Crosslinkable allyl amido polymers

Abstract

The present invention relates to a combination of a poly(2-oxazoline) or poly(2-oxazine) polymer or copolymer with an allyl amido side chain with a crosslinker, a crosslinked composition obtained thereby, and hydrogels thereof. Further, the present invention discloses methods of providing the combinations, compositions and hydrogels described herein and uses thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of polymer chemistry and hydrogels. More specifically, the present invention relates to a combination comprising a polymer having allyl amido side chains and a crosslinker, a crosslinked composition obtained therefrom and a hydrogel thereof. Furthermore, methods of providing the combination, composition and hydrogel described herein and uses thereof are disclosed.

[0002] The present invention particularly relates to a combination of a poly(2-oxazoline) or poly(2-oxazine) polymer or copolymer having allyl amido side chains and a crosslinker, a crosslinked composition obtained therefrom and a hydrogel thereof. Furthermore, methods of providing the combination, composition and hydrogel described herein and uses thereof are disclosed. BACKGROUND

[0003] Hydrogels are physically or chemically crosslinked polymer networks capable of absorbing large amounts of water. In other words, hydrogels are compositions comprising a natural or synthetic polymer matrix. In nature, types of hydrogels include collagen, hyaluronic acid and others. In the past decades, scientists have focused on improving the properties of natural hydrogels and also on providing synthetic hydrogels for various applications. Hydrogels currently have a wide range of applications in the food and pharmaceutical industry and have proven useful for bioengineering applications such as tissue engineering, where hydrogels are required to be chemically stable under physiological conditions and have compatible mechanical properties.

[0004] As previously mentioned, hydrogels are characterized by the presence of a polymer network or matrix that provides the swelling properties. Said polymer network is obtained by crosslinkable groups attached to the polymer backbone (homopolymer, copolymer) through crosslinking. To accomplish crosslinking, various crosslinking methods exist.

[0005] The crosslinking methods in the prior art can be mainly divided into two categories: physical methods and chemical methods. Among these methods, chemical crosslinking methods provide the formation of covalent bonds between polymer chains, which leads to more stable hydrogels and more controllable mechanical properties. In particular, the use of photo-crosslinking strategies is of particular interest as these methods are typically characterized by relatively mild conditions that allow for, for example, cell encapsulation in hydrogels. Photo-crosslinking can be achieved by exposing various types of photo-reactive functional groups to electromagnetic radiation, for example UV light. Among the various chemical species available, thiol-ene chemistry has gained interest in the past decades due to its versatility.

[0006] Thiol-ene chemistry is a versatile tool for creating carbon-sulfur bonds and has been widely used to create cross-linked structures with both commercial and research value. Thiol-ene coupling reactions are advantageous because (1) they are thought to be insensitive to oxygen inhibition, (2) can be performed in a single step under a wide range of conditions, including in aqueous media, (3) can be performed in the presence of cells without deleterious effects, and can be formed from any range of free thiols and available vinyl groups.

[0007] In thiol-ene coupling reactions for the formation of hydrogels, it is useful to start with macromolecular precursors of moderate to high molar mass. These should contain thiol or ene groups (e.g. olefinic or allyl moieties) and cross-link with a second small molecule or macromolecule containing corresponding reactive thiol groups.

[0008] In the production of hydrogels, the choice of the polymer backbone of the cross-linked polymer network determines the final properties of the hydrogel. Based on the desired application of the hydrogel, one polymer backbone can be more suitable than another. Some desirable target attributes when developing new cross-linkable polymers for biomedical applications are cell compatibility, minimal foreign body reaction (FBR), high-yield fast cross-linking under mild conditions, few or no side reactions, simple formulation, and availability of inexpensive and readily available or easily synthesized starting materials. The polymer backbone can comprise natural polymers such as collagen and gelatin, or synthetic polymers such as PEG, polysaccharides, proteins, peptides, growth factors, and others.

[0009] Taking into account many of these properties, previous work by Hoogenboom et al., 2009 aimed at developing new hydrogels based on poly(2-alkyl-2-oxazoline)s (PAOx). The underlying rationale behind the use of PAOx relative to other non-ionic hydrophilic materials is their rich chemistry, relatively straightforward synthesis, and potential biocompatibility. A more detailed discussion highlighting the attractiveness of PAOx as a base material for hydrogels has recently been published (Dargaville et al., 2018). Polymeric materials based on poly(2-oxazines) (PAOzi) have also been highlighted in the literature as promising materials in drug delivery systems (DDS) and polymeric therapeutics. PAOzi offers a wider synthetic variability than PAOx, allowing for more precise design of the polymer carrier structure to enable control over its biological behavior. The superior hydrophilicity of both PAOx and PAOzi polymers, in particular PMeOx and PMeOzi, leads to their better antifouling performance compared to PEG, see Sedlacek, O et al., 2020.

[0010] In the past few years, Hoogenboom et al. have developed hydrophilic PAOx copolymers incorporating alkene-terminated alkyl side chains using 2-decenyl-2-oxazoline (DecenOx) or 2-butenyl-2-oxazoline (ButenOx) copolymerized with 2-methyl-2-oxazoline (MeOx) or 2-ethyl-2-oxazoline (EtOx). These polymers can be crosslinked by means of thiol-ene coupling, through any number of dithiol molecules.

[0011] Dargaville et al., 2016 describe the synthesis of PAOx-based hydrogels. These hydrogels have been found to be advantageous in many applications, particularly biomedical applications, playing a key role in the construction of systems for drug / gene delivery or tissue engineering. In particular, PAOx offers full control over the polymer structures that can be achieved, including block, gradient and star structures. Furthermore, the properties of PAOx can be highly tuned by varying the side chain groups as well as by copolymerization of different monomers. Dargaville et al., 2016 describe that the hydrophobic crosslinkable group containing a terminal double bond, i.e. decenyl (providing DecenOx), can cure faster than those with shorter, more hydrophilic groups, i.e. butenyl (providing ButenOx). Furthermore, Dargaville et al. consider that the faster curing of the hydrophobic crosslinkable group can be a result of the hydrophobic association of this hydrophobic crosslinkable group, which determines a higher local double bond concentration, thus providing faster crosslinking.

[0012] Although Dargaville et al., 2016 disclose groups that are able to cure faster, their hydrophobic nature makes them less compatible with polar solvents, such as water, thus providing a reduced compatibility with direct curing in said polar solvents. In bioengineering applications, a higher compatibility of the photocrosslinkable functional group with polar solvents is particularly desirable, where water or aqueous solutions are the choice of biocompatible solvents. In other words, a drawback of these materials is that the hydrophobic side chains incorporating alkenes significantly contribute to the overall hydrophobicity of the polymer, which means that in order to remain water-soluble, they should be copolymerized with more hydrophilic MeOx monomers, or their concentration in the polymer should be kept low.

[0013] Therefore, there is a need to provide hydrogels, compositions and combinations thereof and methods thereof that overcome the drawbacks of the prior art. Furthermore, the present invention aims to provide hydrogels and compositions and combinations thereof with improved curing properties and improved biocompatibility. SUMMARY

[0015] In a first aspect, the present application provides a combination comprising a polymer or copolymer having one or more allyl amido side chains; and a crosslinker, wherein the polymer or copolymer is selected from a poly(2-oxazoline) or a poly(2-oxazine). It has surprisingly been found that the combination according to the present application provides faster crosslinking. This finding is surprising because based on the prior art it was expected that allyl side chain moieties provide slower curing compared to moieties comprising a terminal double bond of increased length, such as decenyl and butenyl. Dargaville et al., 2016, considered that faster curing of more hydrophobic crosslinkable groups such as decenyl can be the result of hydrophobic association of such hydrophobic crosslinkable groups, which determines a higher local double bond concentration and thus provides faster crosslinking. Thus, polymers comprising e.g. decenyl (providing DecenOx) can cure faster than those with shorter, more hydrophilic groups, more specifically butenyl (providing ButenOx).

[0016] In a further embodiment, the crosslinker comprises two or more thiol groups.

[0017] In a further embodiment, the polymer or copolymer comprises monomeric units selected from the group consisting of 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-methyl-2-oxazine, 2-ethyl-2-oxazine and 2-propyl-2-oxazine.

[0018] In one embodiment according to the present application, the combination comprises a copolymer comprising a first 2-oxazoline or 2-oxazine monomer having one or more allyl amido side chains and a second 2-oxazoline or 2-oxazine monomer not having an allyl amido side chain in a ratio of about 95-5 to 5-95, preferably 70-30 to 10-90, more preferably 40-60 to 10-90.

[0019] In a further embodiment of the present application, the polymer in the combination is represented by formula (I):

[0020] (X-Z) n - main chain (I),

[0021] wherein:

[0022] X represents an allyl amido side chain;

[0023] Z represents a direct bond or a spacer; and

[0024] the main chain is a poly(2-oxazoline) or poly(2-oxazine) polymer or copolymer main chain;

[0025] and n is an integer, wherein n > 2.

[0026] In one specific embodiment of the invention, the degree of polymerization of the polymer or copolymer in the combination is about 50 to 1000, preferably 100 to 800, and more preferably 200 to 500.

[0027] In a second aspect, the present invention provides a composition comprising the combination according to the invention, wherein the allylamino side chain and the crosslinking agent are crosslinked with each other.

[0028] In a third aspect, the present invention provides a hydrogel comprising a composition as described in embodiments of the present invention.

[0029] In a fourth aspect, the present invention provides a method for providing a composition according to the invention, the method comprising the steps of: a) providing a combination as defined in the present invention, and b) curing the polymer with a crosslinking agent to obtain the composition.

[0030] In another aspect, the present invention provides (bio)inks comprising combinations according to the invention, and further, the use of the (bio)inks for 3D printing, two-photon polymerization, bioprinting, or biomaterials.

[0031] In another aspect, the present invention provides combinations or compositions or hydrogels as described in other embodiments of the invention for use in human medicine or veterinary medicine.

[0032] In another aspect, the present invention provides for the use of combinations or compositions or hydrogels as described in other embodiments of the invention in one of the following: food industry, cosmetics, drug delivery, cell delivery, bioengineering applications. Brief description of the attached diagram

[0034] Referring now specifically to the accompanying drawings, it is emphasized that the details shown are merely illustrative and for the purpose of discussing different embodiments of the invention only. They are presented to provide the most useful and readily apparent description of what is considered to be the principles and concepts of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than necessary for a basic understanding of the invention. The description taken in conjunction with the accompanying drawings makes it clear to those skilled in the art how several forms of the invention will be embodied in practice.

[0035] Appendix Figure 1 Also abbreviated as Figure 1 The mechanism of cationic ring-opening polymerization (CROP) of EtOx and C3MestOx using oxazoline onion salt (2-phenyl-2-oxazoline onion tetrafluoroborate (HPhOx-BF4)) as an initiator and piperidine as a terminator is shown.

[0036] Appendix Figure 2 Also abbreviated as Figure 2This illustrates the allyl acylation of the methyl ester side chain of P(EtOx-C3MestOx) using 6 equivalents of allylamine and TBD as catalysts in CH3CN.

[0037] Appendix Figure 3 Also abbreviated as Figure 3 The storage modulus (G′) of PEAOx solutions with 10% different thiol:alkene ratios is shown before and during irradiation with 365 nm UV light.

[0038] Appendix Figure 4 Also abbreviated as Figure 4 This demonstrates the dependence of the thiol-olefin ratio on the maximum storage modulus.

[0039] Appendix Figure 5A Also abbreviated as Figure 5A The photocuring behavior of decenyl-functionalized poly(2-oxazoline) (P1DecenOx) and the allyl amide-containing polymer according to the present invention (P2EAOx) is shown under the same conditions in the time range of 0 to 500 s, thus clearly revealing the much faster curing behavior of the latter. Appendix Figure 5B Also abbreviated as Figure 5B It shows the relationship with Figure 5A The same polymer described herein exhibits photocuring behavior under the same conditions within a short time range of 0 to 200 s.

[0040] Appendix Figure 6A Also abbreviated as Figure 6A The curing behavior of three storage moduli of P1DecenOx was determined: G′-A at the beginning of curing, G′-B at the intermediate curve, and G′-C before reaching the plateau G′(max). (Appendix) Figure 6B Also abbreviated as Figure 6B This shows that for P1DecenOx and P2EAOx, the following is achieved: Figure 6A The differences in gelation time between G′-A, G′-B, and G′-C are shown in the figure.

[0041] Appendix Figure 7A Also abbreviated as Figure 7A Experimental results comparing the curing properties of poly(allylacrylamide) and poly(pentenylacrylamide) copolymers are shown, with the percentage of olefin (allyl or pentenyl) at 3%. The results indicate that the polymer containing pentenyl-terminated double bonds crosslinks faster than the polymer containing the allyl moiety. (See attached image.) Figure 7B Also abbreviated as Figure 7B The results of a similar experiment are shown, in which the percentage of olefin (allyl or pentenyl) is 10%. Invention Details

[0043] The invention will now be described further. In the following paragraphs, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other one or more aspects unless explicitly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous. When describing the compounds of the invention, the terminology used should be interpreted according to the following definitions unless the context otherwise requires.

[0044] When referring to measurable values ​​such as parameters, quantities, durations, etc., the terms “about” or “approximately” as used herein mean a variation of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, provided that such variation is suitable for implementation in the disclosed invention. It should be understood that the numerical values ​​referred to by the modifier “about” or “approximately” are themselves specifically and preferably disclosed.

[0045] As used in the specification and appended claims, the singular forms “a,” “an,” and “the” include a plural of indicators, unless the context clearly specifies otherwise. For example, “polymer” means one or more polymers.

[0046] The compounds of the present invention can be prepared according to the reaction schemes provided in the examples below; however, those skilled in the art will understand that these are for illustrative purposes only, and the compounds of the present invention can be prepared by any of several standard synthetic methods commonly used by those skilled in the art of organic chemistry.

[0047] In a first aspect, the present invention provides a combination comprising a poly(2-oxazoline) polymer or copolymer having two or more allylamido side chains; and a crosslinking agent. In the context of the present invention, the term "combination" as used herein refers to a selection of two or more chemical compositions or compounds. Therefore, the combination of the present invention may thus comprise a polymer or copolymer as defined herein and a crosslinking agent.

[0048] In the context of this invention, a poly(2-oxazoline) polymer or copolymer is a polymer or copolymer comprising a polymer backbone of a ring-opening polymerization (ROP) product derived from 2-oxazoline or a 2-oxazoline derivative thereof. In the context of this invention, the 2-oxazoline derivative may be 2-alkyl-2-oxazoline (AOx).

[0049]

[0050] In the context of this invention, a poly(2-oxazine) polymer or copolymer is a polymer or copolymer comprising a polymer backbone derived from a ring-opening polymerization (ROP) of 5,6-dihydro-4H-1,3-oxazine or a 5,6-dihydro-4H-1,3-oxazine derivative thereof. 5,6-dihydro-4H-1,3-oxazine is also simply referred to herein as 2-oxazine. In the context of this invention, a 2-oxazoline derivative may be 2-alkyl-2-oxazine (AOZi).

[0051]

[0052] Therefore, in one specific embodiment of the present invention, the poly(2-oxazoline) or poly(2-oxazine) backbone may also be represented by the following formula:

[0053]

[0054] The above formula can be unified by formula Y of the present invention:

[0055]

[0056] The carbon atoms of the monomer units belonging to the polymer backbone can be 2 or 3, wherein when the atom is 2 carbon atoms, it represents a poly(2-oxazoline) backbone, and when the atom is 3 carbon atoms, it represents a poly(2-oxazoline) backbone, and wherein the wavy bond shown in Formula Y is connected to any other atom or molecule, such as a spacer group.

[0057] In the context of this invention, the term "side chain" as used herein refers to a chemical group connected to the main chain.

[0058] In the context of this invention, the term "allylamino" as used herein refers to a portion having the following formula:

[0059]

[0060] The wavy bond connects to any other atom or molecule, such as the polymer or copolymer backbone or spacer group.

[0061] In the context of this invention, the term "crosslinking agent" as used herein refers to one or more molecules comprising portions that can be crosslinked according to various crosslinking methods (e.g., but not limited to thiol-ene crosslinking). Thiol-ene crosslinking refers to polymer crosslinking techniques that utilize thiol-ene chemistry to form covalently bonded polymer networks. Thiol-ene chemistry, in a broad sense, refers to the reaction of thiol-containing compounds with olefins or "enes". Thiol-ene chemistry is preferred because it has a number of advantages, such as, but not limited to: i) they proceed rapidly under mild conditions, which makes them compatible with cells and other biomolecules; ii) they have well-defined and well-characterized reaction mechanisms and products; and iii) they facilitate the introduction of thiol and olefin functional groups into polymers compared to other functional groups.

[0062] In a further embodiment, the crosslinking agent comprises two or more thiol groups. For example, dithiothreitol can be used. Further thiol-containing crosslinking agents that can be used according to embodiments of the invention are: PEG-dithiol, oligoPEG-dithiol, (oligo)peptides containing two or more cysteine ​​groups, further polymers having thiol side chains such as PEG-trithiol and PEG-tetrathiol, thiolated gelatin, and PAOX having thiol side chains.

[0063] In one embodiment, the present invention provides a combination as defined herein, wherein the polymer or copolymer comprises a monomer unit selected from the group consisting of 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-methyl-2-oxazine, 2-ethyl-2-oxazine, and 2-propyl-2-oxazine, wherein 2-propyl-2-oxazoline is selected from 2-n-propyl-2-oxazoline, 2-isopropyl-2-oxazoline, and 2-sec-propyl-2-oxazine, and wherein 2-propyl-2-oxazine is selected from 2-n-propyl-2-oxazine, 2-isopropyl-2-oxazine, and 2-sec-propyl-2-oxazine.

[0064] Therefore, in a further embodiment, the present invention provides a combination as defined herein, wherein the copolymer comprises a first 2-oxazoline or 2-oxazine monomer having one or more allylamide side chains and a second 2-oxazoline or 2-oxazine monomer not having allylamide side chains, in a ratio of about 95-5 to 5-95, preferably 70-30 to 10-90, more preferably 40-60 to 10-90.

[0065] In the case of copolymers provided by the present invention, the allylamino-containing 2-oxazoline monomer can be considered as a "first" monomer. Therefore, in the context of the present invention, the term "first monomer" as used herein refers to a monomer of a polymer having an allylamino moiety on its side chain.

[0066] In the context of this invention, the term "second monomer" as used herein refers to a monomer of a polymer that does not have an allylamido moiety on its side chain.

[0067] More specifically, the polymer according to the invention does not necessarily contain a second monomer and is therefore a copolymer, but may also be a homopolymer consisting only of monomers containing allyl amide.

[0068] In a further embodiment of the invention, the polymer in the combination is represented by formula (I):

[0069] (XZ) n -Y(I)

[0070] in:

[0071] X represents the allyl amide side chain;

[0072] Z represents a direct bond or a spacer group; and

[0073] Y represents a poly(2-oxazoline) or poly(2-oxazine) backbone; particularly a poly(2-oxazoline) polymer or copolymer;

[0074] n are integers, where n≥2, meaning there should be at least two side chains containing allylamido moiety.

[0075] In the context of this invention, the term "main chain" as used herein refers to the polymer or copolymer backbone; in other words, the backbone is the longest series of covalently bonded atoms that together form a continuous chain of polymer or copolymer. The backbone of this invention is particularly a poly(2-oxazoline) or poly(2-oxazine) backbone.

[0076] In the context of this invention, the term "spacer group" refers to a portion intended to provide a (flexible) hinge between two other elements of a molecule it comprises, thereby spatially separating said elements. Possible spacer groups include alkyl spacer groups and ethylene oxide (PEG) spacer groups. The term "alkyl" itself, or as part of another substituent, refers to formula C x H 2x+1 A fully saturated hydrocarbon, where x is a number greater than or equal to 1. Typically, the alkyl groups of the present invention comprise 1 to 20 carbon atoms. The alkyl groups can be straight-chain or branched and can be substituted as shown herein. When a subscript is used herein after a carbon atom, the subscript indicates the number of carbon atoms that the named group may contain. Thus, for example, C 1-4 Alkyl groups refer to alkyl groups with 1 to 4 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, butyl and their isomers (e.g., n-butyl, isobutyl, and tert-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers. C1-6 Alkyl groups include all straight-chain, branched, or cyclic alkyl groups having 1 to 6 carbon atoms, and therefore include methyl, ethyl, n-propyl, isopropyl, butyl and their isomers (e.g., n-butyl, isobutyl, and tert-butyl); pentyl and its isomers, hexyl and its isomers, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl or 4-methylcyclopentyl, cyclopentylmethylene, and cyclohexyl.

[0077] For example, in the polymer / copolymer according to the invention, Z can be an alkyl spacer group, such as a C2 alkyl or C3 alkyl spacer group. Those skilled in the art will appreciate that various spacer groups can be used in the context of this invention, the choice of which will depend on the monomer used and the provided allylamide side chain. For example, in the case where the polymer according to the invention has a main chain that is a poly(2-oxazoline) backbone and is therefore covered by formula Y as defined above, the first monomer is allylamidized 2-methoxycarboxypropyl-2-oxazoline (C3MestOx), as described below, and the second monomer is 2-ethyl-2-oxazoline (EtOx), not shown, where m represents the number of monomer units. The polymer / copolymer according to the invention comprises at least one allylamide side chain, in this specific case present in the first monomer. In the first monomer, X is an allylamide side chain and Z is a spacer group, more specifically:

[0078]

[0079] In one specific embodiment of the invention, the degree of polymerization of the polymer or copolymer in the combination is about 50 to 1000, preferably 100 to 800, more preferably 200 to 500. Typically, the degree of polymerization is determined by size exclusion chromatography using a multi-angle light scattering detector to measure the absolute molecular weight.

[0080] In a second aspect, the present invention provides a composition comprising the combination according to the invention, wherein the allylamino side chain and the crosslinking agent are crosslinked with each other.

[0081] In a third aspect, the present invention provides hydrogels comprising combinations or compositions as described in embodiments of the invention. A hydrogel can be obtained by crosslinking the combination to obtain the composition and contacting the composition with a swelling agent absorbed by said composition. In other words, a method of providing a hydrogel is described herein, comprising the step of swelling the crosslinked composition as defined according to the invention with a swelling agent. Several swelling agents can be used in the context of the invention, such as, but not limited to: water, serum, intravenous fluid, glucose solution, Hartmann's solution, stem cell solution, plasma, phosphate buffer, HEPES, and saline solution.

[0082] In the context of this invention, the term "hydrogel" as used herein refers to a polymer composition comprising a polymer network capable of absorbing or retaining liquids within the network.

[0083] In a fourth aspect, the present invention provides a method for providing a composition according to the invention, the method comprising the steps of: a) providing a combination as defined in the present invention; b) curing the polymer with a crosslinking agent to obtain the composition. Step b) of curing the polymer with a crosslinking agent to obtain the crosslinked composition can be performed using various techniques of the prior art. According to a specific embodiment of the invention, the curing step b) is performed by UV curing or thermal curing, preferably UV curing.

[0084] Furthermore, in one specific embodiment of the invention, the curing step b) is carried out in the presence of a photoinitiator, such as a photoinitiator selected from the non-limiting list including: 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]2-methyl-1-propanone (Irgacure 2959), (4-benzoylphenoxy)-2-hydroxy-N,N,N-trimethyl-1-propanium chloride with methyldiethanolamine (Q-BPQ+MDEA), hydroxyalkylacetone (APi-180), sodium and lithium salts of monoacylphosphine oxide (Na-TPO and Li-TPO), and sodium and lithium salts of diacylphosphine oxide (BAPO-OLi and BAPO-ONa). Other suitable photoinitiators not described herein will be apparent to those skilled in the art.

[0085] In a further aspect, the present invention provides (bio)inks comprising combinations according to the invention, wherein the (bio)inks are used for 3D printing, two-photon polymerization, bioprinting, or biomaterials.

[0086] In the context of this invention, the term "(bio)ink" as used herein refers to a material suitable for being shaped into filaments or droplets, for example, by extrusion through a printing nozzle or needle, and which can maintain shape fidelity after deposition.

[0087] When the material is in droplet form, jet printing techniques such as piezoelectric jetting, thermal jetting, microvalve jetting, and acoustic jetting can be used. Alternatively, a polymer solution can be converted into a cross-linked 3D object using two-photon polymerization.

[0088] In another aspect, the present invention provides combinations or compositions or hydrogels as described in other embodiments of the invention for use in human medicine or veterinary medicine.

[0089] In another aspect, the present invention provides for the use of combinations or compositions or hydrogels as described in other embodiments of the invention in one of the following: food industry, cosmetics, drug delivery, cell delivery, bioengineering applications.

[0090] More specifically, the combinations or compositions or hydrogels according to the invention can be used in cosmetic procedures, large-volume tissue reconstruction, small-volume tissue reconstruction, fat grafting, fat filling, burns, dental applications, contact lenses, cartilage and bone tissue engineering, soft tissue engineering such as fat, spine, heart tissue engineering, muscle and tendon tissue engineering, as creams or ointments or gelling agents or thickeners, as extracellular matrix mimics.

[0091] Example 1

[0092] In this embodiment, a novel allyl amidated polymer according to the present invention, referred to as PEAOx, is described. The synthesis of PEAOx begins with copolymerization of 2-methoxycarboxypropyl-2-oxazoline (C3MestOx) with 2-ethyl-2-oxazoline (EtOx), followed by direct allyl amidation of the methyl ester of C3MestOx to produce a highly water-soluble allyl-containing polymer for crosslinking. The photo-hydrogelling and cytotoxicity kinetics of the precursor are described along with a first in vivo evaluation of the FBR (foreign body reaction) of the PEAOx hydrogel, using a polyethylene glycol hydrogel as a benchmark, to provide crucial animal safety data, thereby laying the foundation for further biomaterial applications.

[0093] Materials and methods

[0094] Unless otherwise stated, all materials used in the synthesis of the polymer were obtained from Merck. 2-Ethyl-2-oxazoline was donated by PolymerChemistry Innovations and distilled on BaO and ninhydrin prior to use, and stored in a glove box under inert and dry conditions. The synthesis of 2-phenyl-2-oxazoline ontium tetrafluoroborate (HPhOx-BF4) was carried out according to the procedure described in Monnery et al., 2018. Piperidine was distilled on CaH2 prior to use. Dry solvents were obtained from a solvent purification system with an alumina drying tower and nitrogen flow from JCMeyer. The deuterated solvent used for 1H NMR spectroscopy, i.e., chloroform-d (CDCl3, ≥99.8% D, water <0.01%), was purchased from Euriso-top. Irgacure 2959 (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone) was a gift from BASF and used as is. Based on the previously reported process, PJM Bouten et al. prepared C3MestOx in 2015.

[0095] synthesis

[0096] Copolymerization of C3MestOx and EtOx

[0097] Using improved literature methods, and based on Figure 1 The synthetic scheme shown describes the copolymerization of 2-ethyl-2-oxazoline (EtOx) with 10 mol% C3MestOx. All glassware was cleaned and dried in a 200°C oven before silanization with trimethylchlorosilane (TMS-Cl) to remove any water that could cause premature termination of polymer chains and thus increase polymer dispersion. Next, 2-phenyl-2-oxazoline onium tetrafluoroborate (a, 60.6 mg, 0.258 mmol, 0.003 equivalent) was added as an initiator to the flask and heated under active vacuum (1.6 × 10⁻⁶). -1 Melting was performed at 1 mbar. The silanization flask was transferred to a glove box under an inert and dry atmosphere, where monomers EtOx (7.85 mL, 77.76 mmol, 0.9 equivalents) and C3MestOx (1.29 mL, 8.64 mmol, 0.1 equivalents) (meaning a 9:1 EtOx:C3MestOx ratio) were used, and a dry solvent (acetonitrile, 8.87 mL) was added. The mixture was vigorously stirred, and the conversion was tracked by gas chromatography (GC) and 1H-NMR spectroscopy using a t=0 sample as the starting point. To obtain a P(EtOx-C3MestOx) copolymer with a target DP of 300 at 91.5% conversion, the reaction mixture was placed in an oil bath at 60 °C for 60 hours. After the reaction, 51 μL of piperidine was added at 0 °C, and the resulting mixture was stirred overnight. The copolymer was purified by precipitation in ice-cold diethyl ether, followed by dialyzing (MWCO = 3.5 kDa) and subsequent lyophilization to obtain P(EtOx-C3MestOx) (Mw = 23 kDa) as a colorless, fluffy powder. (See b). Gas chromatography, size exclusion chromatography, and... 1 The H-NMR spectrum was used for complete characterization.

[0098] Modification was achieved by polymerization of P(EtOx90-stat-C3MestOx10) directly amidated with allylamine.

[0099] The synthesis of allylamidized polyoxazoline described in this invention is as follows: Figure 2As shown. The synthesized P(EtOx-C3MestOx) copolymer contained 10 mol% (30 units) of methyl ester side chains, which were functionalized by amidation with allylamine in the post-polymerization modification step. The previously synthesized P(EtOx-C3MestOx) copolymer (a, 2 g, 0.0719 mmol) containing 2.156 mmol of functional methyl ester groups (1 equivalent) was dissolved in 15.4 mL of acetonitrile using 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD, 0.5 equivalent, 1.078 mmol, 150 mg) as a catalyst. Subsequently, allylamine (6 equivalent, 12.9 mmol, 0.97 mL) was added and the mixture was reacted at 70 °C for 30 h to completely convert to PEAOx, b. Purification was performed by precipitation in ice-cold diethyl ether, followed by dialyzing (MWCO = 1 kDa) and subsequent lyophilization. 1 H-NMR spectroscopy and size exclusion chromatography (Mw = 29 kDa, It was confirmed that the methyl ester side chain was completely modified into an allyl amide side chain.

[0100] Characterization

[0101] Instruments and Meters

[0102] The monomer conversion was determined by gas chromatography (GC) of the sample, based on the ratio of the integrals from the monomer and the reaction solvent. GC was performed on an Agilent Technologies 7890A system equipped with a VWR Carrier-160 hydrogen generator and an Agilent Technologies HP-5 column with a length of 30 m and a diameter of 0.320 mm. An FID detector was used, and the inlet was set to 250 °C with a split injection ratio of 25:1. Hydrogen was used as the carrier gas at a flow rate of 2 mL / min. The oven temperature was set to 20 °C / min. -1 Increase the temperature from 50°C to 120°C, then increase it at 50°C / min. -1 Heating from 120°C to 300°C.

[0103] Size exclusion chromatography (SEC) was performed on an Agilent 1260 series HPLC system equipped with a 1260 online degasser, a 1260 ISO pump, a 1260 automated liquid sampler (ALS), a 50°C thermostatic column chamber (TCC), a 1260 diode array detector (DAD), and a 1260 refractive index detector (RID). The thermostatic column chamber was equipped with two PLgel 5μm mixed-D columns and a pre-column in tandem. The eluent used was N,N-dimethylacetamide (DMA) containing 50 mM LiCl at a flow rate of 0.5 mL / min. -1The SEC elution profile was analyzed using Agilent ChemStation software with GPC enabled. The molar mass values ​​were calculated relative to the PMMA standard from PSS. value.

[0104] Freeze-drying was performed on a Martin Christ freeze dryer (model Alpha 2-4LSCplus).

[0105] Monomer and polymer mixtures were stored and prepared in the VIGOR Sci-Lab SG 1200 / 750 glove box system to achieve purity levels with both water and oxygen content below 1 ppm.

[0106] Nuclear magnetic resonance (NMR) spectra were recorded at room temperature using a Bruker Avance 400MHz spectrometer. Measurements were taken in chloroform-d(CDCl3) purchased from Euriso-top. 1 H NMR spectrum.

[0107] Photorheology

[0108] Gelation kinetics were investigated by performing small-strain oscillatory shear experiments at 30 °C on an Anton Paar MCR302 rheometer with a 10 mm parallel plate-plate geometry. Samples were irradiated using an Omnicure Series 1000 UV light source equipped with a 365 nm filter and an optical fiber probe mounted beneath the quartz base of the rheometer. An example of polymer sample preparation is as follows: To prepare a 10% PEAOx hydrogel with a 1:1 stoichiometry of thiol to olefin, 75 μL of a 12% wt / vol PEAOx aqueous solution was mixed with 6.4 μL of a 10% DTT solution, 4.5 μL of a 2% I2959 solution, and 4.1 μL of distilled water to prepare a total of 90 μL. A 28 μL aliquot of this solution was pipetted onto a quartz plate, and the UV source was turned on to begin testing after collecting baseline data for 30 or 60 seconds. After irradiation, the sample was recovered, washed in water, freeze-dried, and weighed to determine the swelling ratio.

[0109] Cytotoxicity

[0110] Human fetal fibroblasts were seeded at a rate of 50,000 per cell in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and L-glutamine (2 mM). After incubation overnight at 37°C in 5% CO2, the medium was replaced with fresh DMEM, and FBS was replaced with 0.1% bovine serum albumin (BSA). H2O2 (200 mM; negative control) or a soluble polymer (0.25 to 2 mg / mL) was added to the cells in the medium, and the cells were incubated for 6 hours. The medium was discarded, and the cells were washed in PBS and then added with CellTiter diluted 1:10 in clear DMEM. Aqueous MTS solution (Promega, Cat#G3582). After incubation for 1 hour, absorbance was measured at 490 nm. Data: Average values ​​expressed as sem are percentage changes in absorbance relative to the control after background correction for the MTS solution alone.

[0111] Hydrogel microsphere formation

[0112] A stock solution containing PEAOx (60 mg, 1.684 mmol) and dithiothreitol (DTT) (3.9 mg, 25.2 mmol, 0.5 equivalences relative to the olefin of PEAOx) was prepared in 510 μL of PBS (pH 7.3). 30 μL of 2% w / v I2959 aqueous solution was added just before loading the solution into the syringe. The polymer solution was then added dropwise through a 29G needle to 10 mL of poly(dimethylsiloxane) oil stirred at 400 rpm with a 1.5 cm magnetic stir bar in a 25 mL round-bottom flask. The suspension was then irradiated with UV light (Omnicure S2000, 365 nm) for 600 seconds with continued stirring. The resulting hydrogel spheres were washed with 200 mL of dichloromethane and filtered five times, followed by washing with acetone (5×) and ethanol (5×) sequentially. Finally, before implantation into mice, the hydrogel was washed with ultrapure ethanol (1×) and sterile PBS (5×) under sterile conditions in a laminar flow hood.

[0113] In vivo assay of foreign body reaction

[0114] Experiments involving animals were conducted in accordance with Australian guidelines for animal care and use for scientific purposes and Queensland University of Technology’s Research Conduct Guidelines, and were approved by the university’s Animal Ethics Committee. A total of six 8-week-old male C57BL / 6 mice (weight, 23 ± 1 g) were purchased from the Animal Resources Centre (WA, Australia). Animals were given free access to water and fed an irradiated rodent diet. Mice were housed in a specific pathogen-free environment (filter rack, Tecniplast) under a 12-hour light / dark cycle at the Medical Engineering Research Facility (Queensland University of Technology, Australia). Mice were anesthetized with isoflurane (Laser Animal Health) and preemptively administered meloxicam (1 mg / kg) and buprenorphine (0.05 mg / kg) subcutaneously as analgesics. With the mice in a ventral recumbent position, the upper and lower back areas were clamped and brushed with 10% povidone-iodine (Bio-O-I), followed by four longitudinal incisions (approximately 3 mm) and the formation of subcutaneous pouches through blunt dissection. Using forceps, two hydrogel samples—two sets of 10×PEAOx balls—were placed in the bag. The wound was sutured. Tramadol (25 mg / L) was administered via drinking water for 5 days postoperatively as a postoperative analgesic. Mice were monitored daily for 28 days during euthanasia by CO2 asphyxiation in an appropriate chamber, and hydrogel samples were collected and processed for histological analysis to examine in vivo FBR.

[0115] Histology

[0116] The tissue explants were immersed in 4% paraformaldehyde overnight and then embedded in paraffin using a standard embedding protocol. Each embedded tissue sample was cut into 5 μm sections and stained with H&E using a standard protocol.

[0117] Results and discussion

[0118] The copolymerization of C3MestOx monomer with commercially available 2-ethyl-2-oxazoline (EtOx) in a 9:1 molar ratio (9:1EtOx:C3MestOx) was achieved at 60°C using 2-phenyl-2-oxazoline tetrafluoroborate as an initiator and conventional heating, with a target DP of 300, thus providing a P(ETOx90-stat-C3MestOx10) copolymer. See the synthesis protocol. Figure 1 Size exclusion chromatography (SEC) of the copolymer showed a dispersion of 1.35.

[0119] To introduce an allyl group into the side chain for thiol-olefin crosslinking, a simple amidation reaction using excess allylamine was chosen; see [link to relevant documentation]. Figure 2 The synthesis scheme in [the text]. 1 1H NMR spectroscopy confirmed the consumption of methyl ester and the presence of allyl and secondary amines.

[0120] Rheology was used to investigate the hydrogelation of PEAOx via thiol-olefin photocrosslinking using dithiothreitol (DTT) in real time. Gelization kinetics showed rapid crosslinking approximately 15 seconds after UV light irradiation; see [link to relevant documentation]. Figure 3 However, when thiols are not used, gelation does not occur. Figure 3 Representative curves of the storage modulus (G′) of 10% PEAOx solutions with different thiol:olefin ratios are shown before and during irradiation with 365 nm UV light. This contradicts our previous findings on the hydrogelation of poly(2-methyl-2-oxazoline-co-2-decenyl-2-oxazoline) copolymers, where homopolymerization of vinyls leads to gelation even in the absence of thiols. This is explained by the aggregation of hydrophobic decenyl side chains. Similar aggregation should not exist in PEAOx due to the more polar allyl-amidOx monomers, thus reducing homopolymerization. Another advantage of using allyl-amidOx is that the copolymer with EtOx is water-soluble; comparing this to 2-decenyl-2-oxazoline copolymers, where EtOx copolymers are water-insoluble, would limit its use in aqueous systems to copolymerization with very hydrophilic monomers such as MeOx. PEAOx also dissolves rapidly in water (within seconds) and has low surfactant-like properties, meaning it is easy to pipette without generating bubbles, resulting in a defect-free hydrogel. By varying the thiol to olefin ratio, the final modulus was observed to be relatively insensitive to the amount of thiol used, although a maximum was observed near a molar ratio of 0.5. Furthermore, Figure 4 This shows the dependence of the thiol-olefin ratio on the maximum storage modulus. It is speculated that at higher thiol ratios, significant disulfide bond formation occurs, thereby reducing the storage modulus.

[0121] To test the toxicity of PEAOx, human fetal fibroblasts were exposed to solutions at concentrations up to 2 mg / ml. Based on standard MTS metabolic assays (data not shown), the solution was found to be non-toxic at these concentrations. This is likely due to the structural similarity between PEAOx and PETOx, which is known to be non-toxic over a wide concentration range. Furthermore, to evaluate the FBR response of crosslinked PEAOx, the polymer was formulated into spherical geometries. For this study, spheres were prepared by dropping a solution of PEAOx, DTT, and I2959 into stirred silicone oil and irradiating with UV light until stable spheres formed. All spheres were thoroughly washed with ethanol until silicone was undetectable by NMR spectroscopy.

[0122] The size distribution of the spheres was measured using an optical microscope, and for PEAOx spheres, the range was 0.75–1.75 mm (data not shown). The average diameter of PEAOx was 1.3 mm. In this embodiment, PEAOx consisted of an allylated copolymer in a 9:1 molar ratio (9:1 EtOx: C3MestOx). The equilibrium swelling ratio of the PEAOx spheres was 10.0 ± 0.8 (n = 3).

[0123] Approximately ten PEAOx hydrogel spheres were subcutaneously implanted into immune-active C57BL / 6 mice, with four implantation sites per animal—one group each of the shoulder and hip. After 28 days, the animals were sacrificed, and the tissue surrounding the hydrogel spheres was removed. In all but one case, the hydrogel was recovered, with no visual signs of degradation (23 or 24 hydrogel implants). This lack of degradation contrasts with Lynn et al., 2010, who recovered only 20% of the 5 × 1 mm PEG-acrylate discs from mice after 28 days. In their cases, it was hypothesized that the presence of cleavable esters in the acrylate groups was the source of the initial degradation products leading to macrophage recruitment and subsequent complete degradation. PEAOx hydrogels lack degradation sites. Previous studies examining simulated biological oxidative stress have shown that reactive oxygen species can degrade poly(2-ethyl-2-oxazoline). However, the good integrity of the recovered PEAOx spheres implies no substantial degradation during the experimental timeframe.

[0124] Analysis of the tissue surrounding the recovered hydrogel spheres was based on fluorescence and bright-field stereomicroscopy images of the spheres, as well as z-stacked confocal microscopy images of the same spheres. The spheres were stained for cell nuclei (DAPI), myofibroblast markers (α-smooth muscle actin, α-SMA), and F-actin. Staining of the PEAOx spheres followed by fluorescence stereomicroscopy and confocal microscopy revealed the presence of cell deposits (DAPI, F-actin) and myofibroblast markers (α-smooth muscle actin, α-SMA). The presence of α-SMA indicates that the fibroblasts have become fibrotic (data not shown). These results clearly demonstrate the biocompatibility of the PEAOx hydrogel beads.

[0125] Figures 5 and 6 illustrate the curing behavior of the compositions according to the invention compared to prior art. More specifically, Figures 5 and 6 provide a comparison between the curing behavior of PEAOx (based on a 9:1 EtOx:C3MestOx) designated P2EAOx and decenyl-functionalized poly(2-oxazoline) designated P1DecenOx. Photocuring behavior was investigated under the same conditions, more specifically, at a polymer concentration of 10 wt%, an olefin to DDT ratio of 1:1, and a photoinitiator concentration of 0.1% Irgacure2959 (I-2959).

[0126] Furthermore, the sample was irradiated with 80% Omnicure at a distance of 10 mm from the tip to the quartz plate. The rheometer used was then set to a temperature of 5 °C, a speed of 8 rad / s, and a strain of 0.2%.

[0127] Specifically, Figure 5A The photocuring behavior of decenyl-functionalized poly(2-oxazoline) (P1DecenOx) and the allylamide-containing polymer according to the present invention (P2EAOx) are shown under the same conditions in the time range of 0 to 500 s, clearly revealing the much faster curing behavior of the latter. Then, Figure 5B It shows the relationship with Figure 5A The same polymer described herein exhibits photocuring behavior over a short time range of 0 to 200 seconds under the same conditions. Furthermore, Figure 6A Three storage modulus values ​​were determined for the curing behavior of P1DecenOx: G′-A at the start of curing, G′-B at the midpoint of the curve, and G′-C before reaching the maximum storage modulus G′(max) plateau. Figure 6A The curve presented in the middle is also Figure 5A As shown in the image.

[0128] Figure 6B This shows that for P1DecenOx and P2EAOx, the following is achieved: Figure 6A The differences in gelation time shown for G′-A, G′-B, and G′-C are illustrated. Based on Figure 6B The information shown clearly indicates that the gelation time required for P2EAOx to reach the same storage modulus values ​​G′-A, G′-B, and G′-C is always shorter than the corresponding gelation time for P1DecenOx.

[0129] Example 2

[0130] In addition to Example 1, we also prepared copolymers of 2-methoxycarbonylethyl-2-oxazoline (C2MestOx) and EtOx, as well as copolymers of C2MestOx and 2-n-propyl-2-oxazoline (EtOx), using a similar process to that described in Example 1. nCopolymers of PrOx. After amidation of these copolymers with allylamine, we obtained the following allylamide-functionalized copolymers, denoted as P(EtOx-co-C2AamOx) and P( n PrOx-co-C2AamOx).

[0131]

[0132] Using a process similar to that described in Example 1, a transparent hydrogel was successfully prepared by irradiating a 10% by weight solution of the copolymer in water with P(EtOx-co-C2AamOx) in the presence of DTT or 2,2′-(ethylenedioxy)diethylthiol (0.5 equivalents compared to allyl) as a crosslinking agent and Irgacure2959 (10 mol% compared to DTT) as a photoradioactive agent.

[0133] Thermosensitive hydrogels with a volumetric phase transition temperature of approximately 15 °C were successfully prepared using P(PrOx-co-C2AamOx). These hydrogels were prepared by irradiating a 10 wt% solution of the copolymer in ethanol in the presence of DTT (0.5 equivalences compared to allyl) or pentaerythritol tetra(3-mercaptopropionate) (0.25 equivalences compared to allyl) as a crosslinking agent and Irgacure 2959 (10 mol% compared to DTT) at a concentration of 365 nm. The ethanol was then exchanged with water to obtain the hydrogel.

[0134] Example 3 - Comparative Example

[0135] The inventors further investigated the curing properties of other polymers containing allyl amide side groups linked to poly(2-oxazoline); more specifically, poly(allylacrylamide). Experiments were conducted to compare the curing properties of poly(allylacrylamide) copolymers (see Formula A on the left) and poly(pentenylacrylamide) copolymers (see Formula B on the right). More specifically, the curing properties of copolymers having the following formula:

[0136]

[0137] The results showed that polymers containing pentenyl-terminated double bonds crosslinked faster than polymers containing allyl moieties. This finding is explained by the hydrophobic association of this hydrophobic crosslinkable group (pentenyl), which determines a higher local double bond concentration, thus providing faster crosslinking. Simultaneously, these findings illustrate the existence of a surprising technical effect achieved through the combination according to the invention, wherein the polymer contains an allylamide side chain; a crosslinking agent; and wherein the polymer contains a first monomer having said allylamide side chain, the first monomer being 2-oxazoline. In particular, following the discovery of poly(allylacrylamide) and previous literature on polymers containing poly(2-decenyl-2-oxazoline), a slower crosslinking rate was expected for the more hydrophilic allylamide-containing polymers. Conversely, we found that these allylamide-containing poly(2-oxazoline) polymers exhibited much faster crosslinking rates (see Example 1).

[0138] Materials and methods

[0139] Material

[0140] The following chemicals were purchased from their respective suppliers and used as is: triazabicyclodecene (TBD, 98%, TCI), ethanolamine (99%, TCI), allylamine (99%, Sigma-Aldrich), DL-dithiothreitol (DTT) (>98%, Sigma-Aldrich). 50W X8 hydrogen-type strong acid 50-100 mesh (Sigma-Aldrich), acetone (≥99%, Sigma-Aldrich). 2959 was a generous donation from BASF. PMA was purchased from Scientific Polymer Products (40.08% toluene solution, Mw: approximately 40,000 g / mol). -1 4-Pentenamine was synthesized according to a publicly available method (see Byrne, J. et al., 2016). Deuterated water (D2O) was purchased from Eurisotop.

[0141] Instruments and Meters

[0142] Measurements were taken at room temperature using a Bruker Avance 300MHz Ultrashield. 1 H-NMR ( 1¹H-NMR spectra were obtained, and chemical shifts were given as parts per million (δ) relative to tetramethylsilane. Size exclusion chromatography (SEC) was performed on an Agilent 1260 series HPLC system equipped with a 1260 in-line degasser, a 1260 ISO pump, a 1260 automated liquid sampler (ALS), a temperature-controlled column chamber (TCC) set at 50 °C, which was equipped with two PLgel 5 μm Mixed-D columns (7.5 mm × 300 mm) and a pre-column in series, a 1260 diode array detector (DAD), and a 1260 refractive index detector (RID). The eluent used was N,N-dimethylacetamide (DMA) containing 50 mM LiCl at a flow rate of 0.5 mL / min. Molar mass values ​​and molar mass distribution, i.e., dispersion, were calculated relative to polymethyl methacrylate standards from PSS. Values. FT-IR spectra were measured on a Perkin-Elmer 1600 series FT-IR spectrometer and expressed as wavenumbers (cm²). -1 Report. Centrifugation was performed using 50 mL screw-capped centrifuge tubes from VWR or 15 mL high-transparency polypropylene conical tubes from Falcon on a ThermoScientific ALC multi-speed refrigerated centrifuge PK 121R. Photoinitiated thiols-enes were performed using an Anton Paar MCR302 rheometer equipped with a UV lamp source via in-situ photocrosslinking rheology.

[0143] synthesis

[0144] Preparation processes of A and B

[0145] Weigh PMA (0.5 g, 40 kDa, 0.0125 mmol, corresponding to approximately 5.81 mmol of methyl ester groups) in a 5 mL flask (5 mL microwave tube). Introduce an appropriate amount of amine (6 equivalents of amine per methyl ester group) at a predetermined ratio (molar ratio 1:1 or 2:1) into the flask, and cool the solution to 0 °C and degas by bubbling with argon for 10 minutes. Flask 1A, molar ratio 2:1, ethanolamine (23.25 mmol, 1.39 mL) / allylamine (11.6 mmol, 1.03 mL). Flask 2A, molar ratio 1:1, ethanolamine (17.43 mmol, 1.04 mL) / allylamine (17.43 mmol, 1.54 mL). Flask 1B, molar ratio 2:1, ethanolamine (23.25 mmol, 1.39 mL) / 4-pentenamine (11.6 mmol, 1.16 g). Flask 2B, molar ratio 1:1, ethanolamine (17.43 mmol, 1.04 mL) / 4-pentenamine (17.43 mmol, 1.75 g). TBD (81 mg, 0.58 mmol, 0.1 equivalent per methyl ester) was then added to the mixture, and the flask was rinsed with argon, capped, and heated at 80 °C for 24 hours. After returning to room temperature, the mixture was poured into 30 mL of cold acetone to precipitate the polymer. The solution was centrifuged, and the liquid supernatant was discarded. The polymer was further precipitated three times by dissolving it in a minimal amount of methanol (2–3 mL) and pouring it into cold acetone (30 mL). To remove TBD and residual trace amine, the resulting polymer was dissolved in water, and for each sample, Dowex (160 mg, twice the mass of TBD) was added. After stirring for 5 hours and filtering to remove Dowex, water was removed by freeze-drying, and the resulting solid was dried overnight in a vacuum oven at 40 °C to obtain the desired pure polymer as a white powder.

[0146] Curing experiment

[0147] In-situ photocrosslinking experiments were conducted using a 10 wt% solution of the polymer in water as a solvent, wherein each double bond (allyl, pentenyl) of the polymer contained 0.5 equivalents of DDT and a 10 mol% concentration of photoinitiator (Irgacure 2959) relative to each DDT. The solution (approximately 0.4 mL) was deposited onto a rheometer glass plate with a fixed gap of 0.4 mm (upper profile of 25 mm diameter). Storage modulus and loss modulus were measured over a total time period of 665 seconds, with a γ amplitude of 0.1% for (oscillatory) shear deformation at a deformation frequency of 1 Hz. Baselines were measured over 1 minute, and then the solution was irradiated at room temperature with a UV lamp (filtered at 365 nm, irradiated through an optical fiber at the bottom of the glass plate).

[0148] Results and discussion

[0149] Figure 7A and Figure 7B The results of curing experiments comparing the curing properties of poly(allylacrylamide) and poly(pentenylacrylamide) copolymers are shown. More specifically, Figure 7A and Figure 7B The storage modulus G′ and loss modulus G″ of poly(allylacrylamide) copolymer and poly(pentenylacrylamide) copolymer are shown. Figure 7A In the study, the concentration of the tested olefin (allyl or pentenyl) within the polymer was 3%, as measured by NMR. Figure 7B In the study, the concentration of the tested olefin (allyl or pentenyl) in the polymer was 3%, which was also measured by NMR.

[0150] Using a copolymer concentration of 10 wt%, water as a solvent, 0.5 equivalents of DDT / allyl, and a photoinitiator (Irgacure) / DDT concentration of 10 mol%, the following were performed: Figure 7A and 7B The curing experiment shown.

[0151] based on Figure 7A and 7B The results shown clearly demonstrate that the presence of the pentenyl moiety provides faster curing and a higher final G′ compared to copolymers with allyl moiety.

[0152] References

[0153] 1. Hoogenboom, R. Poly(2-oxazoline)s: A polymer class with numerous potential applications. Angewandte Chemie-International Edition 48, 7978-7994, doi:10.1002 / anie.200901607(2009).

[0154] 2. Dargaville, TR, Park, JR & Hoogenboom, R. Poly(2-oxazoline) Hydrogels: State-of-the-Art and Emerging Applications. Macromolecular Bioscience 18, doi:10.1002 / mabi.201800070(2018).

[0155] 3.Sedlacek,O.and Hoogenboom,R.(2020),Drug Delivery Systems Based onPoly(2-Oxazoline)s and Poly(2-Oxazine)s.Adv.Therap.,3:1900168.

[0156] 4.Dargaville,Tim&Lava,Kathleen&Verbraeken,Bart&Richard,Hoogenboom.Unexpected Switching of the Photogelation Chemistry When Cross-Linking Poly(2-oxazoline)Copolymers.Macromolecules.49.10.1021 / acs.macromol.6b00167(2016).

[0157] 5.Monnery,B.D.et al.Defined High Molar Mass Poly(2-Oxazoline)s.Angewandte Chemie-International Edition 57,15400-15404,doi:10.1002 / anie.201807796(2018).

[0158] 6.P.J.M Bouten,Dietmar Hertsen,Maarten Vergaelen,Bryn D.Monnery,SaronCatak,Jan C.M.van Hest,Veronique Van Speybroek,Richard Hoogenboom,Synthesisof poly(2-oxazoline)s with side chain methyl ester functionalities:Detailedunderstanding of living copolymerization behavior of methyl ester containingmonomers with2-alkyl-2-oxazolines,J.Polym.Sci.,Part A:Polym.Chem.,53,2649-2661,https: / / doi.org / 10.1002 / pola.27733(2015).

[0159] 7.Lynn,A.D.,Kyriakides,T.R.&Bryant,S.J.Characterization ofthe invitro macrophage response and in vivo host response topoly(ethylene glycol)-based hydrogels.J.Biomed.Mater.Res.,Part A 93,941-953,doi:10.1002 / jbm.a.32595(2010).

[0160] 8.Byrne,J.P.;Blasco,S.;Aletti,A.B.;Hessman,G.;Gunnlaugsson,T.,Formation of Self-Templated 2,6-Bis(1,2,3-triazol-4-yl)pyridine[2]Catenanesby Triazolyl Hydrogen Bonding:Selective Anion Hostsfor Phosphate.AngewandteChemie International Edition 2016,55(31),8938-8943.

Claims

1. A composition comprising: - A polymer or copolymer having two or more allylamino side chains, wherein the allylamino side chains have the following chemical formula: - Crosslinking agent, The polymer or copolymer has a poly(2-oxazoline) or poly(2-oxazine) backbone; and the allylamino side chains of the polymer or copolymer and the crosslinking agent are crosslinked with each other.

2. The composition according to claim 1, wherein the crosslinking agent comprises two or more thiol groups.

3. The composition according to claim 1 or 2, wherein the poly(2-oxazoline) or poly(2-oxazine) backbone is represented by the following formula Y:

4. The composition according to claim 1 or 2, wherein the polymer or copolymer comprises a monomer unit selected from the group consisting of 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-methyl-2-oxazine, 2-ethyl-2-oxazine, and 2-propyl-2-oxazine.

5. The composition of claim 4, wherein the copolymer comprises a first 2-oxazoline or 2-oxazine monomer having one or more allylamide side chains and a second 2-oxazoline or 2-oxazine monomer not having allylamide side chains in a molar ratio of 95-5:5-95.

6. The composition according to claim 5, wherein the molar ratio of the first 2-oxazoline or 2-oxazine monomer to the second 2-oxazoline or 2-oxazine monomer is 40-60:10-90.

7. The composition according to claim 3, wherein the polymer or copolymer is represented by formula (I): (X-Z) n -Y(I) in: X represents the allyl amide side chain; Z represents a direct bond or a spacer group; Y represents the poly(2-oxazoline) or poly(2-oxazine) backbone as defined in claim 3; and n is an integer, where n≥2.

8. The composition according to claim 7, wherein Z represents a spacer group.

9. The composition according to claim 1 or 2, wherein the degree of polymerization of the polymer or copolymer is 50 to 1000, wherein the degree of polymerization is determined by size exclusion chromatography using a multi-angle light scattering detector to determine the absolute molecular weight value.

10. The composition according to claim 9, wherein the degree of polymerization of the polymer or copolymer is 200 to 500.

11. A hydrogel comprising the composition according to claim 1 or 2.

12. A method for providing a composition according to any one of claims 1 to 8, the method comprising the steps of: a) Provide - The polymer or copolymer as defined in any one of claims 1 to 8; and - Crosslinking agent as defined in claim 1 or 2; b) Curing the polymer or copolymer with the crosslinking agent to obtain the composition.

13. Use of the composition according to claim 1 or 2 as an ink for 3D printing, two-photon polymerization, bioprinting or biomaterials.

14. Use of the composition according to claim 1 or 2, or of the hydrogel according to claim 11, for use in human medicine or veterinary medicine.

15. Use of the composition according to claim 1 or 2, or the hydrogel according to claim 11, in any of the following applications: drug delivery, cell delivery, or bioengineering.

16. Use of the composition according to claim 1 or 2, or the hydrogel according to claim 11, in any of the food industry or cosmetics.

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