acetyl cysteine (NAC). NAC was then deprotected by endogenous thiols, which leads to the formation of hydrogels in situ. [13] ln this method, however, addition or naturally high levels ofthiols is required, which would interfere with any thiol-mediated cross-linking reaction. [009] A polymer architecture based on poly(2-oxazoline)s bearing protected thiol functionalities, which can be selectively liberated/deprotected by irradiation with UV light, has been tested. [14] Deprotection with UV light can, however, be harmful if cells are present in the polymer architecture. 3 id="p-10" id="p-10" id="p-10"

[0010] Branched po|y(ethy|ene glycol) modified with protected thiol end groups were produced and deprotection of the thiol was achieved using a base, e.g. sodium hydroxide, quantitatively liberating a thiol group and a non-toxic acetate ion. [15] Hydrolysis using NaOH is, however, not cell compatible. id="p-11" id="p-11" id="p-11"

[0011] There is, hence, a need to provide a method for cross-linking hydrogels based on polymers provided with protected thiol groups, which relies on a more cell-compatible chemistry.

SUMMARY OF THE INVENTION id="p-12" id="p-12" id="p-12"

[0012] lt is an object of the present disclosure to provide a method for providing cross-linked hydrogels based on polymers provided with protected thiol groups, which method uses a cell- compatible chemistry. Further objects are to provide a hydrogel-forming mixture and a kit of parts for forming a cross-linked hydrogel. id="p-13" id="p-13" id="p-13"

[0013] The invention is defined by the appended independent patent claims. Non-limiting embodiments emerge from the dependent patent claims, the appended drawings and the following description. id="p-14" id="p-14" id="p-14"

[0014] According to a first aspect there is provided a method of forming a cross-linked hydrogel, comprising providing a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thiol groups of a thiol- containing moiety are protected with enzyme-responsive thiol protection groups, and adding an enzyme to the polymer composition, optionally, activating the enzyme if inactive, the enzyme being configured for enzymatic deprotection of protected thiol groups, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalent cross- linked hydrogel. id="p-15" id="p-15" id="p-15"

[0015] The one or more thiol groups of a thiol-containing moiety of a polymer is provided with an enzyme-responsive thiol protection group, such that no or very little thiol-mediated cross- linking of the polymer molecules takes place in the polymer composition before the enzyme has been added to or been activated in the polymer composition. id="p-16" id="p-16" id="p-16"

[0016] A thiol-containing moiety may for example be a cysteine. id="p-17" id="p-17" id="p-17"

[0017] The thiol protection group should not spontaneously be removed under physiologically relevant conditions, including any conditions of relevance for maintaining or culturing of 4 eukaryotic cells. Cross-linking ofthe polymer molecules is initiated when the enzyme is added to the polymer composition or when the added enzyme is activated. The enzyme being configured for enzymatic deprotection of the protected thiol group, thereby enabling thiol- mediated cross-linking ofthe polymer molecules, forming a covalent cross-linked hydrogel. [0018] The cross-linking is initiated as a result of the spontaneous oxidation ofthiols, resulting in formation of disulphide. id="p-19" id="p-19" id="p-19"

[0019] With hydrogel is here meant a three-dimensional network of hydrophilic polymers that does not dissolve in water but can swell and hold a large amount of water while maintaining its structure. id="p-20" id="p-20" id="p-20"

[0020] A crosslinked hydrogel maintains its structural integrity and controls its mechanical properties. id="p-21" id="p-21" id="p-21"

[0021] Through the enzymatic deprotection, the thiol protection group is completely removed or at least partially removed, such that the thiol group is exposed and available for forming cross-links. id="p-22" id="p-22" id="p-22"

[0022] With the above described method, hydrogel cross-linking can be triggered at a specific time-point and the extent and rate of gelation can be controlled. This can for example be controlled by the number of thiol moieties present in the polymer composition and by the amount of enzyme added to the polymer composition, by the time point at which the enzyme is added to or activated in the polymer composition. Thereby, the cross-linking commences in a controlled way, why controlled processing of the hydrogels, including 3D bioprinting, is possible. id="p-23" id="p-23" id="p-23"

[0023] With the above described method the cross-linking can be performed at physiological ionic strength and pH conditions. Further, no harsh chemicals are needed to perform the cross-linking and the hydrogel is both cell friendly and non-cytotoxic. The hydrogels produced by the above described method are structurally robust due to the covalent cross-links. id="p-24" id="p-24" id="p-24"

[0024] ln one example the thiol protection group is phenylacetylaminomethyl (phacm) and the enzyme may be penicillin acylase or more specifically penicillin G acylase. id="p-25" id="p-25" id="p-25"

[0025] The method may further comprise to add one or more thiol-reactive species selected from one or more of maleimide, methacrylate, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and alkyne to the polymer solution. id="p-26" id="p-26" id="p-26"

[0026] The thiol-reactive species may be added before, after or at the same time as the enzyme is added or activated. id="p-27" id="p-27" id="p-27"

[0027] The thiol reactive cross-linkers are configured to chemically react with more than one thiol group, which after deprotection of the thiol moiety may enable cross-linking of the polymer molecules. This may result in formation of a covalent polymer network.

Due to kinetic factors, not all deprotected thiols may immediately oxidize and contribute to the cross-linking of the hydrogels. Remaining free thiols are thus available for further functionalization using thiol-reactive molecules. The thiol-reactive molecules may rapidly react with free thiols by a Michael addition reaction. The type and amount of the thiol-reactive cross-linker added to the polymer composition, and the time-point at which it is added to the polymer composition can affect the extent and rate of gelation of the hydrogel. id="p-28" id="p-28" id="p-28"

[0028] To activate an inactive enzyme an enzyme activator may be added to the polymer composition. id="p-29" id="p-29" id="p-29"

[0029] ln one embodiment, the enzyme added to the polymer composition may be an inactive enzyme, which is activated by adding an enzyme activator to the composition. The activator may be a substance allosterically regulating the enzyme, a specific ion, an acid/base added to the polymer composition causing a pH change ofthe composition, etc. ln one embodiment the ion is a co-factor. The ion can be Ca". id="p-30" id="p-30" id="p-30"

[0030] A ratio ofthiol-containing moieties per total amount of monomer units building up the polymer molecules in the polymer composition may be 1:10,000 to 1:1. id="p-31" id="p-31" id="p-31"

[0031] A monomer is a molecule that reacts, physically or chemically, with other monomers to form a larger molecule. The resulting macromolecule is called a polymer. id="p-32" id="p-32" id="p-32"

[0032] A ratio ofthiol-containing moieties per total amount of monomer units building up the polymer molecules in the polymer composition may be 1:10,000 to 1:1, 1:5000 to 1:1, 112500 to 1:1, 1:1000 to 1:1, 1:500 to 1:1, 1:250 to 1:1, 1:100 to 1:1, 1:50 to 1:1, 1:10,000 to 1:50, 1:10,000 to 1:100, 1:10,000 to 1:250, 1:10,000 to 1:500, 1:10,000 to 1:1000, 1:10,000 to 1:2500, 1:10,000 to 1:5000, 1:500 to 1:50, 1:1000 to 1:100, or 1:5000 to 1:1000. id="p-33" id="p-33" id="p-33"

[0033] The higher the ratio ofthiol-containing moieties per total amount of monomer units in the composition, the higher degree of cross-linking is possible, and the more robust the formed hydrogel. The ratio used may depend on the specific application of the formed cross- linked hydrogel. id="p-34" id="p-34" id="p-34"

[0034] The polymers may comprise monomer units naturally comprising one or more thiol- containing moieties. Alternatively, or in addition monomer units may be modified to include one or more thiol-containing moieties. 6 id="p-35" id="p-35" id="p-35"

[0035] The polymer composition may comprise a mixture of polymers in which a portion is provided with thiol-containing moieties and another portion does not comprise any thiol- containing moieties. Some polymers may comprise a larger amount of thiol-containing moieties and some a fewer amount of such moieties. id="p-36" id="p-36" id="p-36"

[0036] The polymer composition may comprise one or more polymer types selected from polysaccharides, polypeptides, peptides, synthetic polymers or combinations thereof. id="p-37" id="p-37" id="p-37"

[0037] The method may further comprise to add cells into the polymer composition. id="p-38" id="p-38" id="p-38"

[0038] The cells may be added into the polymer composition before, during or after enzyme addition or activation. ln one example the cells are fibroblasts. As described above, the crosslinking can be performed at physiological ionic strength and pH conditions, without need of any harsh chemicals. The hydrogel is, hence, both cell friendly and non-cytotoxic. id="p-39" id="p-39" id="p-39"

[0039] The method may further comprise a step of forming a shaped matrix from the composition before and/or after adding or activating the enzyme, thereby forming a cross- linked shaped matrix. id="p-40" id="p-40" id="p-40"

[0040] The enzyme can be added or activated at different time points during the formation process. id="p-41" id="p-41" id="p-41"

[0041] The shaped matrix may for example be a wire, a cord, a tube, a mesh, a bead, a sheet, a web, a disc, a cylinder, a coating, an interlayer, or an impregnate. id="p-42" id="p-42" id="p-42"

[0042] Forming the shaped matrix may comprise printing the shaped matrix using the polymer composition as/in a printing ink. id="p-43" id="p-43" id="p-43"

[0043] Alternatively, a shaped matrix may be formed for example by moulding or casting the polymer composition. id="p-44" id="p-44" id="p-44"

[0044] According to a second aspect there is provided a hydrogel-forming mixture, comprising a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thiol groups of a thiol-containing moiety are protected with enzyme-responsive thiol protection groups. id="p-45" id="p-45" id="p-45"

[0045] The hydrogel-forming mixture may further comprise an enzyme, the enzyme being configured to, possibly upon activation thereof, enzymatically deprotect the protected thiol group, thereby enabling thiol-mediated cross-linking of the polymer molecules in the hydrogel-forming mixture, forming a covalently cross-linked hydrogel. id="p-46" id="p-46" id="p-46"

[0046] An inactive enzyme may be activated by adding an enzyme activator to the mixture.

The activator may be an activator as discussed above. The enzyme is configured for enzymatic 7 deprotection of the protected thiol group, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalent cross-linked hydrogel. id="p-47" id="p-47" id="p-47"

[0047] The hydrogel-forming mixture may further comprise one or more thiol-reactive species selected from one or more of maleimide, methacrylate, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and alkyne. id="p-48" id="p-48" id="p-48"

[0048] A ratio ofthiol-containing moieties per total amount of monomer units building up the polymer molecules in the polymer composition may be 1:10,000 to 1:1. id="p-49" id="p-49" id="p-49"

[0049] The polymer composition may comprise one or more polymer types selected from polysaccharides, polypeptides, peptides, synthetic polymers or combinations thereof. id="p-50" id="p-50" id="p-50"

[0050] According to a third aspect there is provided a kit of parts for forming a cross-linked hydrogel, comprising: a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thiol groups of a thiol-containing moiety are protected with enzyme-responsive thiol protection groups, and an enzyme, the enzyme being configured for enzymatic deprotection of the protected thiol group, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalent cross-linked hydrogel. id="p-51" id="p-51" id="p-51"

[0051] The polymer composition and the enzyme may be provided in two separate compartments and mixed upon use. Alternatively, in case the enzyme is an inactive enzyme, the polymer composition and the inactive enzyme may be provided in the same compartment. [0052] The kit of parts may further comprise an enzyme activator for activating the enzyme, if inactive, and enabling the thiol-mediated cross-linking ofthe polymer molecules. id="p-53" id="p-53" id="p-53"

[0053] ln case the enzyme is an inactive enzyme, the polymer composition and the enzyme may be provided in the same compartment, and an enzyme activator is provided in a separate compartment and mixed with the other components upon use. Alternatively, the polymer composition and the enzyme are provided in two separate compartments and mixed upon use. id="p-54" id="p-54" id="p-54"

[0054] The kit may further comprise one or more thiol-reactive species selected from one or more of maleimide, methacrylate, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and alkyne. id="p-55" id="p-55" id="p-55"

[0055] The polymer composition and the thiol-reactive cross-linkers may be provided in the same or in separate containers that are mixed upon use. 8 id="p-56" id="p-56" id="p-56"

[0056] The polymer composition, the enzyme and the thiol-reactive cross-linker may be provided in separate compartments and mixed upon use. Alternatively, in case the enzyme is an inactive enzyme, the polymer composition, the enzyme and the thiol-reactive cross-linker may be provided in the same compartment. ln yet an alternative, the polymer composition and the thiol-reactive cross-linker are provided in the same compartment and the enzyme in a Sepafate COmpaFtmeHt.

BRIEF DESCRIPTION OF THE DRAWINGS id="p-57" id="p-57" id="p-57"

[0057] Fig. 1 schematically illustrates functionalization of alginate with enzyme responsive cysteine residue Boc-L-Cys(Phacm)-NH2 and subsequent release of the amine protection group, Boc. id="p-58" id="p-58" id="p-58"

[0058] Fig. 2 shows a schematic presentation of deprotection of protected sulfhydryl groups on L-Cys(Phacm)-NH2 formed in Fig. 1 using penicillin G acylase (PGA) and formation of disulfide bonds and a hydrogel. id="p-59" id="p-59" id="p-59"

[0059] Fig. 3 shows increase of free thiols, as determined by an Ellman's tests, when deprotecting sulfhydryl groups on L-Cys(Phacm)-NH2 when exposed to PGA at room temperature. id="p-60" id="p-60" id="p-60"

[0060] Fig. 4 shows viscoelastic properties of a hydrogel formed as illustrated in Fig. 2, using different polymer concentrations: 2, 3, 4 and 5 % (w/v), after overnight treatment with the same concentration of PGA at 37°C. id="p-61" id="p-61" id="p-61"

[0061] Fig. 5 shows viscoelastic properties of a hydrogel formed as illustrated in Fig. 2, using the same polymer concentration, 2 % w/v, treated with 1.38 ul, 2.07 ul and, 2.77 ul of PGA, respectively. id="p-62" id="p-62" id="p-62"

[0062] Fig. 6 shows gelation kinetics of a 4 % w/v hydrogel formed as illustrated in Fig. 2 treated with Caz* ions, PGA, or both Caz* and PGA. id="p-63" id="p-63" id="p-63"

[0063] Fig. 7 shows cell viability of encapsulated and cultured human primary fibroblasts for a period of 5 days in the cross-linked hydrogels. id="p-64" id="p-64" id="p-64"

[0064] Fig. 8 shows a schematic presentation of a reaction between maleimide and unreacted thiols in a hydrogel formed as in Fig. 2. 9 id="p-65" id="p-65" id="p-65"

[0065] Fig. 9 shows gelation kinetics of AlgCP - p(I\/|al)4 at a Phacm:Maleimide mole ratio of 3:1 before and after adding 2 units of PGA. The arrow indicates the time point of PGA addition. id="p-66" id="p-66" id="p-66"

[0066] Fig. 10 shows gelation kinetics of AlgCP - p(I\/|al)4, mole ratio Phacm:I\/|alemide = 6:1 in comparison with the gelation kinetics of AlgCP in absence of p(I\/|al)4, both treated with 2 units of PGA. id="p-67" id="p-67" id="p-67"

[0067] Fig. 11 shows how increasing the mole ratio of I\/|aleimide to Phacm groups increases storage modulus of the hydrogel (stiffer gel). **** represents p < 0.0001. id="p-68" id="p-68" id="p-68"

[0068] Fig. 12 shows gel formation, increase in storage modulus (G') with time, in a mixture of an 8-arm-PEG-Boc-Cys(Phacm) (p(BCP)) and an 8-arm-PEG(Maleimide) after addition of PGA.

DETAILED DESCRIPTION id="p-69" id="p-69" id="p-69"

[0069] Engineered hydrogels can be tailored to mimic specific aspects of the native extracellular matrix (ECM). I\/lost ECI\/I-mimicking hydrogels are polymer based. Cross-linking of the polymers is central for maintaining the structural integrity of the materials and for controlling their mechanical properties. Numerous different polymer cross-linking methods have been developed and explored for generating hydrogels, including both covalent and non- covalent chemistries. id="p-70" id="p-70" id="p-70"

[0070] The possibilities to cross-link hydrogels using disulfide bonds have been widely explored for a wide range of different polymers. Different strategies to overcome the obstacles with thiol/disulfide crosslinking has been taken, in which thiol groups have been protected with protection groups and then deprotected to allow crosslinking and hydrogel formation. Such methods are generally, however, based on deprotection ofthiols with a cell- noncompatible chemistry. id="p-71" id="p-71" id="p-71"

[0071] Below is described a method of forming a cross-linked hydrogel, which comprises to provide a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thiol groups of a thiol-containing moiety are protected with enzyme-responsive thiol protection groups, and to add an enzyme to the polymer composition, optionally, activating the enzyme if inactive, the enzyme being configured for enzymatic deprotection of the protected thiol group, thereby enabling thiol-mediated cross- linking ofthe polymer molecules, forming a covalent cross-linked hydrogel. id="p-72" id="p-72" id="p-72"

[0072] With this method, hydrogel cross-linking can be triggered at a specific time-point and the extent and rate of gelation can be controlled. This can for example be controlled by the number of thiol moieties present in the polymer composition, by the amount of enzyme added to the polymer composition, and by the time point at which the enzyme is added to or the enzyme is activated in the polymer composition. Thereby, the cross-linking commences in a controlled way, why controlled processing of the hydrogels, including 3D bioprinting, is possible. Further, the crosslinking can be performed at physiological pH conditions. ln addition, no harsh chemicals are needed to perform the crosslinking and the hydrogel is both cell friendly and non-cytotoxic. Such hydrogels are also structurally robust due to the covalent cross-links. id="p-73" id="p-73" id="p-73"

[0073] The polymer composition may be used for forming a cross-linked shaped matrix before and/or after adding or activating the enzyme. The enzyme can be added or activated at different time points during the formation process. Such forming may be for example by bioprinting using a bioprinter with the polymer composition as/in the printing ink. Another example is forming a shaped matrix using moulding or casting. id="p-74" id="p-74" id="p-74"

[0074] An inactive enzyme may be activated by adding an enzyme activator to the composition. The activator may be a substance allosterically regulating the enzyme, a specific ion, an acid/base added to the polymer composition causing a pH change of the composition, etc. id="p-75" id="p-75" id="p-75"

[0075] Cells, such as fibroblasts, may be added into the polymer composition before, during or after enzyme addition or activation. id="p-76" id="p-76" id="p-76"

[0076] The thiol protection group should meet the requirement not to spontaneously be removed under physiologically relevant conditions. The thiol protection group may for example be a phenylacetamidomethyl group or derivatives thereof and the enzyme may be a penicillin acylase, such as penicillin G acylase. Using derivatives of a thiol protection group may alter selectivity and rate of the thiol deprotection. id="p-77" id="p-77" id="p-77"

[0077] The enzyme used may be one or more types of enzymes with selectivity for the thiol protection group. The enzymes should preferably have a high selectivity, which allow for selective and efficient thiol deprotection and thus triggering thiol-mediated hydrogel cross- linking. id="p-78" id="p-78" id="p-78"

[0078] Thiol-reactive species, such as e.g. maleimides, methacrylates, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and/or alkyne may be added to the polymer 11 composition before, during and/or after the addition or activation of the enzyme. Due to steric constraints, not all deprotected thiols may immediately oxidize and contribute to the cross- linking of the hydrogels. Remaining free thiols are thus available for further functionalization using thiol reactive molecules. The thiol-reactive species rapidly react with free thiols by a Michael addition reaction. Chemical functionalization of hydrogels after formation/bioprinting can allow for tuning of the cell microenvironment. To covalently modify already formed/printed crosslinked hydrogel structures thiol-reactive species can be added, i.e. post- forming/post-printing functionalization of hydrogels is possible. id="p-79" id="p-79" id="p-79"

[0079] ln one example the thiol-reactive species is maleimide (H2C2(CO)2NH) and derivatives thereof (where the NH group is replaced with alkyl or aryl groups, or a small molecule (such as biotin, a fluorescent dye, an oligosaccharide, or a nucleic acid), a reactive group, or a synthetic polymer such as polyethylene glycol, can be used. id="p-80" id="p-80" id="p-80"

[0080] The thiol-reactive species may be added to the polymer composition in amounts of 0.01 nM to 10 mM. id="p-81" id="p-81" id="p-81"

[0081] The polymer composition may comprise one or more polymers selected from polysaccharides, polypeptides, peptides, synthetic polymers or combinations thereof. id="p-82" id="p-82" id="p-82"

[0082] The polysaccharides may be selected from for example alginate, hyaluronan, cellulose, heparin, heparan sulphate, chitosan, and any combination thereof. The polypeptides may be a protein selected from for example collagen, gelatin, laminin, fibrin, elastin, and any combination thereof. The peptide may be any combination of amino acids but comprising at least one protected thiol residue. The synthetic polymers may be selected from for example polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers, any copolymer or combination thereof. id="p-83" id="p-83" id="p-83"

[0083] The polymer composition may for example comprise only one kind of polysaccharide, polypeptide, peptide, or synthetic polymer. Alternatively, the polymer composition may comprise a mixture of different types of polysaccharides, polypeptides, peptides, or synthetic polymers. ln yet an alternative, the polymer composition comprises a combination of one or more types of polysaccharides and/or types of polymers and/or types of peptides and/or types of synthetic polymers. id="p-84" id="p-84" id="p-84"

[0084] The polymer molecules provided with (a) thiol-containing moiety/moieties, may be naturally occurring polymer molecules comprising such thiol moieties. Alternatively, the polymer molecules are functionalized with one or more thiol-containing moieties. ln one 12 example the polymer is conjugated with a thiol-containing moiety using the well-known EDC/NHS Chemistry. id="p-85" id="p-85" id="p-85"

[0085] The thiol-containing moiety could be any thiol-containing moiety. One example is cysteine. The thiol group of the thiol moiety must be possible to protect with an enzyme- responsive/labile protection group. All or a portion/fraction of the polymer molecules in the polymer composition may comprise one or more thiol containing moieties. lf comprising more than one type of polymer molecule, all or some of the polymer molecule types may be provided with thiol-containing moieties. id="p-86" id="p-86" id="p-86"

[0086] A ratio ofthiol-containing moieties per total amount of monomer units building up the polymer molecules in the polymer composition may be 1:10,000 to 1:1. ln one example, 20% of the repetitive monomer units of a polymer molecule may be provided with one or more thiol moieties. The higher the ratio ofthiol-containing moieties per total amount of monomer units in the composition, the higher degree of cross-linking is possible, and the more robust the formed hydrogel. The ratio used may depend on the specific application ofthe formed cross-linked hydrogel. id="p-87" id="p-87" id="p-87"

[0087] The polymer composition comprising polymer molecules may be a solution comprising polymer molecules dissolved therein. The solution may be water or a buffer, such as a phosphate buffer. The amount of polymer molecules in the composition/solution upon addition or activation ofthe enzyme may be 0.1-10%(w/v)//1-100 mg/ml. For an efficient deprotection, the amount of enzyme added to the polymer composition may be in the range of 0.1-1000 units for each micromole of thiols (where 1 unit corresponds to the amount of enzyme which hydrolyzes 1 umole benzylpenicillin per minute at pH 7.6 and 37°C). id="p-88" id="p-88" id="p-88"

[0088] The formed cross-linked hydrogel may be part of an interpenetrating polymer network (IPN) including two or more entangled, interpenetrating polymer networks. The first polymer network may be based on the covalent cross-linked hydrogel and the second polymer network may be comprised of polymer molecules present in the polymer composition but not provided with any thiol containing moieties. id="p-89" id="p-89" id="p-89"

[0089] The method may further comprise to add Caz* to the polymer solution. The Caz* may be added to the polymer composition before or after deprotection of the thiol groups. Caz* can be utilized to dynamically modulate the stiffness and robustness of self -healing hydrogels. Caz* may be used as a temporary cross-linker until covalent binding is obtained. The ionic and enzyme-triggered cross-linking operates at different time scales, where the Ca2+-mediated 13 process occurs within minutes whereas the disulfide bridging is slower and proceeds over several hours. The hydrogel can be formed/printed both with and without Ca2+, as long as the enzyme is added during formation/printing to maintain the integrity ofthe formed structures. Caz* may be added to the polymer composition in amounts of 0.1 nM to 100 mM Ca". The calcium ions may for example be added as a salt. The method may further comprise adding Caz* before or after addition of thiol-reactive cross-linkers. id="p-90" id="p-90" id="p-90"

[0090] ln one embodiment, a polymer molecule provided with a thiol containing moiety may further comprise an amine group protected with an amine protection group. id="p-91" id="p-91" id="p-91"

[0091] A free amine group in the proximity of the thiol group (present in the same moiety as the thiol group or at another position of the polymer molecule) may be used to optimize the formation of disulfides after deprotection of the thiol group. Presence ofthe amine group may give an increased oxidation rate and thereby and increased cross-linking rate ofthe thiol groups. Electron-withdrawing groups, such as primary amines, in the proximity ofthiols decreases the pKa of the sulfhydryl group and stabilize the thiolate ion, resulting in a pronounced increase in the rate of disulfide bond formation. ln one example the thiol containing moiety is a cysteine in which the N-terminus of the cysteine is kept free to enhance the rate of oxidation after deprotection. The amine group may be protected using an amine protection group such as BOC (tert-butyloxycarbonyl) protecting group. Deprotection of a BOC-protected amine is a simple carbamate hydrolysis in acidic conditions. The polymer composition may be treated with dichloromethane and trifluoroacetic acid (TFA) at room temperature to deprotect the amine group. id="p-92" id="p-92" id="p-92"

[0092] The polymer composition discussed above may be provided in a kit of parts for forming a cross-linked hydrogel. An inactive enzyme, being configured for, upon activation thereof, enzymatic deprotection of the protected thiol group, as discussed above, may be in the polymer composition from start. For activation of the enzyme in the composition, an enzyme activator may be added, thereby enabling thiol-mediated cross-linking ofthe polymer molecules, forming a covalent cross-linked hydrogel. ln case an active enzyme is used, the kit may comprise the enzyme in a separate compartment, which enzyme then is added into the polymer composition upon use. The kit of parts may further comprise an enzyme activator for activating the enzyme and enabling the thiol-mediated cross-linking ofthe polymer molecules. [0093] The kit may further comprise thiol-reactive cross-linkers. The thiol-reactive cross- linkers may be provided in the same compartment as the polymer composition or in a 14 separate compartment. When provided in a separate container, the thiol-reactive cross-linkers may be added before or after an active enzyme is added, or in the case of an inactive enzyme is used, before or after the addition of the activator. id="p-94" id="p-94" id="p-94"

[0094] Below is described a specific example of production of such a cross-linked hydrogel, in which the polymer composition comprises alginate functionalized with cysteine (comprising the thio| group and an amine group). The thio| group being protected by phenylacetamidomethyl and the free amine group with a BOC (tert-butyloxycarbonyl) protecting group. Penicillin G acylase is used as the enzyme. The formed cross-linked hydrogel is then analysed in different ways.

Experimental id="p-95" id="p-95" id="p-95"

[0095] Conjugation of Boc-L-Cys(Phacm)-NH2 to alginate (Alg) N-alpha-t-Butyloxycarbonyl-S-(Phenylacetylaminomethyl)-L-cysteine (Boc-L-Cys(Phacm)-OH, 13.6 mmol, Iris Biotech GMBH, I\/larktredwitz, Germany) was dissolved in chloroform and combined with ethyl carbodiimide hydrochloride (EDC, 15.0 mmol, I\/lerck KGaA, Darmstadt, Germany), N-Hydroxy succinimide (NHS, 15.0 mmol, I\/lerck KGaA, Darmstadt, Germany) and diisopropylethylamine (30 mmol, I\/lerck KGaA, Darmstadt, Germany) to generate a cysteine succinimide ester. Then ethylenediamine (1.4 mole, I\/lerck KGaA, Darmstadt, Germany) was added to the mixture and stirred at room temperature overnight. The crude product was washed with 3><100 ml water before being dried, filtered and concentrated. The crude product was purified by a C18 column (ReproSil-Pur C18, Dr.I\/|aisch HPLC GmbH, Ammerbuch, Germany) attached to a semi-preparative high-performance liquid chromatography (HPLC) system (Dionex, Sunnyvale, USA) using an aqueous gradient of acetonitrile with 0.1 % TFA. Purification of Boc-L-Cys(Phacm)-NH2 was evaluated by LC-I\/lass and 1H-NMR (NMR 500Hz, Oxford instruments, Abingdon, United Kingdom). id="p-96" id="p-96" id="p-96"

[0096] Alginate (250 mg, Sigma-Aldrich, I\/lerck KGaA, Darmstadt, Germany) (Alg) was dissolved in MES buffer (22.5 ml, 100 mM, pH 7, Sigma-Aldrich, I\/lerck KGaA, Darmstadt, Germany), and 2.5 ml acetonitrile (Sigma-Aldrich, I\/lerck KGaA, Darmstadt, Germany) was thoroughly mixed with the solution (S1). Boc-L-Cys(Phacm)-NH2 (0.4 molar equivalents to carboxyl groups in Alg), hydroxy benzotriazole (HOBt) (2 molar equivalent to amine groups, Sigma-Aldrich, I\/lerck KGaA, Darmstadt, Germany), and EDC (4 molar equivalent to amine groups, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were dissolved in a mixture of 9 ml MES buffer and 1 ml acetonitrile (S2). S1 and S2 were mixed, and pH was adjusted to pH 7. The reaction was allowed to proceed overnight at room temperature followed by dialyzes against 10 % acetonitrile, 5 % and acetonitrile, and finally MilliQ water using a dialysis tube with 12-14 kDa cutoff. The dialyzed sample was lyophilized and stored in powder form at -20 °C. 1H NMR proved the success of conjugating Boc-L-Cys(Phacm)-NH2 to Alg generating AlgBCP. id="p-97" id="p-97" id="p-97"

[0097] Boc deprotection of AlgBCP To release the Boc group in Boc-L-Cys(Phacm)-NH2 conjugated to Alg (AlgBCP), AlgBCP was treated with a 5 ml dichloromethane (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 10 ml trifluoroacetic acid (TFA, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) mixture for 2 hours at room temperature, generating AlgCP. AlgCP was then concentrated and precipitated in cold diethyl ether (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The resulting pellet was dissolved in MilliQ water, and pH was adjusted to be neutral by 1 M NaOH. AlgCP solution was dialyzed against 2.5 mg/ml, 1.25 mg/ml NaCl, and pure MilliQ water by 14- 16 kDa tubes and freeze dried. id="p-98" id="p-98" id="p-98"

[0098] The degree of substitution of carboxyl groups with the Cys(Phacm) was estimated using an assay for determination of free primary amines. A solution of flurosceamine (FA, 10 mM) in acetone was prepared. 150 ml of sample was mixed with 50 pl of FA for a minute and the fluorescence intensity was recorded using an excitation wavelength of 400 nm and an emission wavelength of 450 nm. A calibration curve was obtained from a serial dilution of L- Cysteine (0.5 to 7.5 uM) in 200 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4. id="p-99" id="p-99" id="p-99"

[0099] Purification and sterilization of Penicillin G acylase Penicillin G acylase (PGA) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany, lot number: BCCF0392). Prior use, the enzyme was purified and sterilized. First, the enzyme purified on a size exclusion chromatography column (PD midi trap TM G-25, Cytiva, Uppsala, Sweden) using 20 mM HEPES buffer pH 7.4 supplemented with 0.5 mM CaClz as an elution buffer. The collected fraction was then concentrated using a 10 kDa spin filter and centrifuged at 10000 rpm at 4 °C three times. The concentrated samples were then sterilized using a 0.2 pm sterile filter. id="p-100" id="p-100" id="p-100"

[00100] Thiol group deprotection assessment by Ellman's test 16 The deprotection of thiol groups after treating AlgCP with PGA was demonstrated utilizing an Ellman's test. [16]. Briefly, 4 mg of Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was dissolved in 1 ml 100 mM phosphate buffer pH 8. A solution of 5 mg/ml AlgCP was prepared in 200 mM HEPES, pH 7.4. A mixture of 20 % (v/v) AlgCP, 2% (v/v) DTNB, and 78 % (v/v) phosphate buffer (100 mM, pH 8) was added to a 96-we|| plate. To each umole of Phacm in the AlgCP sample, 1U of PGA (according to the supplier estimation) was added, and the absorbance changes at a wavelength of 412 nm were recorded over 14 hours using plate reader (Tecan infinite M1000 Pro, Tecan Trading AG, Switzerland). The measurements were done in triplicates. The same experiment was conducted for 400 pIVI BCP as a control for the enzyme activity. id="p-101" id="p-101" id="p-101"

[00101] Oscillatory rheology The rheological properties of 2, 3, 4 and 5% (w/v) AlgCP prepared in 200 mM HEPES pH 7.4 were investigated using a Discovery HR-2 rheometer (TA instruments, New Castle, USA) using an 8 mm parallel plate geometry after 2 hours of swelling in 200 mM HEPES pH 7.4. Frequency sweeps were performed between 1 to 100 rad s'1 at 1 % strain, and amplitude sweeps were done between 0.1 to 50 % at 1 Hz oscillatory frequency. Each rnicrornole ofprotected thiols in the solution Was treated With one unite of Penicillin G acylase or otherwise as stated in the text (2.77 ul to 30 u1of4 % (W/v)A1gCP). The gelation of 4 % (w/v) AlgCP in the presence and absence of 10 mM Ca" was studied using a 20 mm 1° cone geometry at 1 Hz and 1% strain for up to 16 hours. ln all experiments, liquid silicone was applied around the geometry in addition to applying a solvent trap to prevent samples from drying. Samples treated with Caz* were prepared according to the protocol presented by Mooney's group.[17] Briefly, a 100 mg/ml mixture of CaCOg was prepared in 200 mM HEPES and sonicated for 10 minutes in an ultrasound bath at 20 °C, and diluted to 10 mM upon mixing with AlgCP solution. lmmediately after mixing, freshly prepared gluconic acid solution was added to the sample (4 mole equivalents to Caz* ions). The rheological measurement was started directly after adding the gluconic acid. All measurements were done at 37 °C. The gelation kinetics of AlgCP reacted with 4-arm PEG-maleimide (p(I\/|al)4) was measured for 1hr at 1% strain and 1Hz shear stress. A solution of 22.85 mg/ml AlgCP was prepared in 50mM HEPES buffer, pH 7.4 and mixed with a 35 mg/ml p(I\/|al)4 solution in 50 mM HEPES, pH 7.4. The molar ratio between Phacm group on AlgCP backbone and maleimide groups on p(I\/|al)4 was 3 to 1. The gelation kinetics was recorded for this sample without enzyme treatment. Then, 2 pl of enzyme (2 units) was added 17 to the solution and the modules were measured (Fig. 9). The same experiment was repeated while p(I\/|al)4 was substituted with the volume of 50 mM HEPES (Fig. 10). The impact of changing the number of maleimide groups for each Phacm group on the mechanical properties ofthe final gel was studied by mixing 45 pl AlgCP 22.85 mg/ml with different volumes of p(I\/|al)4 35mg/ml solution (the final volume of 60 pl) to have the molar ratios of 1.5:1, 3:1, 6:1, 12:1 of Phacm groups and maleimide groups. All samples were treated with 2 units of PGA and mixed in a PDMS mold (8 mm diameter and 1 mm height). Samples were treated overnight at 37°C. Before transferring the gels to rheometer, they were swelled in 50 mM HEPES for 2 hrs at room temperature. All rheological measurements were done using 8 mm geometry. The frequency sweep and amplitude sweep were done as mentioned earlier (Fig. 11). All measurement were done in triplets. id="p-102" id="p-102" id="p-102"

[00102] A further gelation kinetics experiment was performed in which an 8-arm-PEG- Boc-Cys(Phacm) (p(BCP)) was dissolved in 50 mM HEPES 50 pH 7.4 to prepare a 8 % (w/v) solution. Then 40 pl of p(BCP) was mixed with 10 pl of 8-arm-PEG(Maleimide) 35 mg/ml and then 2 pl of PGA was added to start the gelation. Gelation kinetics was recorded using a rheometer for 3600 seconds at 37°C (Fig. 12). id="p-103" id="p-103" id="p-103"

[00103] Swelling ratio AlgCP hydrogels (n = 4) were prepared as described above and incubated in 200 mM HEPES pH 7.4 to swell for 24 hours. The swelled gels were weighted (Wwef), and after 4 days of drying in a desiccator, dried weight (Wdfy) was recorded. The swelling ratio (JW) was calculated by equation (1): Wwet-Wdry JW: (Eq. 1) Wdry [00104] Self-healing of hydrogels AlgCP solutions (4% w/v, n = 2) were prepared in 200 mM HEPES pH 7.4 and colored with Alcian Blue (blue color) and Alizarin Red S (red color). The hydrogels were then cut into half, and two pieces with different colors were brought together at the incision site, and the integrity of the combined structures was assessed after 5 hours by subjecting the hydrogels to tensile forces. id="p-105" id="p-105" id="p-105"

[00105] Cell culture and analysis 18 Primary human dermal fibroblasts obtained from healthy donors were cultured in high glucose Dulbecco's I\/|odified Eagle Medium (DI\/|EI\/|) with 10% Fetal bovine serum (FBS), 1% penicillin- streptomycin) until 70% confluency. The medium was exchanged every 3 days. Passage 6 cells were used for this study. A solution of 4% (w/v) of AlgCP was prepared in Hanks' Balanced Salt Solution (HBSS, without Caz* and I\/|g2+, 20 mM HEPES pH 7.2) after sterilizing AlgCP by UV light. Cells were trypsinized (0.25 % trypsin-EDTA at 37°C for 10-15 min), counted, pelleted, and 2><106 cells/ml was resuspended in 100 ul ofthe hydrogel and 9.2 ul of sterile purified PGA was added. [00106] Cell viability test ul of the cell-laden hydrogels (n = 3) were added to 96 well by a gentle pipetting. After 1 hour of incubation at 37 °C, 5 % C02, the culture medium was added at the top of the hydrogels. Cell viability and proliferation were assessed by an Alamar blue assay (Thermo Fisher Scientific, Waltham, USA) assay at day 1 and 5. At each time point, the hydrogels were first washed with phosphate buffer saline (PBS) 10 mM pH 7.4 and then incubated for 2 hours in a 10% (v/v) Alamar blue reagent solution. The supernatant was collected and transferred to a 96 well plate. The fluorescence intensity was recorded using an excitation wavelength of 570 nm and an emission wavelength of 580-610 nm. id="p-107" id="p-107" id="p-107"

[00107] 3D Bioprinting in gelatin slurry 3D bioprinting was conducted in a gelatin slurry support bath.[18] A solution of 4.5 % (w/v) of gelatin (Sigma-Aldrich, I\/lerck KGaA, Darmstadt, Germany) was prepared in I\/|illiQwater and then left for overnight at 4 °C. The gelatin gel was topped with cold I\/|illiQwater and then vigorously blended for 120 s to obtain a slurry of gelatin. The slurry was centrifuged at 3000 rpm for 5 minutes at 4 °C and then washed with fresh cold I\/|illiQwater. The washing process was repeated at least for 5 times until no foam formed at the top of the solution after centrifugation. The prepared slurry was then vortexed in MilliQ water and kept at 4 °C for further use. To use the gelatin slurry for 3D bioprinting of AlgCP structures, the slurry was first centrifuged at 4500 rpm for 5 minutes and the supernatant was discarded and a cold solution of 200 mM HEPES and 10 mM CaClz was added and mixed with the slurry. This step was repeated one more time and after discarding the suspension the slurry at the bottom of the tube was used for as a support during 3D bioprinting. The slurry was transferred to a 12 well plate. A solution of 4% (w/v) AlgCP was prepared in 200 mM HEPES pH 7.4 colored with Brilliant Blue to facilitate imaging of the bioprinted hydrogels. AlgCP (100 ul) was mixed with 19 9.2 ul of PGA and then added to the back of a 22-gauge needle. A BioX bioprinter (Cellink AB, Gothenburg, Sweden) was used for printing the 10><10><3 mm lattice structure. The pneumatic pressure was about 1 kPa and the nozzle moving speed was set to 3 mm/s. The printed structures were kept at 4 °C for at least 30 minutes and then washed with warm HEPES 200 mM pH 7.4 solution and incubated at 37°C overnight. id="p-108" id="p-108" id="p-108"

[00108] 3D bioprinting of primary fibroblasts Freeze dried AlgCP was sterilized by UV light and then dissolved in sterile HBSS to obtain a 4 % (w/v) solution. About 4><106 cells were mixed gently with 100 ul of AlgCP solution and then 9.2 ul of sterile PGA was added to this mixture prior transferring the bioink to a 22-gauge needle and connected to the printing head. A lattice of 10><10><0.4 mm was directly printed using 1 kPa pressure and 20 mm/s nozzle moving speed on a six-well plate surface. The printed structure was allowed to be partially cross-linked in the incubator for 2 hours before addition of culture medium and overnight incubation. id="p-109" id="p-109" id="p-109"

[00109] Post-printing cell viability Post-printing viability at 24 hours of the cells was assessed using a Live/Dead assay (Thermo Fisher Scientific, Waltham, USA) and compared to viability of cells in the same bioink subjected to gentle pipetting (50 ul) following the same procedure as extrusion bioprinting. After 24 hours incubation the printed/pipetted bioinks were washed with PBS and immersed in a mixture of 2 uM calcein AM (Biotiurn, Frernont, USA) and 4 uM ethidiurn hornodimer-l (Merck KGaA, Darmstadt, Germany) in PBS for 30 minutes at room temperature. The structures were imaged using confocal microscopy (Zeiss LSM 780, Carl Zeiss, Oberkochen, Germany) with a 10X objective, covering a "80 um stack of 12 slices. The stacks were converted to a 2D image using ImageJ software (FIJI). id="p-110" id="p-110" id="p-110"

[00110] Post-printing functionalization Post-bioprinting functionalization of hydrogels either printed in gelatin slurry or directly on a six-well plate surface was demonstrated by addition of 10 uM Cy3-maleimide (Cy3-mal, Lumiprobe GmbH, Hannover, Germany) in HEPES 200 mM pH 7.4, followed by overnight incubation. The functionalized hydrogels were imaged using confocal microscopy at a wavelength of 550 nm. id="p-111" id="p-111" id="p-111"

[00111] Degradation of bioprinted structures To demonstrate the dynamic formation and disassembly of AlgCP we subjected supporting bath assisted bioprinted hydrogels to 5 mM reduced glutathione (Sigma-Aldrich , I\/|erck KGaA, Darmstadt, Germany) in 200 mM HEPES pH 7.4. The bioprinted hydrogels were first functionalized with Cyanine3 maleimide (Cy3-mal) to facilitate imaging by confocal microscopy.

Results and Discussion [00112] Functionalization of Alginate To functionalize Alg with enzyme responsive cysteine (Cys) residues, Boc-L-Cys(Phacm)-OH was first reacted with ethylenediamine in order to introduce an amine group for further conjugation to Alg (Fig. 1). The resulting Boc-L-Cys(Phacm)-NH2 was subsequently conjugated to Alg using carbodiimide chemistry, yielding AlgBCP. Finally, the Boc protected Cys amine group was deprotected to generate AlgCP (Fig. 1). The percentage of Alg modification was estimated to be about 28 % using primary amine assay. The amine group of Cys was retained to optimize the formation of disulfides after deprotection of the thiol moiety. id="p-113" id="p-113" id="p-113"

[00113] PGA-triggered gelation To trigger deprotection of the thiols in AlgCP and subsequent disulfide-mediated cross-linking, AlgCP was exposed to penicillin G acylase (PGA) to release the Phacm group (Fig. 2), resulting in formation of a transparent hydrogel. The rate of Phcam deprotection was investigated using an Ellman's test for detection of free thiols (Fig. 3). An initially rapid increase in free thiol concentration was followed by a more moderate increase in the number of deprotected thiols.

The analysis was complicated by the simultaneous oxidation ofthe generated thiols, which results in a decrease in concentration of free thiols. The oxidation and formation of disulfide bridges between Alg chains, however, also resulted in a distinct concomitant increase in viscosity. After addition of PGA, AlgCP turned from a viscous liquid into a stiff hydrogel when incubated at 37°C. After 15 hours a storage modulus of 250 Pa was reached. id="p-114" id="p-114" id="p-114"

[00114] The viscoelastic properties of the hydrogels were then investigated using oscillatory shear rheology for different AlgCP concentrations, ranging from 2 to 5 % (w/v), treated with the same concentration of PGA, (Fig. 4). The stiffness (storage modulus, G') increased from about 60 Pa to 240 Pa with increasing AlgCP concentration, which is in the range of soft tissues and a typical stiffness range for weakly cross-linked Alg-based hydrogels. lncreasing the amount of PGA twice did not increase the stiffness further, indicating that the limiting step was not the deprotection of the AlgCP (Fig. 5). 21 [00115] Addition of PGA to a solution of4 % (w/v) AlgCP solution at 37°C resulted in a gradual increase in storage modulus over a period of several hours (Fig. 6). ln contrast, the ionic cross-linking of AlgCP by Caz* resulted in softer hydrogels (G'"5O Pa at 1 Hz and 1% strain) but reached the final stiffness in less than 30 minutes. The two methods for cross- linking thus operate at different time scales. The combination of the fast but highly dynamic Ca2+-induced gelation and the slower disulfide-triggered cross-linking can thus both potentially facilitate syringe extrusion, 3D/4D bioprinting and improve long-term stability the hydrogels and 3D bioprinted structures. These applications, however, also require that the materials are compatible with cells. [00116] Cytocompatibility To investigate the cytocompatibility of the hydrogels and the method for cross-linking, human primary fibroblasts were encapsulated and cultured for a period of 5 days. The cells were combined with 4 % (w/v) AlgCP and the cross-linking was initiated by addition of PGA 1 U per umole of Phacm group in the absence of Ca". The cells maintained their proliferation (Fig. 7) and showed high cell viability (about 95 %) over a period of 5 days, consumption of Alamar blue remained the same over the period, demonstrating that both the materials and PGA are well tolerated by the cells. The cells were evenly distributed in the hydrogels and no cell sedimentation was observed, indicating homogenous and sufficiently rapid gelation. [00117] 3D bioprinting The possibilities to print the AlgCP hydrogels using a 3D bioprinter were explored both in the presence and absence of Caz* and using PGA to covalently lock the printed structures after printing, with and without embedded cells. Printing of AlgCP directly on a solid substrate in the absence of Ca2+, to generate a single layer structure with the shape of a lattice, was first investigated. PGA was added to the biomaterial ink immediately prior printing. The thiol deprotection and concomitant disulfide cross-linking was sufficiently fast to retain the shape of the printed structures with reasonable resolution. Bioprinting of human primary fibroblast using the same strategy was further evaluated. The bioprinted structures were allowed to cross-link in a cell incubator for 2 hours prior addition of cell culture medium to avoid excessive swelling ofthe hydrogels. The structures were imaged after 15 hours by Live/dead staining the cells. The viability of cells after printing remained high (about 85%), which is on par with cells encapsulated in the AlgCP-based bioink and gently extruded through a pipette. 22 [00118] Printing of 3D structures were conducted in the presence of Caz* (10 mM) using a gelatin support bath according to the freeform reversible embedding of suspended hydrogels technique developed by Hinton et al. [19], to maintain the shape of the hydrogels during printing. PGA was added to the AlgCP biomaterial ink immediately prior printing to initiate deprotection ofthe thiol groups and to induce cross-linking. ln contrast to many fast- gelling hydrogel systems, nozzle clogging was not an issue during the printing process. The gelatin support bath in combination with the rapid Ca2+-mediated ionic cross-linking further facilitated the printing process. The printed structures were cured for about 30 minutes at 4°C before melting of the support bath by heating to 37 °C. To further investigate the effect of Caz* on the printed structures, two identical structures were printed as described above. One of the samples was washed and then incubated in HEPES buffer without supplemental Caz* whereas the second sample was incubated in a solution containing 10 mM Ca". Both samples remained structurally stable after overnight incubation at 37°C, however, due to the gradual dissociation ofthe ionic cross-linking, the sample incubated in the absence of Caz* swelled about 150% compared to the sample that retained the Ca". Samples not treated with PGA dissolved during the incubation. The dual ionic and covalent and cross-linking thus allow for both maintaining long-term structural stability and dynamic modulation of the shape/swelling of the printed structures. To further demonstrate the role of the disulfide cross-links on the stability ofthe hydrogels, the printed structures were exposed to glutathione (GSH). GSH is the most abundant low molecular weight thiol produced by cells and is critical for maintaining redox homeostasis and protecting cells from oxidative stress. Printed structures exposed to a buffer containing 5 mM GSH but no Caz* rapidly disintegrated and were completely dissolved in less than 30 minutes. [00119] Post-printing functionalization Due to steric constraints, not all deprotected thiols will immediately oxidize and contribute to the cross-linking of the hydrogels. Remaining free thiols are thus available for further functionalization using thiol reactive molecules. To investigate the possibilities to covalently modify the already printed structures, we subjected them to Cy3-maleimide (Cy3-mal) (Fig. 8) Maleimides rapidly reacts with free thiols by a I\/|ichael addition reaction, resulting in a distinct pink color of the hydrogels due to the conjugated Cy3 dye. Chemical functionalization of hydrogels after bioprinting can allow for tuning ofthe cell microenvironment. id="p-120" id="p-120" id="p-120"

[00120] Self-healing 23 Presence of unreacted thiols in combination with the dynamic nature of the disulfide bonds further allow for dynamic rearrangements in the polymer network. This is well illustrated by the self-healing properties of the hydrogels. Self-healing is the capability of a hydrogel to spontaneously repair the polymer network and reestablish the cross-links after physically breaking and disassembling the hydrogel structure. Two AlgCP hydrogels in two different colors were prepared to facilitate visualization of the healing process. The hydrogels were cut into two halves and then recombined by positioning the two halves in physical contact. After incubation at room temperature for 5 hours the two halves were chemically connected and inseparable as demonstrated by subjecting the materials to lateral strain using two spatulas. [00121] Cross-linking using multi-arm PEG-maleimide ln addition to formation of disulfide bridges, thiol-containing polymers can also be cross-linked using thiol-reactive cross-linkers. Maleimides react specifically with thiols at pH 6.5-7.5, resulting in a stable, non-reversible, thioether linkage. Possibilities to form hydrogels by combining PGA deprotection of AlgCP and addition of multi-arm PEG-maleimide (4-arm PEG- maleimide, p(I\/|al)4) was explored (Fig. 9). No increase in storage and loss modules were seen prior addition of PGA in the presence of p(I\/|al)4, confirming that without enzymatic deprotection ofthe thiol groups, AlgCP did not react with the maleimide cross-linkers. While upon addition of PGA to the solution a fast increase in the measured modulus was recorded. As the reaction between maleimide and free thiols is faster than thiol oxidation, the gelation kinetics in the presence of p(I\/|al)4 was much faster compared to cross-linking by formation of disulfides after addition of PGA (Fig. 10). lncreasing the amount of p(I\/|al)4 relative to protected thiols resulted in stiffer hydrogels due to a higher cross-linking density (Fig. 11). We also showed that the thiol-based enzymatically activated crosslinking can be used to form poly(ethylene glycol) (PEG) hydrogels using 8-arm-PEG-CysPhacm and 8-arm-PEG-maleimide. The two molecules had the same PEG chain molecular weight (xx Da) and the ratio of maleimide to Phacm was set to 1. The addition of PGA resulted in an increase in an increase in both storage- and loss modulus over time (Fig. 12).

Conclusions [00122] ln conclusion, we show a novel strategy to trigger covalent cross-linking of cysteine-functionalized alginate (AlgCP) by enzymatic deprotection of a Phacm-protected thiol moieties using penicillin G acylase (PGA). The resulting free thiols can subsequently oxidize, 24 forming disulfide cross-links resulting in gelation of the alginate or be cross-linked using thiol- reactive cross-linkers, such as multi-arm-PEG-maleimide. AlgCP could also be ionically cross- linked using Ca". The ionic and PGA-mediated disulfide cross-linking operates at different time scales where the Ca2+-mediated process occurs within minutes whereas the disulfide bridging is slower and proceeds over several hours. The PGA-triggered disulfide cross-linking was still rapid enough to allow for efficient cell encapsulation while avoiding cell sedimentation, resulting in high viability ofthe cells. The gelation by multi-arm-PEG-maleimides commence rapidly after addition of PGA. As a bioink, AlgCP could be printed both with and without Ca2+, as long as PGA was added during printing to maintain the integrity of the structures. Printed structures were dynamic and addition or removal of Caz* resulted in macroscopic changes in swelling and stiffness. Due to presence of free thiols after PGA treatment, the printed hydrogels could be further functionalized using thiol reactive molecules post printing. The hydrogels were also self-healing due to the dynamic nature of the disulfides in combination with presence of free thiols that could re-establish the cross-linked polymer network after being physically disconnected. id="p-123" id="p-123" id="p-123"

[00123] The dual and dynamic cross-linking of AlgCP controlled by enzymatic deprotection of a Phacm protected thiols provide new means to fabricate hydrogels for a wide range of biomedical applications.

Claims (1)

1.Claims A method of forming a cross-linked hydrogel, comprising: providing a polymer composition comprising polymer molecules provided with thiol- containing moieties, wherein the one or more thiol groups of a thiol-containing moiety are protected with enzyme-responsive thiol protection groups, adding an enzyme to the polymer composition, optionally, activating the enzyme if inactive, the enzyme being configured for enzymatic deprotection of protected thiol groups, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalently cross-linked hyd rogel. The method of claim 1, further comprising adding one or more thiol-reactive species selected from one or more of maleimide, methacrylate, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and alkyne, to the composition. The method of claim 1 or 2, wherein activating an inactive enzyme comprises to add an enzyme activator to the polymer composition. The method of any of claims 1-3, wherein a ratio of thiol-containing moieties per total amount of monomer units building up the polymer molecules in the polymer composition is 1:1,0000 to 1: The method of any of claims 1-4, wherein the polymer composition comprises one or more polymer types selected from polysaccharides, polypeptides, peptides, synthetic polymers or combinations thereof. The method any of claims 1-5, further comprising to add cells into the polymer composition. 28 The method of any of claims 1-6, further comprising a step of forming a shaped matrix from the composition before and/or after adding or activating the enzyme, thereby forming a cross-linked shaped matrix. The method of claim 7, wherein forming the shaped matrix comprises printing the shaped matrix using the polymer composition as/in a printing ink. A hydrogel-forming mixture, comprising: a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thio| groups of a thiol-containing moiety are protected with an enzyme-responsive thio| protection group. The hydrogel-forming mixture of claim 13, further comprising an enzyme, the enzyme being configured to, possibly upon activation thereof, enzymatically deprotect the protected thio| group, thereby enabling thiol-mediated cross-linking of the polymer molecules in the hydrogel-forming mixture, forming a covalently cross-linked hyd rogel. The hydrogel-forming mixture of claim 9 or 10, further comprising one or more thiol- reactive species selected from one or more of maleimide, methacrylate, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and alkyne. The hydrogel-forming mixture of any of claims 9-11, wherein a ratio of thiol-containing moieties per total amount of monomer units building up the polymer molecules in the polymer composition is 1:10,000 to 1:1. The hydrogel-forming mixture of any of claims 9-12, wherein the polymer composition comprises one or more polymer types selected from polysaccharides, polypeptides, peptides, synthetic polymers or combinations thereof. A kit of parts for forming a cross-linked hydrogel, comprising:a polymer composition comprising polymer molecules provided with thiol-containing moieties, wherein the one or more thiol groups of a thiol-containing moiety areprotected with enzyme-responsive thiol protection groups; and an enzyme, the enzyme being configured for enzymatic deprotection of the protected thiol group, thereby enabling thiol-mediated cross-linking of the polymer molecules, forming a covalent cross-linked hydrogel. The kit of parts of claim 14, further comprising an enzyme activator for activating the enzyme if inactive and enabling the thiol-mediated cross-linking ofthe polymer molecules. The kit of parts of claim 14 or 15, further comprising one or more thiol-reactive species selected from one or more of maleimide, methacrylate, haloacetyls, pyridyls disulfides, norbornene, vinyl sulfone, alkene, and alkyne.