Synthetic copolymers with pendent aldehydes and hydrogels thereof

Abstract

The present invention relates to a copolymer according to Formula I comprising a plurality of ionic monomeric units and a plurality of one or more aldehyde monomeric units. In addition, the invention relates to a functionalized copolymer according to Formula III comprising a plurality of functionalized groups. Furthermore, the invention relates to a method of producing the copolymer using a radical polymerisation, preferably RAFT polymerisation, of one or more ionic monomers according to Formula A and one or more aldehyde monomers according to Formula B and an initiator, preferably a chain transfer agent (CTA), according to formula T1-T2. The invention also relates to a dynamically linked hydrogel comprising one or more (functionalized) copolymers and a crosslinking agent and a method of preparing such a hydrogel and a method of controlled degradation thereof, as well as to the use of the copolymer and the hydrogel and a scaffold prepared therewith.

Description

[0001] TITLE Synthetic copolymers with pendent aldehydes and hydrogels thereof

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to copolymers, functionalized copolymers, methods of preparing these copolymers, hydrogels formed with these copolymers, methods of making these hydrogels, use of said copolymers as well as scaffolds prepared from these copolymers and / or hydrogels.

[0004] Background

[0005] Dynamic hydrogels are increasingly attractive in the field of tissue engineering and regenerative medicine for their ability to mimic key mechanical properties of the extracellular matrix (ECM), which covalent hydrogels cannot accomplish. While covalent hydrogels have been successful at mimicking select ECM properties such as stiffness, they fail to capture the time-dependent dynamic characteristics of the ECM including stress relaxation or spatiotemporal ligand presentation. Notably, temporal control over (co-)presentation of biomolecular cues is a key factor in recapitulating native ECM and dictating cell fates. Successful implementation of aldehydes in natural biopolymers has demonstrated their utility; however, natural polymers are heterogeneous, often unstable, and rarely chemically well-defined. Synthetic polymers overcome these drawbacks, but prior art examples of synthetic polymers with pendent aldehyde groups are synthetically challenging, water-insoluble, or contain non- cytocompatible co-monomer residues, precluding biomedical applications.

[0006] There is therefore a need to a new class of synthetic (co-)polymers having pendent aldehyde groups that allow the formation of dynamic hydrogels.

[0007] Objects

[0008] It is an object of the present invention to provide an improved class of synthetic (co)polymers having pendent aldehyde groups that allow the formation of dynamic hydrogels.

[0009] It is a further object of the present invention to provide water-soluble copolymers with a tunable fraction of pendent aldehyde groups, in particular biocompatible copolymers that are able to form dynamic hydrogels displaying native ECM bioactivity and mechanical properties, such as strain-stiffening.

[0010] It is a further object of the present invention to provide powerful synthetic platforms for functionalized synthetic biomaterials.

[0011] The present invention addresses one or more of these needs and achieves one or more of these objects.

[0012] STATEMENT OF THE INVENTION

[0013] In a first aspect, the invention relates to a copolymer according to claim 1. In a second aspect, the invention relates to a functionalized copolymer according to claim 4. In a third aspect, the invention relates to a method of producing the copolymer according to claim 5. In a fourth aspect, the invention relates to a dynamically linked hydrogel according to claim 8. In a fifth aspect, the invention relates to a method of preparing the dynamically linked hydrogel according to claim 11. In a sixth aspect, the invention relates to a method of preparing the functionalized copolymer according to claim 12. In a seventh aspect, the invention relates to a use of the copolymer according to claim 13. In an eighth aspect, the invention relates to a use of the dynamically linked hydrogels according to claim 14. In a ninth embodiment, the invention relates to a scaffold according to claim 15. In a tenth embodiment, the invention relates to a method of controlled hydrogel degradation according to claim 17.

[0014] Corresponding embodiments for the first aspect are also applicable for the other aspects according to the present invention and vice versa.

[0015] DETAILED DESCRIPTION

[0016] The present invention is elucidated below with a detailed description.

[0017] List of definitions

[0018] The following definitions are used in the present description and claims to define the stated subject matter. Other terms not cited below are meant to have the generally accepted meaning in the field. “random copolymer” as used in the present description means: copolymers with a random distribution of monomers throughout the copolymer.

[0019] “gradient copolymer” as used in the present description means: copolymers in which the change in monomer composition is gradual from predominantly one monomeric unit to predominantly the other monomeric unit.

[0020] “block copolymer” as used in the present description means: copolymers in which the change in monomeric composition is abrupt and which blocks of one monomeric unit are alternated with blocks of another monomeric unit.

[0021] “monomer” as used in the present description means the starting material for the preparation of the copolymer.

[0022] “monomeric unit” as used in the present description means the repeating unit in the copolymer that is derived from a specific monomer.

[0023] “plurality” as used in the present description means two or more.

[0024] “initiator” as used in the present description means a molecule that is able to form a radical, which forms the starting point for the generation of the polymer chain. Initiator according to the present invention is meant to also include chain transfer agents.

[0025] “initiator residue” as used in the present description means part of a radical initiator that has been added at both ends of the copolymer, the initiator forms two residues each of which are at opposite ends of the copolymer.

[0026] “chain transfer agent” as used in the present description is a molecule that has a weak chemical bond and facilitates the chain transfer reaction during the controlled radical polymerization.

[0027] “chain transfer residue” as used in the present description means a residue of a chain transfer agent.

[0028] “functionalized copolymer” as used in the present description means a copolymer according to the present invention in which at least part of the pendant aldehyde groups of the aldehyde monomeric units have been functionalised with a functional group. In particular with a functional group that has one aldehyde reactive group.

[0029] “protecting group” or “protective group” as used in the present description is a reversibly formed derivative of an existing functional group in a molecule, in the present case of an aldehyde. “dynamic bond” as used in the present description means a bond that exists in an equilibrium of bound and unbound states, which are constantly and reversibly exchanged. A dynamic bond according to the invention is a reversible bond.

[0030] “dynamically linked hydrogel” as used in the present description means a hydrogel that is linked via dynamic bonds in contrast to covalently bonded hydrogels.

[0031] “crosslinking agent” as used in the present description means an agent or compound that has at least two aldehyde-reactive group and that can form at least two dynamic bonds with two aldehyde groups within the copolymer chain in order to achieve crosslinking. Hydrogel formation is multi-functionalization with a compound that has at least two aldehyde-reactive groups (crosslinker). The crosslinking agent may be bifunctional (having two aldehyde-reactive groups) or multifunctional (have three or more aldehyde-reactive groups).

[0032] “scaffolds” as used in the present description means a construct or 3D structure prepared from the hydrogel or copolymer of the present invention.

[0033] “hydrogel degradation” as used in the present description means decrosslinking of the dynamic hydrogel meaning that the crosslinking agent is partly displaced or removed in order to at least partly return the hydrogel into the copolymer. This can for example be done using a competing agent or competitive agent.

[0034] “competing agent” or “competitive agent” or “agent that competes” according to the present description means an agent that competes with the binding of the crosslinking agent, in other words has a stronger binding to the pendant aldehyde groups than the crosslinking agent.

[0035] Brief description of drawings

[0036] The present invention is described hereinafter with reference to the accompanying drawings in which embodiments of the present invention are shown and in which like reference numbers indicate the same or similar elements.

[0037] Figure 1A: general chemical structure of the copolymer; Figure 1 B: chemical structure of a specific embodiment of the copolymer and Figure 1C: schematic representation thereof;

[0038] Figure 2A: equilibrium reaction equation when functionalizing the copolymer; Figure 2B schematic representation of functionalized copolymer; Figure 3A: equilibrium reaction equations when functionalizing the copolymer with two different functional groups; Figure 3B schematic representation of functionalized copolymer;

[0039] Figure 4A: shear storage moduli of crosslinked copolymers; Figure 4B normalized differential modulus versus stress of crosslinked copolymers, showing strain-stiffening behaviour;

[0040] Figure 5A: microfluidic printing of a scaffold according to the invention; Figures 5B and 5C scaffolds according to the invention.

[0041] Figure 6 shows a schematic representation of the multiple functionalization of the copolymer;

[0042] Figure 7 shows a series of photographs of hydrogels and degradation thereof;

[0043] Figure 8 shows immunofluorescence staining of human dermal fibroblasts (HDFs) seeded on hydrogels.

[0044] Each aspect of the present invention is elucidated below with a detailed description.

[0045] Copolymers

[0046] In a first aspect, the present invention relates to a copolymer according to Formula I

[0047] Formula I comprising a plurality of ionic monomeric units and a plurality of one or more aldehyde monomeric units, wherein:

[0048] Ti and T2 are initiator residues, for example chain transfer residues, wherein in each of the one or more monomeric units R1 and R2 are each is individually selected from hydrogen and methyl, Xi and X2 are individually selected from O and NH, Z1 and Z2 are each individually a spacer group, preferably an alkyl group or heteroalkyl group, preferably having between 1 and 8 carbon atoms, more preferably between 2 and 5 carbon atoms, W is an ionic group, preferably an anionic group, wherein n is the total number of ionic units in the copolymer and is an integer between 2 and 900, preferably between 40 and 500, such as between 100 and 275, and m is the total number of aldehyde units in the copolymer and is an integer between 2 and 750, preferably between 40 and 500, such as between 85 and 250.

[0049] The present copolymer has a plurality of (an)ionic monomeric units and a plurality of aldehyde monomeric units. The copolymer may be any type of copolymer, such as a random copolymer, a gradient copolymer, or a block copolymer.

[0050] One of the monomeric units forming the copolymer has a pendant aldehyde group. Said monomeric unit may be an acrylate (R2 = hydrogen) or a methacrylate (R2 = methyl (-CH3). Said monomeric unit may have either an amide group (X2 = NH) or an ester group (X2 = O). A combination of two or more different types of aldehyde monomeric units may be used in the present invention in combination with one or more of the (an)ionic monomeric units discussed below, to form the copolymer according to the present invention. Z2 is a spacer group that may be an alkyl group, e.g. denoted by (CH2)qin which q is an integer between 1 and 8, preferably between 2 and 4. Z2 can also be an ethylene glycol moiety denoted by (CH2CH2O)t wherein t is an integer between 1 and 8, a cyclic alkane (CnH2n), or an aromatic-based spacer.

[0051] Specific examples of suitable aldehyde monomeric units are disclosed in Table 1 below.

[0052] Table 1 : Specific examples of suitable aldehyde monomeric units

[0053] The ionic monomeric unit may for example comprise an anionic group W (e.g. a sulphate or phosphate group with a cationic counter ion such as an alkali or alkali earth ion, e.g. potassium (K+) or sodium (Na+)) and a neutral spacer Zi (e.g. an alkyl spacer such as (CH2)P). The ionic monomeric unit may for example also be zwitterionic, e.g. it may comprise an anionic group W (e.g. a sulphate or phosphate group) with a cationic spacer Zi (e.g. a quaternary ammonium alkyl spacer), such as a sulfobetaine such as [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide. The ionic monomeric unit may for example also be cationic, e.g. a quaternary ammonium or choline group. The W group may also be a deprotonated acid end group (-COO-) in which case the spacer may for example be a polyethylene glycol (CH2CH2O). Other examples of zwitterionic monomeric units include 2-methacryloyloxyethyl phosphorylcholine (MPC) having a cationic choline group as W (-NC3H9, where N has a positive charge) and a negatively charged PO4' group in the spacer Zi. The spacer may be an alkyl group, e.g. denoted by (CH2)Pin which p is an integer between 1 and 8, preferably between 2 and 5. Zi can also be a heteroalkyl group, for example a group having the formula (CH2)r-X3-(CH2)swherein r and s are each independently an integer between 1 and 12, such as between 2 and 6, and wherein X3is a ionic group, for example a quaternary ammonium (e.g. -N+(CH3)2) or a phosphate group (e.g. -PCU'). Examples of such groups ((CH2)r-X3-(CH2)s) are sulfobetaines having a sulphate group as W and having a quaternary ammonium group as X3and 2-methacryloyloxyethyl phosphorylcholine (MPC) having a -N+(CH3)3 group as W and having a phosphate group (-PO4"), as X3group.

[0054] Said monomeric unit may be an acrylate / acrylamide (Ri = hydrogen) or a methacrylate / methacrylamide (Ri = methyl (-CH3)). Said monomeric unit may have either have an amide group (Xi = NH) or an ester group (Xi = O). A combination of two or more different types of ionic monomeric units may be used in the present invention in combination with one or more of the aldehyde monomeric unit discussed above to form the copolymer according to the present invention.

[0055] Specific examples of suitable ionic monomeric units are disclosed in Table 2 below

[0056] Table 2: Specific examples of suitable ionic monomeric units

[0057] The present copolymer has a plurality of aldehyde monomeric units (denoted by m) and a plurality of ionic monomeric units (denoted by n). The aldehyde monomeric units are present in an amount of between 1 and 99 mol.% of the total monomeric units comprising the copolymer. If less than 1 mol.% of aldehyde is present, there are not sufficient pendant aldehyde groups present for functionalization or crosslinking. If more than 99 mol.% of aldehyde groups are present, the water solubility is insufficient to form a hydrogel. In an embodiment, one type of aldehyde monomeric units is present. In another embodiment, two or more types of aldehyde monomeric units are present.

[0058] The ionic monomeric units are present in an amount of between 1 and 99 mol.% of the total monomeric units comprising the copolymer. If less than 1 mol.% of ionic groups are present, the water solubility is insufficient to form a hydrogel. If more than 99 mol.% of ionic groups are present, there are not sufficient pendant aldehyde groups present for functionalization or crosslinking. In an embodiment, one type of ionic monomeric units is present. In another embodiment, two or more types of ionic monomeric units are present.

[0059] The combined molar fractions of the aldehyde monomeric units and the ionic monomeric units is set to 100 mol.% based on the combined fraction of the aldehyde monomeric units and the ionic monomeric units, preferably based on the total molar fractions of all monomeric units (excluding the fraction of the initiator residues).

[0060] In an embodiment, the copolymer comprises between 5 and 95 mol.% of one or more aldehyde monomeric units and between 5 and 95 mol.% of one or more ionic monomeric units. In an embodiment, the copolymer comprises between 10 and 90 mol.% of one or more aldehyde monomeric units and between 10 and 90 mol.% of one or more ionic monomeric units. In an embodiment, the copolymer comprises between 15 and 85 mol.% of one or more aldehyde monomeric units and between 15 and 85 mol.% of one or more ionic monomeric units. In an embodiment, the copolymer comprises between 20 and 80 mol.% of one or more aldehyde monomeric units and between 20 and 80 mol.% of one or more ionic monomeric units. In an embodiment, the copolymer comprises between 25 and 75 mol.% of one or more aldehyde monomeric units and between 25 and 75 mol.% of one or more ionic monomeric units. In an embodiment, the copolymer comprises between 30 and 70 mol.% of one or more aldehyde monomeric units and between 30 and 70 mol.% of one or more ionic monomeric units. In an embodiment, the copolymer comprises between 40 and 60 mol.%, such as between 45 and 55 mol.% or even 50 mol.%, of one or more aldehyde monomeric units and between 40 and 60 mol.% such as between 45 and 55 mol.% or even 50 mol.%, of one or more ionic monomeric units.

[0061] In an embodiment, the copolymer is a RAFT copolymer, being a copolymer produced by RAFT polymerization.

[0062] In an embodiment, the copolymer has a dispersity (£>) of between 1.1 and 1.3. Dispersity (£>) is used as a measure of broadness of molecular weight distribution. The larger the £>, the broader the molecular weight. £> of a polymer is calculated as the ratio of the weight average to number average molecular weight.

[0063] In an embodiment, the number average molecular weight ( / Wn) is between 1 kg / mole and 200 kg / mole, such as between 10 and 60 kg / mole.

[0064] In a specific embodiment, the present invention relates to a copolymer prepared from a protected aldehyde monomer / V-(3,3-diethoxypropyl)-methacrylamide (DEPMAm) and the ionic monomer 3-sulfopropyl methacrylate potassium salt (SM). The present inventors have used RAFT polymerization to obtain gradient copolymers with a tunable pendent aldehyde fraction, depending on the monomer feed ratio.

[0065] In this specific embodiment, the copolymer is according to formula II:

[0066]

[0067] Formula II

[0068] While the aldehyde group is protected, DEPMAm had limited aqueous solubility, potentially precluding the formation of a water-soluble polymeric intermediate. The incorporation of a highly polar, charged co-monomer is used to ensure aqueous solubility of the resulting copolymer. To this end, the present inventors have selected 3-sulfopropylmethacrylate (SM) as the co-monomer for this copolymerization. Moreover, as the copolymers are intended to be used for tissue engineering applications, sulfonate groups have been shown to possess useful biological functions. For example, they displayed strong affinity for ECM biopolymers such as fibronection or collagen II, and contributed to the generation of a lubricating surface, which is beneficial for articular cartilage applications. In addition, synthetic sulfonates have also been shown to sequester growth factors, such as transforming growth factor pi or bone morphogenic protein-4, which are known to affect cellular differentiation.

[0069] In an embodiment, T1 is

[0070] More options for initiators T1-T2 will be discussed below. Figure 1A shows a representation of the general chemical structure of the copolymer according to the present invention, wherein Figure 1 B shows a specific copolymer according to the first aspect of the invention. Figure 1C shows a schematic representation thereof that is applicable to the structure of Figure 1A as well as the structure of Figure 1 B with the backbone as a dark grey thick line and the pendant aldehyde groups as light grey arrows. The copolymer backbone is for the purposes of schematic representation is denoted as “COPOL”.

[0071] By strategically selecting a combination of aldehyde and ionic monomers the present inventors have ensured that both aqueous solubility as well as strategic reactivity is obtained.

[0072] These synthetic copolymers were found to display excellent cytocompatibility - a prerequisite for biomaterials applications. Due to the straightforward synthetic procedure, the present invention provides a novel synthetic platform.

[0073] Functionalized copolymers

[0074] In a second aspect, the present invention relates to a functionalized copolymer according to Formula III

[0075] Formula III wherein Ti, T2, R1, R2, Xi, X2, Z1, Z2, W, n, and m are as in claim 1 and wherein Y is selected from the group consisting of O, NH, NH-(C=O), NH-(C=O)- NH, NH-(C=S)-NH wherein -R3 is a functional group, preferably selected from the group consisting of peptides, fluorophores, drugs and one or more combination thereof, and wherein s is the number of functionalized monomeric units and is an integer between 1 and m, in the latter case if s=m, there are no aldehyde monomeric units since m-s equals zero.

[0076] To highlight the versatility of the present synthetic copolymer for a synthetic platform as a biomaterial, the present inventors have tested the functionalization. To functionalize (decorate) the copolymer aminooxy-functionalized ligands were used.

[0077] The invention also relates to a method of preparing a functionalized copolymer comprising reacting the copolymer according to the invention and a functionalized ligand according to Formula VI in an aqueous solution. In Formula VI, R3 is a functional group as defined above and Rs is a discussed below for the crosslinker according to Formula IV or V. Q is a spacer and can be determined by a person skilled in the art depending on the type of functional group (R3) that is used. Formula VI is shown below.

[0078] The copolymer according to the present invention in which at least part of the pendant aldehyde groups of the aldehyde monomeric units have been functionalised with a functional group is called a functionalized copolymer. In an embodiment, at least 1 %, preferably at least 10%, such as at least 40% of the aldehyde groups have been functionalized. One or more functional groups may be used to functionalize the copolymer, in other words, the functionalized copolymer can have a mixture of different functionalized groups. The level of functionalization depends on the use. In case the copolymer is intended for use in preparing of a hydrogel, there need to be sufficient aldehyde groups remaining for the hydrogel formation. In essence, the hydrogel formation is also functionalization but with a compound that has at least two aldehydereactive group (crosslinker).

[0079] In a specific embodiment, the present inventors have tested the functionalization of the copolymer according to the first aspect of the invention. In order to do so they have added a hydrazide compound (R3CONHNH2) to the copolymer with pendent aldehyde groups. Figure 2A shows a reaction equation of the equilibrium that occurs when the copolymer (denoted below as “COPOL-C=O” to show the one of the pluralities of the pendent aldehyde groups) is mixed with a hydrazide compound. Figure 2B shows a schematic representation of a functionalized copolymer according to the second aspect of the present invention.

[0080] The present inventors have prepared copolymers that comprise free aldehyde groups for rapid, dynamic crosslinking to functional groups, while using the highly charged ionic groups to ensure aqueous solubility.

[0081] To demonstrate the dynamic behaviour of the present functionalized copolymers, the present inventors have reviewed the displacement of the hydrazine with an aminooxy compound (R3ONH2). Figure 3A shows a reaction equation of the equilibrium that occurs when an aminooxy compound is added to the functionalized copolymer discussed above in Figure 2. Figure 3B shows a schematic representation of functionalized copolymer according to the second aspect of the present invention, in this specific functionalized copolymer two functional groups are present; when aminooxy group is added, this binds more strongly than the hydrazide groups and the hydrazide groups will be replace by the aminooxy group. This reversible nature of these two dynamic covalent bonds was demonstrated by competitive ligand displacement via fluorescence resonance energy transfer (FRET).

[0082] Method of preparing (functionalized) copolymers

[0083] In a third aspect, the present invention relates to a method of producing said copolymers. Said method comprises a radical polymerisation, preferably RAFT polymerisation, of one or more ionic monomers according to Formula A and one or more aldehyde monomers according to Formula B and an initiator, preferably a chain transfer agent (CTA), according to formula T1-T2

[0084] Formula A Formula B wherein for each monomer Ri, R2, Xi, X2, W, and Z1 and Z2 are individually selected and are as show above and wherein OP is an aldehyde protected by a protecting group.

[0085] One of the monomers used in the preparation of the copolymers according to the present invention is a protected aldehyde monomer. For example, wherein OP is

[0086] Type of polymerization

[0087] The present copolymer may be synthesized by conventional polymerization techniques, preferably radical polymerization, preferably controlled radical polymerization (e.g. Atom Transfer Radical Polymerization (ATRP), Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization, and Nitroxide- mediated Polymerization (NMP), more preferably RAFT polymerization, which is type of chain-growth polymerization in which a chain-transfer agent is used. In an embodiment, the copolymer is produced by RAFT polymerization.

[0088] Initiator

[0089] The copolymer according to the present invention is prepared using an initiator, T1-T2, that in the case of RAFT polymerization is a chain transfer agent (OTA). After polymerization the initiator and / or the CTA ends up as to residues (T1- and -T2) in the copolymer, these initiator residues cap the ends of the copolymer. T1 and / or T2 can also be -H or -CH3, for example in case of free radical polymerization.

[0090] In an embodiment in which the radical polymerization is a RAFT polymerization and a CTA is used, it is preferred that the CTA is a (meth)acrylate or (meth)acrylamide compatible CTA. Examples are dithiobenzoates, trithiocarbonates, dithiocarbates and xanthates. A person skilled in the art will be able to determine the best selection for this. Options for radical initiators are for example azobisisobutyronitrile (abbreviated Al BN), 2,2-azobis(2-methylpropionamidine) (V-50), and 4,4'-azobis(4-cyanopentanoic acid) (AC PA).

[0091] Solvent

[0092] Examples of suitable solvents used during the polymerization are mixture of water and polar aprotic solvents that are miscible with water (e.g. 1 ,4-dioxane). These may be mixed in a ratio to ensure solubility of the monomers as well as the forming copolymer during the complete reaction process, which can be determined by a skilled person. Polymerisation temperatures and times can be selected by a person skilled in the art depending on the type and concentration of the monomers used, the temperature, solvent, chain transfer agent, initiator.

[0093] Hydrogels

[0094] In a fourth aspect, the present invention relates to a dynamically linked hydrogel comprising one or more copolymers according to the invention and / or one or more functionalized copolymers according to the invention and a crosslinking agent, preferably wherein the crosslinking agent is selected from the group consisting of aminooxy, (thio)semicarbazides, hydrazides, hydrazines, and amines, preferably the crosslinking agent is a bifunctional crosslinking agent or a multifunctional crosslinking agent, more preferably a dihydrazide. In an embodiment, the crosslinking agent is according to Formula IV or V below wherein each Rs is individually selected from the groups below, preferably both Rs groups are the same. In the drawing below also Formula VI is shown, which is discussed above for the functionalization of copolymers. R3 is as discussed above in the section of the functionalized copolymer.

[0095] Formula VI wherein v is an integer between 15 and 900, preferably 100 and 455, and wherein w is an integer between 1 and 6, preferably 2 and 4.

[0096] In an embodiment, the hydrogel has critical strain (ac) of between 1 and 5,000 Pascal, preferably between 10 and 2,000 Pascal.

[0097] The copolymers according to the present invention were found by the present inventors to crosslink rapidly. Several embodiments were in addition found to exhibit strain-stiffening behaviour.

[0098] The present inventors have shown that the hydrogels of the present invention have fast gelation kinetics. The present inventors have prepared hydrogels with hydrogel stiffness ranging from about 2 to 20 kPa. In addition, the inventors were able to tune the onset of strain-stiffening towards a biologically relevant regime (oc~ 10 Pa). Method of preparing dynamic hydrogels

[0099] In a fifth aspect, the present invention relates to a method of preparing a dynamically linked hydrogel, comprising reacting the copolymer according to the invention and / or the functionalized copolymer according to the invention and a (dynamic) crosslinking agent in an aqueous solution.

[0100] Hydrogels may be formed in aqueous solutions, such as in water or in buffers, for example phosphate buffered saline (PBS), e.g. at pH 7.4. Hydrogels may be formed with different copolymer concentrations, such as between 0.5 and 15 wt.%, e.g. between 1 and 10 wt.%, such as between 2 and 6 wt.%.

[0101] The ratio between crosslinker and aldehyde groups (in particular on a nonfunctionalized copolymer) may vary between 0.1 and 1.5 equivalent of crosslinker per equivalent of aldehyde, preferably between 0.2 and 0.9 such as between 0.4 and 0.6, e.g. approximately 1 equivalent meaning that for each aldehyde group there is one reactive group for crosslinker. With equivalent of crosslinker is used equivalent of reactive groups. In case a bifunctional crosslinker is used, each crosslinker had two reactive groups. The ratio of aldehyde and crosslinker on a partly functionalized copolymer can be adapted by a person skilled in the art, depending on the amount of functionalization, viz. depending on the number of aldehyde groups that were no longer available for crosslinking.

[0102] The gelation time of the hydrogel can be between 1 and 90 seconds, such as between 1 and 60 seconds, such as between 1 and 10 seconds.

[0103] Use of copolymers and hydrogels

[0104] In another aspect, the present invention relates to a use of the present copolymers and / or functionalized copolymers and crosslinking agent for the preparation of scaffolds, preferably by printing, or for delivery of active molecules.

[0105] In addition, the present invention relates to the use of the dynamically linked hydrogels for the preparation of scaffolds or for delivery of active molecules. The use of a dynamic crosslinker is important to the preparation of dynamic hydrogels. The behaviour and properties of dynamic hydrogels may be modulated - without changing systemic conditions such as the mass content, pH, or temperature - by the addition of a competitive agent, and / or the chemical composition of the reactive group of the dynamic crosslinker thereby altering the binding equilibrium and distribution of bound (crosslinked) and unbound (non-crosslinked) states. In an example, not limiting to the present invention, a hydrazone is used as the crosslinker and as competitive agent an oxime functionalized compound is used; in the controlled degradation two functions are present, namely the oxime and hydrazone. It is possible to use mixed functions, e.g. hydrazone and oxime or other mixtures, for crosslinking in the hydrogel system of the present invention.

[0106] The present invention also demonstrates that the reversibility of the dynamic bond in the described dynamic hydrogels enables the controlled degradation of a formed hydrogel.

[0107] In another aspect, a plurality of dynamic crosslinkers or combinations of dynamic crosslinker and competitive agent may be used, respecting the molar ratio of functions outlined in the method of preparing dynamic hydrogels, to modulate the initial mechanical properties of the resulting hydrogel and the rate of degradation upon addition of an excess of a competitive agent. The present inventors have prepared hydrogels comprising both a competitive agent and a crosslinker and have observed a clear difference in degradation rates / stability, namely an increased rate of hydrogel degradation (from weeks to hours) upon addition of the competitive agent.

[0108] In yet another aspect, the functionality, more specifically the valency, of the dynamic crosslinker may be varied to alter the mechanical properties (such as the rigidity or strain-stiffening response) of a resulting dynamic hydrogel independently from the aforementioned systemic conditions or molar ratio of crosslinker and aldehyde functions.

[0109] Scaffold

[0110] In yet another aspect, the present invention relates to a scaffold prepared of said hydrogels and / or copolymers. The scaffold, preferably a fiber, may obtained by printing and crosslinking one or more copolymers according to the invention and / or one or more functionalized copolymers according to the invention and at least one crosslinking agent, preferably using a microfluidic device.

[0111] The scaffold may comprise a plurality of cells, such as for example human dermal fibroblast and human mesenchymal stem cells, or cell clusters comprising a plurality of multiple cell types such as aggregates and organoids. However, other types of cells can also be provided.

[0112] Hydrogels may be formed into scaffolds after or during the formation of the hydrogel. Hydrogel formation may also be done concurrently with the formation of the scaffold. The present copolymers have a rapid crosslinking times so that they are suitable for use in a microfluidic bioprinter where in situ hydrogels are formed. In this technique, copolymer and crosslinker solutions meet just before entering the printing nozzle, and must crosslink during their short residence time in the nozzle in order to form a printed fiber.

[0113] Method of controlled hydrogel degradation

[0114] In another aspect, the present invention relates to a method of controlled hydrogel degradation of a dynamically linked hydrogel according to invention or prepared according to a method of the invention adding an agent that competes with the dynamic bonding of the crosslinking agent thereby replacing at least part of the crosslinking agent to degrade said hydrogel. The competitive agent is a monofunctional agent, and thus unable to independently form dynamic crosslinks, but binding to the pendant aldehyde groups of the copolymer thereby blocking these functional groups for reaction with a difunctional crosslinker. In other words, the present invention relates to a method of controlled hydrogel degradation of a dynamically linked hydrogel prepared according to the present invention by adding a competitive agent, being an agent that competes with the dynamic bonding of the crosslinking agent, said competitive agent binding to the pendant aldehyde groups thereby replacing at least part of the crosslinking agent to degrade said hydrogel. The competitive agent may be represented by a dynamically binding group with a stronger binding affinity for the pendant aldehyde groups, thus displacing the crosslink, or by an irreversible binding to the aldehyde group and displacement of the crosslinker. In an embodiment as competitive agent a dynamically binding group with a much higher binding affinity is used. In an embodiment, an irreversibly binding competing agent is not used. The present invention demonstrates that the reversibility of the dynamic bond in the dynamic hydrogels enables the controlled degradation of a formed hydrogel. As shown above, the inventors have provided evidence that displacement of one compound by another compound is possible.

[0115] Since it was established that cells are viable and that hydrogels can be degraded, the present inventors believe that the scaffolds of the invention can release cells on demand for future applications in cell-delivery technologies.

[0116] In an embodiment, the method is carried out at a physiological pH. A physiological pH is required for specific applications that are desired, such as biomedical applications wherein the pH may be dictated by a target tissue around the site of application / use. In an embodiment, the method is carried out at a pH of between 6.2 and 7.8, preferably between 7.2 and 7.6. The term "physiological pH" refers to the pH ranges typically found in biological systems, particularly within bodily fluids and tissues under normal conditions. For the purposes of this invention, physiological pH is defined as ranging from approximately 6.2 to 7.8, encompassing the pH of blood (7.35-7.45), interstitial fluid (approx. 7.4), intracellular fluid (approx. 7.2), mucosal surfaces (approx. 6.2 to 7.4) and other relevant biological environments. This range ensures compatibility with biological systems, minimizing adverse effects on cellular function and biochemical processes. Maintaining pH within this range is critical for enzymatic activity, molecular stability, and overall physiological homeostasis.

[0117] In an embodiment, the competitive agent may be selected from the group consisting of mono-reactive compounds having one aminooxy, (thio)semicarbazide, hydrazide, hydrazine, or amine group, preferably an aminooxy group. An example of a competitive agent is an aminooxy compound (R3ONH2) wherein R3 is as disclosed above. A person skilled in the art will be able to select a competitive agent based on the binding strength compared to the crosslinking agent used.

[0118] The combination of highly tunable composition, stiffness, and strain-stiffening, in conjunction with temporal control over ligand exchange / presentation, position the present cytocompatible copolymers as a powerful synthetic platform for the rational design of next generation synthetic biomaterials.

[0119] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. The scope of the present invention is defined by the appended claims. One or more of the objects of the invention are achieved by the appended claims.

[0120] EXAMPLES

[0121] The present invention is further elucidated based on the examples below which are illustrative only and not considered limiting to the present invention.

[0122] Materials

[0123] All chemicals were purchased from commercial suppliers and used as received, unless otherwise specified.

[0124] Synthesis of N-(3,3-diethoxypropyl)-methacrylamide (DEPMAm)

[0125] A protocol reported by Wang et al. (Polyion Complex-Templated Synthesis of Cross- Linked Single-Enzyme Nanoparticles. Macromolecules 2020, 53 (13), 5487-5496) was adapted for the synthesis of DEPMAm. Glassware used for the reaction was oven- dried prior to use. In a dry N2 atmosphere, methacryloyl chloride (5.5 mL, 56 mmol, 1.1 equivalent) was added dropwise to a pre-cooled solution of 1-amino-3,3- diethoxypropane (8.2 mL, 51 mmol, 1.0 equivalent) and triethylamine (7.8 mL, 56 mmol, 1.1 equivalent) in 110 mL anhydrous dichloromethane (DCM) at 0 °C. The reaction was left to stir for 3 hours; the ice-salt bath was not replaced during this time. The crude reaction mixture was washed with i) 100 mL of 0.1 mol / dm3HCI; ii) twice with 100 mL of saturated NaHCOs; iii) twice with 100 mL of H2O; and iv) twice with 100 mL of brine. After drying over anhydrous MgSCU, the DCM was removed under reduced pressure to yield a transparent yellow oil. This oil was passed through a silica plug (approx. 22 g silica) using approx. 300 mL ethyl acetate as the mobile phase, followed by rotary evaporation to remove the solvent. This gave a pale yellow oil (9.33 g, 77.4% crude yield, 80% pure).1H NMR (700 MHz, DMSO-cfe) analysis revealed that two major amide impurities are present. The desired methacrylamide was isolated from the crude product by silica gel column chromatography (9: 1 v / v DCM:Acetone, TLC: Rf = 0.39; UV, KMnC approx. 300 g silica and approx. 1 ,000 mL mobile phase) and collected under reduced pressure as a very pale yellow oil. Final yield = 4.87 g, 40%.1H NMR (700 MHz, DMSO-cfe, 6 in ppm): 5 1.11 (t, 6H, J = 7.1 Hz, CH2CH3), 1.70 (dt, 2H, J = 5.6, 6.0 Hz, CHCH2CH2), 1.84 (t, 3H, J = 1.1 Hz, C=CCH3), 3.14 (dt, 2H, J = 6.0, 6.0 Hz, CH2NH), 3.43 (dq*, 2H, J = 7.1 Hz, CH3CH2O), 3.56 (dq*. 2H, J = 7.1 Hz, CH3CH2O), 4.51 (t, 1 H, J = 5.6 Hz, CHOO), 5.31 (quint, 1 H, J = 1.5 Hz, C=CHb), 5.62 (t, 1 H, J = 1.1 Hz, C=CHa), 7.85 (t, 1 H, J = 5.2 Hz, NH).13C NMR (700 MHz, DMSO- d6, 6 in ppm): 5 15.2 (CH2CH3), 18.5 (C=CCH3), 33.2 (CHCH2CH2), 35.0 (CH2NH), 39.4 (DMSO), 60.6 (CH2O), 100.6 (CHOO), 118.7 (C=CCH3), 139.9 (C=CCH3), 167.2 (NC=O). ATR-FTIR (neat) cm’1: 3335 (w), 1656 (m), 1616 (m), 1527 (m), 1217 (w), 1128 (s), 1055 (s). Note: Peaks at 3.43 and 3.56 ppm are denoted as double quartets, but these groups are two individual overlapping quartets. Magnetic inequivalence arising from the lack of a symmetric plane splits the two hydrogens on the ethyl ether groups (-OCH2CH3). As result, a total of four different microenvironment are present, leading to four quartets. All quartets couple back to the terminal methyl group (J = 7.1 Hz).

[0126] RAFT (co-) polymerizations

[0127] A series of RAFT (co-)polymerizations were performed using 4-Cyano-4- (phenylcarbonothioylthio)pentanoic acid (CPPA) as the chain transfer agent (T1-T2) and using 4,4'-azobis(4-cyanopentanoic acid) (ACPA) as the initiator. Different ratios of the two monomers were used. The monomers that were used were: 3- sulfopropylmethacrylate potassium salt (SM) (commercially obtained) and A / -(3,3- diethoxypropyl)-methacrylamide (DEPMAm) (prepared as above). The scheme below shows the reaction mechanism.

[0128] SOO corresponds to 0 mol.% of ionic monomer (not according to the invention) and S100 corresponds to 100 mol.% of ionic monomer (not according to the invention). Equiv is the number of equivalents with respect to the CTA. The total monomer concentration, the CTA concentration, and initiator concentration were kept constant at 1.0 mol / dm3, 4.0 mmol / dm3, and 1.1 mmol / dm3, respectively, in all (co-)polymerizations.

[0129] Table 3. compositions and yield for each monomer feed ratio studied in this work.

[0130] SM DEPMAm

[0131] For each reaction, SM was first dissolved in 1.8 mL of 1 : 1 vlv dbhOI ,4-dioxane. After determining the new volume with a micropipette to account for differences in monomer solution density, the SM solution and the DEPMAm were added to a round-bottomed flask. Next, 4.54 mg CPPA (16.2 pmol, 1 equivalent) and 1.23 mg ACPA (4.4 pmol, 0.27 equivalent) were added from stock solutions (1 :1 v / v dH2O:1 ,4-dioxane). The final volume was then adjusted to 4.06 mL with 1 :1 v / v dH2O:1 ,4-dioxane. After bubbling dry N2 gas through the solution for 45 min at RT, the flask was submerged in an oil bath at 70 °C for 200 min while maintaining positive dry N2 pressure. Aliquots (approx. 35 pL) were collected under dry conditions with N2-flushed syringes at t = 20 min, 40 min, 60 min, 90 min, 120 min, and 200 min. The aliquots of the crude reaction mixtures were rapidly cooled in air and immediately diluted in 700 pL DMSO-cfe for NMR and GPC analysis; an exception here was the SM100 reaction, which was insoluble in DMSO-cfe so D2O was used. After 200 min, the remaining volume was transferred to a

[0132] 3.5 kDa MWCO Snakeskin dialysis membrane and dialyzed sequentially against 0.1 mol / dm3HCI, 50 mmol / dm3NaCI (periodically neutralized with saturated NaHCOs), 25 mmol / dm3NaCI, and finally dH2O. The resulting (co-)polymers were collected as pale pink to white fluffy solids (depending on residual CTA content) after lyophilization.

[0133] Free radical polymerization were also tested by the present inventors and found to provide the desired polymer.

[0134] To manage the difference in solubility between the two monomers and growing polymer, the present inventors have used a mixed solvent system (1 :1 v / v dioxane:distilled water (dH2O)).

[0135] The present inventors have observed that S100 homopolymerization (not according to the invention) proceeds with a conversion of >90 % whereas the copolymerization according to the present invention have similar conversions of 59-65 %. T able 4 below shows this and other effects of the variation in feed ratio of the two monomers.

[0136] The present inventors found (aligned with the trend in conversion) a decrease from

[0137] 51.5 kg / mol for S100 to 43.5-34.5 kg / mol for the inventive copolymers. As the proportion of aldehyde monomer in the copolymer feed ratio increases, the final molecular weight of the copolymer slightly decreases, which is consistent with the lower monomer conversion observed for the homopolymerization of the aldehyde monomer compared to the ionic monomer. The dispersity observed is < 1.20 for all copolymers which is consistent with a controlled RAFT polymerization.

[0138] The present inventors have observed that during the polymerization reaction deprotection of the aldehyde protection group was observed, this is an in situ deprotection.

[0139] Table 4. The effect of feed ratio on the composition and molecular weight of pSM and pSM-co-OMAm.

[0140] The feed ratio of the ionic monomer ( / SM) and the aldehyde monomer ( DEP MAm) as well as the conversion of the monomers in percentages were determined using1H NMR (DM SO- de). The feed ratio of the copolymers was determined via1H NMR (700 MHz, DMSO-de) at t = 0 min via the integral ratio of the vinylic protons of the methacrylate (1 H, 6.00-6.05 ppm) to the methacrylate and methacrylamide (1 H, 5.64-5.59 ppm) monomer.

[0141] The product was purified by dialysis. The fraction of incorporated aldehyde units (FAM) was determined by1H NMR (D2O), except for SOO* for which DMSO-cfe was used.1H NMR spectra (700 MHz, D2O) of purified products were used to determine the fraction of aldehyde incorporation as well as the Mn. The incorporated fraction of backbone units containing free aldehyde was determined from the integral ratio of the free and hydrated aldehyde (5.05- 5.18(A*) and 9.67-9.77 ppm (A), respectively) to the 2H of the CH2 moiety adjacent to the sulfate group (3.9-4.4 ppm (a)), according to 2(A+A*) / (2(A+A*) + a).

[0142] The theoretical Mn(Mn, theo) was determined according to [M]OP( / SM- / WSM + fDEPMAm- / WDEPMAm) / [CPPA] + / WCPPA, where [M]o is the initial total monomer concentration, and MSM and / WoEPMAm are the molecular weights of SM and DEPMAm. The conversion (p) was set to 1 , and fSM and fDEPMAm denote the initial feed ratios. The expression (fsM- / WsM + fDEPMAm- / WoEPMAm) represents the feed ratio adjusted average molecular weight of the initial monomer composition.

[0143] The Mnwas also determined using NMR ( / WJI. NMR) from the integral ratio of the 3H of the methyl group to the backbone. The Mnwas also determined using GPC data ( / Wn, GPC) from the crude reaction mixture at t = 200 minutes. In addition, the dispersity was determined from the GPC curves (£>).

[0144] Rheological analysis of copolymer hydrogels crosslinked with PEG-HZ

[0145] The present inventors have explored if hydrogels could be formed with these copolymers via dynamic covalent crosslinking. Two copolymers (S50 and S75) were tested using a bifunctional poly(ethyleneglycol) dihydrazide crosslinker according to formula IV having a Mwof approx. 5 kg / mol, wherein v is approximately 110 and wherein both Rs are (C=O)-NH-NH2

[0146] Both copolymers were formed into hydrogels in phosphate buffered saline (PBS) at pH 7.4 with different concentrations between 2 wt% and 6 wt.%. It was found that all formulations gelled rapidly (<10 s), with the exception of 2 wt.% S75 (60 s). Next, the rheological properties of these hydrogels were reviewed. The inventors realized that attempting to maintain an equimolar relationship between the crosslinker and the aldehyde would lead to large difference in overall mass content between samples since crosslinker becomes a significant contributor. To minimize this, the inventors have kept the total concentration of crosslinker constant (10 mmol / dm3, 2.70 wt.%, 1 equivalent hydrazide with respect to S75 aldehyde concentration at 2 wt.%), while varying either the copolymer concentration or the copolymer type. The inventors have observed that increasing the S75 polymer concentration from 2 wt.% to 3 wt.% decreased the gelation onset time (sudden increase in shear storage modulus (G')) from approximately 60 s to < 8 s. A further increase in concentration to 4 wt.% only slightly increased crosslinking speed. In contrast, maintaining a constant 2 wt.% copolymer concentration, and constant crosslinker concentration, while changing the copolymer composition from S75 to S50 and S25 (and thus increasing the aldehyde concentration) had a dramatic impact on crosslinking kinetics. The S75 took approximately 60 s to begin gelling while both the S50 and S25 had already begun gelling < 8 s. Without wishing to be bound to a particular theory, the present inventors believe that this can be attributed to a higher local concentration of aldehyde, enabling faster formation of a contiguous network.

[0147] The inventors further reviewed the final shear moduli (Figure 4A) and found that an increase in S75 concentration from 2 wt.% to 4 wt.% increased G' from 1.9 kPa to 4.9 kPa. However, keeping the copolymer concentration (and crosslinker) constant while changing the composition had a much smaller impact, with S50 and S25 at 2 wt.% reaching 2.6 kPa and 2.4 kPa respectively; though the absolute value of 2.4 kPa for the S25 should be considered with care as the sample is already under load due to its extremely fast crosslinking. Given that both the mass content and crosslinker concentration are constant, the present inventors anticipate that the final moduli being similar. The difference between 1.9 kPa, 2.6 kPa, and 2.4 kPa may be attributed to small differences in network architecture arising from increased aldehyde concentration. To illustrate the potential range of hydrogel stiffnesses accessible with our copolymer systems, the inventors also prepared a 6 wt.% S75 hydrogel with 1 equivalent (with respect to aldehyde) of crosslinker, resulting in a G' of 18 kPa - an order of magnitude increase in stiffness.

[0148] Rheological measurements were performed using a DHR-2 from TA instruments equipped with a Peltier heating element and solvent trap using a 20 mm cone-plate with an angle of 2.002° at 20 °C. A time sweep was performed for 3600 s to follow crosslinking kinetics at 1 rad / s and 1 % strain, followed by a frequency sweep from 1- 100 rad / s1at 1 % stain, and finally a strain sweep from 0.1-1 ,000 % strain at 1 rad / s. Final shear moduli values were taken as the average value of the plateau moduli during frequency sweeps. The differential modulus (K' = da / d ) was determined from the strain sweeps by taking the derivative of the oscillation stress w.r.t. strain. This stiffening index, m, was given by the slope to a linear fit of Log( ') vs Log(o) (as K' <x <jm) for the final 5 points on the stiffening curve prior to rupture. Similarly, the critical strain (ac) was determined from the intersection of the same linear fit with the plateau modulus.

[0149] Figure 4A shows the shear storage moduli for different copolymers (S75 and S50) at different copolymer concentrations (2 wt.%, 3 wt.%, 4 wt.%, and 6 wt.%) at a fixed crosslinker concentration and average values taken from the frequency sweeps performed after crosslinking were reported as the mean ± standard deviation of 2-3 replicates.

[0150] The present inventors, while studying the rheological responses of the hydrogels, noticed that the hydrogels demonstrated strain-stiffening behaviour. Strain-stiffening is common in natural polymers and a fundamental property of the native ECM, playing a key role in mechanotransduction and consequently cell fate. However, this phenomenon is rare in purely synthetic hydrogel systems. Further tests were carried out to evaluate the strain-stiffening properties by determining the differential modulus as a function of stress for the same data (Figure 4B). The hydrogels were found to possess critical stresses (crc; onset of strain-stiffening; a measure of the sensitivity of the material to external force) ranging from 580 Pa (2 wt.%) to 1740 Pa (4 wt.%), increasing with total mass content (and resulting stiffness). The stiffening parameter (m; a measure of the magnitude of the stiffening response) decreases slightly with increasing mass content (Am = 0.08). In contrast to the mass content, changing the copolymer chain composition at a constant 2 wt.% had only a minor impact on crc(S75:580 Pa; S25:860 Pa) and m (Am = 0.12). Without wishing to be bound to any particular theory, the present inventors believe that these results suggest that the network density is the dominant factor for strain-stiffening in the system.

[0151] Figure 4B shows normalized differential modulus (K' = do / d ) versus stress (o) of copolymer hydrogel formulations uncovered the tunability of the strain-stiffening regime. Reducing the copolymer concentration to 1 wt.% and maintaining equimolar hydrazide dramatically decreases the critical stress to approximately 40 Pa while increasing the stiffening index (m = 0.70). Both ocand m display a linear correlation with total wt.%.

[0152] Microfluidic printing scaffolds hydrogels

[0153] S25 and S75 hydrogels were used to prepare scaffolds using a microfluidic bioprinter (RX1 Aspect Biosystems, Canada). Copolymer solutions of either S75 (8.0 wt.%, [aldehyde] = 40 mmol / dm3) or S25 (9.5 wt.%, [aldehyde] = 280 mmol / dm3) with a homobifunctional poly(ethyleneglycol) dihydrazide crosslinker (PEG-HZ, 13.2 wt.%, [hydrazide] = 50 mmol / dm3; Mw= 5,000 g / mol) crosslinking solution. Both the copolymer as well as crosslinker solutions where loaded into the bioprinter using a DUO CORE-SHELL print head allowing inflow of two materials into the printing nozzle. The S75 solution was in the ‘Core A’ channel and the PEG-HZ was in the ‘Shell A’ channel. A pressure of 40 mbar was applied to both the crosslinker and copolymer solution to enable inflow into the print head. The inventors fabricated a rectangular, single structure (width = 40 mm) using a translation speed of 10 mm / min. The S25 and the PEG-HZ were printed used the syringe pumps to enable inflow into the print head. The ‘Buffer’ channel contained PBS (also during printing of the S75) and flowed at a speed of 100 pL / min, while ‘Core A’ contained the S25 and flowed at a speed of 200 pL / min. Finally, in ‘Shell A’, the PEG-HZ was present, which flowed at 50 pL / min. Despite these relatively high polymer concentrations of S75 and S25, the viscosity of these stock solutions was sufficient low to flow freely through the microfluidic channels of the print head under pressure.

[0154] The interfacial gelation kinetics were suited to extrude stable rigid fibers (Figure 5A, scale bar = 1 mm). Moreover, two simple geometries were successfully fabricated (Figure 5B & 5C). Figure 5B shows a microfluidic print of a donut-shaped fiber using S25 (9.5 wt.%; 280 mmol / dm3aldehyde) and PEG-HZ (13.2 wt.%; 50 mmol / dm3hydrazide) solutions. Scale bar = 1 mm. Figure 5C shows a microfluidic printed square (width = 40 mm) using S75 (8.0 wt.%; 40 mmol / dm3aldehyde) and PEG-HZ (13.2 wt.%; 50 mmol / dm3hydrazide) solutions. Scale bar = 10 mm. Given the relatively large difference in aldehyde concentration between S75 and S25, these results indicated that the presented copolymer platform can function as a robust (bio)ink for this printing technology, and offers an avenue for future research.

[0155] Functionalization of copolymers

[0156] The inventive copolymer with pendent aldehydes provides opportunities to add ligands via bio-orthogonal conjugation, in particular via oxime ligation and dynamic covalent hydrazone crosslinking. The present inventors have prepared several functionalized copolymers using a modular approach with aminooxy-functionalized ligands. The inventors have tested both a sequential, and a one-pot synthetic route to attach a cocktail of aminooxy-functionalized molecules onto the S50 copolymer (Figure 6). As model compounds to demonstrate functionalization, the inventors have selected two commercially available aminooxy-functionalized fluorophores, viz. aminooxy- CF™488A (Ox-CF488, 1) and an aminooxy-CF™640R (Ox-CF640, 2), and an aminooxy-functionalized RGD peptide (Ox-RGD, 3).

[0157] Two experimental sequences were tested: (1) aminooxy-CF™488A (Ox-CF488, Sigma-Aldrich, molecular weight = -766, Aexc= 490, Aem= 515, £ = 70,000), (2) aminooxy-CF™640R (Ox-CF640, Sigma-Aldrich, molecular weight = -1107, AeXc= 642, Aem= 662, £ = 105,000), and (3) aminooxy-RGD (Ox-RGD) and 2-1-3.

[0158] Step 1 . The S50 copolymer was pre-dissolved in 3 mL PBS (pH 5.0) with a final concentration of 25 mg / mL, after correction for the increased density due to the polymer presence (=1.04 g / m3). Then 0.92 mL of copolymer stock solution (22.8 mg, 29.1 pmol of aldehyde groups, 1.0 equivalent) was added into 9.1 mL of PBS (pH 7.4). The final polymer concentration was 2.3 mg / mL. For the reactions with dyes 1 and 2, we added 16.8 pL and 24.3 pL of Ox-CF488 (2.61 mmol / dm3, 4.4- 10’2pmol, 1.5- 103equivalent) and Ox-CF640 (1.81 mmol / dm3, 4.4 10'2pmol, 1.5- 10’3equivalent) stock solutions to the reaction mixtures, respectively. We let the solutions stir at room temperature (RT) for 17 h. Crude mixtures were analyzed via GPC after 1 :1 dilution in 0.1 mol / dm3NaNOs. The solutions were then dialyzed in a 3.5 kDa MWCO Snakeskin dialysis membrane against distilled water (dH2O) with 2-3 bath changes over 48 h, and finally freeze-dried.

[0159] Step 2. S50+1 (16 mg, 20.4 pmol of aldehyde groups) and S50+2 (19 mg, 24.3 pmol of aldehyde groups) were again pre-dissolved in PBS (pH 5.0) and diluted to a final concentration of 2.3 mg / mL in PBS (final pH = 7.4). Subsequently, from the stock solutions, 19.8 pL of Ox-CF640 (4.2 10'2pmol, 1.7- 10'3equivalent) and 16.2 pL of Ox- CF488 (3.5 1 O'2pmol, 1.7- 10’3equivalent) were added, respectively. We again let the solutions stir at RT for 17 h. Before GPC analysis, dialysis and lyophilization were performed as described in step 1.

[0160] Step 3. S50+1-2 (11.8 mg, 15.0 pmol of aldehyde groups) and S50+2-1 (11.6 mg, 14.7 pmol of aldehyde groups) were again pre-dissolved in PBS (pH 5.0) and diluted to a final concentration of 2.3 mg / mL in PBS (final pH = 7.4). Subsequently, we added 37.5 pL and 36.9 pL of Ox-RGD from a 20 mmol / dm3stock solution (0.75 pmol, 0.05 equiv, and 0.74 pmol, 0.05 equiv, respectively) to the solution. We again let the solutions stir at RT for 17 h. Before GPC analysis, dialysis and lyophilization were performed as described in Step 1. The final products were collected as green fluffy solids. Final yields (both S50+1-2-3 and S50+2-1-3) were 9.0 mg (39%). A one-pot simultaneous functionalization was also tested.

[0161] The S50 copolymer was pre-dissolved in PBS (pH 5.0) with a final concentration of 24 mg / mL. Then 0.746 mL of the polymer stock solution (18 mg, 22.9 pmol of aldehyde groups, 1.0 equivalent) was added to 6.95 mL of PBS (final pH = 7.4). The final polymer concentration was 2.3 mg / mL. Subsequently, 15.1 pL of Ox-CF488 (2.6 mmol / dm3, 3.9 10'2pmol, 1.7- 10-3equivalent), 21.8 pL of Ox-CF640 (1.8 mmol / dm3, 3.94 10'2pmol, 1.7- 10’3equivalent), and 197 pL of Ox-RGD (2.0 mmol / dm3, 3.9 10'1pmol, 1.7- 1 O'2equivalent) stock solutions were added. The reaction mixture was left to stir at RT for 17 h. Crude mixtures were analyzed via GPC after 1 :1 dilution in 0.1 mol / dm3NaNOs. The solutions were dialyzed in a 3.5 kDa MWCO Snakeskin dialysis membrane against dH2O with 3 bath changes over 48 h, after which the product was freeze-dried, yielding a green fluffy solid (14.5 mg, 81%).

[0162] Figure 6 shows a schematic representation of these sequential and one-pot reactions.

[0163] The aminooxy-conjugated fluorophores (Ox-CF488, 1 and Ox-CF640, 2), as well as aminooxy-RGD (Ox-RGD, 3) were attached onto S50 via oxime ligation using either a sequential (left side of Figure 6) or one-pot reaction route (right side of Figure 6). The present inventors have observed that both routes lead to multi-functionalized copolymers with a high coupling efficiency (> 80%) with only small differences between either sequential or one-pot routes. These results indicate that this copolymer system is a modular platform that enables facile decoration with multiple ligands depending on desired biological application, while maintaining its ability to form a hydrogel via rapid hydrazide crosslinking. Although a one-pot reaction requires less work to obtain a multi-functionalized product, the sequential pathway shows that addition of another biomolecule is possible at a later stage.

[0164] Controlled hydrogel degradation

[0165] A test was carried out to review if the formed hydrogels could be reversibly formed, in other words, if the formed hydrogels could be degraded after formation. Decrosslinking (degradation) of S25 hydrogel was tested by addition of an excess of a competitive agent, in this case Ox-RGD. Two droplets were prepared of S25 copolymer (3.8 wt.% stock, 111 mmol / dm3aldehydes) and PEG-HZ (5 wt.% stock, [hydrazides] = 19 mmol / dm3hydrazides). Hydrogels (50 pL) were prepared at 1 wt.% S25 with 0.20 equivalent hydrazides and 0.001 equivalent Ox-AL647 (Alexa Fluor™ 647 hydroxylamine (= aminooxy) - used as a colouring agent to clearly see if degradation of the colourless hydrogel has occurred - corresponding to a final dye concentration of 28 pmol / dm3. Once formed, hydrogels were transferred to a glass vial and covered with 800 pL of PBS (left vials in photographs in Figure 7) or with 800 pL of 4.1 pmol / dm3aminooxy-RGD (Ox-RGD, approx. 2 equivalent with respect to aldehydes) as a competitive agent (right vials in photographs in Figure 7). Photos were taken to follow the evolution of each gel at 0 h, 5 h and 24 h. The photograph shows that after 5 hours the hydrogel in a solution comprising a competitive agent already starts to degrade as the hydrogel is reduced in size. After 24 hours the hydrogel is complete degraded in the vial on the right hand side.

[0166] Cell viability

[0167] To investigate the biocompatibility of this scaffolds, the inventors have cultured human dermal fibroblasts (HDFs) on 2 wt.% hydrogels of S75, S50, and S25, maintaining a constant crosslinker concentration of 10 mmol / dm3- corresponding to aldehyde equivalents of 1.0, 0.4 and 0.18 respectively. The inventors also investigated whether the presence of 1.0 mmol / dm3Ox-RGD would facilitate cell adhesion. Staining (calcein-AM / ethidium homodimer-1 for live / dead cells) and fluorescent imaging after

[0168] 20 h showed good cell viability, with no apparent effect of the different chain composition or presence of Ox-RGD (Figure 8). The absence of spreading in viable HDFs is possibly related to interactions between free aldehydes and cell membranes as well as RGD surface density. After culturing HDFs for 3 days on S50 hydrogels 1.0 equivalent of O-ethylhydroxylamine was added as a competitor (competitive agent) to the hydrogels. After 7-9 h, it was observed that the hydrogels de-crosslinked, leading to a release of HDFs to the underlying tissue culture plastic (TCP). The HDFs, seeded on hydrogels containing RGD, were growing on the TCP after 3 days. Notably, without RGD present in the initial hydrogel, HDFs did not survive. Consequently, no cell growth on the TCP was observed, which further confirmed that the HDFs remained more viable in the presence of RGD even though cell spreading was absent.

[0169] HDFs were cultured at 37 °C under a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM, gibco) containing high glucose (4.5 g / dm3) and GlutaMax™, supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin (P / S, Gibco). Cells were passaged at approximately 80% confluence and used between passage number 8-14.

[0170] Human dermal fibroblast viability was tested on S25-S75 hydrogels containing either 0 mmol / dm3or 1 mmol / dm3RGD. Hydrogels were prepared in the same manner as the rheology samples (see above), except that stock solutions of 3.8 wt.% for the S75 ([aldehyde] = 19.6 mmol / dm3) and 10.5 wt.% for the PEG-HZ ([hydrazide] = 39.7 mmol / dm3) were used. The inventors prepared 130 pL hydrogels in Ibidi p-Plate (96 Well Black Glass Bottom): First, 68.2 pL of S25-S75 was loaded in the well. Subsequently, a mixture of 33.6 pL of PEG-HZ and 31.4 pL PBS was added. In the gels containing Ox-RGD (stock solution: 20 mmol / dm3), 5 pL was mixed with PEG-HZ and PBS; the Ox-RGD volume was subtracted from the PBS volume in this mixture. Before cell seeding, the gels were incubated with 130 pL serum-free media supplemented with 1% penicillin / streptomycin (P / S) for 3 h. Then, HDFs (P13) were seeded at a density of 15,000 cells / cm2. After 1 day of culture, media was replaced with a solution (1 : 1 (v / v) full media:PBS) containing 1 pmol / dm3calcein and 2 pmol / dm3ethidium homodimer-1 was incubated for 30 min at 37 °C, shielded from light. Afterwards, the staining solution was replaced with fresh media and the cells were imaged using an inverted fluorescence microscope (Nikon Eclipse Ti-e) equipped with a live-cell incubator.

[0171] Culture of HDFs on top of S50 hydrogels and release to underlying substrate via controlled degradation

[0172] S50 hydrogels (100 pL) comprising 0, 2.5, 5.0, and 10 mmol / dm3were prepared using PEG-HZ as crosslinker in an Ibidi black 96-well flat-bottom plate. Gels were formed by first loading 52.5 pL of the S50 stock solution in the wells. Subsequently, a mixture of PEG-HZ, PBS and Ox-RGD was added and hydrogels were left to form overnight at 4 °C. Prior to cell seeding, hydrogels were pre-incubated with serum-free media for 4 h. human dermal fibroblasts (HDFs, passage 14) were seeded at a density of 15,000 cells / cm2. After 3 days, the morphology of the cells was assessed using brightfield microscopy. After assessing morphology (at day 3), the media was changed. We added 10 pL of a 250 mmol / dm3O-ethylhydroxylamine stock solution (1.0 equivalent w.r.t. aldehydes, 25 mmol / dm3final concentration) and » 90 pL of full medium to de-crosslink the hydrogels. The bottom of the wells were imaged using brightfield microscopy after 3 more days of culture (6 days total).

[0173] After 24 h and 48 h, we determined the lactate dehydrogenase (LDH) activity of the HDFs seeded on the top of the hydrogels according to manufacturer’s protocol of the CyQUANT™ LDH Cytotoxicity Assay Kit (Invitrogen). Briefly, 50 pL of the media was collected and transferred to an Ibidi black 96-well flat-bottom plate. Subsequently, we added 50 pL of the ‘Reaction Mixture’ of the kit to each well and left the plate at RT for 30 min, protected from light. Next, we added 50 pL of ‘Stop Solution’ to each well. Then the absorbance at 490 nm and 680 nm was measured. To determine the LDH activity, the absorbance at 680 nm was subtracted from the absorbance at 490 nm. To calculate the cell viability, the data is corrected for the spontaneous LDH release (HDFs seeded on tissue culture plastic) and the maximum LDH release. The latter is determined by lysing cells seeded on tissue culture plastic by adding 10 pL of 10X lysis buffer to 90 pL of media. Finally, the cell viability was calculated, according to the following formula:

[0174] Cell viability (

[0175] Figure 8 shows live / dead staining of human dermal fibroblasts (HDFs) seeded on S75- S25 hydrogels which shows good cytocompatibility after 1 day. HDFs were seeded on copolymer hydrogels (S75-S25) with (A-C) and without (D-F) 1 .0 mmol / dm3Ox-RGD. Scale bars are 300 pm and 100 pm for the overview and insets, respectively. N = 2.

Claims

ormula IFormula I comprising a plurality of ionic monomeric units and a plurality of one or more aldehyde monomeric units, wherein:Ti and T2 are initiator residues, for example chain transfer residues, wherein in each of the one or more monomeric units R1 and R2 are each is individually selected from hydrogen and methyl, Xi and X2 are individually selected from O and NH, Z1 and Z2 are each individually a spacer group, preferably an alkyl group or heteroalkyl group, preferably having between 1 and 8 carbon atoms, more preferably between 2 and 5 carbon atoms, W is an anionic group, wherein n is the total number of ionic units in the copolymer and is an integer between 2 and 900, preferably between 40 and 500, such as between 100 and 275, and m is the total number of aldehyde units in the copolymer and is an integer between 2 and 750, preferably between 40 and 500, such as between 85 and 250.

2. The copolymer according to claim 1 , according to formula II:

4. A functionalized copolymer according to Formula III10 Formula IIIwherein T1, T2, R1, R2, Xi, X2, Z1, Z2, W, n, and m are as in claim 1 and wherein Y is selected from the group consisting of O, NH, NH-(C=O), NH-(C=O)-NH, NH-(C=S)-NH wherein -R3 is a functional group, preferably selected from the group consisting of peptides, fluorophores, and drugs or a combination thereof.

5. A method of producing the copolymer according to any one of the claims 1-3, comprising polymerization, preferably radical polymerization, more preferably controlled radical polymerization, Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization, and Nitroxide-mediated Polymerization (NMP), more preferably RAFT polymerisation, of one or more ionic monomers according to Formula A and one or more aldehyde monomers according to Formula B and an initiator, preferably a chain transfer agent (CTA), according to formula T1-T2Formula A Formula B wherein for each monomer R1, R2, Xi, X2, W, Z1 and Z2 are individually selected and are according to claim 1 and wherein OP is an aldehyde protected by a protecting group.

6. The method according to claim 5, wherein OP is7. The method according to claim 5 or 6, wherein T1-T2 is8. A dynamically linked hydrogel comprising one or more copolymers according to any one of claims 1-4 and / or one or more functionalized copolymers according to claim 5 and a crosslinking agent, preferably wherein the crosslinking agent is selected from the group consisting of aminooxy, (thio)semicarbazides, hydrazides, hydrazines, and amines, preferably the crosslinking agent is a bifunctional crosslinking agent or a multifunctional crosslinking agent, more preferably a dihydrazide.

9. The dynamically linked hydrogel according to claim 8, comprising a crosslinking agent according to Formula IV or V below wherein each Rs is individually selected from the groups below, preferably both R5 groups are the sameFormula V wherein v is an integer between 15 and 900, preferably 100 and 455, and wherein w is an integer between 1 and 6, preferably 2 and 4.

10. The dynamically linked hydrogel according to claim 8 or 9, having a critical strain (ac), determined as disclosed in the description, of between 1 and 5,000 Pascal, preferably between 10 and 2,000 Pascal.

11. A method of preparing the dynamically linked hydrogel according to claim 8-10, comprising reacting the copolymer according to any one of claims 1-3 or the functionalized copolymer according to claim 4 and a crosslinking agent in an aqueous solution.

12. A method of preparing the functionalized copolymer according to claim 4, comprising reacting the copolymer according to any one of claims 1-3 and a functionalized ligand according to Formula VI in an aqueous solutionFormula VI wherein R3 is a functional group as defined in claim 4 and Rs is as discussed in claim 9.

13. A use of one or more copolymers according to any one of claims 1-4 and / or one or more functionalized copolymers according to claim 5 and a crosslinking agent for the preparation of scaffolds, preferably by printing, or for delivery of active molecules.

14. A use of one or more dynamically linked hydrogels according to any one of claims 8-10 for the preparation of scaffolds or for delivery of active molecules.

15. A scaffold, preferably a fiber, obtained by printing and crosslinking one or more copolymers according to any one of claims 1-3 and / or one or more functionalized copolymers according to claim 4 and at least one crosslinking agent, preferably using a microfluidic device.

16. The scaffold according to claim 15 comprising a plurality of cells.

17. A method of controlled hydrogel degradation of a dynamically linked hydrogel prepared according to any one of claims 8-10 or prepared according to a method of claim 11 by adding a competitive agent, being an agent that competes with the dynamic bonding of the crosslinking agent, said competitive agent binding to the pendant aldehyde groups thereby replacing at least part of the crosslinking agent to degrade said hydrogel.

18. The method of claim 17, wherein the method is carried out at a pH of between 6.2 and 7.8, preferably between 7.2 and 7.6.

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