Stimulus responsive hydrogel precursor polymers, formulations and hydrogels

Abstract

Provided are hydrogel precursor polymers derived from poly(aspartamide) (PAspAm) comprising a cleavable and cross-linkable moiety connected to the pendant carboxylic acid group of the poly(aspartamide); as well as a hydrophilic moiety also connected to the pendant carboxylic group, such as an ether-containing moiety obtained by ring-opening polysuccinimide with 2-(2-aminoethoxy) ethanol.

Description

Attorney Docket No. P23-106-WO-PCT STIMULUS RESPONSIVE HYDROGEL PRECURSOR POLYMERS, FORMULATIONS AND HYDROGELS Sequence Listing

[0001] The present application claims the benefit of priority of U.S. provisional patent application no.63 / 582,400, filed on September 13, 2023, the entire contents of which are hereby incorporated by reference in their entirety.

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 24, 2024, is named P23-106-WO- PCT_SL.xml and is 11,456 bytes in size. Field of Invention

[0003] The present invention provides hydrogel precursor polymers based on a poly(aspartamide) (PAspAm) scaffold containing structural units that are cleavable by external stimuli. Such external stimuli include, for example, light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes. Furthermore, the hydrogel precursor polymers of the present invention contain moieties providing and / or enhancing cellular biological functions.

[0004] In addition, the hydrogel precursor polymers are crosslinkable, so that hydrogels with high dimensional stability and structural integrity can be obtained, which are susceptible to a controlled degradation by external stimuli, thereby modulating the spatiotemporal microenvironment of the hydrogel. Said hydrogels exhibit the cellular biological functions of the hydrogel precursor polymers from which they are formed. Hence, they are biocompatible, biodegradable, exhibit high cell viability and promote cellular biological functions such as, for example, attachment, proliferation, differentiation, metabolism, and extracellular matrix disposition.

[0005] There is further provided a formulation for forming a hydrogel comprising the hydrogel precursor polymers of the present invention and a process for forming aAttorney Docket No. P23-106-WO-PCT hydrogel using said formulation. Furthermore, the present invention provides a hydrogel obtainable by said process for forming a hydrogel, wherein said hydrogel is particularly suitable for use in various biomedical applications such as, for example, tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics, and / or other biomedical applications.

[0006] Finally, there is provided a process for forming artificial tissue, wherein said process includes contacting the hydrogel of the present with cells capable of forming artificial tissue, proliferating said cells, and cleaving the hydrogel by exposure to external stimuli. Background

[0007] A hydrogel is a three-dimensional network of hydrophilic polymers that contain a significant amount of water while maintaining their structure due to cross- linking of individual polymer chains. Hydrogels possess highly attractive and easily tunable physicochemical properties that make them excellent candidates for a wide range of biomedical applications.

[0008] The high water content, flexibility, porosity, and diffusivity of small molecules make hydrogels attractive materials for mimicking soft tissue microenvironments. They can also serve as reservoirs and delivery vehicles for cells, biomolecules, and hydrophilic drugs. Hydrogels also offer great potential to mimic the native extracellular matrix (ECM) due to the ease of tailoring their physiochemical and mechanical properties through the incorporation of degradable moieties and orthogonal chemistries.

[0009] Hydrogels that enable control of cell microenvironment properties must meet several physicochemical and biological design criteria that are dictated by the intended application. Some of the key design criteria include biocompatibility, tunability of mechanical properties, ability to crosslink in the presence of cells, mass transport, and controlled degradability. These characteristics of the hydrogels need to be optimized at the hydrogel preparation stage.

[0010] Hydrogels have been prepared using a variety of polymeric materials, whichAttorney Docket No. P23-106-WO-PCT can be divided broadly into two categories according to their origin: natural or synthetic.

[0011] Natural polymers such as polysaccharides and proteins serve as ideal building blocks for preparing hydrogels that can mimic aspects of the structural and biological properties of the cellular microenvironment. However, limited tunability of degradation kinetics, relatively poor mechanical properties, batch-to-batch manufacturing variations, and potential immunogenic reactions can restrict the application of natural polymer-based hydrogels.

[0012] Synthetic polymers, on the other hand, afford tunable physicochemical properties, ease of scale-up, batch-to-batch consistency, and a large scope for chemical modifications, including the introduction of degradable or biochemical moieties into the hydrogel structures. Besides, commercial availability coupled with great flexibility in the working range of pH, ionic strength, temperature, and chemical conditions, make synthetic polymers excellent candidates for hydrogel preparation. However, purely synthetic polymers often exhibit inferior biocompatibility and biodegradability in comparison to natural polymers, which may limit their use in applications where targeted and specific biological activity is desired.

[0013] Many cellular processes in tissues are influenced by the dynamic spatiotemporal changes in the cell microenvironment. Controlled modulation of such spatiotemporal changes is highly desirable for several biomedical applications including soft tissue engineering. For example, to promote cell secretory properties and enable tissue elaboration, or to allow delivery of therapeutics locally or systemically in a tunable manner, spatiotemporal control over the cell microenvironment is required. In hydrogel microenvironments, spatiotemporal changes can be induced through selective, local, and controlled degradation of polymer chains. The focus of the present invention is to provide a platform technology where hydrogel spatiotemporal microenvironment can be modulated using external stimuli.

[0014] Various systems are known from the prior art, but none of them meet the requirements of the present invention.

[0015] Water-soluble, pH-degradable polyacetals with a low critical solutionAttorney Docket No. P23-106-WO-PCT temperature behavior, falling out of aqueous solution upon increase in temperature are described by Samanta et al.[1]

[0016] S. Bhattacharya et al. describes a biodegradable copolymer for stimuli- responsive sustained release of doxorubicin.[2]The copolymer includes a PEG moiety to achieve biocompatibility and aqueous solubility and an acid-sensitive acylhydrazine-based linker to attach doxorubicin to trigger sustained drug-release.

[0017] R. Chandrawati describes enzyme-responsive polymer hydrogels for therapeutic delivery focusing on protease- and glycosidase-based catalyzed reactions.[3]

[0018] H. Zhao et al. describes the use of o-nitrobenzyl (o-NB) alcohol derivatives in polymer and material science such as (i) o-NB-based cross-linkers for photodegradable hydrogels, (ii) o-NB side chain functionalization in (block)copolymers, (iii) o-NB side chain functionalization for thin film patterning, (iv) o-NB for self-assembled monolayers, (v) photocleavable block copolymers, and (vi) photocleavable bioconjugates.

[0019] Hence, there is still a continuous need for the development of new stimulus- responsive hydrogel precursor polymers and hydrogels that can be prepared therefrom, which overcome the disadvantages known from the prior art. In particular, there is a need for improved stimulus-responsive hydrogel precursor polymers, which are crosslinkable to yield hydrogels with high dimensional stability and structural integrity being susceptible to a controlled degradation by external stimuli, thereby modulating the spatiotemporal microenvironment of the hydrogel. Furthermore, there is a need for improved hydrogels which are biocompatible, biodegradable, exhibit high cell viability and promote and / or enhance cellular biological functions and which are particularly suitable for use in various biomedical applications such as, for example, tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics, and / or other biomedical applications. Objective

[0020] The present invention aims to overcome the disadvantages in the prior art.Attorney Docket No. P23-106-WO-PCT Hence, it is an objective of the present invention to develop hydrogel precursor polymers containing structural units which are cleavable by external stimuli. Such external stimuli include, for example, light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes.

[0021] The hydrogel precursor polymers of the present invention can be further formulated into formulations for forming a hydrogel from which hydrogels can be obtained. Said hydrogels contain structural units that are cleavable by external stimuli in their polymer network structure and thus form stimuli-responsive (stimuli- degradable) hydrogels, which are spatially and temporally degradable under the influence of external stimuli.

[0022] It is a further objective of the present invention to develop hydrogel precursor polymers containing crosslinkable groups that are chemically reacted to give hydrogels.

[0023] Yet another objective of the present invention is to develop hydrogel precursor polymers containing moieties that provide and / or enhance cellular biological functions, which are transferred to hydrogels obtained therefrom.

[0024] Yet another objective of the present invention is to provide hydrogel precursor polymers from which formulations for forming hydrogels are obtained and from which hydrogels are obtained that are biocompatible, biodegradable, exhibit high cell viability, and promote and / or enhance cellular biological functions such as, for example, attachment, proliferation, differentiation, metabolism, and extracellular matrix disposition.

[0025] Another objective of the present invention it to provide a process for forming a hydrogel using the formulation for forming a hydrogel.

[0026] Finally, it is an objective of the present invention to provide a hydrogel obtainable by the process for forming a hydrogel, wherein said hydrogel is particularly suitable for use in various biomedical applications including tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices,Attorney Docket No. P23-106-WO-PCT implantable electronics, and / or other biomedical applications. Summary

[0027] The embodiments of the present invention described hereinafter provide a technical solution to the aforementioned objectives.

[0028] In a first embodiment of the present invention, there is provided a hydrogel precursor polymer, wherein the hydrogel precursor polymer contains a repeating unit U1 and a repeating unit U2:, wherein: R1and R2are independently from each other selected from alkylene, preferably from C1-C20alkylene, more preferably from C1-C10alkylene, most preferably from methylene, ethylene and propylene; R11and R22are independently from each other selected from hydrogen or alkyl, preferably from hydrogen or C1-C20alkyl, more preferably from hydrogen or C1-C10alkyl, most preferably from hydrogen, methyl, ethyl and propyl; A1is a moiety that breaks into smaller fragments under the influence of one or more external stimuli; A2is a moiety imparting hydrophilicity to the polymer; B1is a moiety containing at least one reactive group that can be further reacted toAttorney Docket No. P23-106-WO-PCT crosslink two or more polymer chains to form a hydrogel; X1is a chemical bond or a divalent linking moiety, wherein preferably X1is selected from a chemical bond, a divalent aliphatic moiety having 1 to 20 C atoms, preferably 1 to 10 C atoms, or a divalent aromatic moiety having 6 to 14 C atoms, preferably 6 to 10 C atoms; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000.

[0029] In a second embodiment of the present invention, there is provided a formulation for forming a hydrogel, wherein said formulation comprises a hydrogel precursor polymer according to the first embodiment.

[0030] In a third embodiment of the present invention, there is provided a process for forming a hydrogel, wherein the formulation for forming a hydrogel according to the second embodiment is subjected to irradiation and / or thermal treatment to thereby crosslink and form a hydrogel.

[0031] In a fourth embodiment of the present invention, there is provided a hydrogel obtainable or obtained by the process according to the third embodiment.

[0032] In a fifth embodiment of the present invention, there is provided a process for forming artificial tissue, wherein said process comprises the following steps: (a) contacting a hydrogel according to the fourth embodiment with cells capable of forming artificial tissue; (b) proliferating said cells; and (c) cleaving said hydrogel by exposure to one or more external stimuli.

[0033] The present invention is further described in the embodiments followingAttorney Docket No. P23-106-WO-PCT hereinafter in the detailed description. Brief Description of the Figures

[0034] Fig.1: Rheological analysis of time-dependent shear storage modulus (G’) on visible light (405 nm) irradiated PAspAm-Cys-MA (2).

[0035] Fig.2: Dry weight remaining ratio of the PAspAm-Cys-MA (2) hydrogel in PBS and 20 mM GSH / PBS at 37 °C as a function of time.

[0036] Fig.3A: Cell growth (hMSCs) in PAspAm-Cys-MA (2) hydrogel. Live / Dead stained hMSCs encapsulated in PAspAm-Cys-MA (2) hydrogel after 1 and 7 days of culture. Scale bar = 750 μm.

[0037] Fig.3B: Cell growth (hMSCs) in PAspAm-Cys-MA (2) hydrogel. Cell viability calculated as percentage of live cells from the Live / Dead staining images.

[0038] Fig.3C: Cell growth (hMSCs) in PAspAm-Cys-MA (2) hydrogel. Cell metabolism measured by PrestoBlue® fluorescence assay. *p<0.05. Detailed Description Hydrogel Precursor Polymer

[0039] In a first embodiment of the present invention, a hydrogel precursor polymer is provided, wherein the hydrogel precursor polymer contains a repeating unit U1 andAttorney Docket No. P23-106-WO-PCT a repeating unit U2:, wherein: R1and R2are independently from each other selected from alkylene, preferably from C1-C20alkylene, more preferably from C1-C10alkylene, most preferably from methylene, ethylene and propylene; R11and R22are independently from each other selected from hydrogen or alkyl, preferably from hydrogen or C1-C20alkyl, more preferably from hydrogen or C1-C10alkyl, most preferably from hydrogen, methyl, ethyl and propyl; A1is a moiety that breaks into smaller fragments under the influence of one or more external stimuli; A2is a moiety imparting hydrophilicity to the polymer; B1is a moiety containing at least one reactive group that can be further reacted to crosslink two or more polymer chains to form a hydrogel; X1is a chemical bond or a divalent linking moiety, wherein preferably X1is selected from a chemical bond, a divalent aliphatic moiety having 1 to 20 C atoms, preferably 1 to 10 atoms, or a divalent aromatic moiety having 6 to 14 C atoms, preferably 1 to 10 C atoms; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably fromAttorney Docket No. P23-106-WO-PCT 100 to 5,000; and n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000.

[0040] In a preferred embodiment of the present invention, the hydrogel precursor polymer further contains a repeating unit U1’ and U2’:, wherein: m1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and R1, R2, R11, R22, A1, A2, B1and X1have the same definition as in the first embodiment above.

[0041] A1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is a moiety that breaks into smaller fragments under the influence of light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes. A moiety that breaks into smaller fragments under the influence of light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / orAttorney Docket No. P23-106-WO-PCT enzymes means a moiety having at least one chemical group that undergoes bond cleavage by exposure to light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes.

[0042] Preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more photolabile groups, pH labile groups, reactive oxygen species (ROS) labile groups, redox labile groups and / or enzymatically labile groups.

[0043] Preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more photolabile groups, wherein preferably said photolabile groups comprise one or more motifs selected or derived from anthracene, arylmethyl, arylcarbonylmethyl, azobenzene, coumarin, diarylethene, pyrimidine, spiropyran, and nitroaryl, wherein more preferably said photolabile groups comprise one or more motifs selected from anthracene, arylmethyl, arylcarbonylmethyl, azobenzene, coumarin, diarylethene, pyrimidine, spiropyran, and nitroaryl. More preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more photolabile groups, wherein said photolabile groups are nitroaryl groups. Most preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer is represented by -[CH2-CH2-O]a-C(=O)-[CH2]b-O- C6H4(NO2)-CH2-, wherein a is an integer from 1 to 5, preferably 1, 2 or 3, more preferably 2; and b is an integer from 1 to 5, preferably 1, 2 or 3, more preferably 3.

[0044] Preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more pH labile groups, wherein preferably said pH labile groups comprise one or more motifs selected or derived from acetal, azomethine, imine, catechol-boronate, phosphoramidate, silyl ether, hydrazone, N-acyl hydrazone, and carbonate, wherein more preferably said pH labile groups comprise one or more motifs selected from acetal, azomethine, imine, catechol-boronate, phosphoramidate, silyl ether, hydrazone, N-acyl hydrazone, and carbonate. More preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more pH labile groups, wherein said pH labile groups are acetal groups. Most preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer is represented by -[CH2-CH2-O]c-CRcRd-[O-CH2-CH2]d-, wherein Rcand Rdare independently from each other selected from H or C1-5alkyl, preferably H, methyl, ethyl or propyl; and cAttorney Docket No. P23-106-WO-PCT is an integer from 1 to 5, preferably 1, 2 or 3, more preferably 2; and d is an integer from 1 to 5, preferably 1, 2 or 3, more preferably 1.

[0045] Preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more reactive oxygen species (ROS) labile groups, wherein preferably said reactive oxygen species (ROS) labile groups comprise one or more motifs selected or derived from thioketal, and oxalate, wherein more preferably said reactive oxygen species (ROS) labile groups comprise one or more motifs selected from thioketal, and oxalate.

[0046] Preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more redox labile groups, wherein preferably said redox labile groups comprise one or more motifs selected or derived from disulfide, and trimethyl benzoquinone, wherein more preferably, said redox labile groups comprise one or more motifs selected from disulfide, and trimethyl benzoquinone. More preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more redox labile groups, wherein said redox labile groups are disulfide groups. Most preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer is represented by -[CH2]e-S-S-[CH2]f-, wherein e and f are independently from each other integers from 1 to 10,from 1 to 5, more preferably 1, 2 or 3, most preferably 2.

[0047] Preferably, A1in the repeating units U1 and U1’ of the hydrogel precursor polymer comprises one or more enzymatically labile groups, wherein preferably said enzymatically labile groups are peptide sequences. Preferred peptide sequences are selected from matrix metalloproteinase (MMP)-sensitive peptide sequences, collagenase-sensitive peptide sequences, and human neutrophil elastase (HNE) sensitive peptide sequences. More preferred peptide sequences are peptide sequences including GPQGIWGQ (SEQ ID NO: 1), VPMSMRGG (SEQ ID NO: 2) and / or GCRDVPMSMRGGDRCG (SEQ ID NO: 10).

[0048] A2in the repeating units U2 and U2’ of the of the hydrogel precursor polymer according to the present invention is a moiety imparting hydrophilicity to the polymer. A moiety imparting hydrophilicity to the polymer means a moiety that comprises at least one polar group and / or ionic group capable of interacting withAttorney Docket No. P23-106-WO-PCT water by forming secondary bonds such as, for example, hydrogen bonds, dipolar bonds, electrostatic interaction, and / or van-der-Waals interaction with water molecules.

[0049] Preferably, A2in the repeating units U2 and U2’ of the hydrogel precursor polymer comprises one or more groups selected from -OH, -SH, -NH2, -CO-NH2and -CO2H.

[0050] Preferably, A2in the repeating units U2 and U2’ of the hydrogel precursor polymer comprises a water-soluble moiety. Such water-soluble moiety includes water-soluble polymer moieties such as, but not limited to, polyethylene oxide, polyvinyl alcohol, and polyacrylamide moieties.

[0051] Preferably, A2in the repeating units U2 and U2’ of the hydrogel precursor polymer comprises an alkylene unit -(CH2)x- and / or an ethylene oxide unit -(CH2- CH2-O)y-, wherein x and y are independently from each other integers from 1 to 2,000, preferably from 1 to 100, more preferably from 1 to 10, most preferably 1.

[0052] More preferably, A2in the repeating units U2 and U2’ of the hydrogel precursor polymer comprises one or more groups selected from -OH, -SH, -NH2, - CO-NH2 and -CO2H; and an alkylene unit -(CH2)x- and / or an ethylene oxide unit - (CH2-CH2-O)y-, wherein x and y are independently from each other integers from 1 to 2,000, preferably from 1 to 100, more preferably from 1 to 10, most preferably 1.

[0053] Most preferably, A2in the repeating units U2 and U2’ of the hydrogel precursor polymer is selected from the list consisting of -CH2-CH2-OH, -CH2-CH2-O- CH2-CH2-OH, -CH2-CH2-O-CH2-CH2-O-CH2-CH2-OH, -CH2-CH2-O-CH2-CH2- OCH3, -CH2-CH2-O-CH2-CH2-O-CH2-CH2-OCH3, -CH2-CH2-SH, -CH2-CH2-O-CH2- CH2-SH, -CH2-CH2-NH2, -CH2-CH2-O-CH2-CH2-NH2, -CH2-CH2-CO2H, -CH2-CH2- O-CH2-CH2-CO2H, -CH2-CH2-CO2H, and -CH2-CH2-O-CH2-CH2-CO2H.

[0054] Preferably, the reactive group contained in B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from the list consisting of groups capable to undergo polymerization crosslinking reactions, groups capable to undergo Michael-type or click chemistry-type crosslinking reactions, groups capable to undergo oxidative crosslinking reactions,Attorney Docket No. P23-106-WO-PCT and groups capable to undergo redox crosslinking reactions.

[0055] More preferably, the reactive group contained in B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from groups capable to undergo polymerization crosslinking reactions. More preferably, the groups capable to undergo polymerization crosslinking reactions contain one or more aliphatic vinyl, aromatic vinyl and / or allyl groups. Most preferably, the groups capable to undergo polymerization crosslinking reactions contain one or more acrylate, methacrylate or styrene groups. Most preferably, B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from the list consisting of -O-C(=O)-CH=CH2, -O- C(=O)-C(CH3)=CH2, -NH-C(=O)-CH=CH2, and -NH-C(=O)-C(CH3)=CH2.

[0056] More preferably, the reactive group contained in B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from groups capable to undergo Michael-type or click chemistry-type crosslinking reactions. More preferably, the groups capable to undergo Michael-type or click chemistry-type crosslinking reactions contain one or more acrylate, methacrylate, n-alkyne, cyclo-alkyne, amine, azide, carboxyl, hydrazide, hydroxyl, maleimide, norbornene, tetrazine and / or thiol groups. Most preferably, B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from the list consisting of -O-C(=O)-CH=CH2, -O- C(=O)-C(CH3)=CH2, -NH-C(=O)-CH=CH2, and -NH-C(=O)-C(CH3)=CH2.

[0057] More preferably, the reactive group contained in B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from groups capable to undergo oxidative crosslinking reactions, wherein preferably said groups capable to undergo oxidative crosslinking reactions contain one or more aromatic moieties with one or more aromatic hydroxyl groups, wherein more preferably said groups capable to undergo oxidative crosslinking reactions contain one or more mono-phenol, di-phenol, tri-phenol, oligo-phenol and / or poly- phenol moieties, wherein particularly preferably said groups capable to undergo oxidative crosslinking reactions contain a mono-phenol moiety. Most preferably, B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from -CH2-CH2-C6H4-OH and -CH(CO2H)-CH2-C6H4-Attorney Docket No. P23-106-WO-PCT OH. More preferably, the reactive group contained in B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from groups capable to undergo redox crosslinking reactions, wherein preferably said groups capable to undergo redox crosslinking reactions contain one or more thiol and / or amine groups. Most preferably, B1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from glutathione moieties or histidine moieties.

[0059] Preferably, X1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from a chemical bond, a divalent aliphatic moiety having 1 to 20 C atoms, preferably 1 to 10 C atoms, or a divalent aromatic moiety having 6 to 14 C atoms, preferably 6 to 10 C atoms. The divalent aliphatic moiety may contain one or more substituents selected from the list consisting of -NH2, -OH, -SH, -CN, -NHRx, -NRx2, -ORxand -SRx, wherein Rxis C1-10alkyl or C6-10aryl, preferably Rxis methyl, ethyl, propyl or phenyl. The aliphatic moiety may contain one or more functional groups selected from the list consisting of C=C double bond, CŁC triple bond, -NH-, -NHCO-, -O-, -S-, -CO-, -CO-O-, -O-CO- O-, -O-CO-NH-, -NRy-, -NRyCO- and -O-CO-NRy-, wherein Ryis C1-10alkyl or C6-10aryl, preferably Ryis methyl, ethyl, propyl or phenyl. The divalent aromatic moiety may contain one or more substituents selected from the list consisting of -NH2, -OH, - SH, -CN,-NHRz, -NRz2, -ORzand -SRz, wherein Rzis C1-10alkyl or C6-10aryl, preferably Rzis methyl, ethyl, propyl or phenyl.

[0060] More preferably, X1in the repeating units U1 and U1’ of the hydrogel precursor polymer according to the present invention is selected from a chemical bond, a divalent aliphatic moiety having 1 to 10 C atoms or a divalent aromatic moiety having 6 to 10 C atoms, wherein the divalent aliphatic moiety may contain one or more substituents selected from the list consisting of -NH2, -OH, -SH, -CN,-NHRx, -NRx2, -ORxand -SRx, wherein Rxis C1-5alkyl or C6aryl, preferably Rxis methyl, ethyl, propyl or phenyl, wherein the aliphatic moiety may contain one or more functional groups selected from the list consisting of C=C double bond, CŁC triple bond, -NH-, -NHCO-, -O-, -S-, -CO-, -CO-O-, -O-CO-O-, -O-CO-NH-, -NRy-, - NRyCO- and -O-CO-NRy-, wherein Ryis C1-5alkyl or C6aryl, preferably RyisAttorney Docket No. P23-106-WO-PCT methyl, ethyl, propyl or phenyl, and wherein the divalent aromatic moiety may contain one or more substituents selected from the list consisting of -NH2, -OH, -SH, - CN,-NHRz, -NRz2, -ORzand -SRz, wherein Rzis C1-5alkyl or C6-aryl, preferably Rzis methyl, ethyl, propyl or phenyl.

[0061] Most preferably, X1is a chemical bond.

[0062] In a more preferred embodiment of the present invention, the hydrogel precursor polymer further contains (i) a repeating unit U3 and a repeating unit U3’, or (ii) a repeating unit U4 and a repeating unit U4’, or (iii) a repeating unit U3, a repeating unit U3’, a repeating unit U4 and a repeating unit U4’: ,wherein: R3and R4are independently from each other selected from alkylene, preferably from C1-C20alkylene, more preferably from C1-C10alkylene, most preferably from methylene, ethylene and propylene;Attorney Docket No. P23-106-WO-PCT R33and R44are independently from each other selected from hydrogen or alkyl, preferably from hydrogen or C1-C20alkyl, more preferably from hydrogen or C1-C10alkyl, most preferably from hydrogen, methyl, ethyl and propyl; A3is a moiety imparting biological properties to the polymer; and A4is a moiety imparting shear thinning properties to the polymer; wherein in case (i): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and the sum of n1, n2, n3, m1, m2 and m3 is greater than 5, preferably greater than 50, more preferably greater than 500; or wherein in case (ii): n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and the sum of n1, n2, n4, m1, m2, and m4 is greater than 5, preferably greater than 50, more preferably greater than 500, or wherein in case (iii): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000;Attorney Docket No. P23-106-WO-PCT n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and where in case (iii) the sum of n1, n2, n3, n4, m1, m2, m3 and m4 is greater than 7, preferably greater than 50, more preferably greater than 500.

[0063] A3in the repeating units U3 and U3’ of the hydrogel precursor polymer according to the present invention is a moiety imparting biological properties to the polymer. Preferred biological properties are properties that support cellular biological functions such as, for example, attachment, proliferation, differentiation, metabolism and extracellular matrix disposition.

[0064] Preferably, A3in the repeating units U3 and U3’ of the hydrogel precursor polymer contains one or more synthetic functional peptide sequences found in native proteins, wherein preferably said native proteins are selected from fibronectin, laminin, collagen, elastin, albumin, immunoglobulin or silk.

[0065] Preferred synthetic functional peptide sequences are selected from the list consisting of RGD, YIGSR (SEQ ID NO: 3), IKVAV (SEQ ID NO: 4), DGEA (SEQ ID NO: 5), PHSRN (SEQ ID NO: 6), PRARI (SEQ ID NO: 7), (GAGAGS)p(SEQ ID NO: 8), (VPGXG)n(SEQ ID NO: 9), and (GPO)m, wherein p, n and m are independently from each other integers of ^ 1.

[0066] A4in the repeating units U4 and U4’ of the hydrogel precursor polymer according to the present invention is a moiety imparting shear thinning properties to the polymer.

[0067] Preferably, A4in the repeating units U4 and U4’ of the polymer is a moiety imparting shear thinning properties due to supramolecular interactions. Preferred supramolecular interactions are host-guest or self-assembly interactions, wherein such interactions preferably include interactions between cyclodextrin, adamantane, cucurbit[8]uril, cholesterol, polyethylene glycol and ureidopyrimidinone.

[0068] More preferably, A4in the repeating units U4 and U4’ of the polymerAttorney Docket No. P23-106-WO-PCT contains one or more cyclodextrin, adamantane, cucurbit[8]uril, cholesterol, polyethylene glycol or ureidopyrimidinone moieties.

[0069] Most preferably, A4in the repeating units U4 and U4’ of the polymer is a cyclodextrin, adamantane, cucurbit[8]uril, cholesterol, polyethylene glycol or ureidopyrimidinone moiety.

[0070] In a most preferred embodiment of the present invention, the hydrogel precursor polymer is represented by Formula I:wherein: R1, R2, R3and R4are independently from each other selected from alkylene, preferably from C1-C20alkylene, more preferably from C1-C10alkylene, mostAttorney Docket No. P23-106-WO-PCT preferably from methylene, ethylene and propylene; R11, R22, R33and R44are independently from each other selected from hydrogen or alkyl, preferably from hydrogen or C1-C20alkyl, more preferably from hydrogen or C1-C10alkyl, most preferably from hydrogen, methyl, ethyl and propyl; A1is a moiety that breaks into smaller fragments under the influence of one or more external stimuli; A2is a moiety imparting hydrophilicity to the polymer; A3is a moiety imparting biological properties to the polymer; A4is a moiety imparting shear thinning properties to the polymer; and B1is a moiety containing at least one reactive group that can be further reacted to crosslink two or more polymer chains to form a hydrogel; X1is a chemical bond or a divalent linking moiety, wherein preferably X1is selected from a chemical bond, a divalent aliphatic moiety having 1 to 20 C atoms, preferably 1 to 10 C atoms, or a divalent aromatic moiety having 6 to 14 C atoms, preferably 6 to 10 C atoms; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; n3 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10 to 5,000, most preferably from 100 to 5,000; and m3 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10Attorney Docket No. P23-106-WO-PCT to 5,000, most preferably from 100 to 5,000; n4 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10 to 5,000, most preferably from 100 to 5,000; and m4 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10 to 5,000, most preferably from 100 to 5,000; wherein the sum of n1, n2, n3, n4, m1, m2, m3 and m4 is greater than 5, preferably greater than 7, more preferably greater than 50, most preferably greater than 500.

[0071] The repeating units U1, U1’, U2, U2’, U3, U3’, U4 and U4’ can be distributed in the polymer represented by Formula I in any order. The polymer can thus be present, for example, as a random copolymer, as an alternating copolymer, and / or as a block copolymer. The asterisk * denotes a linkage of the two parts of the polymer shown in Formula I.

[0072] Preferred embodiments for the moieties A1to A4, B1and X1in the hydrogel precursor polymer represented by Formula I are as indicated above.

[0073] In a preferred embodiment of the present invention, the hydrogel precursor polymer has a weight average molecular weight (Mw) in the range from 1,000 Da (1 kDa) to 100,000,000 Da (100 MDa), preferably from 10,000 Da (10 kDa) to 10,000,000 Da (10 MDa), more preferably from 20,000 Da (20 kDa) to 5,000,000 Da (5 MDa), most preferably from 30,000 Da (30 kDa) to 1,000,000 Da (1 MDa). The weight average molecular weight (Mw) can be determined by any standard method known to those skilled in the art, such as GPC. Formulation

[0074] In a second embodiment of the present invention, there is provided a formulation for forming a hydrogel, wherein said formulation comprises a hydrogel precursor polymer according to the first embodiment of the present invention.

[0075] Preferably, the mass concentration of the hydrogel precursor polymer in the formulation for forming a hydrogel is from 0.1% (wt / v) to 99% (wt / v), preferably from 1% (wt / v) to 70% (wt / v), more preferably from 2% (wt / v) to 40% (wt / v),Attorney Docket No. P23-106-WO-PCT particularly preferably from 5% (wt / v) to 30% (wt / v), and most preferably from 5% (wt / v) to 20% (wt / v), based on the total volume of the formulation. Depending on the type of application for which the formulation for forming a hydrogel is applied, a person skilled in the art is able to determine suitable mass concentrations of the hydrogel precursor polymer in the formulation.

[0076] In a preferred embodiment of the present invention, the formulation for forming a hydrogel comprises one or more diluents. Such diluents typically act as solvents and / or dispersants diluting the hydrogel precursor polymer in the formulation. Preferred diluents are compounds that are liquid at room temperature. Preferred diluents are water, cell culture media such as, for example, DMEM, RPMI and MEM, or water-based buffer systems such as, for example, phosphate buffered saline (PBS), Hank’s buffer, Earle’s balanced salt solution, Tyrode buffer, HEPES buffer, etc.

[0077] In a preferred embodiment of the present invention, the formulation for forming a hydrogel further comprises one or more selected from crosslinking reagents and crosslinking additives. The crosslinking reagent serves the purpose to crosslink the hydrogel precursor polymers to form a crosslinked three-dimensional hydrogel. The crosslinking agent serves the purpose to react with the hydrogel precursor polymers to form a crosslinked three-dimensional hydrogel.

[0078] Preferred crosslinking reagents are selected from the list consisting of photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes mediating oxidative crosslinking, and agents mediating redox crosslinking.

[0079] Preferred photoinitiators are selected from the list consisting of acetophenone, p-anisil, benzil, benzoin, benzophenone, 2-benzoylbenzoic acid, 4,4’- bis(diethylamino)benzophenone, 4,4’-bis(dimethylamino)benzophenone, benzoin methyl ether, benzoin isopropyl ether, benzoin isobutyl ether, benzoin ethyl ether, 4- benzoylbenzoic acid, 2,2’-bis(2-chlorophenyl)-4,4’,5,5’-tetraphenyl-1,2’-biimidazole, methyl 2-benzoylbenzoate, 2-(1,3-benzodioxol-5-yl)-4,6-bis(trichloromethyl)-1,3,5- triazine, 2-benzyl-2-(dimethylamino)-4’-morpholinobutyrophenone, (±)- camphorquinone, 2-chlorothioxanthone, 4,4’-dichlorobenzophenone, 2,2- diethoxyacetophenone, 2,2-dimethoxy-2-phenylacetophenone, 2,4-Attorney Docket No. P23-106-WO-PCT diethylthioxanthen-9-one, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 1,4- dibenzoylbenzene, eosin Y, 2-ethylanthraquinone, 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-2-methylpropiophenone, 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone, 2-isopropylthioxanthone, lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate, 2-methyl-4’-(methylthio)-2-morpholino- propiophenone, 2-isonitrosopropiophenone, 2-phenyl-2-(p-toluenesulfonyl- oxy)acetophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, tris(2,2’- bipyridyl)dichlororuthenium(II) hexahydrate, and a combination of tris(2,2’- bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate.

[0080] More preferred photoinitiators are selected from the list consisting of eosin Y, 2-hydroxy-4’(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6- trimethylbenzoylphosphinate, tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate, and a combination of tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate.

[0081] Preferred thermal initiators are selected from the list consisting of tert-amyl peroxybenzoate, 4,4-azobis(4-cyanovaleric acid), 1,1’- azobis(cyclohexanecarbonitrile), 2,2’-azobisisobutyronitrile (AIBN), 2,2’-azobis(2- methylpropionamidine)dihydrochloride, benzoyl peroxide, 2,2-bis(tert- butylperoxy)butane, 1,1-bis(tert-butylperoxy)cyclohexane, 2,5-bis(tert-butylperoxy)- 2,5-dimethylhexane, 2,5-bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, bis(1-(tert- butylperoxy)-1-methylethyl)benzene, 1,1-bis(tert-butylperoxy)-3,3,5- trimethylcyclohexane, tert-butyl hydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butyl peroxybenzoate, tert-butylperoxy isopropyl carbonate, cumene hydroperoxide, cyclohexanone peroxide, dicumyl peroxide, lauroyl peroxide, 2,4- pentanedione peroxide, peracetic acid, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0082] More preferred thermal initiators are selected from the list consisting of 4,4- azobis(4-cyanovaleric acid), 2,2'-azobisisobutyronitrile (AIBN), 2,2’-azobis(2- methylpropionamidine)dihydrochloride, benzoyl peroxide, tert-butyl peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0083] Preferred basic initiators are selected from the list consisting of hydroxides,Attorney Docket No. P23-106-WO-PCT amines and amides. More preferred basic initiators are selected from the list consisting of MOH, MOR, NH3, RNH2, and R2NM, wherein M is an alkali metal, preferably Li, Na, K; and R is a carbyl moiety, preferably C1-C6alkyl. Most preferred basic initiators are selected from the list consisting of OH–, NH3and (iPr)2NLi (LDA).

[0084] Preferred oxidative initiators are selected from the list consisting of hydroxyl (HO•) and peroxyl (ROO•), peroxynitrous acid / peroxynitrite (ONOOH / ONOO–), nitrogen dioxide (NO2•), nitrosoperoxycarbonate (ONOOCO2–), carbonate (CO3•–), and lipid hydroperoxides(LOOH), wherein R is a carbyl moiety, preferably C1-C6alkyl, and L is a lipid moiety.

[0085] Preferred enzymes mediating oxidative crosslinking are selected from the peroxidase families, including horseradish peroxidase, myeloperoxidase, laccase, etc.

[0086] Preferred agents mediating redox crosslinking are selected from the list consisting of peroxides, hypochlorous acid, chloramines, hypobromous acid, bromamines, hypothiocyanous acid, nitroxyl, peroxynitrous acid, and other nitrating species.

[0087] In a preferred embodiment of the present invention, the formulation for forming a hydrogel comprises two crosslinking reagents selected from the lists described above.

[0088] Preferred crosslinking additives are compounds comprising two or more polymerizable groups with at least one C=C double bond. Preferred polymerizable groups with at least one C=C double bond are selected from the list consisting of acrylate, methacrylate, allyl, styryl and vinyl groups.

[0089] In a preferred embodiment of the present invention, the concentration of the crosslinking reagents in the formulation for forming a hydrogel is from 0.1 mM to 100 mM, preferably from 0.2 mM to 50 mM, more preferably from 0.5 mM to 40 mM, even more preferably from 1 mM to 30 mM, particularly preferably from 5 mM to 25 mM, and most preferably from 15 mM to 25 mM.

[0090] The formulation for forming a hydrogel according to the present invention is compatible with various human and non-human cell types and therefore useful forAttorney Docket No. P23-106-WO-PCT forming various artificial tissues.

[0091] In conclusion, the hydrogel precursor polymers described herein overcome the disadvantages known from the prior art. In particular, the hydrogel precursor polymers described herein contain structural units that are cleavable by external stimuli. Such external stimuli include, for example, light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes. Furthermore, they contain moieties providing and / or enhancing cellular biological functions. In addition, the hydrogel precursor polymers are crosslinkable, so that hydrogels with high dimensional stability and structural integrity can be obtained, which are susceptible to a controlled degradation by external stimuli, thereby modulating the spatiotemporal microenvironment of the hydrogel. Process for Forming a Hydrogel

[0092] In a third embodiment of the present invention, there is provided a process for forming a hydrogel, wherein the formulation for forming a hydrogel according to the second embodiment of the present invention is subjected to irradiation and / or thermal treatment to thereby crosslink and form a hydrogel.

[0093] Preferred irradiation treatment includes IR irradiation, VIS irradiation, and / or UV irradiation. Preferred IR radiation is in the range from 600 nm to 1 mm, more preferably in the range from 750 nm to 900 nm, and most preferably in the range from 770 nm to 790 nm. Preferred VIS radiation is in the range from 400 nm to 700 nm, more preferably in the range from 400 to 500 nm, and most preferably in the range from 400 to 450 nm. Preferred UV radiation is in the range from 100 nm to 400 nm, more preferably in the range from 300 nm to 400 nm, and most preferably in the range from 350 nm to 400 nm. Preferred thermal treatment includes exposure to elevated temperatures up to 120°C, preferably up to 100°C, more preferably up to 60°C. Most preferably, thermal treatment includes exposure to elevated temperatures between 30°C and 40°C. Depending on the type of formulation for forming a hydrogel and the hydrogel precursor polymer comprised therein, a person skilled inAttorney Docket No. P23-106-WO-PCT the art is able to determine suitable irradiation and / or thermal treatment conditions. Hydrogel

[0094] In a fourth embodiment of the present invention, there is provided a hydrogel obtainable or obtained by the process according to the third embodiment.

[0095] Said hydrogels exhibit the cellular biological functions of the hydrogel precursor polymers from which they are formed. Hence, they are biocompatible, biodegradable, exhibit high cell viability and promote cellular biological functions such as, for example, attachment, proliferation, differentiation, metabolism, and extracellular matrix disposition. The hydrogels are particularly suitable for use in various biomedical applications such as, for example, tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics and / or other biomedical applications.

[0096] Preferably, said hydrogel according to the present invention is for use in tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics and / or other biomedical applications. Process for Forming Artificial Tissue

[0097] In a fifth embodiment of the present invention, there is provided a process for forming artificial tissue, wherein said process comprises the following steps: (a) contacting a hydrogel according to the fourth embodiment with cells capable of forming artificial tissue; (b) proliferating said cells; and (c) cleaving said hydrogel by exposure to one or more external stimuli.

[0098] Preferably, said external stimuli are selected from light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygenAttorney Docket No. P23-106-WO-PCT species (ROS), redox active chemicals and / or enzymes.

[0099] It is to be understood that a skilled person can freely combine the above- mentioned preferred, more preferred, even more preferred, particularly preferred and most preferred embodiments relating to hydrogel precursor polymers and related embodiments of the present invention. Definitions

[0100] As used herein, the term “external stimulus” (or plural term “external stimuli”) refers to an external stimulus that acts on the hydrogel precursor polymer or the hydrogel obtained therefrom. The external stimulus does not emanate from the hydrogel precursor polymer itself or the hydrogel formed therefrom, but rather comes from an external source. Such an external source can be, for example, cells or other components contained in the formulation for forming a hydrogel or in the hydrogel obtained therefrom. Typical external stimuli in the context of this invention are: light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes. These external stimuli cause the A1moiety in the hydrogel precursor polymer or the hydrogel obtained therefrom to be cleaved into smaller fragments.

[0101] As used herein, the term “hydrogel precursor polymer” refers to a precursor compound from which a hydrogel can be prepared. Hydrogel precursor polymers are typically polymeric compounds that are not yet crosslinked to one another, but can be crosslinked to one another by forming covalent bonds.

[0102] As used herein, the term “hydrogel” refers to a three-dimensional crosslinked polymer having covalent crosslinking bonds (chemical hydrogel), which is able to absorb a large amount of water or biological fluids. Typically, the three- dimensional crosslinked polymer forming the hydrogel is a porous, permeable solid that is insoluble in water.

[0103] As used herein, the term “polymer” includes, but is not limited to, homopolymers, copolymers, for example, block, random, and alternating copolymers, terpolymers, quaterpolymers, etc., and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possibleAttorney Docket No. P23-106-WO-PCT configurational isomers of the molecule. These configurations include, but are not limited to isotactic, syndiotactic, and atactic symmetries. A polymer is a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units (i.e. repeating units) derived, actually or conceptually, from molecules of low relative mass (i.e. monomers).

[0104] As used herein, the term “monomer” refers to a molecule which can undergo polymerization thereby contributing constitutional units (repeating units) to the essential structure of a polymer or an oligomer.

[0105] As used herein, the term “copolymer” generally means any polymer derived from more than one species of monomer, wherein the polymer comprises more than one species of corresponding repeating unit. In one embodiment the copolymer is the reaction product of two or more species of monomer and thus comprises two or more species of corresponding repeating unit. It is preferred that the copolymer comprises two, three, four, five or six species of repeating unit. Copolymers that are obtained by copolymerization of three monomer species can also be referred to as terpolymers. Copolymers that are obtained by copolymerization of four monomer species can also be referred to as quaterpolymers. Copolymers may be present as block, random, and / or alternating copolymers.

[0106] As used herein, the term “block copolymer” refers to a copolymer, wherein adjacent blocks are constitutionally different, i.e. adjacent blocks comprise repeating units derived from different species of monomer or from the same species of monomer but with a different composition or sequence distribution of repeating units.

[0107] As used herein, the term “random copolymer” refers to a copolymer in which the probability of finding a given repeating unit at any given site in the chain is independent of the nature of the adjacent repeating units. Usually, in a random copolymer, the sequence distribution of repeating units follows Bernoullian statistics.

[0108] As used herein, the term “alternating copolymer” refers to a copolymer consisting of macromolecules comprising two species of repeating units in alternating sequence.

[0109] As used herein, the term “crosslinking reagent” refers to a reagent thatAttorney Docket No. P23-106-WO-PCT provides a reactive species capable of activating a reactive group to react with another reactive group in a crosslinking reaction. This enables the crosslinking of two or more polymer chains that carry reactive groups. Typical crosslinking reagents in the context of the present invention are photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes mediating an oxidative crosslinking, and agents mediating a redox crosslinking. The crosslinking reagents mentioned either directly provide a reactive species or must be activated for this purpose, for example by irradiation treatment, thermal treatment, etc.

[0110] As used herein, the term “crosslinking additive” refers to a compound that is capable to react with a reactive group in a crosslinking reaction. For example, the crosslinking additives can react with the reactive groups in the polymer, thereby providing additional crosslinking.

[0111] In the context of the present invention, the term “diluent” as used herein, denotes one or more compounds serving as a solvent, suspending agent, carrier and / or matrix for the hydrogel precursor polymer and any other additive comprised in the formulation for forming a hydrogel. Diluents are typically inert compounds that do not react with said polymers and said other additives. Typical diluents are compounds that are liquid at room temperature.

[0112] As used herein, the term “shear thinning” is the non-Newtonian behavior of fluids whose viscosity decreases under shear strain. Bioinks with shear thinning properties enable extrusion at lower extrusion forces thus minimizing damage to cells while bioprinting. The ratio G’’ / G’ of the shear loss modulus (G’’) to the shear storage modulus (G’) in a viscoelastic material is defined as the tan į (loss tangent), which provides a measure of damping the material. Tan į can also be visualized as the tangent of the phase angle į between the storage and loss modulus. Shear thinning means that the viscosity decreases with increasing shear rate. This is critical during extrusion-based printing because extruding highly viscous bioink at a relatively high speed (high shear rate) would result in high shear stress, thus killing cells. If the bioink is shear thinning, which means its viscosity is decreasing during extrusion (high shear rate), it lowers the shear stress applied on cells and thereby protecting cells from damage. To some extent, a shear thinning bioink under extrusion (high shear rate) would become less viscous and more liquid, suggesting higher G” and / orAttorney Docket No. P23-106-WO-PCT lower G’.

[0113] As used herein, the term “supramolecular interactions” refers to a class of molecular interactions categorized by their non-covalent character. Supramolecular interactions include host-guest interactions or self-assembly interactions based, for example, on van-der-Waals forces, pi-pi stacking, hydrogen bonding, hydrophobic interactions, metal-ligand coordination, and / or electrostatic interactions.

[0114] As used herein, the term “alkyl” refers to a saturated hydrocarbon chain, such as, but not limited to, methyl, ethyl, propyl and butyl. Alkyl may be straight- chain or branched-chain. For example, as used herein, propyl encompasses both n- propyl and iso-propyl; butyl encompasses n-butyl, sec-butyl, iso-butyl and tert-butyl, and so forth. Divalent alkyl is also referred to as "alkylene" in the present application. Those skilled in the art are familiar with this nomenclature.

[0115] As used herein, the term “allyl” refers to a substituent with the structural formula R-CH2-CH=CH2, where R is any other group of atoms or H. It consists of a methylene ) attached to a vinyl group (-CH=CH2).

[0116] As used herein, the term “vinyl” refers to a substituent with the structural formula R-C(R’)=CH2, where R and R’ are independently from each other any other group of atoms or H. Preferably, it consists of a vinyl group (-CH=CH2).

[0117] As used herein, the term “aryl” refers to an aromatic hydrocarbon group. Aryl includes, e.g., phenyl, biphenyl, naphthyl, anthracenyl.

[0118] The term “aliphatic moiety” as used herein, relates to a linear, branched, cyclic or bridged cyclic aliphatic unit which forms part of a structure of a chemical compound. An aliphatic moiety may contain heteroatoms selected from N, O, S and P.

[0119] The term “aromatic moiety” as used herein, relates to a monocyclic or polycyclic aromatic unit which forms part of a structure of a chemical compound. Polycyclic aromatic units include two or more connected aromatic ring systems which are fixed in one plane. Aromatic moieties contain an aromatic ring structure made ofAttorney Docket No. P23-106-WO-PCT carbon atoms.

[0120] The present invention is further illustrated by the examples following hereinafter which shall in no way be construed as limiting. A skilled person will acknowledge that various modifications, additions and alternations may be made to the invention without departing from the spirit and scope of the invention as defined in the appended claims. Examples

[0121] Example 1: Synthesis of pH-responsive hydrogel precursor polymer (1)

[0122] The pH-responsive hydrogel precursor polymer (1) is synthesized by incorporating an acid cleavable acetal functional group into a poly(aspartamide) structure as shown in the following Scheme 1.

[0123] Scheme 1: Synthesis of pH-responsive hydrogel precursor polymer (1)

[0124] Polysuccinimide (10 g) is dissolved in 100 ml of anhydrous dimethylform- amide (DMF) under nitrogen. To this solution 2-(2-aminoethoxy)ethanol (20 g) was added and it was stirred for 12 h at 40°C. The intermediate polymer (1a) was isolatedAttorney Docket No. P23-106-WO-PCT by precipitation into a copious amount of diethyl ether, followed by filtration.

[0125] The intermediate polymer (1a) (5 g) was further reacted with vinyloxyethyl methacrylate (10 g) in DMF using pyridinium p-toluenesulfonate as a catalyst at ambient conditions for 12 h. The polymer (1) was isolated by precipitation with a copious amount of diethyl ether. It was further purified by dialysis and isolated by freeze drying.

[0126] Example 2: Synthesis of redox-responsive hydrogel precursor polymer (PAspAm-Cys-MA) (2)

[0127] The redox-responsive hydrogel precursor polymer (PAspAm-Cys-MA) (2) is synthesized by incorporating a reductive cleavable disulfide linkage group into a poly(aspartamide) structure as shown in the following Scheme 2.

[0128] Scheme 2: Synthesis of redox-responsive hydrogel precursor polymer (PAspAm-Cys-MA) (2)

[0129] Polysuccinimide (1 g) was dissolved in 10ml of anhydrous dimethylform- amide (DMF) under nitrogen. To this solution 2-(2-aminoethoxy)ethanol (0.84 mL)Attorney Docket No. P23-106-WO-PCT was added and it was stirred for 24 h at room temperature. Then, cystamine (2 g) was added to the reaction solution and it was stirred for another 24 h at room temperature. The intermediate polymer (2a) was isolated by precipitation with a copious amount of diethyl ether followed by filtration.

[0130] The intermediate polymer (2a) (1 g) was dissolved in 10 mL of phosphate buffered saline (PBS). To this solution methacrylic anhydride (0.42 mL) was added and it was stirred at ambient conditions for 24 h. The aqueous reaction solution was dialyzed against water for 72 h and the polymer (PAspAm-Cys-MA) (2) was isolated by freeze drying.

[0131] Rheological characterization

[0132] Rheology measurements were performed at 25 °C on a Discovery HR-2 hybrid Rheomoter (TA instruments) fitted with a 20 mm diameter plate-plate geometry. The PAspAm-Cys-MA hydrogel precursor polymer (2) was photocrosslinked in-situ, and oscillatory time sweeps were performed under a shear strain of 1% and frequency of 1 Hz for 5 min. The samples were irradiated with visible light (OmniCure LX500, 405 nm, 8.8 mW / cm2) after the time sweep test started for 60 s. About 320 μL PAspAm-Cys-MA (2) solution (10% wt / v in PBS) containing 0.5% wt / v photoinitiator, lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate (LAP) was used for each test and geometry gap was set at 1 mm.

[0133] As shown in Figure 2, 10% PAspAm-Cys-MA (2) solution can form gel rapidly and its shear moduli can reach a plateau at 2.4 kPa within 30 s under light exposure.

[0134] In vitro degradation of the PAspAm-Cys-MA (2) hydrogel

[0135] The hydrolytic and reductive degradation of the PAspAm-Cys-MA (2) hydrogel were measured in PBS and glutathione (GSH) / PBS solutions, respectively. 0.5 mL 10% PAspAm-Cys-MA (2) solution with 0.5% wt / v LAP was gelled in a cylindrical mold and then transferred to a 20 mL glass vial for lyophilization. The lyophilized gels were weighed (W0). Then they were immersed in PBS or 20 mM GSH / PBS solution at 37 °C. The hydrogels were taken out at predetermined timeAttorney Docket No. P23-106-WO-PCT points (day 3 and day 7) and rinsed by deionized water, then freeze-dried, and weighed again (W1). Three samples were measured at each time point. The dry weight remaining was calculated as follow: Dry weight remaining ratio (%) = W1 / W0 x 100%.

[0136] The PAspAm-Cys-MA (2) hydrogel can be hydrolytically degraded at a slow rate. However, in the presence of reductant GSH, its degradation can be accelerated (see Figure 3). In PBS, the hydrogel remained 87.5 ± 7.0 % of its initial mass after 7 days of degradation. In contrast, in GSH solution, the mass remaining of the hydrogel was 35.2 ± 4.8 %. The hydrogel degradation was significantly faster in GSH solution than that in PBS which confirmed the redox-response of the PAspAM- Cys-MA polymer (2) in the presence of the reductant GSH.

[0137] Cell encapsulation and cell viability

[0138] Human bone marrow-derived mesenchymal stem cells (hMSCs; ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Corning) and 8 ^g / mL fibroblast growth factor-basic human (hBFGF, Sigma) in a humidified atmosphere with 5% CO2at 37 °C. PAspAm-Cys-MA polymer (2) together with the photoinitiator LAP were dissolved in PBS and filtered through a 0.2 μm filter, then mixed with the cell suspension. The final concentrations for each component included 10% wt / v of PAspAm-Cys-MA polymer (2), 0.5% wt / v LAP, 0.7 M / mL of hMSCs, respectively. The polymer / cell mixture was loaded to a 96-well plate and a 24-well-plate at 33 μL per well and 190 μL per well, respectively. Then they were cured at 405 nm light (8.8 mW / cm2) for 30 s. The cell viability in the cell- laden PAspAm-Cys-MA (2) hydrogel was quantified by PrestoBlue® cell viability agent (Invitrogen) at 1 and 7 days (n = 4). A Live / Dead cell staining kit (Live, calcein-AM; Dead, propidium iodide; MilliporeSigma) was used to visualize the cell growth in the PAspAm-Cys-MA (2) hydrogel.

[0139] Live / Dead staining and resazurin assay were used to evaluate the cell viability qualitatively and quantitatively in the PAspAm-Cys-MA (2) hydrogel over 7 days of culture (see Figures 4A-C). Live&Dead staining images showed the hMSCs inside the hydrogels were considerably viable and a small portion of cells started toAttorney Docket No. P23-106-WO-PCT elongate at day 7 (see Figure 4A). The cell survival rates of the hMSCs inside the PAspAm-Cys-MA (2) hydrogels were all above 95% from day 1 to day 7 (see Figure 4B). The PrestoBlue® assay showed the cell metabolism in the PAspAm-Cys-MA (2) hydrogel significantly increased in 7 days of culture (see Figure 4C), which suggests that the PAspAm-Cys-MA polymer (2) has no obvious cell toxicity, thus can be potentially used in biomedical applications.

[0140] Example 3: Synthesis of enzyme-responsive hydrogel precursor polymer (3)

[0141] The enzyme-responsive hydrogel precursor polymer (3) is synthesized by incorporating a matrix metalloproteinases (MMPs) cleavable peptide sequence GCRDVPMSMRGGDRCG (SEQ ID NO: 10) into the poly(aspartamide) structure as shown in the following Scheme 3.

[0142] Scheme 3: Synthesis of enzyme-responsive hydrogel precursor polymer (3). Scheme 3 discloses SEQ ID NOS 11 and 10, respectively, in order of appearance.

[0143] Polysuccinimide (1 g) was dissolved in 10 ml of anhydrous dimethylformamide (DMF) under nitrogen. To this solution Į-amino-^-methacrylate GCRDVPMSMRGGDRCG (SEQ ID NO: 10) peptide (500 mg) was added and stirred for 12 h at 40°C. Then, 2-[2-(2-aminoethoxy)ethoxy]ethanol (1 g) was added to the reaction solution and it was stirred for another 12 hours at 40°C. The polymer (3) was isolated by precipitation with a copious amount of diethyl ether followed byAttorney Docket No. P23-106-WO-PCT filtration. It was further purified by dialysis followed by freeze drying.

[0144] Example 4: Synthesis of light-responsive hydrogel precursor polymer (4)

[0145] The light-responsive hydrogel precursor polymer (3) is synthesized by incorporating a photo-cleavable o-nitrobenzyl moiety as shown in the following Scheme 4.

[0146] Scheme 4: Synthesis of light-responsive hydrogel precursor polymer (4)

[0147] The intermediate polymer (4a) was synthesized as described in Example 1 above. Then, in a second step intermediate polymer (4a) was reacted at ambient conditions for 12 h with 4-(3-(methacryloyloxymethyl)-4-nitrophenoxy)-butanoic acidAttorney Docket No. P23-106-WO-PCT chloride (2 g) in THF. The polymer (4) was isolated by precipitation with a copious amount of diethyl ether. It was further purified by dialysis and isolated by freeze drying.

[0148] The examples provided herein are not intended in any way to limit the scope of the invention as set forth in the claims. References

[0149] [1] S. Samanta, D.R. Bogdanowicz, H.H. Lu, J.T. Koberstein, Macromolecules, 2016, 49, 1858-1864.´

[0150] [2] S. Bhattacharya, M.N. Ganivada, H. Dinda, J.D. Sarma, R. Shunmugam, ACS Omega, 2016, 1, 108-117.

[0151] [3] R. Chandrawati, Experimental Biology and Medicine, 2016, 241, 972- 979.

[0152] [4] H. Zhao, E.S. Sterner, E.B. Coughlin, P. Theato, Macromolecules, 2012, 45, 1723-1736.

Claims

Attorney Docket No. P23-106-WO-PCT We claim:

1. A hydrogel precursor polymer, wherein the hydrogel precursor polymer contains a repeating unit U1 and a repeating unit U2:, wherein: R1and R2are independently from each other selected from alkylene, preferably from C1-C20alkylene, more preferably from C1-C10alkylene, most preferably from methylene, ethylene and propylene; R11and R22are independently from each other selected from hydrogen or alkyl, preferably from hydrogen or C1-C20alkyl, more preferably from hydrogen or C1-C10alkyl, most preferably from hydrogen, methyl, ethyl and propyl; A1is a moiety that breaks into smaller fragments under the influence of one or more external stimuli; A2is a moiety imparting hydrophilicity to the polymer; B1is a moiety containing at least one reactive group that can be further reacted to crosslink two or more polymer chains to form a hydrogel; X1is a chemical bond or a divalent linking moiety, wherein preferably X1is selected from a chemical bond, a divalent aliphatic moiety having 1 to 20 C atoms, preferablyAttorney Docket No. P23-106-WO-PCT 1 to 10 C atoms, or a divalent aromatic moiety having 6 to 14 C atoms, preferably 6 to 10 C atoms; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000.

2. The hydrogel precursor polymer according to claim 1, wherein the hydrogel precursor polymer further contains a repeating unit U1’ and U2’:, wherein: m1 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m2 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and R1, R2, R11, R22, A1, A2, B1and X1have the same definition as in claim 1.

3. The hydrogel precursor polymer according to claim 1 or 2, wherein A1is a moiety that breaks into smaller fragments under the influence of light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes.Attorney Docket No. P23-106-WO-PCT 4. The hydrogel precursor polymer according to one or more of claims 1 to 3, wherein A1comprises one or more photolabile groups, wherein preferably said photolabile groups comprise one or more motifs selected or derived from anthracene, arylmethyl, arylcarbonylmethyl, azobenzene, coumarin, diarylethene, pyrimidine, spiropyran, and nitroaryl.

5. The hydrogel precursor polymer according to one or more of claims 1 to 4, wherein A1comprises one or more pH labile groups, wherein preferably said pH labile groups comprise one or more motifs selected or derived from acetal, azomethine, imine, catechol-boronate, phosphoramidate, silyl ether, hydrazone, N- acyl hydrazone, and carbonate.

6. The hydrogel precursor polymer according to one or more of claims 1 to 5, wherein A1comprises one or more reactive oxygen species (ROS) labile groups, wherein preferably said reactive oxygen species (ROS) labile groups comprise one or more motifs selected or derived from thioketal, and oxalate.

7. The hydrogel precursor polymer according to one or more of claims 1 to 6, wherein A1comprises one or more redox labile groups, wherein preferably said redox labile groups comprise one or more motifs selected or derived from disulfide, and trimethyl benzoquinone.

8. The hydrogel precursor polymer according to one or more of claims 1 to 7, wherein A1comprises one or more enzymatically labile groups, wherein preferably said enzymatically labile groups are peptide sequences, wherein more preferably said peptide sequences are selected from matrix metalloproteinase (MMP)-sensitive peptide sequences, collagenase-sensitive peptide sequences, and human neutrophil elastase (HNE) sensitive peptide sequences, wherein most preferably said peptide sequences are peptide sequences including GPQGIWGQ (SEQ ID NO: 1), VPMSMRGG (SEQ ID NO: 2) and / or GCRDVPMSMRGGDRCG (SEQ ID NO: 10).

9. The hydrogel precursor polymer according to one or more of claims 1 to 8, wherein A2comprises one or more groups selected from -OH, -SH, -NH2and -CO2H.Attorney Docket No. P23-106-WO-PCT 10. The hydrogel precursor polymer according to claim 9, wherein A2comprises an alkylene unit -(CH2)x- and / or an ethylene oxide unit -(CH2-CH2-O)y-, wherein x and y are independently from each other integers from 1 to 2,000.

11. The hydrogel precursor polymer according to one or more of claims 1 to 10, wherein the reactive group contained in B1is selected from the list consisting of groups capable to undergo polymerization crosslinking reactions, groups capable to undergo Michael-type or click chemistry-type crosslinking reactions, groups capable to undergo oxidative crosslinking reactions, and groups capable to undergo redox crosslinking reactions.

12. The hydrogel precursor polymer according to one or more of claims 1 to 11, wherein the reactive group contained in B1is selected from groups capable to undergo polymerization crosslinking reactions, wherein preferably said groups capable to undergo polymerization crosslinking reactions contain one or more aliphatic vinyl, aromatic vinyl and / or allyl groups.

13. The hydrogel precursor polymer according to one or more of claims 1 to 12, wherein the reactive group contained in B1is selected from groups capable to undergo Michael-type or click chemistry-type crosslinking reactions, wherein preferably said groups capable to undergo Michael-type or click chemistry-type crosslinking reactions contain one or more acrylate, methacrylate, n-alkyne, cyclo-alkyne, amine, azide, carboxyl, hydrazide, hydroxyl, maleimide, norbornene, tetrazine and / or thiol groups.

14. The hydrogel precursor polymer according to one or more of claims 1 to 13, wherein the reactive group contained in B1is selected from groups capable to undergo oxidative crosslinking reactions, wherein preferably said groups capable to undergo oxidative crosslinking reactions contain one or more aromatic moieties with one or more aromatic hydroxyl groups, wherein more preferably said groups capable to undergo oxidative crosslinking reactions contain one or more mono-phenol, di- phenol, tri-phenol, oligo-phenol and / or poly-phenol moieties, wherein particularly preferably said groups capable to undergo oxidative crosslinking reactions contain a mono-phenol moiety.Attorney Docket No. P23-106-WO-PCT 15. The hydrogel precursor polymer according to one or more of claims 1 to 14, wherein the reactive group contained in B1is selected from groups capable to undergo redox crosslinking reactions, wherein preferably said groups capable to undergo redox crosslinking reactions contain one or more thiol and / or amine groups.

16. The hydrogel precursor polymer according to one or more of claims 1 to 15, wherein the hydrogel precursor polymer further contains (i) a repeating unit U3 and a repeating unit U3’, or (ii) a repeating unit U4 and a repeating unit U4’, or (iii) a repeating unit U3, a repeating unit U3’, a repeating unit U4 and a repeating unit U4’: ,wherein: R3and R4are independently from each other selected from alkylene, preferably from C1-C20alkylene, more preferably from C1-C10alkylene, most preferably from methylene, ethylene and propylene;Attorney Docket No. P23-106-WO-PCT R33and R44are independently from each other selected from hydrogen or alkyl, preferably from hydrogen or C1-C20alkyl, more preferably from hydrogen or C1-C10alkyl, most preferably from hydrogen, methyl, ethyl and propyl; A3is a moiety imparting biological properties to the polymer; and A4is a moiety imparting shear thinning properties to the polymer; wherein in case (i): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and the sum of n1, n2, n3, m1, m2 and m3 is greater than 5, preferably greater than 50, more preferably greater than 500; or wherein in case (ii): n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and the sum of n1, n2, n4, m1, m2, and m4 is greater than 5, preferably greater than 50, more preferably greater than 500, or wherein in case (iii): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000;Attorney Docket No. P23-106-WO-PCT n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, more preferably from 100 to 5,000; and where in case (iii) the sum of n1, n2, n3, n4, m1, m2, m3 and m4 is greater than 7, preferably greater than 50, more preferably greater than 500.

17. The hydrogel precursor polymer according to claim 16, wherein A3contains one or more synthetic functional peptide sequences found in native proteins, wherein preferably said native proteins are selected from fibronectin, laminin, collagen, elastin, albumin, immunoglobulin or silk.

18. The hydrogel precursor polymer according to claim 16 or 17, wherein A3contains one or more synthetic functional peptide sequences selected from the list consisting of RGD, YIGSR (SEQ ID NO: 3), IKVAV (SEQ ID NO: 4), DGEA (SEQ ID NO: 5), PHSRN (SEQ ID NO: 6), PRARI (SEQ ID NO: 7), (GAGAGS)p(SEQ ID NO: 8), (VPGXG)n (SEQ ID NO: 9), and (GPO)m, wherein p, n and m are independently from each other integers of ^ 1.

19. The hydrogel precursor polymer according to one or more of claims 16 to 18, wherein A4is a moiety imparting shear thinning properties due to supramolecular interactions.

20. The hydrogel precursor polymer according to claim 19, wherein the supramolecular interactions are host-guest or self-assembly interactions, wherein such interactions preferably include interactions between cyclodextrin, adamantane, cucurbit[8]uril, cholesterol, polyethylene glycol and ureidopyrimidinone.

21. The hydrogel precursor polymer according to claim 19 or 20, wherein A4contains one or more cyclodextrin, adamantane, cucurbit[8]uril, cholesterol, polyethylene glycol or ureidopyrimidinone moieties.

22. A formulation for forming a hydrogel, wherein said formulation comprises a hydrogel precursor polymer according to one or more of claims 1 to 21.Attorney Docket No. P23-106-WO-PCT 23. The formulation for forming a hydrogel according to claim 22, wherein the mass concentration of the hydrogel precursor polymer is from 0.1% (wt / v) to 99% (wt / v), preferably from 1% (wt / v) to 70% (wt / v), more preferably from 2% (wt / v) to 40% (wt / v), particularly preferably from 5% (wt / v) to 30% (wt / v), and most preferably from 5% (wt / v) to 20% (wt / v), based on the total volume of the formulation.

24. The formulation for forming a hydrogel according to claim 22 or 23, further comprising one or more selected from crosslinking reagents and crosslinking additives.

25. The formulation for forming a hydrogel according to claim 24, wherein the crosslinking reagents are selected from the list consisting of photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes mediating oxidative crosslinking, and agents mediating redox crosslinking.

26. The formulation for forming a hydrogel according to claim 24 or 25 , wherein the crosslinking additives are molecules comprising two or more polymerizable groups with at least one C=C double bond, wherein preferably said polymerizable groups with at least one C=C double bond are selected from the list consisting of acrylate, methacrylate, allyl, styryl and vinyl groups.

27. A process for forming a hydrogel, wherein the formulation for forming a hydrogel according to one or more of claims 22 to 26 is subjected to irradiation and / or thermal treatment to thereby crosslink and form a hydrogel.

28. A hydrogel obtainable by the process according to claim 27.

29. The hydrogel according to claim 28, wherein said hydrogel is for use in tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics, and / or other biomedical applications.

30. A process for forming artificial tissue, wherein said process comprises the following steps:Attorney Docket No. P23-106-WO-PCT (a) contacting a hydrogel according to claim 28 with cells capable of forming artificial tissue; (b) proliferating said cells; and (c) cleaving said hydrogel by exposure to one or more external stimuli.

31. The process according to claim 30, wherein the external stimuli are selected from light, temperature, electric field, magnetic field, ultrasound waves, pH, ionic concentration, reactive oxygen species (ROS), redox active chemicals and / or enzymes.

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