Crosslinked hydrogels with controlled permeability

Abstract

There is provided a method of producing a hydrogel. A first polymer functionalized with a homocysteine thiolactone of formula I is reacted to open the ring and obtain a pendant thiol group, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen;. (I) The pendant thiol group of the first polymer are then crosslinked with a pendant vinyl group on a second polymer in the presence of a gel former to obtain the hydrogel.

Description

CROSSLINKED HYDROGELS WITH CONTROLLED PERMEABILITYCROSS-REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U.S. provisional application number 63 / 575,213 filed on April 5, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of hydrogels, particularly semi-permeable hydrogels that are useful in 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 tissue, 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] Control over the pore dimensions of hydrogels allows control over the permeability properties of the hydrogels and their adaptability to various applications. For example, when hydrogels are used to encapsulate cells for transplantation purposes, the porosity is an important parameter that has a direct role in the diffusion of molecules through the hydrogels and in the protection of encapsulated cells against the immune system. Other factors that are important in designing hydrogels for implantation are how they interact with tissues at their 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 the encapsulated therapeutic cells. Another consideration involves mechanical resilience of the hydrogels, which ideally are designed to persist for extended periods of time in vivo. Accordingly, improvements in hydrogels are desired, particularly with respect to the control of their porosity and thus permeability to components of the immune system, and their ability to retain cells.SUMMARY

[0005] In one aspect, there is provided a method of producing a hydrogel, the method comprising: reacting a first polymer functionalized with a homocysteine thiolactone of formula I to open the ring and obtain a pendant thiol group, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen;

[0006] crosslinking the pendant thiol group of the first polymer with a pendant vinyl group on a second polymer in the presence of a gel former to obtain the hydrogel.

[0007] In some embodiments, the gel former is selected from alginate, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose or elastin.

[0008] In some embodiments, the first polymer and the second polymer have a backbone selected from 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.

[0009] In some embodiments, the reacting step is a hydrolysis or an aminolysis.

[0010] In some embodiments, the pendant vinyl group is a pendant vinylsulfone.

[0011] In some embodiments, the second polymer is PMM-cysteamine vinyl sulfone (PMM-CVS).

[0012] In some embodiments, R2 is H.

[0013] In some embodiments, the first polymer is PMM-homocysteine thiolactone (PMM-HTL) which becomes PMM-homocysteine after ring opening.

[0014] In some embodiments, the method further comprises coating the hydrogel with polycations.

[0015] In some embodiments, the polycation is a polycation polymer with monomers selected from 2-aminoethyl (meth)acrylate, 2-aminoethyl (meth)acrylamide), 3-aminopropyl (meth)acrylate, 3-aminopropyl(meth)acrylamide, 4-aminobutyl (meth)acrylate, 4-aminobutyl (meth)acrylamide, (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 (CBM), [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide (SBMA), N,N-dimethylaminoethylacrylate (DMAEA), N,N- dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl (meth)acrylamide, 3-N,N- dipropylaminopropyl (meth)acrylate, 4-N,N-dimethylaminopropyl (meth)acrylamide, 1,3- bis(dimethylamino)propyl (meth)acrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate and combinations thereof.

[0016] In some embodiments, the polycation is poly[APM-co-DMAEA-co-SBMA] (PADS).

[0017] In some embodiments, the PADS has an initial molar feed ratios of 1:1:2 of APM, DMAEA, and SBMA, with each value in the ratio varying by ±20%.

[0018] In a further aspect, there is provided a functionalized polymer, comprising a functional group as follows:Polymer Backbone

[0019] wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen.

[0020] In some embodiments, the backbone polymer is selected from 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.

[0021] In some embodiments, all of the R2 are H.

[0022] In still a further aspect, there is provided a functionalized polymer, comprising a functional group as follows: polymer backbone

[0023] wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen, and wherein X is a primary, secondary or tertiary amine, a hydroxyalkyl, a hydroxyl or -O'.

[0024] In some embodiments, the backbone polymer is 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, and methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides.

[0025] In some embodiments, all of the R2 are H.

[0026] In one aspect, there is provided a hydrogel comprising alginate, and poly(methyl vinyl ether-alt-maleic anhydride) homocysteine crosslinked to poly(methyl vinyl ether-alt-maleic anhydride) cysteamine vinyl sulfone as per formula A

[0027] wherein X is as defined as a primary, secondary amine, a hydroxyalkyl, a hydroxyl or -0; and wherein the hydrogel has pores having a size of less than 18 nm. In some embodiments, the pores exclude IgG antibodies when the hydrogel is in a hydrated state in saline at a pH of 7.4.

[0028] In one aspect, there is provided a method of producing a homocysteine thiolactone polymer, the method comprising: performing a radical-mediated polymerization with a homocysteine thiolactone methacrylamide monomer and / or a homocysteine thiolactone acrylamide monomer and a radical initiator in a solvent optionally under heating.

[0029] In some embodiments, the solvent is an aqueous solvent (preferably at low pH such as 3-4) or an organic solvent. In some embodiments, the organic solvent is toluene, tetrahydrofuran, dioxane, ethylacetate or chloroform. In some embodiments, the radical initiator is azobisisobutyronitrile (AIBN) or a Vazo™ initiator. In some embodiments, the homocysteine thiolactone polymer has a molecular weight of 5,000 to 100,000 g / mol.

[0030] In some embodiments, the method further comprises adding a monomer selected from the group consisting of N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl (meth)acrylamide, 3-N,N-dipropylaminopropyl (meth)acrylate, 4-N,N-dimethylaminopropyl (meth)acrylamide, 1 ,3-bis(dimethylamino)propyl (meth)acrylate, and 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate, 2- hydroxyethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, 3-hydroxypropyl (methacrylamide), polyethyleneglycol methacrylates, methyl (meth)acrylate, 2-methacryloyloxyethyl phosphorylcholine (MPC), [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (SBMA), carboxybetaine methacrylates, (meth)acrylic acid, 2-acrylamido-2-methylpropane sulfonic acid, and 3-sulfopropyl (meth)acrylate, to produce a copolymer.

[0031] 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

[0032] FIG. 1 is a1H nuclear magnetic resonance (NMR) spectrum of polymethyl methacrylate homocysteine thiolactone (PMM-HTL) in D2O.

[0033] FIG. 2 is a1H NMR spectrum of poly[APM-co-DMAEA-co-SBMA] in D2O.

[0034] FIG. 3 is a schematic showing the formation of crosslinked capsules from PMM-HTL and PMM-cysteamine vinyl sulfone (PMM-CVS).

[0035] FIG. 4 is a schematic showing illustrations of hydrogel capsules, strings, or patch filaments with thiolactone groups used in three example configurations. Homocysteine thiolactone groups, depicted as pentagons, can be incorporated within the hydrogel (configuration 1), onto the hydrogel surface as part of a coating (configuration 2), and both within and on the hydrogel (configuration 3).

[0036] FIG. 5A is a microscopy image showing hydrogel capsules (about 1 mm diameter) formed with 2 wt.% total PMM-CVS / PMM-HTL (1 : 1 wt. ratio) with 1 % alginate, coated with 0.3 wt.% PADS (formulation labeled sample “A”).

[0037] FIG. 5B is a microscopy image showing human donor islets encapsulated in the hydrogel capsules of Fig. 5A.

[0038] FIG. 6A is a confocal microscopy image of “A” hydrogel capsules showing the fluorescein isothiocyanate (FITC) channel for PMM-CVS-FITC.

[0039] FIG. 6B is a confocal microscopy image of “A” hydrogel capsules showing the tetramethylrhodamine (TRITC) channel for PADS.

[0040] FIG. 6C is a confocal microscopy image combining the FITC and TRITC channels of Figs. 6A and 6B.

[0041] FIG. 7A is a brightfield microscopy image of “A” as formed.

[0042] FIG. 7B is a brightfield microscopy image of “A” after treatment with sodium citrate.

[0043] FIG. 7C is a brightfield microscopy image of “A” after treatment with sodium citrate and then sodium hydroxide.

[0044] FIG. 8 is a microscopy image showing hydrogel capsules formed with 4 wt.% total PMM-CVS / PMM-HTL (1 :1 wt ratio) with 1 % alginate, coated with 1 wt.% PADS (formulation labeled sample “B”).

[0045] Fig. 9 is a microscopy image showing islets encapsulated in “B”of Fig. 8.

[0046] FIG. 10 is a microscopy image showing hydrogel capsules formed with 2 wt.% totalPMM-CVS / PMM-HTL (1 :1 wt ratio) with 1 % alginate, coated with 1 wt.% PADS with 25 kDa instead of 15 kDa (formulation labeled sample “C”).

[0047] FIG. 11 A is a confocal cross-sectional image of “C” capsules during lgG-alexafluor488 in-diffusion study after about 24 h incubation with the TRITC-channel showing PADS coating forming the permeability-reducing membrane in the outer capsule shell.

[0048] FIG. 11 B is a confocal cross-sectional image of “C” capsules during lgG-alexafluor488 in-diffusion study after about 24 h incubation which combines Fig. 11 A with FITC-channel showing lgG-alexafluor488 largely excluded from capsule.

[0049] FIG. 12 is a graph showing the in-diffusion of IgG across hydrogel capsules after 24 hours (control (Ctrl) is an uncoated hydrogel control capsule).

[0050] FIG. 13A is a confocal cross-sectional image of capsules (formulation “D”) taken in the TRITC channel showing thiolactone containing PHMS polymer as the final coating.

[0051] FIG. 13B is a confocal microscopy image (formulation “E”) showing a doubly coated PAD and PHMS60-25k capsule.

[0052] FIG. 13C is a brightfield microscopy image showing the encapsulation of human donor islets in the capsules of formulation “E”.

[0053] FIG. 14A is a brightfield image of a coated hydrogel patch formulation.

[0054] FIG. 14B is a stitched fluorescence image of the coated hydrogel patch formulation, showing fluorescence of the PADS coating (TRITC channel).

[0055] FIG. 15A is a confocal cross-sectional image of the coated hydrogel patch, showing the PADS polymer coating in the TRITC channel.

[0056] FIG. 15B is a brightfield cross-sectional image of the coated hydrogel patch.

[0057] FIG. 16A is a brightfield cross-sectional image of the coated hydrogel patch after treatment with sodium citrate followed by sodium hydroxide.

[0058] FIG. 16B is a confocal microscopy image of the coated hydrogel patch of Fig. 16A.

[0059] FIG. 17A is a confocal microscopy image of the double coated hydrogel patch.

[0060] FIG. 17B is a brightfield microscopy image of the double coated hydrogel patch of Fig.17A.DETAILED DESCRIPTION

[0061] There is provided a hydrogel for producing capsules, strings, patches and the like. The hydrogel is crosslinked with thiol-ene chemistry between two polymers, one functionalized with a thiol group and one functionalized with a vinyl group. The polymer functionalized with a thiol group has a pendant thiol group, such as a homocysteine as shown below where X can be a primary or secondary amine, a hydroxyalkyl, a hydroxyl or -O'. X can be linked to a polymer backbone.polymer backbone

[0062] Although the present disclosure provides examples with the backbone polymers being poly(methyl vinyl ether-alt-maleic anhydride) (PMM), other alternatives are possible. Indeed, alternatives to PMM include but are not limited to other copolymers of maleic anhydride with other vinyl ethers (e.g. ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxyethyl vinyl ether, and methoxy ethyl vinyl ether). Other alternatives include itaconic anhydride or citraconic anhydride with vinyl ether comonomers such as methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxyethyl vinyl ether, and methoxy ethyl vinyl ether, and copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides. In some embodiments, the backbone polymer is a homopolymer of methacrylic anhydride, acrylic acid, methacrylic acid, or a copolymer of acrylic acid and methacrylic acid.

[0063] To obtain the pendant thiol group, a cysteamine thiolactone group is first functionalized onto the polymer and then the ring is opened via hydrolysis or aminolysis. In one example, a thiolactone group was functionalized onto PMM by reaction of the amino group of homocysteine thiolactone (HTL) with the anhydride groups of PMM. The homocysteine thiolactone functionalized PMM polymers (PMM-HTL) can then undergo a ring opening reaction to obtain PMM-homocysteine having reactive free thiol groups. One example of a scheme to functionalize PMM with homocysteine thiolactone is presented below.Scheme 1.

[0064] The term “homocysteine thiolactone” as contemplated by the present disclosure, is of formula I where three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen.

[0065] Accordingly, in some embodiments, PMM-homocysteine as per formula Ila or lib below is used in the formation of hydrogels. X is as defined above, or preferably X is O' or NHR where R is a C1-C4 alkyl. It should be noted that R can be a linker or directly linked to a backbonepolymer which could be PMM or a polyacrylate or polymethacrylate polymer bearing primary amine groups.

[0066] When the ring opening is performed by aminolysis, X is NHR:

[0067] Aminolysis can involve small amines, but preferably large molecules and most preferably polymers containing multiple amine groups. These polyamines are electrostatically attracted to the anionic HTL polymer, and by virtue of their relatively high amine density are likely to contain a mixture of protonated and non-protonated primary amines, even at physiological pH, due to the well known polyelectrolyte effect. These two effects: electrostatic attraction and presence of non-protonated primary amines, can speed aminolysis and hence the overall crosslinking reaction.

[0068] The ring opening by aminolysis is performed based on the following scheme:Scheme 2

[0069] On the other hand, when the ring opening is performed by hydrolysis, X in formula lib is O' or O-H (as shown in the structure below). Hydrolysis and subsequent crosslinking of the generated thiol can be performed in vitro in presence of 10% serum, and therefore can also proceed in vivo under similar conditions. In one example, the capsules can crosslink in vitro over a span of a two hours or longer while at pH 7.4-7.6 in buffered solutions. This time may be shortened by carrying out this reaction at slightly elevated temperatures, such as 37 °C. These time spans work well with typical time periods between encapsulation and implantation. As usual, care must be taken to ensure proper oxygenation of encapsulated cells during this period between encapsulation and implantation, through means known to people skilled in the art. Hydrolysis and / or aminolysis, together with subsequent crosslinking, may continue even after implantation in vivo.

[0070] The pendant thiol group can react with a vinyl group to form a crosslinked hydrogel. Preferred vinyl groups are vinylsulfones. This is because vinylsulfones are strongly activated electrophiles, able to react with accessible thiol-functional and amine-functional groups on, e.g polymers such as PMM-homocysteine. While pendant vinylsulfone groups on polymer backbones deactivate by spontaneous hydration to form 2-hydroxyethylsulfone groups, a hydrophilic and largely benign group, some of the residual vinylsulfone groups can also be used to bind other thiol- and amino- functional groups, such as cysteine, mercaptoethanol, and cysteamine to modify hydrophilicity and charge density of the hydrogel for example.

[0071] Accordingly, although the present disclosure provides PMM-cysteamine vinyl sulfone (PMM-CVS) as an example of vinyl group, this is not a limitative example as not only polymers other than PMM can be used, but also other pendant activated vinyl or preferably vinylsulfone groups can be used. A preferred embodiment, which is exemplified in the Example section below, is the formation of a hydrogel between PMM-cysteamine vinyl sulfone (PMM-CVS) and PMM- homocysteine thiolactone (PMM-HTL) 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 initial gel former can be provided in a weight percentage of from 0.5 to 10 wt. %, and preferably 1 to 4 wt. %, and most preferably 1 .5 to 3 wt. %, depending on the viscosity and the type of initial gel former. If the initial gel former is alginate, it is mixed with the synthetic gel formers, and the overall gel is formed by a one-pot two-stage process where the mixture (optionally containing cells) is extruded into a bath containing calcium, strontium, barium, iron, or zinc ions that rapidly gel the alginate to providestructural integrity with ionic crosslinking while the thiolactone covalent crosslinking takes place over the subsequent time of handling, washing steps until implantation, on the order of typically one to three hours.

[0072] The chemical formula of PMM-CVS is presented below:

[0073] As explained above, it should be understood that other vinylsulfone groups and other polymer backbones are suitable. For example, the vinyl functional polymer is more generally a cysteamine vinylsulfone modified polymer as shown below:Polymer backbone

[0074] PMM-homocysteine generated from PMM-HTL as shown in Scheme 2 reacts with PMM-CVS, a thiol-ene reaction between the pendant thiol of PMM-homocysteine and the pendant vinyl group of PMM-CVS to form the structure shown below (X is as defined as a primary, secondary amine, a hydroxyalkyl, a hydroxyl or -O'):

[0075] The aminolysis of thiolactone groups has also been used here to covalently crosslink polycations while coating them onto hydrogel capsule cores (gels). In the context of the present disclosure the term “coat” or “coating” refers to the coating of a layer or the grafting of chemical species or functional groups on a surface, for example the shell of a particle. This process involves reaction of primary or secondary amines on these polycations, with residual homocysteine thiolactone groups on the core-hydrogel, and thus allows to post-modify capsules formed from PMM-CVS and PMM-HTL for example, with primary-amine functional copolymers. In addition to polycations, this process may also be carried out with polyampholytes comprising primary or secondary amines. The polyampholytes can be stoichiometric (50-50 anionic and cationic), close to stoichiometric (45-55 or 55-45 anionic to cationic respectively) or non-stoichiometric (70% cationic and 30 % anionic). This approach allows improved control over final pore-size and hydrogel exclusion properties. It also optionally enables introducing immune-modulating functions into the shell. Finally, it would provide another route to reinforce the outer shell, to enhance mechanical resilience and cell retention. These benefits would similarly apply to other hydrogel geometries such as strings and patches comprising this hydrogel chemistry.

[0076] One of the polycations that was developed for coating hydrogels is a poly[APM-co- DMAEA-co-SBMA] terpolymer (PADS). APM is 3-aminopropylmethacrylamide which contains a primary amine to react with a thiolactone. DMAEA is N,N-dimethylaminoethylacrylate and SBMA is [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide. A key formulation is the PADS112 series of ternary copolymer, where 112 or 1 :1 :2 represents initial molar feed ratios of 1 : 1:2 of APM, DMAEA, and SBMA. The molar ratios can vary by ±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 cation-rich end of the polymer to the anionic hydrogel, while leaving the SBMA- rich end to be tissue-facing. The amine of APM contributes reactivity as well as cationic density for coating onto anionic PMM-CVS / PMM-HTL core hydrogels. DMAEA (N,N-dimethylaminoethyl acrylate) has a tertiary amine in the pendant group which also contributes cationic charge densityto 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).

[0077] With PADS which has a primary amine to react as the pendant amine (NHa+) for the aminolysis, the structure of PMM-HTL - PMM-CVS - PADS is as follows:

[0078] 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.(that can be a statistical distribution and not necessarily in the order shown in the structure below).

[0079] A PMM-HTL - PMM-CVS - PAD can also be produced as explained above for PADS and would have the following structure

[0080] Polycations other than PADS and PAD are also contemplated herein for the coating of the hydrogels, for example other copolymers with primary amino containing comonomers like APM, such as 2-aminoethyl (meth)acrylate, 2-aminoethyl (meth)acrylamide), 3-aminopropyl (meth)acrylate, 3-aminopropyl(meth)acrylamide, 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 (CBM), [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide (SBMA), 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-dipropylaminopropyl (meth)acrylate, 4-N,N- dimethylaminopropyl (meth)acrylamide, 1 ,3-bis(dimethylamino)propyl (meth)acrylate, 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate and combinations thereof.

[0081] The overall composition, net cationic charge, and molecular weight of the polycation used are key parameters for hydrogel coatings. Where the hydrogel is coated with a single coating polymer, the composition of the polycation is preferably 30-80 mol% net cationic in charge, with the remaining 70-20 mol% of the coating polymer containing hydrophilic, charge-neutral comonomer. Of the 30-80 mol% cationic charge, preferably at least 20 mol% is primary amine containing, with the remaining cationic charge from comonomers containing tertiary or quaternary and optionally charge-shifting ammonium cations. Molecular weight of the polycation is also key to coating the hydrogels and forming permeability-controlling membranes, and can be 5,000- 50,000 g / mol, but more preferably 15,000-40,000 g / mol.

[0082] The use of small molecule amines such as lysine, glycinamide, and the like as reagents to ring open thiolactone groups was tested. However, it was found that the rate of aminolysis using small molecule amines at physiologically reasonable concentrations was relatively slow, and became a limiting factor for feasibility of the crosslinking timeline, given encapsulation of cells. Accordingly, although using small amines is feasible it is preferable, especially for large scale processes, to use larger amines such as polymers containing multiple amine groups. Without wishing to be bound by a theory, it is also hypothesized that the pKa of small molecules amines (pKa ~10) are a limiting factor for aminolysis, especially when the pH for hydrogels comprising cells is typically buffered at pH 7.4.

[0083] One advantage of utilizing polycation coatings is that the polycations are held in place electrostatically which facilitates reaction with thiolactone groups bound on polyanionic polymers including but not limited to PMM. Within a polycation, ammonium groups may have a range of pKa’s (pKa 7-10), significantly lower than the pKa of their small molecule analogues, which also may facilitate the aminolysis reaction with the thiolactones.

[0084] Homocysteine thiolactone may also be formed into a monomer via reaction with (meth)acryloyl chloride or (meth)acrylic anhydride to form homocysteine thiolactone (meth)acrylamide (HTL(M)A). The reaction scheme to form the homocysteine thiolactone methacrylamide monomer is as follows:

[0085] The reaction scheme to form homocysteine thiolactone acrylamide is as follows:

[0086] The monomers homocysteine thiolactone methacrylamide and homocysteine thiolactone acrylamide can be used to produce a homocysteine thiolactone polymer as an alternative route to functionalization of polymer backbones to include the homocysteine thiolactone groups. The two monomers can be employed as sole monomers (homopolymerization) or with each other (copolymerization). The homocysteine thiolactone polymer is obtained by radical-mediated polymerization aided by a radical initiator, optionally under heating. One example of a radical initiator is azobisisobutyronitrile (abbreviated AIBN) which can be operated in organic solvents (e.g. tetrahydrofuran, toluene, dioxane, ethylacetate, chloroform) at 60-80 °C. Vazo™ initiators such as Vazo-56TMcan be used in aqueous solvents for a controlled radical polymerization. In another example, V501™ radical initiator can be used.

[0087] Polymerization of homocysteine thiolactone monomers under aqueous conditions and with amine-containing components is compatible at low pH, preferably in the range of pH 3-4, where hydroxide concentration is low and amine functional groups are in the cationic ammonium form where they are non-nucleophilic.

[0088] It is important to note that a wide variety of polymer compositions can be prepared by using various comonomers, neutral, zwitterionic, anionic, and cationic comonomers. Examples of neutral comonomers include but are not limited to 2-hydroxyethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, 3-hydroxypropyl (methacrylamide), polyethyleneglycol methacrylates, and methyl (meth)acrylate. Examples of zwitterionic comonomers include but are not limited to 2-methacryloyloxyethyl phosphorylcholine (MPC), [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (SBMA), and carboxybetaine methacrylates. Examples of cationic comonomers include but are not limited to N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl (meth)acrylamide, 3-N,N-dipropylaminopropyl (meth)acrylate, 4-N,N- dimethylaminopropyl (meth)acrylamide, 1 ,3-bis(dimethylamino)propyl (meth)acrylate, 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate, and combinations thereof.

[0089] In the case of copolymerization, preferred compositions include from 5 to 40 mol% of homocysteine thiolactone (meth)acrylamide. The composition of the other comonomers can be adjusted to tune anti-fibrotic properties if these HTLMA copolymers are used as antifibrotic coatings. For example, 20-70 mol% zwitterionic comonomer can be employed for preferred compositions for anti-fibrotic properties may be incorporated. In some embodiments, the copolymer also incorporates 10-40 mol% of anionic comonomer for polymers that are used to coat cationic surfaces.

[0090] Homocysteine thiolactone monomers can be copolymerized to generate homocysteine thiolactone based polymers that can be used both as gel formers, similar to PMM- HTL, in addition to polymers for coatings. HTL(M)A can be homo- and copolymerized with anionic and zwitterionic monomers such as (meth)acrylic acid, 2-acrylamido-2-methyl-1-propane sulfonic acid, 3-sulfopropyl methacrylate, -[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (SBMA), methacryloyloxyethyl phosphorylcholine (MPC), and carboxybetaine methacrylates (CBM) as well as other betaines. Homocysteine thiolactone based copolymers 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 HTL-functional polymers can also be covalently crosslinked to surfaces and coatings that have primary amino groups. A reaction scheme to form homocysteine thiolactone functional polymers through (co)polymerization is provided for poly[homocysteine thiolactone methacrylamide-co-methacrylic acid-co-[2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (PHMS) is provided as an example as follows:EXAMPLESynthesis of PM M- homocysteine thiolactone (PMM-HTL)

[0091] PMMAn (1 g, 6.41 mmol) was dissolved into 20 mL of acetonitrile in a 100 mL round bottom flask equipped with a magnetic stir-bar stirring at 700 rpm. To the stirring reaction mixture, triethylamine (TEA) (0.36 mL, 2.59 mmol) was added. Homocysteine thiolactone (197 mg, 1.28 mmol) that was dissolved in 4 mL of a 1 :1 v / v solvent mixture of dimethylsulfoxide (DMSO):acetonitrile (see Scheme 3 below). Once fully solubilized, the homocysteine thiolactone solution was added dropwise to the reaction mixture over 1-2 min. The reaction mixture was left overnight at room temperature (22 °C) and subsequently transferred into 12-14 kDa molecular weight cut-off (MWCO) cellulose acetate dialysis tubing and dialyzed against 1 % sodium chloride in distilled water for 2 days with twice daily changes, followed by one day of distilled water bath changed twice daily. It should be noted that the pH of the dialysis water bath and polymer solution are inherently at pH 4-4.5, and hydrolysis of the thiolactone groups are minimized during purification at this pH. The dialyzed polymer solution was freeze-dried, resulting in PMM-HTL20 as a solid, obtained in 80-85 % yield.1H NMR spectrum of PMM-HTL was recorded in D2O on a Bruker™ Advance Neo 600 MHz spectrometer (Fig. 1).Scheme 3 dl-homocysteme thiolactone (20 moft TEA, 2 mol eq►acetonitrile, rt. o / n dl-homocysteinePMMAn thiolactoneSynthesis of PMM-CVS

[0092] The synthesis of PMM-CVS was performed similarly as previously described above for PMM-HTL by replacing HTL with CVS.Fluorescent Labeling of PMM-CVS and PMM-HTL Polymers

[0093] The fluorescent labelling of PMM-CVS and PMM-HTL was performed in a similar manner, using the following general procedure. PMMAn (1 g, 6.41 mmol) was dissolved in 20 mL of acetonitrile and transferred to a 100 mL round bottom flask equipped with a magnetic stir bar. Triethylamine (0.36 mL, 2.59 mmol), followed by fluorescein cadaverine (18 mg), were added to the reaction mixture stirring at 700 rpm at room temperature. The reaction mixture was left to reactfor 2 h before further post-functionalization reactions with either CVS or homocysteine thiolactone as described above.Synthesis of PADS Copolymers (poly[APM-co-DMAEA-co-SBMA])

[0094] PADS copolymers of various compositions were synthesized by reversible additionfragmentation chain-transfer (RAFT) polymerization, targeting molecular weights (MWs) from 5,000 to 50,000 g / mol at about 80 % total monomer conversion. To prepare PADS at a 1 :1 :2 APM:DMAEA:SBMA molar feed ratio at 15,000 g / mol, the following procedure was used. APM (0.406 g, 2.27 mmol) was dissolved in 4.4 mL of distilled water with 1.15 mol eq of HCI added with reference to the mol of DMAEA. DMAEA (0.325 g, 2.27 mmol) was added to the reaction mixture that was cooled in an ice-water bath, followed by pH adjustment to pH 3-4. SBMA (1.27 g, 4.55 mmol) was added to the reaction mixture, followed by a solution of 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTP) (30 mg, 0.108 mmol) and 4,4'-azobis(4- cyanovaleric acid) (V501) (6 mg, 0.021 mmol) in 2.2 mL of 1,4-dioxane, for a 2:1 water: 1 ,4- dioxane v / v ratio as the polymerization solvent mixture. The reaction vessel was equipped with a magnetic stir bar, sealed with a rubber septum, and purged with N2 at room temperature while stirring for 1 h. The reaction mixture was then placed into a 70 °C oil bath for 2 h, followed by quenching in an ice-water bath and exposure to air. Aliquots were sampled for1H NMR analysis in D2O to measure conversion. The polymerization mixture was transferred to 3.5 kDa MWCO dialysis tubing and was dialyzed against distilled water at pH 3 for a total of 2 days with twice daily water bath changes, using UV-vis to measure the absorbance of the dialysate to confirm no monomer or other small molecules were eluting from the polymer mixture. The polymer solution was freeze dried, resulting in PADS-112-15k as a light pink solid in 77 % yield.1H NMR analysis showed 89 % monomer conversion, and a copolymer composition of 23 % APM, 20 % DMAEA, and 57 % SBMA based on monomer consumption.1H NMR spectrum of PADS-112-15k was recorded in D2O on a Bruker Avance Neo 600 MHz spectrometer (Fig. 2).Fluorescent Labeling of PADS Copolymers

[0095] PADS copolymers were fluorescently labelled using either fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC) using the following protocol. To label PADS-112- 15k with RBITC at a targeted degree of 0.5 mol% (relative to total monomer units), the PADS copolymer (150 mg) was first dissolved in 15 mL of distilled water in a 20 mL vial equipped with a stir bar. The pH was adjusted to pH 7.5 followed by the addition of RBITC (180 pL of 1 % solutionin dimethyl formamide (DMF), 3.4 pmol) to the vigorously stirring reaction mixture. After 30 min, the reaction mixture was pH adjusted to pH 3-4 and transferred to 3.5 kDa MWCO dialysis tubing for purification as similarly described above. The polymer was then freeze dried, resulting in PADS-112-15k-RBITC as a magenta solid in about 80-85% yield.Synthesis of Homocysteine Thiolactone Based Copolymers

[0096] Homocysteine thiolactone methacrylamide was copolymerized to generate thiolactone reactive polymers for coating hydrogels anchored by covalent crosslinking with polyamines. As an example, homocysteine thiolactone methacrylamide (HTLMA) was copolymerized with methacrylic acid and SBMA to form poly[HTLMA-co-methacrylic acid-co-SBMA] (PHMS) copolymers.

[0097] PHMS copolymers of varying compositions were synthesized by reversible-addition- fragmentation chain-transfer polymerization (RAFT) polymerization targeting molecular weights ranging from 5,000 to 50,000 g / mol at about 80 % total monomer conversion. To prepare PHMS with a targeted molar feed ratio of 1 :1 :3 for HTLMA:MAA:SBMA at a targeted MWof 25,000 g / mol, called PHMS-113-25k, the procedure was similar to the RAFT polymerization as described above to PADS copolymers. PHMS-113-25k by RAFT polymerization reached 91 % total monomer conversion after 2.5 h at 70 °C. PHMS copolymers were used as thiolactone-functional polymers to prepare covalently crosslinked hydrogel doubly coated capsules.Preparation of Thiolactone-Vinyl Sulfone Hydrogel Capsules

[0098] Gel forming solutions were prepared by combining PMM-CVS and PMM-HTL at 1 to 5 wt.% loadings with sodium alginate (NovaMatrix™, MVG) at 0.5 to 3 wt.% in 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffered saline (HBS). Sodium alginate was used partly as a gel forming processing aid to rapidly form an initial gel scaffold upon exposure to a gelling bath containing multi-valent cations such as calcium, barium, strontium, zinc, and iron. To prepare the core hydrogel capsules at 4 % total PMM-CVS / HTL and 1 % alginate at about 1000 pm in diameter, the following procedure was used. PMM-CVS (40 mg) and PMM-HTL (40 mg) were dissolved in 1 mL of HBS, adjusted to pH 7.2-7.7. To the polymer solution, 1 mL of 2 % sodium alginate in HBS was added, and the mixture was vortexed for 1-2 min, followed by filtering through a 0.22 pm syringe filter. The gel forming solution was then loaded into a 3 mL syringe and placed into a vertically oriented syringe pump (Harvard Apparatus), attached with a coaxial needle (Rame-Hart) with an 20G inner needle and 14G outer needle. The polymer solution was extrudedinto a calcium chloride gelling bath with liquid flow rate set to 15 mL / h and the coaxial air flow rate to 2.65 LPM. The hydrogel capsules were left to cure under stirring in the gelling bath for another 15 min, followed by washing steps with saline, and / or HBS solutions containing 0 to 100 mM calcium chloride and / or barium chloride, prior to subsequent coating steps.Coating of Hydrogel Capsules with Permeability Reducing Membrane

[0099] Hydrogel capsules were coated with polycations containing primary amino groups to electrostatically form covalently crosslinked permeability reducing (size-exclusion) membranes. The amino groups of the polycation subsequently react with the thiolactone groups of the polymer in the hydrogel capsule or polymer coating to form a covalently linked network (described in greater detail below).

[0100] To form PADS coated hydrogels with thiolactone groups in the core hydrogel capsule, the following procedure was used as a representative example. Thiolactone containing hydrogel capsules (4 wt.% PMM-CVS / HTL polymers, 1% alginate), in form of a ca 1.5 mL dense suspension of capsules as formed as described above, were exposed to 4.5 mL a solution of 1 wt.% PADS-112-15k in HBS containing 100 mM calcium chloride for 6 min with occasional swirling. The capsules were then left to settle and the supernatant was removed. The capsules were washed twice with saline and stored in saline for approximately 2 h at room temperature, for covalent crosslinking by aminolysis of thiolactone groups and subsequent thiol / ene reaction of the generated homocysteine thiols with vinylsulfone groups (Scheme 4).Scheme 4- omocysteine

[0101] To form hydrogel capsules with thiolactone groups in both the core hydrogel and in the coating of the capsule, the following was used as a representative example. Thiolactone- containing hydrogel capsules (2 wt. % total PMM-CVS / HTL polymers, 1 wt.% alginate) formed as described above, were exposed to a solution of 0.5 wt.% PAD75-25k (poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate]) in HBS containing CaCh for 6min with occasional swirling where 75 means 75% of DMAEA and 25k is the molecular weight in kDa. The capsules were left to settle, the supernatant was removed, and the capsules were washed twice with HBS containing CaCh. The capsules were then exposed to 0.5 wt.% PHMS60- 25k in HBS containing CaCh for 6 min with occasional swirling. The capsules were then washed twice with HBS and stored in HBS for approximately 2 hours at room temperature to allow for covalent crosslinking by aminolysis of thiolactone groups and subsequent thiol / ene reaction of homocysteine thiols with polymer-bound vinylsulfone groups.Preparation of Hydrogel Patches

[0102] To prepare hydrogel patches (LifeRafts™), the gel forming solution was prepared similarly to the solution for hydrogel capsules, with a representative example described as follows. PMM-CVS / HTL (50 mg each) were dissolved in 0.83 mL of HBS and combined with 4.17 mL of 2.4 wt.% alginate (NovaMatrix™, MVG), for a final concentration of 2 % total PMM-CVS / HTL and2 % alginate, pH adjusted to pH 7.2-7.5. The gel forming solution was filtered through a 0.22 urn syringe filter and loaded into a 3 mL syringe.

[0103] The gel forming solution was used to print a 15 mm x 15 mm patch using a BioX Bioprinter from Cellink™. These patches were generated at room temperature in a 6-well plate, with ~2 mL of LifeSupport™ printing bath in each well, using a rectilinear pattern with a 30% infill density, where the polymer was extruded at 4.25 pL / s and the print head speed was 10 mm / s. 2-3 mL of 12.5 mM calcium gelling bath was added to the well containing the construct and left to cure for 5 min. Following the calcium gelation step, the LifeSupport™ support bath was liquefied by placing the plate in a 37°C hybridizer oven for 30 min. The hydrogel construct was removed and placed into a well containing 3 mL of a 10 mM barium chloride gelling bath for 5 min, followed by 2 washes with 3 mL of saline and stored in saline prior to coatings.Single Coating of Hydrogel Patches

[0104] Hydrogel constructs containing thiolactone groups were coated with polycations to form permeability reducing membranes, similar to hydrogel capsules and strings. A representative example of coating a hydrogel patch is provided as follows and shown in Fig. 3. Following formation, as described above, the hydrogel construct was placed into a 6-well plate and washed twice with 3 mL of HBS containing calcium chloride. Once the supernatant was removed from the patch, 3 mL of 1 % PADS-112-25k (fluorescently labelled with rhodamine B) was added to the well and the plate was placed on an orbital shaker for gentle agitation (approx. 20-25 rpm) for 6min. The supernatant was removed, and the coated hydrogel patch was washed 3 times with 3 mL of saline, and subsequently cured in saline for 2 h to allow for covalent crosslinking via aminolysis of thiolactone groups and thiolene reaction with homocysteine free-thiols with vinylsulfone groups.Double Coating of Hydrogel Patches

[0105] Hydrogel constructs containing thiolactone groups were doubly coated to form permeability reducing membranes, similar to hydrogel capsules and strings. A representative example of a doubly coated hydrogel patch is similar to the protocol as described above for single coated patches with an additional coating step following the first polycation coating. Briefly, following formation of the hydrogel construct, the hydrogel was places in a 6-well plate and washed with 3 mL of HBS containing calcium chloride. Once the supernatant was removed, 3 mL of 1% of PAD75-25k as the polycation was used to coat and react with the thiolactone containing hydrogel for 6 min with gentle agitation on an orbital shaker at 20-25 rpm. The supernatant was then removed, and the hydrogel was washed twice with 3 mL of HBS. For the second coating, 3 mL of 1% PHMS60-25k (rhodamine B labelled) was added to the hydrogel construct for 6 min with gentle agitation on an orbital shaker at 20-25 rpm. The supernatant was removed and the doubly coated hydrogel construct was washed twice with HBS, and supsequently cured for 1-2 hours to allow for covalent crosslinking via aminolysis of the thiolactone groups and thiolene reaction with resulting homocysteine free-thiols with vinylsulfone groups.Chemical Robustness Tests

[0106] To assess the coated hydrogels for covalent crosslinking, the hydrogel capsules, strings, and patches were subject to chelation of calcium using sodium citrate followed by an increase in pH to about pH 12 using 1 M NaOH to disrupt electrostatic complexation. Following this citrate-NaOH treatment, the samples were assessed by microscopy to determine if the hydrogels remained intact, or partially intact, which confirmed the presence of covalent crosslinkages.

[0107] As a representative example, the procedure for a citrate-NaOH test on coated hydrogel capsules is provided as follows. An aliquot (0.25 mL) of hydrogel capsule suspension with a 3:1 capsules to saline ratio, was added to a 4 mL glass vial, followed by 2 mL of 70 mM sodium citrate. The suspension was incubated with occasional mixing for at least 1 h before imaging for survival analysis.Microscopy

[0108] Brightfield and fluorescence images were taken with a Nikon T / Eclipse inverted microscope. Confocal laser scanning microscopy images were taken with a Nikon A1 Confocal T / Eclipse microscope.Membrane Permeability Studies

[0109] To test the permeability of the polymer membranes formed, the coated hydrogels were incubated with fluorescently labelled IgG and dextrans of various molecular weights. The procedure for the in-diffusion of IgG with hydrogel capsule formulations is described as follows. To a well in a glass-bottom 96-well plate, approximately 10-15 capsules were transferred, and the supernatant was removed. To the capsules, 150 pL of 500 pg / mL solution of lgG-alexafluor488 in HBS was added, followed by pipette aspiration for mixing of the supernatant. The capsules were imaged by confocal scanning laser microscopy immediately after sample preparation, and at various time intervals thereafter.

[0110] In-diffusion was measured by selecting multiple circular regions of interest (ROIs) from both within the capsules, and outside the capsules. The mean fluorescence intensity in the FITC channel of the ROIs inside the capsule was divided by the corresponding intensity of the ROIs outside of the capsule to obtain the in-diffusion ratio.Results and Discussion:

[0111] As discussed above, cell therapies such as type 1 diabetes therapies involve transplantation of cells. In the case of type 1 diabetes, the encapsulated cells are beta cells and they require exclusion of auto-antibodies IgG in order to stop the auto-immune response from eliminating the newly transplanted cells as well. The present hydrogels fulfil the need to more tightly control the permeability of the resulting encapsulating hydrogel in order to exclude at least some of the larger auto antibodies known to pose specific dangers to transplanted beta cells.Formation of Coated Hydrogels with Permeability Reducing Membrane

[0112] Coated hydrogels with covalent crosslinking based on thiolactone chemistry were prepared with reactive groups used in the configurations shown in Fig. 4.

[0113] Hydrogel capsules containing homocysteine thiolactone groups in the core were prepared using by using PMM-HTL and PMM-CVS at varying total wt.% loadings along with 1 wt.% alginate in the gel forming solutions (Fig. 4 Configuration 1).

[0114] As an example, 2 % PMM-CVS / HTL (1 :1 wt ratio) and 1 % alginate hydrogel capsules were coated with 0.3 wt.% PADS-112-15k, called formulation “A”. (Fig. 5A). Formulation “A” was used to encapsulate human donor islets (Fig. 5B). The islets within “A” appeared to survive the encapsulation and coating processes, suggesting promising islet and cell compatibility with the thiolactone-polyamine based approach.

[0115] Formulation “A” was prepared with fluorescent derivatives of the polymers, using PMM-CVS-FITC (fluorescein) and PADS-112-15k-RBITC (rhodamine B). The capsules were imaged by confocal laser scanning microscopy and cross-sectional images were obtained to observe the distribution of the polymer components within the hydrogel network (Figs. 6A-6C).

[0116] Formulation “A” appeared to have a relatively homogeneous core consisting of PMM- CVS as visualized in the FITC channel (Fig. 6A). PADS-112-15k appeared to form a polymer membrane, with higher PADS concentration at the surface of the hydrogel and some diffused throughout the core. It is anticipated that the polymer within the core of the hydrogel may not be as cyto-toxic as polycations in solution state as it is complexed with the polyanionic hydrogel of PMM-CVS / HTL and calcium alginate, thus de-risking the cytotoxicity of the polymer within the hydrogel. The permeability of the membrane of formulation “A” was assessed and presented further below.

[0117] Formulation “A” hydrogel capsules were subjected to an aggressive chemical stress test using sodium citrate and sodium hydroxide to probe the presence of covalent crosslinking of the hydrogel network (Figs. 7A-7C). Formulation “A” survived treatment with sodium citrate, which chelated the calcium from the hydrogel core containing calcium alginate. The capsules were able to swell and withstand the osmotic stress during that process. Following citrate treatment, 1 M sodium hydroxide was added to the sample to increase the pH to approximately 12 or higher to disrupt the electrostatic interactions of the PADS copolymer with the hydrogel core by deprotonating the ammonium cations. Some capsules appeared to ruptured after the addition of NaOH, however the capsule membrane appeared to have remained intact, which suggests the that PADS copolymer had indeed reacted with homocysteine thiolactone groups of PMM-HTL in the core hydrogel capsule. These results demonstrate that covalently crosslinked hydrogels withthiolactone-polyamine chemistry are a viable platform to generate permeability-controlled membranes for hydrogels.

[0118] In another experiment, hydrogel capsules were prepared using 4 wt.% total PMM- CVS / HTL (1 :1 wt. ratio), 1 % alginate, and coated with 1 wt.% PADS-112-15k, called formulation “B” (Fig. 8), which was also used to encapsulate human donor islets (Fig. 9).

[0119] Brightfield images of formulation “B” appeared much more opaque relative to “B”, due to the higher polymer loading of the PMM-CVS / HTL polymer in the hydrogel gel network with alginate and likely nano-scale phase-separation of the polymers at these higher concentrations. This phase separation of the PMM-CVS / HTL polymer within the calcium alginate core is thought to aid in covalent crosslinking by increasing the local concentration of the PMM polymers and thus increasing the rate of reaction between free-thiols of ring-opened thiolactone groups and vinyl sulfone groups.

[0120] In another experiment, coated hydrogel capsules were made with 2 % total PMM- CVS / HTL (1 :1 wt. ratio), 1 % alginate, and were coated with 1 % PADS-112-25k (Fig. 10). These higher molecular weight PADS polyamines (25k, versus 15k as used in “A” and “B”) were anticipated to be more restricted to the surface of the hydrogel during the coating process, forming shell membranes that may be thinner, but denser, thus further decreasing shell permeability.

[0121] The permeability of coated hydrogel capsule formulations “A”, “B”, and “C” were tested using fluorescently labelled IgG. The permeability of the coated capsules were compared to uncoated capsule formulation Ctrl. As a representative example, a confocal cross-sectional image of “C” is shown after 24 h of incubation with lgG-alexafluor488 in Figs. 11A-11 B. The in-diffusion of IgG was analyzed by measuring the fluorescence intensity of IgG within the capsule and dividing that value by the fluorescence intensity in the supernatant, using representative regions of interest (ROIs) as described above. The results for this analysis of the capsules incubated with IgG for about 24 h were compiled in Fig. 12.

[0122] The data in Fig. 12 show the progressive exclusion of IgG due to the membranes formed from the polymer coating formulations based on thiolactone-polyamine crosslinking chemistry. This demonstrates the ability to reduce the permeability and thus increase the exclusion of 150 kDa antibodies, using the thiolactone-polyamine crosslinking chemistry.

[0123] It is common in the hydrogel science to use permeability of defined molecular weight probes, such as fluorescently labelled dextrans or IgG, to measure pore dimensions under physiological conditions, e.g. in hydrated state and at pH 7.4 in saline. Dextrans and IgG of 150,000 kDa are known to have hydrodynamic diameters of 18 nm and 12.7 nm, respectively, and the almost complete exclusion of these probes from the capsule interior after 24 hrs sets an upper diameter limit of 12.7 nm for the pore dimensions of the capsule shells.

[0124] In a further example, coated hydrogel capsules were prepared with thiolactone containing polymers as part of a doubly coated capsule formulation. The doubly coated capsule formulation may be beneficial as adding a final layer to the permeability controlled membrane may be used to improve the anti-fibrotic properties of the surface of the capsules. Formulation “D” was coated with PHMS-60-25k that was fluorescently labelled with rhodamine B. Confocal fluorescence imaging of “D” in the TRITC channel showed a distinct layer on the surface of the capsule (Fig. 13A).

[0125] In another example, a doubly coated capsule formulation, Formulation “E”, was prepared using fluorescently labelled PAD (fluorescein) and PHMS60-25k (rhodamine B). Confocal imaging of “E” in the FITC and TRITC channels show the distinct double coating (Fig. 13B). Fig. 13B is a confocal image showing the double coating of the polyamine (FITC channel) and PHMS60-25k (TRITC channel). This same formulation was used to encapsulate human donor islets (Fig. 13C).Hydrogel Patches

[0126] In another experiment, the gel forming polymers of the hydrogel capsule formulations were used to demonstrate the translation of the thiolene-polyamine crosslinking chemistry to other hydrogel shapes. PMM-CVS / HTL (2 wt.%) and alginate (2 wt.%) were combined and extruded using a 3D bioprinter to form a hydrogel construct herein called a hydrogel patch. The thiolactone containing hydrogel patch was prepared, forming a 15 x 15 mm rectilinear pattern, 3-layers thick. The patch was coated with PADS-112-25k (1 wt.%, rhodamine B labeled), forming coated patch formulation P-001. Overview stitched images of P-001 were captured by brightfield (Fig. 14A) and fluorescence microscopy (Fig. 14B).

[0127] Patch P-001 was imaged by confocal laser scanning microscopy to visualize the distribution of the PADS polymer coating onto the hydrogel patch filaments (Fig. 15A). Since the patch was printed in a support bath, the filaments appear rough on the surface due to theimprinting of the support bath particles onto the hydrogel (Fig. 15B). Regardless, the PADS copolymer appeared to coat the filaments of the patch, with greater density at the surface and limited diffusion into the hydrogel.

[0128] The hydrogel patch P-001 was treated with sodium citrate followed by sodium hydroxide to test whether the coated patch was covalently crosslinked (Figs. 16A-16B). The filaments of the hydrogel patch coating appeared to survive this chemical stress test, as seen by the remaining fluorescence of the patch.

[0129] Hydrogel constructs containing thiolactone groups were doubly coated to form permeability reducing membranes, similar to hydrogel capsules and strings. A representative example of a doubly coated hydrogel patch is similar to the protocol as described above for single coated patches with an additional coating step following the first polycation coating. Briefly, following formation of the hydrogel construct, the hydrogel was places in a 6-well plate and washed with 3 mL of HBS containing calcium chloride. Once the supernatant was removed, 3 mL of 1% of PAD75-25k as the polycation was used to coat and react with the thiolactone containing hydrogel for 6 min with gentle agitation on an orbital shaker at 20-25 rpm. The supernatant was then removed, and the hydrogel was washed twice with 3 mL of HBS. For the second coating, 3 mL of 1% PHMS60-25k (rhodamine B labelled) was added to the hydrogel construct for 6 min with gentle agitation on an orbital shaker at 20-25 rpm. The supernatant was removed and the doubly coated hydrogel construct was washed twice with HBS, and supsequently cured for 1-2 hours to allow for covalent crosslinking via aminolysis of the thiolactone groups and thiolene reaction with resulting homocysteine free-thiols with vinylsulfone groups.

[0130] In another experiment, the hydrogel patch was doubly coated with thiolactone reactive polymers both in the core as PMM-HTL, and in the second coating layer as PHMS. The hydrogel patch P-002 was imaged by confocal laser scanning microscopy to visualize the double coating with PHMS (rhodamine B labelled) on the surface of the hydrogel (Fig. 17A). Fig. 17A shows a confocal image of double coated hydrogel construct P-002 in the TRITC channel showing a crosssection of the filament with PHMS (rhodamine B labelled) shown on the surface of the hydrogel filament. The corresponding brightfield image of the double coated hydrogel filament is shown in Fig. 17B.

Claims

WHAT IS CLAIMED IS:1 . A method of producing a hydrogel, the method comprising: reacting a first polymer functionalized with a homocysteine thiolactone of formula I to open the ring and obtain a pendant thiol group, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen;crosslinking the pendant thiol group of the first polymer with a pendant vinyl group on a second polymer in the presence of a gel former to obtain the hydrogel.

2. The method of claim 1 , wherein the gel former is selected from alginate, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose or elastin.

3. The method of claim 1 or 2, wherein the first polymer and the second polymer have a backbone selected from 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.

4. The method of any one of claims 1 to 3, wherein the reacting step is a hydrolysis or an aminolysis.

5. The method of any one of claims 1 to 4, wherein the pendant vinyl group is a pendant vinylsulfone.

6. The method of any one of claims 1 to 5, wherein the second polymer is PMM-cysteamine vinyl sulfone (PMM-CVS).

7. The method of any one of claims 1 to 6, wherein R2 is H.

8. The method of any one of claims 1 to 7, wherein the first polymer is PMM-homocysteine thiolactone (PMM-HTL) which becomes PMM-homocysteine after ring opening.

9. The method of any one of claims 1 to 8, further comprising coating the hydrogel with polycations.

10. The method of claim 9, wherein the polycation is a polycation polymer with monomers selected from 2-aminoethyl (meth)acrylate, 2-aminoethyl (meth)acrylamide), 3- aminopropyl (meth)acrylate, 3-aminopropyl (meth)acrylamide, 4-aminobutyl(meth)acrylate, 4-aminobutyl (meth)acrylamide, (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 (CBM), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl (meth)acrylamide, 3-N,N-dipropylaminopropyl (meth)acrylate, 4-N,N-dimethylaminopropyl (meth)acrylamide, 1,3- bis(dimethylamino)propyl (meth)acrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate, and combinations thereof.

11. The method of claim 10, herein the polycation is poly[APM-co-DMAEA-co-SBMA] (PADS).

12. The method of claim 11, wherein the PADS has an initial molar feed ratios of 1:1:2 of APM, DMAEA, and SBMA, with each value in the ratio varying by ±20%.

13. A functionalized polymer, comprising a functional group as follows:Polymer Backbonewherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen.

14. The functionalized polymer of claim 13, wherein the backbone polymer is selected from 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.

15. The functionalized polymer of claim 13 or 14, all of the R2 are H.

16. A functionalized polymer, comprising a functional group as follows: polymer backbonewherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen, and wherein X is a primary, secondary or tertiary amine, a hydroxyalkyl, a hydroxyl or -O'.

17. The functionalized polymer of claim 16, wherein the backbone polymer is selected from 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.

18. The functionalized polymer of claim 16 or 17, all of the R2 are H.

19. A hydrogel comprising alginate, and poly(methyl vinyl ether-alt-maleic anhydride) homocysteine crosslinked to poly(methyl vinyl ether-alt-maleic anhydride) cysteamine vinyl sulfone as per formula Awherein X is as defined as a primary, secondary amine, a hydroxyalkyl, a hydroxyl or -0; and wherein the hydrogel has pores having a size of less than 18 nm.

20. The hydrogel of claim 19, wherein the pores exclude IgG antibodies when the hydrogel is in a hydrated state in saline at a pH of 7.4.

21. A method of producing a homocysteine thiolactone polymer, the method comprising: performing a radical-mediated polymerization with a homocysteine thiolactone methacrylamide monomer and / or a homocysteine thiolactone acrylamide monomer and a radical initiator in a solvent optionally under heating.

22. The method of claim 21 , wherein the solvent is an aqueous solvent or an organic solvent.

23. The method of claim 22, wherein the organic solvent is toluene, tetrahydrofuran, dioxane, ethylacetate or chloroform.

24. The method of any one of claims 21 to 23, wherein the radical initiator is azobisisobutyronitrile (Al BN) or a Vazo™ initiator.

25. The method of any one of claims 21 to 24, wherein the homocysteine thiolactone polymer has a molecular weight of 5,000 to 100,000 g / mol.

26. The method of any one of claims 21 to 25, further comprising adding a monomer selected from the group consisting of N,N-dimethylaminoethylacrylate (DMAEA), N,N- dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl (meth)acrylamide, 3-N,N- dipropylaminopropyl (meth)acrylate, 4-N,N-dimethylaminopropyl (meth)acrylamide, 1 ,3- bis(dimethylamino)propyl (meth)acrylate, and 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, 3-hydroxypropyl (methacrylamide), polyethyleneglycol methacrylates, methyl (meth)acrylate, MPC, [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (SBMA), carboxybetaine methacrylates, (meth)acrylic acid, 2-acrylamido-2-methylpropane sulfonic acid, 3-sulfopropyl (meth)acrylate and combinations thereof, to produce a copolymer.

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