Click chemistry polymer hydrogels

Abstract

There is provided a polymer system for producing a hydrogel. The system has two components. A first component comprising a first synthetic polymer and a gel former and a second component comprising a second synthetic polymer. The first synthetic polymer is functionalized with a first click chemistry group and the second synthetic polymer is functionalized with a second click chemistry group that reacts with the first click chemistry group to form a covalent link by click chemistry. One of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, an oxime, a nitrone or nitrile-oxide. The gel former has a gelation mechanism that is independent from the click chemistry.

Description

CLICK CHEMISTRY POLYMER HYDROGELSCROSS REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims priority from U.S. provisional application number 63 / 662,748 filed on June 21 , 2024, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of polymer hydrogels, particularly in the context of click chemistry for gel formation by crosslinking for cellular applications, such as cell encapsulation.BACKGROUND OF THE ART

[0003] Hydrogels have long been recognized as useful materials to interface with cells and tissues as it is recognized that hydrogels can mimic certain properties of natural tissues, and can hence be thought of as synthetic extracellular matrix (ECM) materials. Examples range from synthetic crosslinked hydrogels based on hydroxyethyl methacrylate (HEMA) used as contact lenses, to Matrigel™, a commercially available ECM derived from mouse cancer cell-lines that allows culturing stem cells without inducing differentiation, and many forms of polyethylene glycol- based hydrogels.

[0004] Hydrogels can also manage molecular traffic between transplanted cells and the host, by forming semi-permeable barriers to diffusion that may for example allow good flow of nutrients and oxygen to the cell as well as out-diffusion of therapeutic actives, but at the same time prevent or limit in-diffusion of undesired molecular and / or cellular host immune components including, e.g., antibodies and macrophages. Examples range from natural macromolecules such as alginate that can be ionically crosslinked using bivalent cations such as calcium or barium, as well as synthetic hydrogels based on, e.g., derivatives of polyethylene glycol or other polymers, often modified with functional groups that permit covalent crosslinking to enhance resilience and longevity of the hydrogels.

[0005] Hydrogels contain polymers that are gelled with different techniques such as ionic gelation or covalent gelation. In order to enable better control over the properties of the hydrogel, the hydrogel can contain different polymers that are gelled in different ways (for example combining both ionic gelation and covalent gelation). Moreover, rapid gelation of the hydrogel withthe cell payload is essential to maintain cell viability and hydrogel geometric integrity. Formation of crosslinked hydrogels can be carried out using a number of approaches: network formation between one polymer and a small molecule crosslinker, such as glutaraldehyde.

[0006] These approaches are however generally hampered by toxicity of small molecule reactive crosslinkers that can easily enter cells and disrupt biological processes within the cells, such as glutaraldehyde and some light-activated radical initiators.

[0007] Crosslinking of such hydrogels can palliate this and typically involves formation of new covalent bonds between different polymer chains. Generally, the formation of hydrogels with covalent bonds is complex and costly. The chemical reactions can lead to undesirable secondary reactions and have undesirable cross reactivity. Improvements in the incorporation of covalent bonding in the formation of hydrogels is therefore desired. Indeed, covalent bonding provides the opportunity to better control the physicochemical properties of the hydrogels and improve the control over their gelation.SUMMARY

[0008] In one aspect, there is provided a polymer system for producing a hydrogel, the system comprising: a first component comprising a first synthetic polymer and a gel former; and a second component comprising a second synthetic polymer; wherein the first synthetic polymer is functionalized with a first click chemistry group and wherein the second synthetic polymer is functionalized with a second click chemistry group that reacts with the first click chemistry group to form a covalent link by click chemistry; wherein one of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, an oxime, a nitrone or nitrile-oxide; and wherein the gel former has a gelation mechanism that is independent from the click chemistry.

[0009] In some embodiments, the gel formers are selected from the group consisting of alginate, pluronic, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose or elastin.

[0010] In some embodiments, the first synthetic polymer and the second synthetic polymer have the same polymer backbone.

[0011] In some embodiments, the first synthetic polymer and the second synthetic polymer have a different polymer backbone.

[0012] In some embodiments, the first synthetic polymer and the second synthetic polymer have a backbone that is independently selected from the group consisting of poly(methyl vinyl ether-alt-maleic anhydride) (PMM), homopolymer of methacrylic anhydride, homopolymer of polyacrylic acid, homopolymer of polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides, and lysine homopolymers.

[0013] In some embodiments, the first synthetic polymer and the second synthetic polymer have a backbone that is comprised of monomeric units selected from the group consisting of acrylic acid, methacrylic acid, sodium acrylate, sodium methacrylate, sodium ethacrylate, acrylamide, methacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-methacryloyloxyethyl phosphorylcholine, N-(carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N- (carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3- methacrylamido-N,N-dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N- dimethylpropan-1-aminium, 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / \ / -[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2-(methacryloyloxy)ethanesulfonic acid.

[0014] In some embodiments, the first synthetic polymer and / or the second synthetic polymer have a molecular weight of 10,000 to 100,000 Da.

[0015] In some embodiments, the cycloalkene is a C3-C10 cycloalkene.

[0016] In some embodiments, the cycloalkyne is a C7-C9 cycloalkyne.

[0017] In some embodiments, the heterocycloalkyne is a 7 to 9 membered ring and the heteroatoms are selected from N, O, and S.

[0018] In some embodiments, one of the first synthetic polymer or the second synthetic polymer is functionalized with the cycloalkene or the heterocycloalkene and the other is functionalized with the tetrazine, the triazine or the pyridazine.

[0019] In some embodiments, one of the first synthetic polymer or the second synthetic polymer is functionalized with the cycloalkyne or the heterocycloalkyne and the other is functionalized with the azide, the oxime, the tetrazine, the triazine, the pyridazine, the nitrone or the nitrile-oxide.

[0020] In some embodiments, the first synthetic polymer has a polyanionic, a polyampholyte or a neutral backbone and the second synthetic polymer has a polycationic backbone. For example, the polycationic backbone comprises monomeric units selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3- aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2-hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2-hydroxyethylmethacrylate (HEMA), 2- (methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxybetaine methacrylate (CBM), carboxy betaine methacrylamide, N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl acrylamide, 3-N,N- dimethylaminopropyl methacrylamide, 3-N,N-dimethylaminopropyl acrylate, 3-N,N- dimethylaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N- dimethylaminobutyl methacrylamide, 1,3-bis(dimethylamino)propyl acrylate, 1 ,3- bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate and 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate.

[0021] In some embodiments, the first synthetic polymer has a PMM backbone and the second synthetic polymer has a PLL backbone.

[0022] In a further aspect, the polymer system of the present disclosure has a hydrogel core that can comprise a PMM polymer functionalized with vinylsulfone groups (PMM-CVS), a second PMM polymer functionalized with homocysteine thiolactone (PMM-HTL), mercaptopyridine (PMM-SPy) and / or cystamine (PMM-cystamine) which can further crosslink with polymerscontaining IEDDA functionalities (e.g., a tetrazine or an alkene). It should be understood that polymers containing tetrazine can freely react with PMM-HTL, PMM-SPy and PMM-cystamine to covalently crosslink the coating to the hydrogel core. For example, HTL groups can be hydrolyzed or aminolyzed to generate free thiol groups to react with tetrazine. Polymers containing norbornene can react with free thiols via thiol-ene click reaction. The same IEDDA chemistry can be applied generally to any coat or core formulation containing at least one polymer functionalized with tetrazine or norbornene, and at least one other polymer functionalized with free thiols or derivatives that can generate free thiols. In some embodiments, polymers containing unsubstituted tetrazines can react with thiol derivatives to afford thioether adducts. In such embodiments, unreacted tetrazines can be quenched by adding thiol-functionalized groups, or serve as handles for other motifs.

[0023] In a further aspect, there is provided a method for producing a hydrogel, the method comprising: providing the first component of the polymer system as defined herein; gelling the gel former of the first component to provide an intermediate gel containing the first synthetic polymer; contacting the second component of the polymer system as defined herein with the intermediate gel and allowing a click chemistry reaction to occur between the first synthetic polymer and the second synthetic polymer.

[0024] In some embodiments, the gel former is alginate and the step of gelling is performed by extruding the first component in a gelling bath containing calcium, strontium, barium, iron, or zinc ions.

[0025] In some embodiments, the extrusion performed is to provide droplets into the gelling bath to produce capsules.

[0026] In some embodiments, the extrusion performed is continuous fashion in order to produce hydrogel strings. In one embodiment, the hydrogel strings are laid down in overlapping fashion to form high surface area grids or patches, partial fusion and covalent crosslinking of the overlap points between string segments in adjacent layers provide strength and result in formation of permanent high surface area grids or patches.

[0027] In some embodiments, the method further comprises, before the gelling step, a step of providing a payload material in the first component.

[0028] In some embodiments, the payload material is live cells or cell clusters.

[0029] In some embodiments, the step of contacting is performed at least partially concurrently with the gelling step.

[0030] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1A is a schematic of a hydrogel according to one embodiment of the present disclosure where synthetic crosslinked by click chemistry are encased in a capsule of gel former.

[0032] FIG. 1 B is a schematic of a hydrogel according to one embodiment of the present disclosure where synthetic crosslinked by click chemistry are coating a capsule of gel former.

[0033] FIG. 1C is a schematic of a hydrogel according to one embodiment of the present disclosure that combines both the core of Fig. 1A and the coating of Fig. 1 B on a gel former capsule.

[0034] FIG. 1D is a schematic showing linear structure, or multi-arm polymers that contain a functional diene or dienophile at the end of the short polymer chains, as well as throughout the short polymer chains.

[0035] FIG. 2A is a1H nuclear magnetic resonance (NMR) spectra in D2O recorded on a 600 MHz spectrometer of poly[methylvinylether-a / f-maleic anhydride] (PMM) functionalized with a tetrazine moiety.

[0036] FIG. 2B is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of poly- L-lysine (PLL) functionalized with a norbornene moiety.

[0037] FIG. 2C is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMM- norbornene.

[0038] FIG. 2D is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMM- trans-cyclooctene.

[0039] FIG. 2E is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMM- N-methylimidazole.

[0040] FIG. 2F is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMM- dibenzocyclooctyne.

[0041] FIG. 2G is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMM- azide.

[0042] FIG. 2H is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMS60- tetrazine.

[0043] FIG. 2I is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMS60- Norbornene.

[0044] FIG. 2J is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMS60- trans-cyclooctene.

[0045] FIG. 2K is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PMS60- dibenzocyclooctyne.

[0046] FIG. 2L is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of Poly[(3- aminopropylmethacrylamide-graft-methyltetrazine)-co-N,N-(dimethylamino)ethyl acrylate] (PAD- mTet).

[0047] FIG. 2M is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PAD- Norbornene.

[0048] FIG. 2N is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PAD- frans-cyclooctene.

[0049] FIG. 20 is a1H NMR spectrum in D2O recorded on a 600 MHz spectrometer of PAD- azide.

[0050] FIG. 3 is a microscopy image of PMM-tetrazine / alginate core hydrogels coated with PLL-norbornene.

[0051] FIG. 4 is a microscopy image of the PMM-tetrazine / PLL-norbornene coated hydrogel capsules after citrate / NaOH treatment.

[0052] FIG. 5 is a fluorescent microscopy image of PMM-tetrazine / PLL-norbornene hydrogel capsules in citrate / NaOH.

[0053] FIG. 6 is a microscopy image of 2 wt.% PMM-Tetrazine / PMM-Norbornene 1 wt.% sodium alginate hydrogel strings imaged in basal DM EM media.

[0054] FIG. 7 is a microscopy image of 2 wt.% PMM-Tetrazine / PMM-Norbornene 1 wt.% sodium alginate hydrogel string after treatment with 70 mM sodium citrate.

[0055] FIG. 8A is a fluorescent microscopy showing Remaining tetrazine reactive groups stained with AF-488 TCO of wt.% PMM-Tetrazine / PMM-Norbornene 1 wt.% sodium alginate hydrogel string.

[0056] FIG. 8B is a fluorescent microscopy showing remaining Norbornene reactive groups stained with Cyanine5 Tetrazine of wt.% PMM-Tetrazine / PMM-Norbornene 1 wt.% sodium alginate hydrogel string.

[0057] FIG. 9 is a microscopy image showing 1 wt.% PMM-Tetrazine 1 wt.% sodium alginate hydrogel capsule covalently crosslinked by in-diffusion of 0.33 wt.% PMM-TCO imaged in HBS.

[0058] FIG. 10 is a microscopy image showing 1 wt.% PMM-Tetrazine 1 wt.% sodium alginate hydrogel capsule covalently crosslinked by in-diffusion of 0.33 wt.% PMM-TCO after treatment with 70 mM sodium citrate.

[0059] FIG. 11A is a microscopy image showing hMSC clusters encapsulated in 2 wt.% PMM- Tetrazine / PMM-Norbornene 1 wt.% sodium alginate capsules.

[0060] FIG. 11B is a microscopy image showing hMSC clusters encapsulated in 2 wt.% PMM- Tetrazine / PMM-Norbornene 1 wt.% sodium alginate strings.

[0061] FIG. 12A is a fluorescent microscopy image showing hMSC clusters (stained for live, dead and nuclei of unencapsulated hMSC clusters showing mostly live cells and only 4 dead).

[0062] FIG. 12B is a fluorescent microscopy image showing hMSC clusters (stained for live, dead and nuclei of encapsulated hMSC clusters in capsules showing mostly live cells and only 3 dead).

[0063] FIG. 12C is a fluorescent microscopy image showing hMSC clusters (stained for live, dead and nuclei of encapsulated hMSC clusters in strings showing mostly live cells and only 2 dead).

[0064] FIG. 12D is a fluorescent microscopy image showing multiple hMSC clusters (stained for live, dead and nuclei of unencapsulated hMSC clusters showing a majority of live cells) one day after encapsulation.

[0065] FIG. 12E is a fluorescent microscopy image showing hMSC clusters (stained for live, dead and nuclei of encapsulated hMSC clusters in capsules one day after encapsulation showing a majority of live cells)

[0066] FIG. 12F is a fluorescent microscopy image showing hMSC clusters (stained for live, dead and nuclei of encapsulated hMSC clusters in strings one day after encapsulation showing a majority of live cells).

[0067] FIG. 13A is a microscopy image showing hMSC cell cluster containing PMM- Tetrazine / PMM-Norbornene crosslinked capsules after treatment with sodium citrate.

[0068] FIG. 13B is a microscopy image showing hMSC cell cluster containing PMM- Tetrazine / PMM-Norbornene crosslinked string segment after treatment with sodium citrate.

[0069] FIG. 14A is a microscopy image showing 0.1 wt.% PAD-Norbornene 0.2 wt.% PMS80- Tetrazine coated calcium alginate capsules in HBS.

[0070] FIG. 14B is a microscopy image showing 0.1 wt.% PAD-Norbornene 0.2 wt.% PMS80- Tetrazine coated calcium alginate capsules in 70 mM sodium citrate.

[0071] FIG. 14C is a microscopy image showing 0.1 wt.% PAD-Norbornene 0.2 wt.% PMS80- Tetrazine coated calcium alginate capsules in 70 mM sodium citrate and 1 M NaOH.

[0072] FIG. 15A is a fluorescent microscopy image showing cross sectional image of a PAD- Norborene / PMS80-Tetrazine covalently crosslinked coated alginate capsule stained with Cyanine5 Tetrazine and AF-488 TCO.

[0073] FIG. 15B is a magnification of Fig. 15A.

[0074] FIG. 16A is a schematic showing alginate core capsules with a covalently crosslinked shell by sequential coatings of a click reactive polycation and a click reactive polyanion.

[0075] FIG. 16B is a schematic showing core covalently crosslinked capsules by encapsulation of click reactive synthetic the first synthetic polymer 1 and indiffusion of low molecular weight click reactive second synthetic polymer 2.

[0076] FIG. 16C is a schematic showing covalently crosslinked coated shell by sequential coatings of a click reactive polycation and a click reactive polyanion on a core covalently crosslinked capsules by encapsulation of click reactive synthetic polymer 1 and indiffusion of low molecular weight click reactive polymer 2.

[0077] FIG. 16D is a schematic showing covalently crosslinked core by static mixer-based extrusion of click reactive synthetic polymer 1 and click reactive synthetic polymer 2.

[0078] FIG. 16E is a schematic showing covalently crosslinked coated shell by sequential coatings of a click reactive polycation and a click reactive polyanion on a core covalently crosslinked capsules by static.

[0079] FIG. 16F is a schematic showing hydrogel configurations according to embodiments of the present disclosure.

[0080] FIG. 17 is a microscopy image showing 2 wt.% PMM-Azide / PMM-DBCO 1 wt.% sodium alginate hydrogel capsules imaged in HBS.

[0081] FIG. 18 is a microscopy image showing 2 wt.% PMM-Azide / PMM-DBCO 1 wt.% sodium alginate hydrogel capsules after treatment with 70 mM sodium citrate.

[0082] FIG. 19 is a microscopy image showing 2 wt.% PMM-Azide / PMM-DBCO 1 wt.% sodium alginate hydrogel capsules coated with 0.2 wt.% PAD-Azide and 0.2 wt.% PMS60-DBCO imaged in HBS.

[0083] FIG. 20A is a microscopy image showing 2 wt.% PMM-Azide / PMM-DBCO 1 wt.% sodium alginate hydrogel capsules coated with 0.2 wt.% PAD-Azide and 0.2 wt.% PMS60-DBCO imaged in 70 mM Sodium Citrate.

[0084] FIG. 20B is a microscopy image showing 2 wt.% PMM-Azide / PMM-DBCO 1 wt.% sodium alginate hydrogel capsules coated with 0.2 wt.% PAD-Azide and 0.2 wt.% PMS60-DBCO imaged in NaOH.

[0085] FIG. 21 is a microscopy image showing 1 wt.% PMM-Tetrazine 1 wt.% sodium alginate hydrogel capsule covalently crosslinked by in-diffusion of 2.1 wt.% poly[sulfobetaine methacrylate]-bi(norbornene) imaged in HBS.DETAILED DESCRIPTION

[0086] There is provided herein a hydrogel for producing capsules, strings, patches and the like. The hydrogel comprises at least two polymers, synthetic polymers which have been crosslinked with click chemistry groups functionalized therein as described in greater detail below and a gel former. In optional embodiments, the hydrogel consists of the synthetic polymers and the gel former. The hydrogel can have a payload such as cells as described in greater details below. In non-limitative embodiments, the hydrogel consists of the synthetic polymers and the gel former as well as a payload (e.g. cells) encapsulated therein. The synthetic polymers and the gel former together form a hydrogel that can have various configurations. For example, as illustrated in Fig. 1A, the hydrogel 10 can be a capsule of gel former 11 that encapsulates in its core a network of the synthetic polymers (which has been covalently crosslinked with the functional click chemistry group functionalized therein). In another embodiment, as illustrated in Fig. 1 B, the hydrogel 20 comprises a capsule 21 that is coated with a layer of the synthetic polymers 22. The two described embodiments can also be combined as illustrated in Fig. 1C to have a hydrogel 30 with both a core containing the synthetic polymers 32 in a gel former capsule 31 and a coating of the synthetic polymers 33 around the capsule 31. It should be understood that although the embodiments of Figs. 1A-1C are presented as a capsule, the same configurations are possible when providing a string or patch of the hydrogel.

[0087] The term “gel former” as used herein refers to a biocompatible polymer and that has a method of gelation that is independent and different from the click chemistry of the functionalized synthetic polymer. The gel formers are for example selected from alginate, pluronic, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose or elastin, and may optionally contain synthetic polymers capable of spontaneous thermal, ionic or covalent gelation during extrusion, or analogous induced gelation post-extrusion, or a combination of both of these. The gel former in the present disclosure is not functionalized with click chemistry groups incontrast with the synthetic polymer in order to produce together a system with two gelation techniques that synergize to improve and better control the properties of the hydrogel particularly in the context of cell encapsulation (e.g. improving cell viability during the gelation and also when implanted in vivo).

[0088] The gel former can be gelled based on chemical and / or physical processes. In preferred embodiment, a gel of the gel former is first formed (optionally with cells or another payload) along with a first synthetic polymer that has been functionalized with a first click chemistry group. This yields a hydrogel of the gel former with the first synthetic polymer incorporated therein. Then, the hydrogel is contacted with a second synthetic polymer that is functionalized with a second click chemistry group that reacts with the first synthetic polymer to undergo a click reaction. The second synthetic polymer may be provided with additional gel former that would further enrich the hydrogel. This results in the formation of a covalent network between the two synthetic polymers within the gel former hydrogel, whether in the shell or the core of the hydrogel. The penetration and diffusion of a synthetic polymer in the shell or the core depends on its capacity to diffuse in. This can be controlled, at least in part, by the molecular weight of the two synthetic polymers as well as their charge. In some embodiments the second synthetic polymer has a degree of polymerization that is smaller than the first synthetic polymer by a factor of at least 2, at least 3, at least 5, at least 10, from 2 to 100, from 2 to 50 or from 2 to 20. In some embodiments, the second synthetic polymer has a degree of polymerization of from 10 to 100 monomer units. In some embodiments, the second synthetic polymer has a molecular weight of up to 15 kDa, up to 14 kDa, up to 13 kDa, up to 12 kDa, up to 11 kDa or up to 10 kDa. The size difference between the second synthetic polymer and the first synthetic polymer facilitates sufficient diffusion of the second synthetic polymer into the hydrogel to react with a majority of the click chemistry groups of the first synthetic polymer (e.g. more than 90%, 95% or 98% of the groups). This can lead to the formation of a synthetic crosslinked interpenetrating network within the gel former hydrogel network. In such embodiments, the second synthetic polymer has a net positive charge and is polycationic, to promote its diffusion. In such embodiments, the second synthetic polymer may have a larger or similar molecular weight to the first synthetic polymer. In still further embodiments, these embodiments are combined (i.e. the second synthetic polymer has both a lower molecular weight and is cationic).

[0089] In embodiments where the gel former is alginate, it can be mixed with synthetic polymers functionalized with a click chemistry group, and the gel is formed by a process where the mixture (optionally containing cells or other payload) is extruded into a bath containingcalcium, strontium, barium, iron, or zinc ions that rapidly gel the alginate to provide structural integrity with ionic crosslinking. In a second step, the hydrogel containing the synthetic polymer is exposed to a solution comprising a second synthetic polymer which has been functionalized with a click chemistry group that reacts with the aforementioned synthetic polymer. As explained above, the second synthetic polymer is preferably a low molecular weight polymer and is optionally net cationic to enhance diffusion. This results in the formation of a covalent network between the two synthetic polymers within the hydrogel, whether in the shell or the core of the hydrogel, depending on the molecular weights of the two synthetic polymers as well as other factors as described below.

[0090] In some further embodiments a static mixer is used to combine the first and second synthetic polymers functionalized with the click chemistry groups, this could be during an extrusion process for example. In another embodiment, the output of the static mixer can be affixed to a coaxial needle or suitable microfluidic device, and air or another fluid in the outer channel used to shear the viscous mixture from the static mixer, optionally containing cells, into droplets that are directed into a gelling bath. The use of the static mixer is described below for an alginate gel former but is not limited to alginate. The explanation is thus provided as an example only. A static mixer is a device that is capable of continuous distributive mixing of two or more fluids flowing through the mixing elements of the static mixer, with the degree of mixing being determined by the number of mixing elements contained in the static mixer and the residence time in the mixer. The fast reaction kinetics of the click reactions inhibits the ability to premix the complementary reactive polymers in a gel former mixture prior to extrusion because of premature reaction between the first and second synthetic polymers before extrusion can proceed. More specifically, the static mixer is connected to two separate syringes that each contain either the first or the second synthetic polymer, with an extrusion nozzle attached to the end of the static mixer. Even more specifically, two separate extrusion mixtures can be prepared 1) alginate with the first synthetic polymer and 2) alginate with the second synthetic polymer. The mutually click reactive polymers are mixed during the extrusion process as they are flowed through a static mixer just prior to being extruded into an alginate gelling bath suitably containing calcium, strontium, barium, iron, or zinc ions where the alginate is gelled by ionic crosslinking to provide structural integrity and the first and second click reactive polymers gel concurrently.

[0091] The term “capsule” as used herein, generally refer to particles between 200 and 2000, or between 400 and 1400 pm in diameter.

[0092] This technology is also amenable to formation of other geometries, such as high surface area hydrogel devices formed by 3D bioprinting, including extrusion 3D printing.

[0093] The term “covalently crosslinked” as used herein with respect to a polymer matrix refers to the formation of covalent bonds between polymer chains that hold together the polymer matrix. It is not possible for the polymer matrix to undergo facile dissolution into individual polymer chains when the covalent crosslinks are present. Covalent crosslinks here include the different click chemistry techniques described in greater detail below.

[0094] The term “biocompatible” as defined herein refers to compounds or microparticles that are compatible with in vitro or in vivo prolonged contact with cells and / or specific biological tissues. Biocompatible compounds or microparticles do not elicit a significant negative effect on the cell survivability, cell function, and / or tissue function, whereby biocompatibility is usually specified in terms of being compatible with a particular tissue or cell environment.

[0095] The term “jammed” as used herein in the context of gels and microgels, refers to close- packed microgel particles that form a shear-thinning medium that appears solid at rest but can be temporarily mobilized by moderate shear forces. The microgels can be provided in the printing bath or within the extruded mixture.

[0096] The synthetic polymers (or simply the polymers) of the present disclosure can be polycationic polymers, polyanionic polymers, polyampholyte polymers, polyzwitterionic polymers or neutral charge polymers. In some embodiments, the synthetic polymers have a backbone selected from poly(methyl vinyl ether-alt-maleic anhydride) (PMM), homopolymer of methacrylic anhydride, homopolymer of acrylic acid, homopolymer of methacrylic acid, or copolymers of acrylic acid and methacrylic acid, or copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides, poly-ornithine, epsilon polylysine, poly-L-lysine, poly-D-lysine, aminoethylmethacrylamide, dimethylaminoethyl methacrylate, and homopolymers of dimethylaminoethyl acrylate. The polymer backbone can also include synthetic polycations such as those described in US10407527 which is incorporated herein by reference (e.g. poly(aminopropylmethacrylamide) (poly(APM))), as well as polyampholyte polymers and polybetaines for example as described in US10369227 which is incorporated herein by reference. In one embodiment, the synthetic polymer includes monomeric units selected from 2-aminoethylacrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3- aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2- hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N- dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3-bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2- (acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N- dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2- (dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / \ / -[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2- (methacryloyloxy)ethanesulfonic acid.

[0097] One advantage of utilizing a synthetic polymer such as those described herein is that synthetic polymers have less batch- to- batch variability over naturally occurring polymers. Additional advantages of synthetic polymers include the control over their chemical composition and molecular weight.

[0098] The click chemistry contemplated in the present disclosure includes the use of Inverse Electron Demand Diels-Alder (I EDDA), strain-promoted azide-alkyne cycloaddition (SPAAC) crosslinking, strain-promoted alkyne-nitrone cycloaddition (SPANG), or strain-promoted alkynenitrile oxide cycloadditions (SPANOC). The term “click chemistry group” therefore includes the pairs of groups that perform IEDDA, SPAAC, SPANC and SPANOC reactions.

[0099] Another consideration when forming crosslinked hydrogels optionally comprising cells, involves the use of mutually reactive, cell-compatible natural or synthetic polymers. Backbones including alginate, gelatin, dextrans, as well as PMM and acrylic polymers are contemplated herein. These systems offer significant benefits over lower molecular weight (MW) gel formers or coupling agents. Their higher MW of typically 20,000 - 200,000 Da limits or prevents uptake into cells, rendering them much less cytotoxic in comparison with small molecule analogs. In addition, their extended backbone reduces the number of crosslinking steps required to form crosslinked networks. Furthermore, they are often easily accessible by either copolymerization of suitable monomers using free or controlled radial copolymerization, or by post-modification of pre-formed polymers.

[0100] A challenge with higher molecular weight polymers is their correspondingly lower diffusional mobility, which can manifest in self-limiting crosslinking reactions where a substantial percentage of the reactive groups remain unreacted in the final gel. It has been presently found that coupling between mutually reactive polymers can be facilitated by combining the mutually reactive polymers with a gel former such as sodium alginate, such that upon exposure to calcium chloride gelling bath, the reactive polymers phase-separate from the forming calcium alginate on a 10s to 100s of nanometer scale (determined by turbidity / opacity), which leads to higher local concentration and hence higher reaction rates and higher degree of reaction between the two polymers. There is thus a synergistic effect in improving the gelation and reaction kinetics of the click chemistry. A similar synergistic effect can be exerted by placing mutually reactive groups on oppositely charged polymers, which allows electrostatic attraction to enhance local concentrations of these mutually reactive groups. Accordingly, in preferred embodiments, the synthetic polymer crosslinked polymer matrix is formed by crosslinking a cationic synthetic polymer functionalized with a click chemistry group and a polyampholyte or polyanionic polymer functionalized with a corresponding click chemistry group. Alternatively, or in combination, the second synthetic polymer is provided as having a smaller molecular weight as defined above. This yields to higher crosslinking rates and higher degrees of crosslinking. At the same time these features yield a hydrogel with a reduced or limited number of residual unreacted click chemistryfunctional groups. In addition, approaches to mitigate the undesired presence of residual functional groups in the crosslinked hydrogel involve capping reactions using lower molecular weight deactivating agents. Preferably, this is not required with the present hydrogels thereby removing a step while maintaining biocompatibility.

[0101] The lower diffusion rates of such gel forming polymers also offer benefits in that they enable combining these mutually reactive pairs of polymers in advantageous ways. One approach involves combining solutions of one or both of the first and second synthetic polymers with another, initial gel former such as sodium alginate. Present experiments have shown that solutions comprising 1 to 2 wt. % alginate, together with synthetic polymers such as reactive PMM or acrylic polymers, can be extruded by, e.g. air shearing into droplets, into an ionic gelling bath comprising calcium, strontium or barium ions. The rapid gelation of the alginate as, e.g., calcium alginate, allows significant immobilization of the synthetic gel former(s) which increases the time available for them to subsequently covalently crosslink, forming a second, interpenetrating network within the calcium alginate initial scaffold. Typically, polymers with molecular weight of above 100,000 g / mol are significantly retained by physical means within calcium alginate gels. In some cases, ionic interactions with, e.g., calcium ions may further reduce the ability of these synthetic polymers to diffuse out of the formed calcium alginate gels.

[0102] Given the rapid reaction between the first and second click chemistry groups (particularly I EDDA pairs), the present method provides that the first and second polymers are introduced sequentially (to avoid having them reacting with each other when it is not desired). For example, the first functionalized polymer, which may be a tetrazine-functionalized polymer (an IEDDA example), is admixed with sodium alginate and optionally, a therapeutic cell payload, and extruded as capsules, strings or patches into a gelling bath comprising calcium, strontium or barium ions to ‘set’ the alginate components and immobilize the first functionalized polymer. Subsequently, the hydrogels are exposed to a solution comprising the second functionalized polymer, in this example functionalized with an electron-rich vinyl component to react with tetrazine. The second functionalized polymer may have a neutral or net neutral backbone including polyampholytes and polybetaines, or a cationic or net cationic backbone such as PLL, poly[3-aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD) or poly[APM-co- DMAEA-co-SBMA] (PADS). Net polycationic polymers used as the second functionalized polymers may have the advantage of facilitating electrostatic pre-concentration of the two reactive polymers in the shell. The first or second polymer may have a higher molecular weight of 30,000 to 200,000 Daltons, while the other polymer would have a somewhat lower MW of 10,000 to100,000 Daltons in order to facilitate diffusion through the hydrogel to participate in forming crosslinks.

[0103] It should be understood that the functional groups added to the first and second polymer are interchangeable. Generally, the “first” synthetic polymer refers to the polymer that is added first in embodiments where they are not combined at the same time with the gel former. For example, the first polymer can be functionalized with an electron-rich vinyl component whereas the second polymer is functionalized with tetrazine.

[0104] Other scenarios involve pre-forming calcium (or strontium, or barium) alginate capsules containing cells, that are subsequently coated with a first, net cationic, polymer comprising either diene or dienophile groups, followed by coating with a second, net anionic polymer comprising dienophile or diene groups, respectively. Molecular weights of both polymers need to be adjusted to enable appropriate interaction with, and in-diffusion into, the pre-formed alginate bead. For example, these two polymers may have molecular weight between 10,000 to 200,000 Daltons, and preferably between 30,000 and 100,000 Daltons. In some embodiments, the second synthetic polymer has a molecular weight of less than 100,000 Daltons whereas the first has a molecular weight of from 100,000 Daltons to 300,000 Daltons in order to better entrap it in the calcium alginate core. Similarly, their net charge balances can be controlled to ensure effective binding of the first, optionally net anionic polymer to the alginate bead, and effective binding of the second, optionally net cationic polymer to the first polymer. For example the first polymer may comprise between 10 and 100 mol%, and preferably between 25 and 60 excess mol% anionic groups. Similarly, the second, net cationic polymer may comprise between 10 and 100 mol%, and preferably between 15 and 60 mol% cationic groups.

[0105] Control over the porosity of the initial calcium alginate hydrogels as well as the final hydrogel comprising an interpenetrating network in at least the shell region, is desirable in order to provide control over key properties of the hydrogels such as permeability, which is desired to mitigate the host immune response while ensuring proper diffusion across the hydrogel. For example, when hydrogels are used to encapsulate cells for transplantation, the hydrogel permeability affects the diffusion of molecules across the hydrogels, including both molecules needed for cell survival and function, and components of the immune system that could negatively affect cell health.

[0106] Another important factor in designing such hydrogels for implantation is how they interact with tissues at the implantation site. Various aspects need to be optimized, including minimization of inflammatory responses that could lead to fibrotic overgrowth of the hydrogel, causing hypoxia of encapsulated therapeutic cells. Polymers with balanced charges, such as polyampholytes and polybetaines, have shown benefit in reducing inflammatory responses. Another consideration involves the mechanical resilience of the hydrogels, which should be able to persist for extended periods of time in vivo. Finally, retention of transplanted cells is important, both to ensure their protection from immune detection, as well as to prevent in migration of immune cells. Both of the latter considerations are improved through use of covalent crosslinks within the hydrogel networks.

[0107] In one aspect, there is provided a method of producing a hydrogel, the method comprising: reacting a first polymer functionalized with a first click chemistry group such as an electron-poor diene such as tetrazine, with a second polymer comprising a corresponding click chemistry group such as an electron-rich alkene e.g norbornene or frans-cyclooctene (TCO). Conversely, the first polymer may comprise an electron-rich alkene while the second polymer carries an electron-poor diene. Either the first or the second polymer, which should have net anionic charge, is admixed with 0.5 to 10, and preferably 0.7 - 4 wt% alginate solution and extruded as droplets or strings or overlapping strings, into a suitable ionic gelling bath comprising calcium, strontium or barium ions or a mixture of them, to form initial hydrogels by ionic gelation. These initially formed hydrogels are then exposed to a solution of the second reactive polymer or oligomer. The second reactive polymer may be net anionic, neutral or net cationic. Molecular weight of both components can be controlled to control and limit the rate of out-diffusion of the first polymer from the calcium alginate gel, and the rate of in-diffusion of the second polymer into the initial calcium alginate gel comprising the first polymer. For instance, PMM is available in a range of MW can be used as the backbone for both the first and second polymers. Proper control of the mobility (indirectly controlled by the choice of MW) of both polymers allows control over the shell thickness, crosslink density, pore diameters and size exclusion properties.

[0108] Covalent reaction to form a synthetic polymer network, interpenetrating with the calcium alginate, takes place in the mixing zone of the first and second synthetic polymers within the capsule or hydrogel shell. This mixing zone depends on the relative molecular weight of the first and second polymers. It can include the whole volume of the hydrogel in cases where one polymer is strongly immobilized while the other is significantly mobile. In other cases, where both polymers have at least some diffusional mobility within the calcium alginate matrix, the mixingzone may be limited to an area corresponding to the outer shell of the capsule or hydrogel. In other words, the location within the hydrogel, as well as the properties of this second, covalently crosslinked network, depends on, and can be controlled through, the ability of both polymeric components to counter-diffuse down their respective concentration gradients until captured by the click chemistry reaction with the matching reactive groups on the other polymer. Finally, it is also possible to sequentially coat a pre-formed alginate hydrogel with both polymers, to effect formation of a crosslinked network in the shell of the hydrogel.

[0109] Since relative rates of in- and out-diffusion of both polymers determines the location of the crosslinked network, diffusion of both components can be tracked in real time using confocal microscopy in case of fluorescently labelled polymers, and can be finely tuned through factors including molecular weights of each component, alginate type and loading, geometry of the calcium alginate gel, as well as presence of divalent salts such as calcium, strontium and barium.

[0110] In some embodiments, the separate initial gel former is selected from alginate, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, pluronic, or elastin.

[0111] In some embodiments, the first polymer and the second polymer have a backbone independently selected from poly-ornithine, poly-L-lysine (PLL), epsilon-polylysine, poly(methyl vinyl ether-alt-maleic anhydride) (PMM), homopolymer of methacrylic anhydride, homopolymer of polyacrylic acid, homopolymer of polymethacrylic acid, or copolymers of acrylic acid and methacrylic acid, or copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, and methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides. The first polymer and second polymer can have the same backbone but in preferred embodiments one of them is a cationic polymer and the other is a polyampholyte / anionic polymer. The polymer backbone can also include synthetic polycations such as those described in US10407527, as well as polyampholyte polymers for example those described in US10369227, both incorporated herein by reference.

[0112] Accordingly, although the present disclosure provides PMM-TZ as an example of a backbone functionalized with an electron-poor diene, this is not a limitative example as not only polymers other than PMM can be used, but also other pendant electron-poor diene groups can be used as well as other click chemistry techniques. A preferred embodiment, which is exemplifiedin the Example section below, is the formation of a hydrogel between PADS-TCO with PMM-TZ, together with a separate initial gel former such as alginate. The initial gel former is for example alginate, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, pluronic, or elastin. The gel former can be provided in a weight percentage of from 0.5 to 10 wt. %, and preferably 1 to 4 wt. %, depending on the viscosity and the type of gel former.

[0113] The chemical formula of PMM-tz is presented below:PMMAn Tetrazine-amine

[0114] As explained above, it should be understood that other electron-poor diene groups and other polymer backbones are suitable which are described in further detail below.

[0115] For example, the IEDDA alkene-functional polymer is more generally a modified polymer as shown below, with TCO given as representative electron-rich alkene.

[0116] I EDDA is a fast process based on using reactive groups that can react by the so called Inverse Electron Demand Diels-Alder reaction. A key example of this class of functional groupsare tetrazine and norbornene, representing, respectively, electron-poor dienes and electron-rich alkene groups. These two functional groups react with each other by a concerted 4+2 I EDDA coupling that is extremely rapid and can reach completion within seconds. Fast, selective, catalyst-free coupling reactions are key features of I EDDA. Another benefit of this approach is the inherent bio-orthogonality of this reaction, as biological tissue does not comprise functional groups able to react with either of these two groups. As a result of these features, lEDDA-mediated crosslinking to form hydrogels is considered a highly biocompatible process.

[0117] In one particular example, there is provided an advantageous hydrogel system that combines the benefits of polymeric gel formers, with the rapid gelation inherent in Inverse Electron Demand Diels Alder chemistry. In particular, there is provided the formation of pairs of polymers, based e.g. on either (meth)acrylic copolymers or PMM or polylysines or other polycations and polyampholytes and polybetaines or a combination thereof, modified each with an electron-poor diene such as tetrazine or with an electron-rich alkene such as norbornene, forming a pair of reactive polymers capable of crosslinking spontaneously under physiological conditions through the IEDDA mechanism. IEDDA comprises a [4+2] cycloaddition between an electron-rich dienophile such as norbornene or trans-cyclooctene (TCO), and an electron-poor diene, e.g. tetrazine. In some embodiments, these two mutually reactive groups are attached to net polycation backbones of 10,000 to 200,000 Da, which are for example (meth)acrylic, PMM, or other synthetic backbones as defined above. Optionally, one of the two mutually reactive polymers may be an oligomer or polymer with number-average molecular weight between 200 and 100,000, more preferably between 500 and 50,000, and most preferably between 2000 and 30,000 that would be able to more easily diffuse into a pre-formed hydrogel containing the other, higher molecular weight reactive polymer immobilized within a calcium alginate matrix. Degrees of functionalization for each component can range from 1 to 40, preferably from 5 to 30, and most preferably from 10 to 25 mol% of monomer units on the two reactive polymers carrying one of these two reactive groups.

[0118] In some embodiments, ionic charges can be used to further manipulate location and mobility of the two click chemistry reactive polymers. For instance, the polymer used for coating / in- diffusion, can be given net cationic charge in order to further attract the polymer to and into the initial hydrogel formed from calcium alginate and comprising the other reactive polymer. These cationic charges may be introduced by copolymerization with cationic (meth)acrylic monomers, or by post-modification of, e.g., PMM with cationic groups, or by modification of polylysines or other pre-formed polycations.

[0119] One of the synthetic polymers that was developed for coating hydrogels is a poly[APM- co-DMAEA-co-SBMA] terpolymer (PADS) which is a type of PMM polymer. APM is 3- aminopropylmethacrylamide which contains a primary amine to react with a click chemistry group to functionalize the polymer. DMAEA is N,N-dimethylaminoethylacrylate and SBMA is [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide. A key formulation is the 1 :1 :2 ternary copolymer, where 1 :1 :2 represents initial molar feed ratios of 1 :1 :2 of APM, DMAEA, and SBMA. The molar ratios can vary by up to ±50%, ±20%, ±10%, ±5% or ±3% each, and additional comonomers can be contemplated. In one example, to obtain the 1 :1 :2 ratio, a set of copolymers with molecular weights (Mn) of 15,000, 25,000 and 40,000 g / mol respectively of APM, DMAEA, and SBMA were prepared using reversible addition-fragmentation chain-transfer (RAFT) polymerization. The three comonomers have significantly different reactivity ratios, which introduces drift during copolymerization - however, due to the controlled (‘living’) nature of this polymerization, this is translated into gradient copolymer chains where each chain has more SBMA at the initiator end, and more cationic comonomers at the other end. An advantage of the gradient distributions is that it facilitates electrostatic binding and covalent reaction of the cationrich end of the polymer to the anionic hydrogel, while leaving the SBMA-rich end to be tissuefacing. The amine of APM contributes reactivity as well as cationic density for coating onto anionic core hydrogels and / or for functionalization of the PADS. DMAEA (N,N-dimethylaminoethyl acrylate) has a tertiary amine in the pendant group which also contributes cationic charge density to assist with coating onto capsules. DMAEA has the additional benefit that it undergoes chargeshifting by hydrolysis with a half-life measured in hours to days. As a result, its initial cationic charge density facilitates binding to capsule and the anionic, HTL-functional polymer network, but after covalent crosslinking and implantation, its charge density decreases by hydrolysis of the DMAEA group and loss of dimethylaminoethanol. This process reduces the overcall cationic charge density of the shell of the capsule and improves anti-fouling properties. Finally, SBMA is [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, a zwitterionic monomer that further improves the antifouling properties of the coating by increasing the hydrophilicity of the materials. The structure of PADS is shown below where n, m, and x represent repeating subunits (that can be a statistical distribution and not necessarily in the order shown in the structure below).

[0120] As indicated above, the primary amine of PADS is leveraged to functionalize the PADS with a click chemistry. In the exemplary reaction shown below, the TCO group is added to the PADS to produce PADS-TCO. Other click chemistry groups can be functionalized therein and TCO is only provided as an example.

[0121] A polymer derived of PADS was also developed by excluding SBMA, namely poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD). PAD retains the advantage of APM and DMEAE as explained above and can be employed in the same manner as PADS.(that can be a statistical distribution and not necessarily in the order shown in the structure below).

[0122] Synthetic polymers other than PADS and PAD are also contemplated herein, for example other copolymers with primary amino containing monomeric units like APM, such as 2- aminoethyl (meth)acrylate, 2-aminoethyl (meth)acrylamide), 3-aminopropyl (meth)acrylate, 4- aminobutyl (meth)acrylate, 4-aminobutyl (meth)acrylamide. The primary amino containing monomer may be copolymerized with comonomers such as (meth)acrylic acid ((M)AA), 2- hydroxypropyl(meth)acrylamide (HP(M)A), 2-hydroxyethyl(meth)acrylate (HE(M)A), 2- (methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxybetaine methacrylate or methacrylamide (CBM), and other anionic, neutral, and zwitterionic monomers, as well as DMAEA as temporary cation. Other cationic comonomers are also contemplated, both temporary and permanent cations, such as N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl (meth)acrylamide, 3-N,N-diaminopropyl (meth)acrylate, 4-N,N- dimethylaminobutyl (meth)acrylamide, 1 ,3-bis(dimethylamino)propyl (meth)acrylate, and 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate.

[0123] Although the present disclosure describes a “first” and “second” polymer, the backbone of these polymers can be the same backbone composition with the only difference being that different click chemistry groups are functionalized on each which are adapted to react to each other. The backbone of the “first” and “second” polymer can also be different. It should also be noted that the synthetic polymer (either the first or the second) can include multiple different polymers. For example, a mixture of the same chemical polymer but with different molecular weights can be used, or even different polymers.

[0124] More generally, the polymers described herein can be functionalized though a primary amine as shown below with TOO as an exemplary click chemistry group in the first and second reactions shown below and a norbornene for the third.

[0125] When one of the synthetic polymers used for the click chemistry crosslinking is a cationic polymer, it is provided as a coating. The overall composition, net cationic charge, and molecular weight of the polycation used are affect the parameters for the hydrogel coating. Where the hydrogel is coated with a single coating polymer, the composition of the polycation is preferably 10-100 mol% net cationic (i.e. it may be 100% net cationic), more preferably 30-60 mol% net cationic in charge with the remaining 90-0 mol% of the coating polymer containing hydrophilic, charge-neutral comonomer. Of the 30-60 mol% cationic charge, preferably 20 mol% is primary amine containing, with the remaining cationic charge from comonomers containingtertiary or quaternary ammonium cations. Molecular weight of the polycation is also plays a role in the coating’s permeability and control over the permeability, and can be 5,000-50,000 g / mol, but more preferably 15,000-40,000 g / mol.

[0126] One advantage of utilizing polycation coatings is that the polycations are held in place electrostatically which facilitates reaction with the matching I EDDA groups bound on polyanionic polymers including but not limited to PMM.

[0127] It is important to recognize that, when making reference to a coating, the polymers used in these coatings do not actually add to the surface of these very permeable hydrogel, but are more typically absorbed into the outer layers of the hydrogel. As a result, these coating processes do not tend to increase the diameter of the hydrogel, but rather increase density of the outer layers. Further, both initial hydrogel components and coating polymers coexist in these outer layers, and, given correct chemical compositions described herein, may react with each other both ionically and covalently. This concept is key to using reactive polycation / polyanion pairs to coat, modify permeability to effect tighter molecular weight exclusion, and otherwise reinforce the outer layers of these hydrogels. The number of layers, in addition to the concentration of the polymer in each layer, can therefore be used as another method to control the overall permeability of the hydrogel coating.

[0128] TCO, tetrazine or other click chemistry may also be formed into a monomer via reaction with acryloyl chloride, methacryloyl chloride, acrylic anhydride or methacrylic anhydride. The monomers can be copolymerized to generate lEDDA-capable polymers that can be used as the polycation coating polymers. This can involve copolymerization with anionic and zwitterionic monomers such as acrylic acid, methacrylic acid, 2-acrylamido-2-methyl-1 -propane sulfonic acid, 3-sulfopropyl methacrylate, SBMA, MPC, and carboxybetaine methacrylates or methacrylamides (CBM) as well as other betaines. I EDDA based polymers with a net anionic charge can be used as a coating polymer to coat hydrogels with a net cationic surface charge, such as polycation coated hydrogels. These I EDDA functional polymers can also be covalently crosslinked to surfaces and coatings that have primary amino groups.

[0129] In some embodiments, the polycation is poly-L-lysine of 15,000 to 30,000 MW, modified with 5 to 40, and preferably 10 to 30 mol% of a click chemistry group such as norbornene or TCO (see functionalization example below).

[0130] In some embodiments, the synthetic polymer is poly[APM-rco-co-DMAEA-co-SBMA] (PATCODS), where APMTCO stands for APM in the copolymer that carries 5 to 40, and preferably 10 to 30 mol% TCO group.

[0131] In some embodiments, the PATCODS has an initial molar feed ratios of 1 : 1 :2 of APMTCO,DMAEA, and SBMA, with each value in the ratio varying by ±50%.

[0132] In some embodiments, the synthetic polymer is poly[APMNB-co-DMAEA-co- SBMA],(PANBDS), where APMNB stands for APM in the copolymer that carries 5 to 40, and preferably 10 to 30 mol% norbornene groups.

[0133] In some embodiments, the synthetic polymer is poly-L-lysine (PLL) of 3,000 to 60,000, and preferably 15,000 - 30,000 Dalton molecular weight, functionalized with 5 to 40, and preferably 10 to 30 mol% norbornene groups or between 10 and 40 mol% TCO as shown below.

[0134] In a further embodiment, there is provided a functionalized polymer called PMM-tz, comprising a functional tetrazine group as follows:PMMAn Tetrazine-amine

[0135] In some embodiments, the PMM-tz may also comprise cationic groups such as obtained by reaction of initially formed PMM-tz with N,N-dimethylaminopropylamine or analogous molecules comprising additional tertiary amine groups, to form a tz-functional polymer with cationic to anionic charge ratios ranging from 90:10 to 60:40.

[0136] Accordingly, the present disclosure provides a hydrogel for producing capsules, strings, patches and the like. The hydrogel is crosslinked using click chemistry (IEDDA, SPAAC, SPANC or SPANOC) between two polymers, each functionalized with corresponding click chemistry groups that react with each other.

[0137] In the above, IEDDA was used as the example for click chemistry groups and functionalization for simplicity, however the same functionalization can be done with click chemistry groups corresponding to SPAAC and SPANC as well as other IEDDA groups not discussed above.

[0138] More generally with regards to the possible click chemistry functional groups, one of the polymers is functionalized with a first click chemistry group and the second polymer is functionalized with a second click chemistry group. The first click chemistry group is a cycloalkene, heterocycloalkene, cycloalkyne or heterocycloalkyne click chemistry group that reacts with the second click chemistry group comprising a nitrogen or an oxygen that reacts with the double bond of the cycloalkene or heterocycloalkene, or the triple bond of the cycloalkyne or heterocycloalkyne to form a covalent link between the two click chemistry groups. Alternatively, the second click chemistry group is a cycloalkene, heterocycloalkene, cycloalkyne or heterocycloalkyne click chemistry group and the first click chemistry group comprises a nitrogen or oxygen that reacts with the double bond of the cycloalkene or heterocycloalkene, or the triple bond of the cycloalkyne or heterocycloalkyne to form a covalent link between the two click chemistry groups. The click chemistry group comprising the nitrogen is for example an azide, a tetrazine, a triazine, a pyridazine, a nitrone or nitrile-oxide.

[0139] The term “cycloalkene” as used herein can be defined as a C3-C10 cycloalkene group, comprising a carbon - carbon double bond that is sterically available. Preferably the carbon atoms of the carbon - carbon double bond are not substituted. The C3-C10 cycloalkene group can be substituted or unsubstituted. Examples of a C3-C10 cycloalkene include, but are not limited to, cyclopropene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene (cis or trans), cyclononene, and cyclodecene. Examples of cycloalkene containing compounds that can be functionalized onto a polymer include trans-cyclooctene — NHS ester (TCO-NHS), TCO-NHS with a polyethylene glycol spacer arm (PEG) such as TCO-PEGn-NHS ester where n is an integer of from 1 to 15, preferably 4 to 12, trans-cyclooctene — COOH, and trans-cyclooctene-PEGn-COOH where n is an integer of from 1 to 15, preferably 4 to 12.TCO-NHS ester, specifically (E)-Cyclooct-4-enyl 2,5- dioxo- 1 -pyrrolidinyl carbonate.

[0140] The TCO-NHS ester as shown above reacts by carbodiimide chemistry with a primary amine on the polymer as shown above. Moreover, similar reactions can be performed with othercompounds to provide the cycloakene, heterocycloalkene, cycloalkyne or heterocycloalkyne groups on the polymers.

[0141] Other examples of cycloalkene can be norbornene such as

[0142] The term “heterocycloalkene” refers to a three to ten membered ring containing at least one heteroatom and the remainder being carbon atoms, the heterocycloalkene comprising a carbon - carbon double bond that is sterically available. Preferably the carbon atoms of the carbon - carbon double bond are not substituted. The heteroatoms are for example one or more of N, S or O. The heterocycloalkene can be substituted or unsubstituted.

[0143] The term “cycloalkyne” as used herein refers to a C7-C9 carbon ring comprising a carbon - carbon triple bond. The cycloalkyne can be substituted or unsubstituted. Preferably the cycloalkyne is cyclooctyne. Examples of cycloalkyne include but are not limited to:

[0144] The term “heterocycloalkyne” refers to a 7 to 9 membered ring containing at least one heteroatom and the remainder being carbon atoms and comprising a carbon - carbon triple bond. The heteroatoms are for example one or more of N, S or O. The heterocycloalkyne can be substituted or unsubstituted. Examples of heterocycloalkyne include but are not limited to:amine where n is an integer from 1 to 6, preferably from 2 to 4)

[0145] One particular example of the click chemistry that was emphasized above is the reaction between the two functional groups trans-cyclooctene (TOO) and tetrazine. Another example is the reaction between an azide and dibenzocyclooctyne (DBCO) such as sulfo-DBCO amine (shown below). This reaction is a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, typically a strained cyclooctyne reacting with an aliphatic azide or a pendant azide. TheI EDDA, SPAAC and SPANOC reactions (which can be done with alternative functional groups as explained herein) are preferable due to their reaction kinetics and compatibility with living cells.(sulfo-DBCO amine)

[0146] In some embodiments, when the first polymer is functionalized with a click chemistry group comprising a cycloalkene or a heterocycloalkene, the second polymer is functionalized with a tetrazine, a triazine or a pyridazine. On the other hand, when the first polymer is functionalized with a click chemistry group comprising a cycloalkene or a heterocycloalkene, then it is the second polymer that is functionalized with a tetrazine, triazine or a pyridazine. The azide group can also react with the cycloalkene or heterocycloalkene with Deep Eutectic Solvents (DES). In some embodiments, when the first polymer is functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the second polymer is functionalized with a click chemistry group comprising a diazenediyl (-N=N-). Alternatively, when the first polymer is functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the second polymer is functionalized with a click chemistry group comprising a diazenediyl (-N=N-). The diazenediyl containing click chemistry group is preferably an azide, a tetrazine, a triazine or a pyridazine. The azide can be a pendant azide group: -N=N+=N'. Exemplary structures are shown below where R is a C1-C6 alkyl, a C6 aryl, a C5-C6 heteroaryl, a halogen or a C1-C6 haloalkyl.

[0147] Strain-promoted alkyne-nitrone cycloaddition (SPANG) reactions are also contemplated herein. Accordingly, in SPANG embodiments, when the first polymer is functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, then the second polymer is functionalized with a click chemistry group comprising a nitrone. When the first polymer is functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the second polymer is functionalized with a click chemistry group comprising a nitrone. The nitrone is of the formula R-C=N+(R’)-O' where R and R’ are alkyl or combine together to form a heterocycloalkyl. The alkyl is preferably an optionally substituted Ci-Ce alkyl or is H. The heterocycloalkyl is preferably a 5 membered or 6 membered ring. One example of a linear nitrone is R-C=N+(Me)-O'. Exemplary cyclic nitrones are as illustrated below. Cyclic nitrones are preferred as they are more stable in aqueous solvents.

[0148] SPANOC is similar to SPAAC and utilizes a cycloalkyne or hetercycloalkyne as described as the first click chemistry groups and combines it with an oxime moiety (e.g. -CH=N- OH) instead of a triazide. The pendant oxide can react much faster than the azide.

[0149] In general, when the first polymer is functionalized with a cycloalkene, heterocycloalkene, cycloalkyne, or heterocycloalkyne, then the second polymer is functionalized with a suitable click chemistry group comprising a nitrogen that reacts with the double bond or the triple bond to form a covalent link between the two groups (and vice versa, i.e. when the first polymer is functionalized with the suitable click chemistry group comprising a nitrogen then the second polymer is functionalized with a cycloalkene, heterocycloalkene, cycloalkyne, or heterocycloalkyne click chemistry group).

[0150] SPAAC, SPANC and SPANOC reactions do not require the use of a catalyst, minimizing potential toxicity or adverse effects on cells. The azide and alkyne functional groups are also non-reactive to most biologically relevant molecules, reducing the incidence of nonspecific reactions, allowing the reactive moieties to participate in crosslinking. SPAAC reactions proceed efficiently under physiological conditions, making them well-suited for cell encapsulation. SPAAC crosslinking also tends to have fast reaction kinetics, which allows for rapid formation of covalent networks. The triazole linkages formed via SPAAC crosslinking are highly stable and resistance to hydrolysis or degradation. This stability supports long term stability and integrity of the hydrogel matrix, which is critical for therapeutic cell transplant.

[0151] Many different functional groups have been explored for this purpose. Some approaches involve nucleophile - electrophile reactions between, e.g., amines and anhydrides attached to different polymers. Strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry offers a bioorthogonal reaction that proceeds without the need for metal catalysts and under physiological conditions, making it ideal for applications involving living cells. Accordingly, SPAACchemistry is leveraged to create synthetic polymer matrices for cell encapsulation, providing a novel approach to improve the efficacy of therapeutic cell-delivery applications.

[0152] Some exemplary functionalization are presented belowExample 1 : Functionalize PMM Anhydride with 20 mol% 3-azido-1-propamine to form PMM-AZ .Example 2: Functionalize PLL with 20 mol% dibenzocyclooctyne-ZV-hydroxysuccinimidyl ester to form PLL-DBCO.Example 3: Functionalize PMM Anhydride with 20 mol% dibenzocyclooctyne-amine to form PMM- DBCO.Example 4: Functionalize DBCO onto primary amines such as that of PADS or PLL.Example 5: functionalize an amine azide (AZ-amine) onto a PMM polymer to obtain PMM-AZ

[0153] In some embodiments, the synthetic polymer is poly-L-lysine (PLL) of 3,000 to 60,000, and preferably 15,000 - 30,000 Dalton molecular weight, functionalized with 5 to 40, and preferably 10 to 30 mol% azide groups or a corresponding cycloalkyne as shown below.

[0154] In some embodiments, the PMM-DBCO or PMM-AZ may also comprise cationic groups such as obtained by reaction of initially formed PMM-DBCO or PMM-AZ with N,N-dimethylaminopropylamine or analogous molecules comprising additional tertiary amine groups, to form a DBCO- or AZ-functional polymer with cationic to anionic charge ratios ranging from 90:10 to 60:40.

[0155] Accordingly, although the present disclosure provides PMM-DBCO and PMM-AZ as examples of a backbone functionalized with SPAAC moieties, this is not a limitative example as synthetic polymers other than PMM can be used, but also other pendant SPAAC-reactive groups can be used. A preferred embodiment, which is exemplified in the Example section below, is the formation of a hydrogel between PADS-DBCO with PMM-AZ, together with a separate initial gel former. The initial gel former is for example alginate, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, pluronic, or elastin. The gel former can be provided in a weight percentage of from 0.5 to 10 wt. %, and preferably 1 to 4 wt. %, depending on the viscosity and the type of gel former.

[0156] PMM-AZ and PLL-DBCO can hence react means of the SPAAC mechanism to form the structure shown below:

[0157] In some embodiments, the hydrogels formed in the present disclosure are provided as a coating for hydrogel comprising a core made from a thiol-ene crosslinked system. An example includes a first PMM polymer functionalized with vinylsulfone groups (PMM-CVS) and a second PMM polymer functionalized with SPy or PMM -cystamine, which can both be combined with alginate and air-sheared into a calcium chloride gelling bath. The initially formed calcium alginate gel is thought to physically retain the two gelformers, enabling mutual crosslinking upon subsequent exposure to reducing agents such as TCEP or THPP that can deprotect the SPy disulfide or cystamine disulfide to liberate a free thiol that subsequently reacts with a vinylsulfone group on the second polymer.

[0158] In another embodiment, the hydrogels formed in the present disclosure are provided as a coating in the hydrogel comprising a core made from a homocysteine thiolactone / vinyl sulfone crosslinked system. An example includes a first PMM polymer functionalized with vinyl sulfone groups (PMM-CVS) and a PMM polymer functionalized with homocysteine thiolactone (PMM-HTL), which can be combined and used to prepare hydrogels as described previously. The homocysteine thiolactone (HTL) groups can be hydrolyzed or aminolyzed to generate free thiol groups to react with vinyl sulfone groups of PMM-CVS for thiolene crosslinking. The hydrogels can be coated with cationic polymer such as PLL-norbornene, where the amino groups of PLL aminolyze the HTL groups to covalently crosslink the coating to the hydrogel core. The PLL- norbornene coated hydrogels can then be coated with an anionic polymer such as PMM-tetrazine to implement the click crosslinking via IEDDA chemistry in the hydrogel coatings, as an example.

[0159] In a further embodiment, the click chemistry described in the present disclosure can be used to prepare core hydrogels. For example, a first polymer containing either diene or dienophile can be admixed with alginate and extruded in a gelling bath to form a hydrogel. This hydrogel can then be treated with an oligomeric or polymeric functional polymer with complimentary diene or dienophile that diffuses into the hydrogel to activate the click covalent crosslinking chemistry through the entire hydrogel core and shell. These oligomeric or polymer species can be a linear structure, or multi-arm polymer that contains the functional diene or dieneophile at the end of the short polymer chains, as well as throughout the short polymer chains. The oligomers / polymers can be anionic or neutral in charge to allow for diffusion throughout the hydrogel. Examples of the linear and multi-arm functionalized oligomers / polymer are shown in Fig. 1 D, with the octagons depicting the location of the diene or dienophile functional groups.EXAMPLESynthesis of PMM-tetrazine (PMM-tz15)

[0160] Poly[methylvinylether-a / f-maleic anhydride] (PMMAn) was conjugated with a tetrazine derivative, targeting 15 mol% degree of functionalization, with respect to the repeat unit. PMMAn, (116 mg, 0.745 mmol) was dissolved in 5 mL of acetonitrile in a 20 mL glass vial equipped with a magnetic stir-bar. To the stirring reaction mixture, triethylamine (29 mg, 0.287 mmol) was added, followed by the drop-wise addition of (4-(1 ,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (25 mg, 0.112 mmol) dissolved in 2 mL of a 1 :1 DMSO:acetonitrile (v / v) solvent mixture. The reaction mixture was left to stir at 700 rpm at room temperature, overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 12-14 kDa molecular weight cut-off. The polymer was dialyzed against distilled water, with twice daily water bath changes for 3 days. The polymer solution was then lyophilized, yielding PMM-tetrazine as a bright pink / red solid at 97 % yield.1H NMR of PMM-tetrazine was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2A) showing approximately 13.5 mol% degree of functionalization.PMMAn Tetrazine-amineSynthesis of PLL-norbornene

[0161] Poly-L-lysine (PLL) was functionalized with a norbornene derivative, targeting 15 mol% degree of functionalization, with respect to the monomer lysine repeat units. PLL (15-30 kDa, hydrobromide salt) (106 mg, 0.507 mmol) was dissolved in 5 mL of N,N-dimethylformamide (DMF) with triethylamine (40 pL, 0.287 mmol) in a 20 mL glass vial equipped with a magnetic stir bar. 5-Norbornene-2-acetic acid succinimidyl ester (19.1 mg, 0.0766 mmol) was dissolved in 1 mL of DMF and the solution was drop-wise added to the stirring reaction mixture. The reaction mixture was left to stir overnight at room temperature, followed by purification by dialysis using 3.5 kDa molecular weight cut-off tubing. The polymer solution was dialyzed against distilled waterwith twice daily water bath changes for 3 days. PLL-norbornene was lyophilized, resulting in a white solid in 65 % yield.1H NMR of PLL-norbornene was recorded in D2O with a Bruker Avance Neo 600 MHz spectrometer (Fig. 2B) showing approximately 7 mol% degree of functionalization.Synthesis of PLL-norbornene (PMM-Nor10)

[0162] PMMAn was conjugated with a norbornene derivative, targeting 10 mol% degree of functionalization with respect to the repeat unit. PMMAn (1.00 g, 6.40 mmol) was dissolved in 20 mL of acetonitrile in a 50 mL round bottom flask equipped with a magnetic stir-bar. To the stirring reaction mixture, triethylamine (162.03 mg, 1.60 mmol) was added, followed by the drop-wise addition of a solution of 5-norbornene-2-methanamine (80.5 mg, 0.641 mmol) dissolved in 2 mL of acetonitrile. The reaction mixture was left to stir at 600 rpm at room temperature, overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 12-14 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 3 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was then lyophilized, yielding PMM-Norbornene as a pale purple solid at 92 % yield.1H NMR of PMM-Norbornene was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2C) showing approximately 9 mol% degree of functionalization.Synthesis of PMM-trans-cyclooctene (PMM-TCO10)

[0163] PMMAn was conjugated with a trans-cy clooctene derivative, targeting 10 mol% degree of functionalization with respect to the repeat unit. PMMAn (305.4 mg, 1.95 mmol) was dissolved in 6 mL of acetonitrile in a 20 mL glass vial equipped with a magnetic stir-bar. Triethylamine (39 mg, 0.39 mmol) was added and the reaction was allowed to stir for 5 min. (E)- cyclooct-4-en-1-yl (3-aminopropyl)carbamate hydrochloride (50 mg, 0.193 mmol) was dissolved in 0.3 mL of DMSO and added dropwise to the reaction mixture. The reaction was allowed to stir at 600 rpm at room temperature, overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 12-14 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 3 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was then lyophilized, yielding PMM-TCO as a pale purple solid at 98 % yield.1H NMR of PMM-TCO was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2D) showing approximately 9 mol% degree of functionalization.Synthesis of PMM- / V-methylimidazole (PMM-MIM10)

[0164] PMMAn was conjugated with an / V-methylimidazole derivative, targeting 10 mol% degree of functionalization with respect to the repeat unit. PMMAn (1.003 g, 6.40 mmol) was dissolved in 20 mL of acetonitrile in a 50 mL round bottom flask equipped with a magnetic stirbar. To the stirring reaction mixture, triethylamine (155.9 mg, 1.54 mmol) was added. Separately, (1-methyl-1 H-imidazol-2-yl)methanamine dihydrochloride (143.0 mg, 0.777 mmol) and triethylamine (155.9 mg, 1.54 mmol) were dissolved in 2 mL of DMF. The solution containing the / V-methylimidazole derivative was added dropwise to the stirring solution containing PMMAn. The reaction mixture was left to stir at 600 rpm at room temperature, overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 12-14 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 3 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was then lyophilized, yielding PMM-MIM as a pale purple solid at 96 % yield.1H NMR of PMM- MIM was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2E) showing approximately 13.6 mol% degree of functionalization.Synthesis of PMM-Dibenzocyclooctyne (PMM-DBCO10)

[0165] PMMAn was conjugated with a DBCO derivative, targeting 10 mol% degree of functionalization with respect to the repeat unit. PMMAn (1.003 g, 6.40 mmol) was dissolved in 20 mL of acetonitrile in a 50 mL round bottom flask equipped with a magnetic stir-bar. To the stirring reaction mixture, triethylamine (155.9 mg, 1.54 mmol) was added. Separately, DBCO-NH2 (37.0 mg, 0.0724 mmol) was dissolved in 0.45 mL of acetonitrile and added dropwise to the stirring solution containing PMMAn. The reaction mixture was left to stir at 600 rpm at room temperature, overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 12-14 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 3 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was then lyophilized, yielding PMM-DBCO as a pale purple solid (99%).1H NMR of PMM-DBCO was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2F) showing approximately 10.33 mol% degree of functionalization.Synthesis of PMM-azide (PMM-N3)

[0166] PMMAn was conjugated with a functional group bearing an azide functionality, targeting 20 mol% degree of functionalization with respect to the repeat unit. PMMAn (780 mg,5.00 mmol) was dissolved in 15.6 mL of acetonitrile in a 50 mL round bottom flask equipped with a magnetic stir-bar. To the stirring reaction mixture, triethylamine (203 mg, 2.01 mmol) was added. Separately, 3-azido-1-propanamine (100 mg, 0.999 mmol) was dissolved in 1.56 mL of 1 :1 DMSO:acetonitrile. The solution containing azide was added dropwise to the stirring solution containing PMMAn. The reaction mixture was left to stir at 600 rpm at room temperature, overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 12-14 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 3 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was then lyophilized, yielding PMM-N3 as a pink solid (96%).1H NMR of PMM-N3 was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2G) showing approximately 21.6 mol% degree of functionalization.Synthesis of Poly[sulfobetaine methacrylate]-dicarboxylate (p(SBMA-(COOH)2))

[0167] Poly[sulfobetaine methacrylate] chain-terminated with carboxylic acids was synthesized targeting an Mnof 10 kDa. In a 20 mL glass vial equipped with a magnetic stir bar, sulfobetaine methacylate (SBMA) (1.000 g, 3.58 mmol) was dissolved in 2.22 mL of 0.5 M saline (pH = 6.32). In a separate 20 mL glass vial, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (25.2 mg, 0.09 mmol) and 4,4’-azobis(4-cyanovaleric acid) (5.02 mg, 0.018 mmol) were dissolved in 1.11 mL of dioxane. The solution containing the RAFT agent / initiator was added in one portion to the solution containing SBMA ([SBMA] = 30 wt. %). The reaction mixture was sparged with nitrogen for 45 minutes at room temperature. The reaction was allowed to stir at 600 rpm at 70 °C under a nitrogen atmosphere for 30 minutes. Separately, 4,4’-azobis(4-cyanovaleric acid) (502 mg, 1.8 mmol) was dissolved in 1.17 mL of dioxane and sparged with nitrogen for 15 minutes and added to the reaction under nitrogen in one portion at 30 minutes. The reaction was allowed to stir for overnight at 70 °C and was then terminated by cooling the vessel in an ice bath for 10 minutes, followed by exposure of the reaction to air. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with distilled water with four daily water bath changes for 2 days. The polymer solution was then lyophilized, yielding p(SBMA)-dicarboxylic acid as an orange solid (79%). Mn(GPC) = 9580 g / mol, D = 1.073.Synthesis of Poly[sulfobetaine methacrylate]-di(norbornene) (p(SBMA)-NOR2)

[0168] In a 20 mL glass vial equipped with a magnetic stir bar, p(SBMA-(COOH)2 (249.4 mg, 0.026 mmol, 0.052 mmol eq. COOH) was dissolved in 1 mL of DI and adjusted to pH 8.09 using 1 M NaOH. AMP-Py (66.3 mg, 0.182 mmol) was added in one portion, and the mixture wasallowed to stir for 5 minutes at room temperature. 5-norbornene-2-methanamine (18.5 mg, 0.15 mmol) was added in one portion, and the reaction was allowed to stir at 50 °C for overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with distilled water with four daily water bath changes for 2 days. The polymer solution was lyophilized, yielding p(SBMA)-NOR2 as an orange solid (80%).Synthesis of Poly[methacrylate-co-sulfobetaine methacrylate] (PMS)

[0169] Poly[methacrylate-co-sulfobetaine methacrylate] (PMS) was synthesized targeting varying mol% composition of the methacrylate monomer for subsequent functionalization with click-reactive groups.Synthesis of PMS20, PMS40, PMS60, PMS80

[0170] For PMS40, in a 20 mL glass vial, sodium methacrylate (MA) (823 mg, 18.97 mmol) and sulfobetaine methacylate (SBMA) (3.184 g, 11.4 mmol) were dissolved in deionized water (DI), adjusted to pH 4.24 using 6M HCI, and balanced to 8.89 mL of DI. In a separate 20 mL glass vial, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (37.1 mg, 0.133 mmol) and 4,4’-azobis(4- cyanovaleric acid) (7.3 mg, 0.026 mmol) were dissolved in 4.45 mL of dioxane. The two solutions were combined in a 50 mL round bottom flask equipped with a magnetic stir bar, and the reaction mixture was sparged with nitrogen for 45 minutes at room temperature. The reaction was allowed to stir at 600 rpm at 70 °C under a nitrogen atmosphere for 5.33 h. The reaction was terminated by cooling the round bottom flask in an ice bath for 10 minutes, followed by exposure of the vessel to air. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with distilled water with twice daily water bath changes for 4 days. The polymer solution was then lyophilized, yielding PMS40 as a pink solid (80%). Mw(GPC) = 25581 , D = 1.187.

[0171] PMS20, PMS60, and PMS80 were synthesized in the same manner as PMS40, using instead MA:SBMA feed ratios of 20:80, 40:60, and 80:20 mol%, respectively.Synthesis of 4-methoxy-T-methyl-[1,2'-bipyridine]-1,1'-diium chloride iodide (AMP-Py)

[0172] In a 100 mL round bottom flask equipped with a magnetic stir bar, 2-chloro-1- methylpyridinium iodide (3.00 g, 11.74 mmol) and 4-methoxypyridine (2.56 g, 23.58 mmol) were dissolved in 60 mL of acetonitrile. The reaction was allowed to stir for 3 hours at roomtemperature. The mixture was subsequently vacuum filtered to yield a precipitate which was washed with 3 x 15 mL of ice-cold acetonitrile and 3 x 15 mL of ice-cold diethyl ether. The product was isolated as a yellow powder (3.67 g, 86%).1H NMR spectrum matched the literature available data on AMP-Py.Synthesis of PMS60-tetrazine (PMS60-Tet10)

[0173] PMS60 was conjugated with a tetrazine derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 17.9 mol% degree of functionalization with respect to the methacrylate monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PMS60 (250 mg, 56% MAA: 44% SBMA, 0.762 mmol MAA equiv.) was dissolved in 3.84 mL of DI and adjusted to pH 7 using 1 M NaOH. AMP-Py (68.9 mg, 0.174 mmol) was added in one portion, and the mixture was allowed to stir for 5 minutes at room temperature. (4-(1 ,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (30.6 mg, 0.137 mmol) and triethylamine (13.8 mg, 0.136 mmol) were added in one portion, and the reaction was allowed to stir at 40 °C for overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 2 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was lyophilized, yielding PMS60-Tet as a bright pink solid (91%).1H NMR of PMS60-Tet was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2H) showing approximately 2.92 mol% degree of functionalization.Synthesis of PMS60-Norbornene (PMS60-Nor10)

[0174] PMS60 was conjugated with a norbornene derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 17.9 mol% degree of functionalization with respect to the methacrylate monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PMS60 (250 mg, 56% MAA: 44% SBMA, 0.762 mmol MAA equiv.) was dissolved in 3.84 mL of DI and adjusted to pH 6.93 using 1 M NaOH. AMP-Py (68.7 mg, 0.173 mmol) was added in one portion, and the mixture was allowed to stir for 5 minutes at room temperature. 5-norbornene-2-methanamine (16.79 mg, 0.136 mmol) was added in one portion, and the reaction was allowed to stir at 40 °C for overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 2 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution waslyophilized, yielding PMS60-Nor10 as a pale pink solid (93%).1H NMR of PMS60-Nor was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2I) showing approximately 3.1 mol% degree of functionalization.Synthesis of PMS60-trans-cyclooctene (PMS60-TCO)

[0175] PMS60 was conjugated with a TCO derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 17.9 mol% degree of functionalization with respect to the methacrylate monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PMS60 (250 mg, 56% MAA: 44% SBMA, 0.762 mmol MAA equiv.) was dissolved in 3.84 mL of DI and adjusted to pH 7 using 1 M NaOH. AMP-Py (69.9 mg, 0.176 mmol) was added in one portion, and the mixture was allowed to stir for 5 minutes at room temperature. (E)-cyclooct-4-en-1-yl (3-aminopropyl)carbamate hydrochloride (37.1 mg, 0.142 mmol) and triethylamine (14.3, 0.142 mmol) were added in one portion, and the reaction was allowed to stir at 40 °C for overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 2 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was lyophilized, yielding PMS60-TCO as a pale pink solid (95%).1H NMR of PMS60-TCO was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2J) showing approximately 3.4 mol% degree of functionalization.Synthesis of PMS60-dibenzocyclooctyne (PMS60-DBCO)

[0176] PMS60 was conjugated with a DBCO derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 17.9 mol% degree of functionalization with respect to the methacrylate monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PMS60 (250 mg, 56% MAA: 44% SBMA, 0.762 mmol MAA equiv.) was dissolved in 3.84 mL of DI and adjusted to pH 7 using 1 M NaOH. AMP-Py (69.5 mg, 0.191 mmol) was added in one portion, and the mixture was allowed to stir for 5 minutes at room temperature. DBCO-NH2 (39.9 mg, 0.144 mmol) was added in one portion, and the reaction was allowed to stir at 40 °C for overnight. The reaction mixture was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with saline (0.27 M) with twice daily water bath changes for 2 days, followed by dialysis with distilled water with twice daily water bath changes for 2 days. The polymer solution was lyophilized, yielding PMS60-DBCO as a whitesolid (90%).1H NMR of PMS60-TCO was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2K) showing approximately 5.01 mol% degree of functionalization.Synthesis of Click-Functionalized PMS20 / 40 / 80 Polymers

[0177] Click-functionalization reactions with PMS20, PMS40, and PMS80, respectively, were performed in the same manner as with PMS60.Synthesis of Poly[(3-aminopropylmethacrylamide-graf -methyltetrazine)-co- / V, / V- (dimethylamino)ethyl acrylate] (PAD-mTet)

[0178] Poly[3-aminopropylmethacrylamide-co-N,N-(dimethylamino)ethyl acrylate] (PAD) was conjugated with a methyltetrazine derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 48 mol% degree of functionalization with respect to the 3-aminopropylmethacrylamide monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PAD (248.2 mg, 23 mol% aminopropylmethacrylamide, 0.294 mmol monomer eq.) and triethylamine (177.10 mg, 1.75 mmol) were dissolved in 5 mL dichloromethane. 2,5-Dioxo-1- pyrrolidinyl 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzeneacetate (46.1 mg, 0.141 mmol) was added in one portion and the reaction was allowed to stir at 600 rpm at room temperature for overnight. The reaction mixture was rapidly dispersed into 45 mL of acetate buffer (0.5 M, pH 3.6). The aqueous phase was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with 1 mM HCI with twice daily water bath changes for 4 days. The polymer solution was lyophilized, yielding PAD-mTet as a bright red solid (79%).1H NMR of PAD-mTet was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2L) showing approximately 13 mol% degree of functionalization.Synthesis of PAD-Norbornene (PAD-Nor)

[0179] PAD was conjugated with a norbornene derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 48 mol% degree of functionalization with respect to the 3-aminopropylmethacrylamide monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PAD (250 mg, 23 mol% aminopropylmethacrylamide, 0.294 mmol monomer eq.) and triethylamine (145 mg, 1.43 mmol) were dissolved in 5 mL dichloromethane and allowed to stir at 600 rpm for 10 minutes. In a 4 mL glass vial equipped with a stir bar, 5-norbornene-2-carboxylic acid (19.5 mg, 0.141 mmol) and pyridine (38.8 mg, 0.492 mmol) were dissolved in 1.5 mL dichloromethane. / V, / V, / V\ / V'-tetrarnethylchloroformamidiniumhexafluorophosphate (TCFH) (51.1 mg, 0.182 mmol) was added in one portion and the reaction was allowed to stir at 600 rpm for 2 minutes. The solution containing the norbornene derivative was added to the solution containing PAD in one portion, and the reaction was allowed to stir at 600 rpm, at room temperature, for overnight. The reaction mixture was rapidly dispersed into 45 mL of acetate buffer (0.5 M, pH 3.6). The aqueous phase was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with 1 mM HCI with twice daily water bath changes for 4 days. The polymer solution was lyophilized, yielding PAD-mTet as an orange solid (75%).1H NMR of PAD-Nor10 was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2M) showing approximately 9.6 mol% degree of functionalization.Synthesis of PAD-trans-cyclooctene (PAD-TCO)

[0180] PAD was conjugated with a TCO derivative, targeting 5 mol% degree of functionalization with respect to the polymer chain, equivalent to 17.5 mol% degree of functionalization with respect to the 3-aminopropylmethacrylamide monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PAD (100 mg, 29 mol% aminopropylmethacrylamide, 0.162 mmol monomer eq.) and triethylamine (67.7 mg, 0.67 mmol) were dissolved in 5 mL dimethylformamide and allowed to stir at 600 rpm for 10 minutes. (E)-cyclooct-4-en-1-yl (3- aminopropyl)carbamate hydrochloride (7.5 mg, 0.057 mmol) was added in one portion and the reaction was allowed to stir at 600 rpm for 1 hour. The reaction mixture was rapidly dispersed into 45 m L of acetate buffer (0.5 M, pH 3.6). The aqueous phase was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with 1 mM HCI with twice daily water bath changes for 4 days. The polymer solution was lyophilized, yielding PAD-TCO as an off-white solid (72%).1H NMR of PAD-TCO10 was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 2N) showing approximately 9.6 mol% degree of functionalization.Synthesis of PAD-azide (PAD-N3)

[0181] PAD was conjugated with an azide derivative, targeting 10 mol% degree of functionalization with respect to the polymer chain, equivalent to 35 mol% degree of functionalization with respect to the 3-aminopropylmethacrylamide monomer. In a 20 mL glass vial equipped with a magnetic stir bar, PAD (300 mg, 35 mol% aminopropylmethacrylamide, 0.485 mmol monomer eq.) and triethylamine (196.4 mg, 1.94 mmol) were dissolved in 15 mL of 1 :1DMF:acetonitrile and allowed to stir at 600 rpm for 10 minutes. 2,5-dioxopyrrolidin-1-yl 2- azidoacetate (33.7 mg, 0.170 mmol) was added in one portion and the reaction was allowed to stir at 600 rpm for 1 hour. The reaction mixture was rapidly dispersed into 35 mL of acetate buffer (0.5 M, pH 3.6). The aqueous phase was transferred into cellulose acetate dialysis tubing with 3.5 kDa molecular weight cut-off. The polymer was dialyzed with 1 mM HCI with twice daily water bath changes for 4 days. The polymer solution was lyophilized, yielding PAD-N3 as an off-white solid (87%).1H NMR of PAD-N3 was recorded in D2O with a Bruker Advance Neo 600 MHz spectrometer (Fig. 20) showing approximately 10.01 mol% degree of functionalization.Capsule Formation

[0182] PMM-tetrazine was combined with sodium alginate as the gel former to form hydrogel capsules. PMM-tetrazine and sodium alginate (MVG, NovaMatrix™) were dissolved in 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS) and combined at a final concentration of 1 wt.% each of the polymers at pH 7.4. The polymer solution was filtered through a 0.22 pm filter and loaded into a 1 mL syringe. The syringe was fitted with a coaxial needle was placed into a vertically oriented syringe pump (Harvard Apparatus). The hydrogel capsules were prepared from air-shearing extrusion into a gelling bath containing calcium chloride. The capsules were left to cure in the gelling bath for 15 min, followed by washes with HBS. The hydrogel capsules containing PMM-tetrazine were stored in HBS for subsequent coating and covalent crosslinking with activated alkene derivatives, such as PLL-norbornene, for I EDDA chemistry.Cross-Linked Coatings via Tetrazine-Norbornene IEDDA Chemistry - PMM-tetrazine / PLL- norbornene Coated Hydrogel Capsules

[0183] PMM-tetrazine containing hydrogel capsules were coated with PLL-norbornene. As a polycation, PLL-norbornene electrostatically complexes with anionic PMM-tetrazine / calcium alginate capsules to form a membrane shell. The tetrazine and norbornene components of PMM and PLL, respectively, undergo spontaneous covalent crosslinking through IEDDA chemistry to form a permanent hydrogel network. More specifically, 1 wt.% PMM-tetrazine and 1 wt.% calcium alginate hydrogel capsules were exposed to 0.1 wt.% PLL norbornene in HBS for 6 min with occasional swirling. Following the coating, the capsules were left to settle and washed twice with HBS in a 3:1 saline to capsule ratio. The coated hydrogel capsules were imaged by microscopy and then subjected to chemical stress tests to test for covalent crosslinking.Chemical Stress Tests

[0184] PMM-tetrazine / PLL-norbornene coated hydrogel capsules were subjected to sodium citrate to chelate calcium from the core hydrogel, followed by the addition of NaOH to increase the pH to disrupt electrostatic interactions of the coating. Following these treatments, the remaining hydrogel confirms the presence of covalent linkages by I EDDA of PMM-tetrazine and PLL-norbornene polymers. The procedure for a citrate-NaOH test on coated hydrogel capsules is provided as follows. An aliquot of hydrogel capsules (0.25 mL) of 3:1 capsules to saline ratio suspension was added to a 4 mL glass vial, followed by 2 mL of 70 mM sodium citrate. The suspension was incubated on an orbital shaker (20-25 rpm) for at least 1 h before imaging for survival analysis post-citrate. To the vial, 0.5 mL of 1 M NaOH was added to the citrate treated sample and the vial was inverted to ensure the supernatant was well mixed. A drop of the supernatant was placed on pH paper to confirm the mixture was at pH 11-12. The sample was imaged for survival analysis post citrate-NaOH steps.Coated Hydrogel Capsules Covalently Crosslinked with IEDDA Chemistry

[0185] Tetrazine functionalized synthetic polymers were prepared by post-modification of PMM (anhydride) with an amino derivative of a tetrazine compound. PMM-tetrazine was used, in combination with sodium alginate as a gel former, to form tetrazine functionalized hydrogel cores when exposed to calcium containing gelling baths.

[0186] PMM-tetrazine and sodium alginate (1% each) were gelled in a calcium chloride containing gelling bath and extruded to form spherical hydrogel capsules. The tetrazine containing hydrogel capsules were then exposed to a solution of norbornene functionalized PLL (PLL- norbornene) to form a capsule membrane. This membrane is driven by the electrostatic complexation of cationic PLL-norbornene and anionic PMM-tetrazine and alginate in the core hydrogel which then allows for IEDDA click chemistry to occur, thereby covalently crosslinking the core and the membrane coating. The coated hydrogel capsules formed are shown in Fig. 3.

[0187] To test for covalent crosslinking of the tetrazine-norbornene coating, the hydrogel capsules were exposed to sodium citrate to chelate the calcium from the hydrogel core, in addition to increasing the pH with sodium hydroxide to eliminate electrostatic interactions by deprotonating the ammonium groups of PLL-norbornene. PMM-tetrazine / PLL-norbornene coated hydrogel capsules remained intact after the citrate / NaOH treatment which confirms the presence of covalent crosslinking through IEDDA chemistry (Fig. 4).

[0188] Fluorescence images of the PMM-tetrazine / PLL-norbornene coated hydrogel capsules were taken in the TRITC channel as it is known that tetrazine functional groups have autofluorescence properties in this range. Fig. 5 shows the remaining fluorescent hydrogel capsules after the citrate / NaOH treatment steps which provides additional evidence PMM- tetrazine crosslinking with PLL-norbornene.Core Covalently Crosslinked Hydrogel Strings by Static Mixer with IEDDA Chemistry

[0189] PMM-Tetrazine / PMM-Norbornene core covalently crosslinked strings were prepared by a dual-syringe extrusion through a static mixer. In separate extrusion mixtures, PMM-Tetrazine and PMM-Norbornene were combined with sodium alginate as the gel former. The respective PMM polymers and sodium alginate (MVG, NovaMatrix™) were dissolved in basal DMEM media, supplemented with glutamine and glucose (DMEM++), at a concentration of 2 wt.% (PMM) and 1 wt.% (sodium alginate) and pH adjusted to 7.4. Both polymer extrusion mixtures were filtered through 0.22 pm filters and loaded into separate 3 mL syringes. The 3 mL syringes were connected by a twin-syringe delivery system fitted with a 16 mixing element static mixer and 18 g blunt tipped needle. Hydrogel strings were formed by extrusion of the dual-syringe set-up by a vertically oriented syringe pump at a flow rate of 7.5 mL / hr into a calcium chloride gelling bath. After extrusion, the hydrogel strings were cured for 5 mins in the calcium chloride gelling bath before being washed three times in a 3:1 volume ratio of DMEM++ to extrusion volume. The formed strings are shown in (Fig. 6).Chemical Stress Tests

[0190] Formation of a covalently crosslinked PMM-tetrazine / PMM-norbornene gel network was assessed chemically by chelation of calcium ions with sodium citrate to dissolve the alginate gel network, with the remaining hydrogel string after treatment confirming the formation of a IEDDA covalently crosslinked network. The chemical stress test was performed on cut string segments with a 70 mM pH 9.0 sodium citrate solution for 1 hour before being imaged by optical microscopy to confirm the presence of a remaining gel network (Fig. 7).Fluorescent Staining of Residual IEDDA Reactive Groups in Core Covalently Crosslinked Hydrogel Strings

[0191] To visual polymer distribution of the covalently crosslinked PMM-Tetrazine / PMM- Norbornene hydrogel strings, residual reactive groups were stained with IEDDA reactivefluorescent labels and imaged by confocal laser scanning microscopy (CLSM). In separate wells of a 24-well glass bottom plate remaining tetrazine and norbornene groups of 2 wt.% PMM- Tetrazine / PMM-Norbornene 1 wt.% sodium alginate string segments were stained separately for 1 hour in 1 mL of 20 pM AF-488 TCO and 1 mL of 10 pM Cyanine5 Tetrazine prepared in HBS. Cross sectional CLSM images of stained hydrogel strings were taking by on a Nikon Ti eclipse confocal microscope (Figs. 8A-8B).Core Covalently Crosslinked Tetrazine-Trans Cyclooctene Hydrogel Capsules by Indiffusion of Crosslinker

[0192] Hydrogel capsules were prepared by combining PMM-Tetrazine and sodium alginate as the gel former. A combined mixture of PMM-Tetrazine 1.0 wt.% and 1.0 wt.% sodium alginate (MVG, NovaMatrix™) were dissolved in HBS at a final pH of 7.4. The combined extrusion mixture was filtered through a 0.22 pm filter and loaded into a 1 mL syringe. The syringe was fitted with a coaxial needle and placed on a vertically oriented syringe pump. Hydrogel capsule extrusion was then performed by air-shearing droplets into a calcium chloride gelling bath. The formed hydrogel capsules were cured for 5 minutes after extrusion in the calcium gelling bath before being washed twice in a 3:1 volume ratio of a calcium chloride containing solution to capsule volume ratio. The washed capsules were then covalently crosslinked by treating the capsules in a 3:1 volume ratio of a 0.33 wt.% solution of PMM-TCO in a calcium chloride containing solution for 6 mins. After crosslinking for 6 mins, the capsules were washed twice in a 3:1 volume ratio of HBS to capsule volume and then imaged as seen in Fig. 9.Chemical Stress Test

[0193] PMM-tetrazine / PMM-TCO core crosslinked hydrogel capsules were tested for covalent crosslinking by chelation of calcium with sodium citrate. In a 24-well glass bottom plate an aliquot of the 1 wt.% PMM-Tetrazine 1 wt.% sodium alginate capsule were added and the HBS supernatant was removed. To the capsule containing well, 2 mL of a 70 mM sodium citrate (pH 9.0) was added and incubated for 1 hr prior to imaging. The presence of capsules after calcium chelation confirms the formation of a covalently crosslinked PMM-tetrazine / PMM-TCO hydrogel network (Fig. 10).Encapsulation of Human Mesenchymal Stem Cell (hMSC) Clusters in Core Covalently Crosslinked Hydrogel Strings and Capsules by Static Mixer with IEDDA ChemistryMSC Cell Culture and Cell Cluster Formation

[0194] HMSC cells were maintained in a 37°C 5% CO2 incubator in supplemented StemMACS™ expansion media (Miltenyi Biotec). At 80% confluency the hMSC cells were washed with calcium and magnesium free phosphate buffered saline (PBS) and then detached by treatment with TrypLE™. MSC cells were seeded at a density 200 of into the wells of a 6- well AggreWell™ plate and cultured for 48 hours to allow for cluster formation prior to collection.Encapsulation of hMSC Clusters

[0195] hMSC cluster containing PMM-Tetrazine / PMM-Norbornene core covalently crosslinked strings and capsules were prepared by a dual-syringe extrusion through a static mixer. In separate extrusion mixtures, PMM-Tetrazine and PMM-Norbornene (2 wt.% each) were combined with 1 wt.% sodium alginate (MVG, NovaMatrix™) dissolved in DMEM++ and pH adjusted to 7.4 before being sterilely filtered through 0.22 pm filters. 10,000 hMSC clusters were resuspended in 1.5 mL of 2 wt.% PMM-Norbornene 1 wt.% sodium alginate and loaded into a separate 3 mL syringe. The 2 wt.% PMM-tetrazine 1 wt.% sodium alginate mixture was loaded into a separate 3mL syringe. The loaded were connected by a twin-syringe delivery system fitted with a 16 mixing element static mixer and placed on a vertically oriented syringe pump. For one half of the extrusion mixture, the static mixer was fitted with a coaxial needle and capsules were formed by air shearing droplet formation into a calcium chloride gelling bath. The second half of the extrusion mixture was extruded as a string through a 18g blunt-tipped needle attached to the static mixer submerged in a calcium chloride gelling bath. The capsules and strings were both cured for 5 minutes after extrusion before being washed twice in a 3:1 volume ratio of DMEM++ to extrusion volume. Aliquots of capsules and string segments were taken for fluorescent LIVE / DEAD staining of encapsuled hMSCs on the day of encapsulation. The formed hydrogel capsules and strings were then transferred into vented capped sterile containers before being placed into a 5% CO2 37 C cell culture incubator for culture overnight. Fig. 11A-11 B shows the formed capsules and stringsFluorescent LIVE / DEAD Staining of Encapsulated hMSC Clusters

[0196] Encapsulated hMSC viability was assessed using fluorescent triple LIVE / DEAD staining on encapsulated hMSCs in both strings and capsules on day of encapsulation and the day after encapsulation. Calcein-AM (green) was used as a live stain that stains metabolically active cells with an intact membrane, Ethidium-homodimer (red) was used as a dead stain that stains the nuclei of clusters with a compromised cellular membrane, and Hoescht 33342 (blue) is a total cell stain that stains the nuclei of all the cells. Unencapsulated clusters and hMSC clusters in capsules and string segments were added to wells of a 96-well glass bottom plate. Unencapsulated clusters were stained with 200 pL of 0.5 pM Calcein-AM, 4 pM Ethidium Homodimer, and 40 pM Hoechst 33342. HMSC clusters encapsulated in capsules and strings were stained with 200uL of 1.0 pM Calcein-AM, 4 pM Ethidium Homodimer, and 40 pM Hoechst 33342. Confocal z-stacks were taken on representative clusters to qualitatively assess post encapsulation viability (Figs. 12A-12F).Chemical Stress Test hMSC Cluster Containing Strings and Capsules

[0197] To confirm the formation of a covalently crosslinked hydrogel network in the presence of cells, PMM-Tetrazine / PMM-Norbornene capsules and string segments were treated with sodium citrate to chelate and dissolve the gel former sodium alginate network. Briefly, an aliquot of crosslinked capsules and string segments were added to wells of 24-well glass bottom plate. To the hydrogel containing wells, 2 mL of 70 mM sodium citrate was added, and the crosslinked gels were incubated for 1 hour prior to imaging to confirm the presence of the remaining gel network (Figs. 13A-13B).Covalently Cross-Linked Shell Coatings via Tetrazine-Norbornene IEDDA Chemistry - PAD-Norbornene / PMS80-TetrazineCapsule Formation and Covalently Cross-Linked Shell Coatings

[0198] Anionic gel former hydrogel capsules were prepared with sodium alginate. Briefily, 1 wt.% sodium alginate (MVG, NovaMatrix™) was dissolved in HBS at a pH of 7.4. The sodium alginate solution was filtered through a 0.22 pm filter and loaded into a 1 mL syringe. The syringe was fitted with a coaxial needle was placed into a vertically oriented syringe pump (Harvard Apparatus). The hydrogel capsules were prepared from air-shearing extrusion into a gelling bath containing calcium chloride and allowed to cure for 5 mins. The formed sodium alginate capsules were collected and washed twice with HBS in a 3:1 volume ratio of wash solution to capsule extrusion volume. The anionic sodium alginate capsules were first coated with polycationsynthetic polymer 1 , PAD-Norbornene, through polyelectrolyte complexation of the anionic alginate and the cationic PAD-Norbornene polymer. The second coating with the polyanionic, PMS80-Tetrazine, then proceeds through polyelectrolyte complexation to exposed polycationic patches of the first PAD-Norbornene coating, with covalent crosslinking occurring between the norbornene and the tetrazine groups after complexation of the two reactive polymers. Specifically PAD-Norbornene was dissolved at a 0.1 wt% concentration in HBS and filtered through a 0.22 pm filter before being added to the sodium alginate capsules in a 3:1 volume ratio for 6 mins under swirling agitation. The now PAD-Norbornene coated capsules were then washed twice in a 3:1 volume ratio with HBS. The PMS80-Tetrazine was dissolved at a 0.2 wt% concentration in HBS and similarly filtered through a 0.22 pm filter and added to the PAD-Norbornene coated capsules in a 3: 1 volume ratio for 6 mins under swirling agitation and then subsequentially washed twice in a 3:1 volume ratio with HBS.Chemical Stress Test

[0199] Formation of a covalently crosslinked shell was assessed first by chemical treatment of the coated capsules with sodium citrate to chelate calcium ions, dissolving the calcium crosslinked alginate gel core of the coated capsules. After treatment with sodium citrate, 1 M NaOH is added to the coated capsules to increase the pH of the solution and neutralizing the cationic amines of the polycation coating and disrupting the polyelectrolyte complexation of the PAD-Norbornene with the alginate and PMS80-Tetrazine. Specifically, an aliquot of PAD- Norbornene / PMS80-Tetrazine coated capsules were added to a well of a 24-well glass bottom plate. To the aliquot of capsules 1 mL of 70 mM sodium citrate (pH 9.0) was added and the capsules were incubated for 1 hour and then 1 mL of 1 M NaOH was added. The treated capsules were then imaged to confirm the presence of a remaining covalently cross-linked shell network (Figs. 14A-14C).Fluorescent Staining of Residual IEDDA Reactive Groups in PAD-Norbornene / PMS80- Tetrazine Covalently Crosslinked Shells.

[0200] To visualize the coating layers of the covalently crosslinked PAD-Norbornene / PMS80- Tetrazine shell, residual reactive groups were stained with IEDDA reactive fluorescent labels and imaged by confocal laser scanning microscopy (CLSM). An aliquot of coated capsules was added to a well of a 24-well glass bottom plate and were first stained with 1 mL of 10 pM Cyanine5 Tetrazine prepared in HBS for 1 hour and washed with HBS before being stained with mL of 20pM AF-488 TCO. Cross sectional CLSM images of stained coated capsules were taking by on a Nikon Ti eclipse confocal microscope (Figs. 15A-15B). Figs. 16A-16F show a schematic representation of the configurations of hydrogels.Core Covalently Crosslinked Hydrogel Strings by Static Mixer with SPAAC Chemistry

[0201] PMM-Azide / PMM-DBCO core covalently crosslinked capsules were prepared with a dual-syringe extrusion through a static mixer. In separate extrusion mixtures, PMM-Azide and PMM-DBCO were combined with sodium alginate as the gel former. The respective PMM polymers and sodium alginate (MVG, NovaMatrix™) were dissolved in HBS at a concentration of 2 wt.% (PMM) and 1 wt.% (sodium alginate) and pH adjusted to 7.4. Both polymer extrusion mixtures were filtered through 0.22 pm filters and loaded into separate 3 mL syringes. The 3 mL syringes were connected by a twin-syringe delivery system fitted with a 16 mixing element static mixer and 20g coaxial needle. Hydrogel capsules were formed by extrusion of the dual-syringe set-up by a vertically oriented syringe pump at a flow rate of 7.5 mL / hr into a calcium chloride gelling bath with an air sheath flow in the coaxial needle to shear off droplets. After extrusion, the hydrogel capsules were cured for 5 mins in the calcium chloride gelling bath before being washed three times in a 3:1 volume ratio of HBS to extrusion volume. The formed hydrogels are shown in (Fig. 17).

[0202] Formation of a covalently crosslinked PMM-DBCO / PMM-Azide gel network was assessed by chelation of the alginate gelling calcium ions with sodium citrate to dissolve the alginate gel network. The remaining hydrogel capsules after treatment confirmed the formation of a SPAAC covalently crosslinked network. The chemical stress test was performed on an aliquot of capsules in a well of a 24-well glass bottom plate with a 70 mM pH 9.0 sodium citrate solution for 1 hour before being imaged by optical microscopy to confirm the presence of a remaining gel network (Fig. 18).Core and Coated Covalently Crosslinked Hydrogel Strings by Static Mixer with SPAAC Chemistry

[0203] PMM-Azide / PMM-DBCO core covalently crosslinked capsules were prepared with a dual-syringe extrusion through a static mixer. Seperate extrusion mixtures of PMM-Azide and PMM-DBCO were combined with sodium alginate as the gel former. The respective PMM polymers and sodium alginate (MVG, NovaMatrix™) were dissolved in HBS at a concentration of 2 wt.% (PMM) and 1 wt.% (sodium alginate) and pH adjusted to 7.4 and filtered through 0.22 pmfilters and loaded into separate 3 mL syringes. The syringes were connected with twin-syringe delivery system fitted with a 16 mixing element static mixer and 20 g coaxial needle. Hydrogel capsules were formed by extrusion of the dual-syringe set-up by a vertically oriented syringe pump at a flow rate of 7.5 mL / hr into a calcium chloride gelling bath with a droplet shearing coaxial outer air flow. After extrusion, the hydrogel capsules were cured for 5 mins in the calcium chloride gelling bath before being washed two times in a 3:1 volume ratio of a CaCh containing solution to extrusion volume. The anionic core crosslinked capsules were first coated with polycation, PAD- Azide, by polyelectrolyte complexation of the anionic core crosslinked hydrogel with the cationic PAD-Azide polymer. The second coating with the polyanionic, PMS60-DBCO, was introduced by polyelectrolyte complexation to polycationic patches of the first coating. Covalent crosslinking then proceeded between the mutually reactive groups after complexation. Specifically, the first coating, PAD-azide, was dissolved at a 0.2 wt% concentration in calcium chloride containing solution and filtered through a 0.22 pm filter before being added to the core covalently crosslinked capsules in a 3:1 volume ratio for 6 mins under swirling agitation. The coated capsules were then washed twice in a 3:1 volume ratio with HBS. The second coating PMS60-DBCO was dissolved at a 0.2 wt% concentration in calcium chloride containing solution and similarly filtered through a 0.22 pm filter and added to the polycation coated capsules in a 3:1 volume ratio for 6 mins under swirling agitation and then subsequentially washed twice in a 3:1 volume ratio with HBS. The formed shown are shown in (Fig. 19).

[0204] Formation of a covalently crosslinked shell and core was assessed first by chemical treatment of the coated core-crosslinked capsules with sodium citrate to chelate calcium ions and dissolve the crosslinked alginate gel core of the coated capsules. After treatment with sodium citrate, 1 M NaOH is added to the coated capsules to increase the pH of the solution and neutralizing the cationic amines of the polycation coating and disrupting the polyelectrolyte complexation of the PAD-Azide with the anionic covalently crosslinked capsules and PMS60- DBCO Specifically, an aliquot of PAD-Azide / PMS60-DBCO coated core crosslinked capsules were added to a well of a 24-well glass bottom plate. To the aliquot of capsules 1 mL of 70 mM sodium citrate (pH 9.0) was added and the capsules were incubated for 1 hour and then 1 mL of 1 M NaOH was added. The treated capsules were then imaged to confirm the presence of a remaining covalently cross-linked shell network (Figs. 20A-20B).Core Covalently Crosslinked IEDDA Hydrogel Capsules by In-diffusion of Oligomeric Crosslinker

[0205] Core covalently crosslinked hydrogel capsules were prepared by combining PMM- Tetrazine and sodium alginate as the gel former. A combined mixture of PMM-Tetrazine 1.0 wt.% and 1.0 wt.% sodium alginate (MVG, NovaMatrix™) were dissolved in HBS at a final pH of 7.4. The combined extrusion mixture was filtered through a 0.22 pm filter and loaded into a 1 mL syringe. The syringe was fitted with a coaxial needle and placed on a vertically oriented syringe pump. Hydrogel capsule extrusion was then performed by air-shearing droplets into a calcium chloride gelling bath. The formed hydrogel capsules were cured for 5 minutes after extrusion in the calcium gelling bath before being washed twice in a 3:1 volume ratio of HBS containing calcium chloride to capsule volume ratio. The formed PMM-tetrazine containing alginate capsule were covalently crosslinked by in diffusion of a 7kDa oligomeric poly(sulfobetaine methacrylate) containing bifunctional norbornene end groups. Specifically, the capsules were incubated for 1 hour with a 2.1 wt.% solution of the bifunctional poly(sulfobetaine methacrylate) oligomer in a 3:1 volume ratio, targeting an approximate molar ratio of 4:1 norbornene:tetrazine reactive groups. The crosslinked capsules were then washed twice in a 3:1 volume ratio of HBS to capsule extrusion volume and then imaged as seen in Fig. 21.

[0206] PMM-tetrazine / -Poly[sulfobetaine methacrylate]-di(norbornene) core crosslinked hydrogel capsules were tested for covalent crosslinking by chelation of calcium with sodium citrate. In a 24-well glass bottom plate an aliquot of the covalently crosslinked capsule were added and the HBS supernatant was removed. To the capsule containing well, 2 mL of a 70 mM sodium citrate (pH 9.0) was added and incubated prior to imaging. The presence of capsules after calcium chelation confirms the formation of a covalently crosslinked PMM-tetrazine / Poly[sulfobetaine methacrylate]-di(norbornene) hydrogel network (Fig. 22).

Claims

WHAT IS CLAIMED IS:1 . A polymer system for producing a hydrogel, the system comprising: a first component comprising a first synthetic polymer and a gel former; and a second component comprising a second synthetic polymer; wherein the first synthetic polymer is functionalized with a first click chemistry group and wherein the second synthetic polymer is functionalized with a second click chemistry group that reacts with the first click chemistry group to form a covalent link by click chemistry; wherein one of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, an oxime, a nitrone or nitrile-oxide; wherein the gel former has a gelation mechanism that is independent from the click chemistry.

2. The polymer system of claim 1 , wherein the gel formers are selected from the group consisting of alginate, pluronic, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose or elastin.

3. The polymer system of claim 1 or 2, wherein the first synthetic polymer and the second synthetic polymer have the same polymer backbone.

4. The polymer system of claim 1 or 2, wherein the first synthetic polymer and the second synthetic polymer have a different polymer backbone.

5. The polymer system of any one of claims 1 to 4, wherein the first synthetic polymer and the second synthetic polymer have a backbone that is independently selected from the group consisting of poly(methyl vinyl ether-alt-maleic anhydride) (PMM), homopolymer of methacrylic anhydride, homopolymer of polyacrylic acid, homopolymer of polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides, and lysine homopolymers.

6. The polymer system of any one of claims 1 to 5, wherein the first synthetic polymer and / or the second synthetic polymer have a molecular weight of 10,000 to 100,000 Da.

7. The polymer system of any one of claims 1 to 6, wherein the second synthetic polymer has a molecular weight that is at least 2 times smaller than that of the first synthetic polymer.

8. The polymer system of any one of claims 1 to 7, wherein the cycloalkene is a C3-C10 cycloalkene.

9. The polymer system of any one of claims 1 to 8, wherein the cycloalkyne is a C7-C9 cycloalkyne.

10. The polymer system of any one of claims 1 to 9, wherein the heterocycloalkyne is a 7 to 9 membered ring and the heteroatoms are selected from N, O, and S.

11. The polymer system of any one of claims 1 to 10, wherein one of the first synthetic polymer or the second synthetic polymer is functionalized with the cycloalkene or the heterocycloalkene and the other is functionalized with the tetrazine, the triazine or the pyridazine.

12. The polymer system of any one of claims 1 to 10, wherein one of the first synthetic polymer or the second synthetic polymer is functionalized with the cycloalkyne or the heterocycloalkyne and the other is functionalized with the azide, the oxime, the tetrazine, the triazine, the pyridazine, the nitrone or the nitrile-oxide.

13. The polymer system of any one of claims 1 to 12, wherein the first and the second synthetic polymer have a backbone that comprises monomeric units selected from2- aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3-aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2-hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethylphosphate (MPC), carboxybetaine methacrylate (CBM), N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N-diaminopropyl acrylate, 3-N,N- diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N- dimethylaminobutyl methacrylamide, 1 ,3-bis(dimethylamino)propyl acrylate, 1 ,3- bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, acrylic acid, methacrylic acid, sodium acrylate, sodium methacrylate, sodium ethacrylate, acrylamide, methacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 1-propanaminium, N,N-dimethyl-N-[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]-3-sulfo-, 2- methacryloyloxyethyl phosphorylcholine, N-(carboxymethyl)-2-(acryloyloxy)-N,N- dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1- aminium, N-(carboxymethyl)-3-methacrylamido-N,N-dimethylpropan-1-aminium, N- (carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / \ / -[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2- (methacryloyloxy)ethanesulfonic acid.

14. The polymer system of any one of claims 4 to 13, wherein the first synthetic polymer has a PMM backbone and the second synthetic polymer has a PLL backbone.

15. A method for producing a hydrogel, the method comprising: providing the first component of the polymer system as defined in any one of claims 1 to 14; gelling the gel former of the first component to provide an intermediate gel containing the first synthetic polymer;contacting the second component of the polymer system as defined in any one of claims 1 to 14 with the intermediate gel and allowing a click chemistry reaction to occur between the first synthetic polymer and the second synthetic polymer.

16. The method of claim 15, wherein the gel former is alginate and the step of gelling is performed by extruding the first component in a gelling bath containing calcium, strontium, barium, iron, or zinc ions.

17. The method of claim 16, wherein the extrusion performed is to provide droplets into the gelling bath to produce capsules.

18. The method of claim 16, wherein the extrusion performed is continuous fashion in order to produce hydrogel strings.

19. The method of claim 18, wherein the hydrogel strings are laid down in overlapping fashion to form high surface area grids or patches, partial fusion and covalent crosslinking of the overlap points between string segments in adjacent layers provide strength and result in formation of permanent high surface area grids or patches.

20. The method of any one of claims 14 to 19, further comprising, before the gelling step, a step of providing a payload material in the first component.

21. The method of claim 20, wherein the payload material is live cells or cell clusters.

22. The method of any one of claims 14 to 21 , wherein the step of contacting is performed at least partially concurrently with the gelling step.

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