Poly(isocyanopeptide) cryogels

Abstract

Disclosed are acrylic functionalized poly (isocyanop ep tides). These poly(isocyanopeptides) can be subjected to crosslinking. This can be accomplished in an aqueous solution, which can be subjected to cryopolymerization using a suitable crosslinker. Thus cryogels having interesting properties are obtained. Particularly, these cryogels allow tuning mechanical properties without thereby necessarily affecting cryogel architecture, such as pore size and pore size distribution.

Description

[0001]P137009PC00 Title: POLY(ISOCYANOPEPTIDE) CRYOGELS Field of the Invention The invention pertains to cryogels suitable for bio-medical applications such as in the fields of tissue engineering and in vitro cell culture. Particularly, the invention pertains to crosslinkable poly(isocyanopeptides). Background of the invention Over the years, synthetic hydrogels have proven remarkably useful as cell culture matrix to elucidate the role of the extracellular matrix (ECM) on cell behavior. Yet, their lack of interconnected macropores undermines the widespread use of hydrogels in biomedical applications. To overcome this limitation, cryogels, a class of macroporous hydrogels, are rapidly emerging. A challenge in this field is that adjusting the mechanical properties, such as stiffness, of such gels, generally also affects the gel architecture, notably pore size. It will be understood that both stiffness and pore size can be key parameters affecting the suitability of a gel for a desired bio-medical applications. I.e., it would be desired to present a suitable cryogel-forming material that allows decoupling these properties from each other. Background art in the field of hydrogels includes poly(isocyanopeptides), generally abbreviated as PIC. Poly(isocyanopeptides) comprise an all-carbon backbone provided with peptide (typically dipeptide) side groups. By virtue of hydrogen-bonding between peptide groups, poly(isocyanopeptide) polymer chains assume a helical structure. The resulting helical polymers form thermoreversible gels in aqueous solutions; at low temperatures, the polymer dissolves and upon heating, they bundle together to form a gel with a fibrous architecture that is similar to biogels like collagen and fibrin. PIC hydrogels exhibit mechanical properties that closely mimic the native cell environment. Furthermore, by providing the PIC gels with side chains having azide end-groups, they can be readily functionalized with biochemical cues through bio-orthogonal click chemistry via such azide groups, typically using trans-cyclooctene (e.g. DBCO)-modified biomolecules. A background disclosure on such azide-modified poly(isocyanopeptides) is EP 2454302. Herein oligo-ethylene glycol substituted poly(isocyanopeptides) are disclosed, that may be obtained by co-polymerization with azide functional monomers, thus resulting in PIC polymer chains having part of its side-chains carrying azide groups. Although the PIC hydrogels have excellent properties, improvement is desired. Particularly, it is desired to provide microporous gels that can be produced in such a way as to decouple mechanical properties from gel architecture. Summary of the Invention In order to better address the foregoing desires, the invention presents, in one aspect, a poly(isocyanopeptide) polymer comprising side chains provided with an oligo (alkylene glycol) group positioned between an oligopeptide moiety and a terminal functional group, wherein the terminal functional groups in the polymer comprise azide groups and acrylic groups, and preferably also alkoxy groups. In another aspect, the invention provides a poly(isocyanopeptide) obtainable by subjecting isocyanide comonomers to Ni(II) catalyzed random copolymerization, wherein the isocyanide monomers comprise, and preferably are consisting of: (a) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an azide terminal functional group; (b) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an acrylic terminal group; (c) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an alkoxy terminal group, preferably C1 to C3alkoxy, more preferably methoxy. In a further aspect, the invention provides a process for the preparation of poly(isocyanopeptide), the process comprising subjecting isocyanide comonomers to Ni(II) catalyzed random copolymerization, wherein the isocyanide monomers comprise: (a) isocyanide substituted with an oligo(alkylene glycol) functionalized peptide side chain having an azide terminal functional group; (b) isocyanide substituted with an oligo(alkylene glycol) functionalized peptide side chain having an acrylic terminal group; (c) isocyanide substituted with an oligo(alkylene glycol) functionalized peptide side chain having an alkoxy terminal group, preferably methoxy. In a still further aspect, the invention presents a poly(isocyanopeptide) obtainable by the aforementioned process. In yet another aspect, the invention resides in a cryogel mixture comprising an aqueous solution of a poly(isocyanopeptide) according to the present invention, a crosslinking agent for crosslinking acrylic functional group, and an acrylic crosslinking catalyst, in a process for preparing a cryogel by subjecting such cryogel to cooling to a temperature below 0°C, preferably in a range of from -80°C to -10°C, and a cryogel obtainable by such process. Brief description of the drawings Fig.1 presents a reaction scheme for the preparation of exemplified acrylate-functionalized monomers according to the invention. Fig.2 presents a reaction scheme for an exemplified poly(isocyanopeptide) of the invention. Fig.3 presents graphs displaying the pore size and pore size distribution, as well as the swelling ratio, of two embodiments of cryogels of the invention. Fig.4 presents graphs displaying the pore size and pore size distribution of cryogels of the invention made with three different polymer concentrations, as well mechanical properties (Young’s moduli and swelling ratios) of these cryogels. Fig.5 presents graphs displaying the pore size and pore size distribution of three cryogels of the invention made with polymers of different lengths, and mechanical properties (Young’s moduli and swelling ratios) of these cryogels. Detailed description of the Invention The invention is based on the judicious insight to adapt poly(isocyanopeptides), notably oligo(ethylene glycol) modified poly(isocyanopeptides), in such a way as to make them suitable for cryogel formation. To this end, the poly(isocyanopeptides) of the invention are obtained from isocyanopeptide co-monomers that include acrylate- functionalized co-monomers. Polyisocyanides, also known as polyiminomethylenes, are prepared by the polymerization of isocyanides, according to reaction equation (i): (i). The driving force for this polymerization reaction is the transformation of a formally divalent carbon atom in the monomer, to a tetravalent carbon atom in the polymer. Essentially, polyisocyanides thus comprise an all-carbon backbone, whereby each carbon atom carries a substituent. In this disclosure, these substituents are referred to with the term “side-chain.” In poly(isocyanopeptides), the side chains all carry a peptide moiety, generally a peptide comprising 2 to 4 amino acids, preferably 2-3 aminoacids, i.e., a dipeptide or a tripeptide motif. Preferred amino acids are Alanine, Arginine, Asparagines, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Thryptophan, Tyrosine, and Valine. Alanine is a preferred amino acid, and dialanine or trialanine are preferred peptide moieties to be included in poly(isocyanopeptide) side chains. In order to provide alanine-containing side chains, it is generally preferred to start from N-protected alanine. Typically, this involves (L)- and (D)-N-Boc protected alanine moieties, Boc referring to the well-known t- butyloxycarbonyl protecting group. After the introduction of the desired dialanine motifs, the Boc-protecting end-group is cleaved off. The poly(isocyanopeptides) of the present invention are oligo(alkylene glycol) functionalized. This means that all of the side chains have glycol ether groups attached to the peptide moieties. Reference is made to EP2454302 for a disclosure on these materials. The poly(isocyanopeptides) of the present invention comprise three types of side chains: (A)oligo(alkylene glycol) functionalized peptide side chains having an azide terminal functional group; these groups allow the poly(isocyanopeptide) to be coupled, employing bio-orthogonal click chemistry, to any desired biomolecule; (B)oligo(alkylene glycol) functionalized peptide side chains having an acrylic terminal group; these groups provide the poly(isocyanopeptide) with a functionality allowing the formation of cryogels to take place, via crosslinking of acrylate groups; (C)oligo(alkylene glycol) functionalized peptide side chains having oligo(alkylene glycol) functionalized peptide side chains with a terminal alkoxy group, i.e., the non-functional end-group of an oligo-ether; these groups, preferably C1to C3alkoxy, more preferably methoxy, are inert for the coupling to biomolecules, as well as for the cryogel-forming reaction, and serve to provide the desired space, the size of which depends on the size of biomolecules to be coupled and / or the pore size to be achieved by acrylate crosslinking. Said types of side-chains are introduced as comonomers in the poly(isocyanide) polymerization. These comonomers thus comprise, and preferably consist of: (a) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an azide terminal functional group; (b) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an acrylic terminal group; (c) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an alkoxy terminal group. The ratio between such comonomers can be varied, generally 0 to 5 mole % of (a), 1 to 10 mole % of (b), and 85 to 99 mole % of (c), preferably 1-3 mole % of (a), 2-5 mole % of (b), and 92-97 mole % of (c). In order to enable the preparation of cryogels, it is required that the poly(isocyanopeptides) are soluble in water. This puts a limit on the fraction of monomers having acrylate groups. Preferably at most 5 mole% of such monomers is applied, more preferably at most 2 mole %. The monomers preferably comprise a di-, tri-, tetra- or more peptidic motif substituted at the C terminal with the desired oligo(alkylene glycol) chains. The chains may be linear, branched, dendronized oligo(alkylene oxide) based. Preferably the chain is linear and composed of ethylene glycol. The peptidic segment can be of different compositions determined by the sequence of natural or non-natural and expended amino- acids or mixture thereof. Preferably the isocyanopeptides are functionalized with at least 3 ethylene glycol units to lead to water soluble materials after polymerization. According to the number of ethylene glycol units and to the terminal substituents of the (alkylene glycol) side chains the general physical properties of the resulting materials can be systematically varied. Examples of suitable alkylene glycols are ethylene-, propylene-, butylene- or pentylene glycol. Preferably the alkylene glycol is ethylene glycol. These chains are preferably terminated with only one free hydroxy or free amine end group to permit the direct coupling to a desired amino acid with an appropriate coupling strategy. Example of coupling protocols for alcohols derivatives are disclosed in EP2454302. Examples of amino acids that advantageously may be used in the method as described herein are N-protected Alanine, Arginine, Asparagines, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Thryptophan, Tyrosine, Valine. After coupling the first amino-acid residue to the (alkylene glycol) chain, a sequential peptidic coupling strategy can be used to introduce the desired number of amino-acids substituents with the desired sequence. The acrylate-functionalized monomers (b) can be prepared by initially coupling the desired oligo (alkylene glycol) ether by means of such ether having a protected end-group, e.g., tetraethylene glycol monobenzyl ether instead of tetraethylene glycol. This is depicted in the reaction scheme of Fig 1, wherein “Bn” stands for benzyl. Acrylic groups refer to the residue of an acrylic carboxylic compound, typically acrylic acid, an acrylic ester, acrylic anhydride, or acrylic acid chloride. The term “acrylic” as used herein encompasses methacrylic groups, which refers to the residue of a methacrylic carboxylic compound, typically methacrylic acid, a methacrylic ester, methacrylic anhydride, or methacrylic acid chloride. Typical acrylic groups are the residues of methyl(meth)acrylate, ethyl(meth)acrylate, butyl(meth)acrylate, 2-ethylhexyl(meth)acrylate, (meth)acrylic anhydride, glycidyl(meth)acrylate. It will be understood that, all of the acrylic groups can be the same, or different. Methacrylate groups are preferred. In preparing the poly(isocyanopeptides) of the invention, a mixture of the comonomers, (A), (B), and (C) is subjected to random copolymerization using a suitable catalyst, preferably a Ni2+catalyst. Suitable catalysts may be selected from the group consisting of Nickel chloride; nickel perchlorate, nickel tetra tertiary butyl isocyanide and others with which the skilled person is familiar. The length of the polymer chain can be tuned by means of the catalyst to monomer ratio. Generally, a higher monomer ratio will result in a longer chain. Non-limiting examples are ratio’s (catalyst : monomer): 1:1000 for P1, 1:3000 for P2 and 1:10000 for P3, which yielded polymers of average lengths of 209, 346 and 562 nm, respectively. The acrylic functionalized poly(isocyanides) of the invention can be turned into cryogels via a cryopolymerization process, i.e., generally conducted at a temperature below 0°C, preferably in a range of from -80°C to -10°C, more preferably -30°C to -10°C, using free-radical polymerization. In cryopolymerization, a hydrogel precursor solution is cooled to sub-zero temperatures, causing a large part of the solvent to crystallize, which forces gel forming components such as monomers and polymers to concentrate in the non-frozen microphase, where chemical crosslinking takes place. Thus, effectively, a higher concentration of reactants results. This so-called cryo-concentration of these constituents accelerates the formation of a macroporous gel network. Subsequent thawing of the resulting ice-crystals results in the formation of a highly interconnected macroporous hydrogel network. Since the ice crystals function as porogens, there is no need to remove potentially harmful micron-sized templates. Generally, this will involve crosslinking agents suitable for crosslinking acrylic functionalized monomers. In a typical experiment, not limiting to the invention, an aqueous reaction mixture containing methacrylate-functionalized polymers and crosslinking agents (ammonium persulfate, APS, and tetramethylethylenediamine, TEMED) was cooled to the polymerization temperature (e.g. –20 °C) and the crosslinking reactions was allowed to take place overnight. Then, the ice crystals were thawed and the cryogel was thoroughly washed with water to remove unreacted residual ingredients. Suitable crosslinking agents are known to the skilled person. E.g., an alternative to APS is potassium persulfate, which is preferably applied in conjunction with TEMED, riboflavin or riboflavin phosphate. Reference is further made to Elizabeth A Pumford et al., ACS Appl Bio Mater, 2024 Mar 14 “Nontoxic Initiator Alternatives to TEMED for Redox Hydrogel Polymerization.” In a preferred embodiment, an acrylic chain extender is added to the cryopolymerization reaction mixture. This typically is a monofunctional acrylate, such as a monohydroxy acrylate or methacrylate, preferably hydroxyethyl methacrylate (HEMA). This was found to generally reduce the pore size, which can be desirable, and in a narrower pore size distribution, which is generally advantageous. An advantage of the judicious choice of cryopolymerization of poly(isocyanides) is that several parameters are available to control the eventual structure of the cryogel obtained. The skilled person will be able, also when apprised of the non-limiting examples in this disclosure, to finetune the poly(isocyanides) and the cryopolymerization process as desired. The main parameters that the skilled person will be able to take into account are: -the length of the polyisocyanides backbone; generally this willvary from 100 to 600 monomeric units, preferably 250-500 monomeric units; -the ratio of the aforementioned types of monomers (a), (b), and(c); -the optional presence of a chain extender such as HEMA and, ifso, the amount thereof; -the polymer concentration in the mixture from which a cryogelis made; generally [2.0-11.0 mg / mL] preferably [3.5-9.5 mg / mL] -the amount of crosslinking agent; such as for APS generally (0.01-0.05 mM, preferably 0.015-0.020 mM; such as for TEMED generally 0.005-0.025 mM, preferably 0.007-0.010 mM); -the cryopolymerization temperature;- the freezing rate;By virtue of swelling ratio experiments, the inventors observed a trend that is opposite to what is previously reported. While for most cryogels, the addition of an acrylate comonomer decreases the swelling ratio, we observe, see Fig.3(c), that cryogels containing HEMA exhibit much higher swelling ratio (6149%) than cryogels without HEMA (1517%). The degree of crosslinking within a cryogel network can affect the density of the polymer walls in the cryogel, which in turn can affect the ability of a cryogel to absorb water. Usually, higher swelling ratios are associated with cryogels with lower degree of crosslinking and lower density of polymer walls. However, the observed increase in swelling ratio for PIC cryogels with HEMA could not be attributed to the cryogels’ polymer wall thickness. Instead, without wishing to be bound by theory, the inventors attribute the increased swelling ratio to the introduction of hydrophobic groups in the cryogel network by addition of HEMA. Advantageously, the addition of HEMA gave rise to cryogels with a homogenous interconnected macroporous structure and excellent swelling behavior. Cryogelation temperature influences cryogel architecture and stiffness The cryogelation process is characterized by a balance between the crosslinking rate and the rate of ice crystal formation; to obtain a macroporous structure, the crosslinking rate should be slower than the rate of crystallization. Furthermore, too fast crosslinking can lead to formation of heterogeneous cryogel networks. Consequently, the temperature at which cryopolymerization takes place influences the nucleation and crystallization rate as well as the crosslinking rate, which means that the cryogelation temperature has a substantial impact on cryogel features such as pore size, structural homogeneity and polymer wall thickness. The inventors found that compositional changes (i.e., the PIC molecular weight and the PIC concentration) primarily affect the mechanical properties and not the pore size, which the inventors believe to be unique for PIC-based cryogels. Accordingly, PIC cryogels are a new and highly tailorable class of cryogels that can be used in biomedical applications where influence of pore size and matrix stiffness is of high importance. Thus the poly(isocyanopeptide) cryogels of the invention can be broadly applied inter alia, in in vitro or ex vivo systems for cell culturing. In sum, disclosed herein are acrylic functionalized poly(isocyanopeptides). These poly(isocyanopeptides) can be subjected to crosslinking. This can be accomplished in an aqueous solution, which can be subjected to cryopolymerization using a suitable crosslinker. Thus cryogels having interesting properties are obtained. Particularly, these cryogels allow tuning mechanical properties without thereby necessarily affecting cryogel architecture, such as pore size and pore size distribution. The invention will be illustrated with reference to the following non-limiting examples. Example 1 Synthesis methacrylate functionalized isocyanopeptide monomer. The synthesis route was started with tetraethylene glycol monobenzyl ether, which was deprotected and functionalized with a methacrylate group. The divergent steps in the synthesis route are described below. An overview of the full synthesis route is depicted in scheme (ii) referred to above. Synthesis of tetraethylene glycol monobenzyl ether (1a). Tetra ethylene glycol (TEG) (17.26 mL, 100 mmol) was added dropwise to a cooled (0oC ) solution of NaH (1.03 g 60% in mineral oil, 25.8 mmol) in tetrahydrofuran (75 mL) under Schlenk conditions and stirred for 45 min. A solution of BnBr (4.490 g, 25.8 mmol) in THF (125 mL) was added and the resulting mixture was warmed to r.t. The reaction mixture was stirred for 72h at r.t. and subsequently concentrated in vacuo. The crude product was dissolved in EtOAc (100 mL) and washed with water (3x50 mL). The combined aqueous layers were washed with EtOAc (3x50 mL). Combined organic layers were dried with NaSO4 and concentrated in vacuo. The product was isolated via column chromatography (SiO2 , EtOAc) 1 as a yellow oil (5.16 g, 18.2 mmol, 70.3%) TLC (EtOAc): Rf = 0.46,1H NMR (400 MHz, CDCl3) δ 7.32 – 3.47 (m, 16H, OCH2), 2.80 (s, 1H, -OH).13C NMR (101 MHz, CDCl3) δ 138.06 (Ar-CCH2), 128.17 (Ar-CH), 127.56 (Ar-CH), 127.39 (Ar-CH), 73.02 (Ar-CH2), 72.37 (OCH2), 70.44 (OCH2), 70.41 (OCH2), 70.38 (OCH2), 70.14 (OCH2), 69.25 (OCH2), 61.47 (OCH2). Synthesis of 1-phenyl-2,5,8,11-tetraoxatridecan-13-yl formyl- D-alanyl-L-alaninate (2). The following steps towards synthesis of the methacrylate monomer are previously described in literature. In short, a Boc- protected L-alanine ester was formed through reaction of 1 with Boc-L- Ala using 4-dimethylaminopyridine (DMAP) and N,N'-dicyclohexylcarbodi- imide (DCC) as coupling reagents. After Boc-deprotection using HCl (4M in Dioxane), a similar condensation reaction with Boc-D-Alanine was performed using DMAP, DCC and N-hydroxybenzotriazole (HOBt). Subsequent deprotection with HCl (4M in dioxane) afforded the free amine, which was formylated with formate to afford 2 as a yellow oil (626 mg, 1.38 mmol, 26% over 5 steps).1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H, HC=ONH), 7.35 – 7.24 (m, 5H, Ar-H), 6.85 (d, J = 7.8 Hz, 1H, NH), 6.66 (d, J = 7.9 Hz, 1H, NH), 4.64 – 4.51 (m, 4H, Ar-CH2, 2 x CHCH3), 4.36 – 4.18 (m, 2H, OCH2), 3.72 – 3.61 (m, 14H, OCH2), 1.40 (ddd, J = 16.3, 7.1, 0.9 Hz, 6H, 2 x CH3).13C NMR (101 MHz, CDCl3) δ 172.43 (C=OCHCH3), 171.32 (C=OCHCH3), 161.33 (HC=ONH), 138.12 (Ar-CCH2), 128.37 (Ar-CH), 127.75 (Ar-CH), 127.65 (Ar-CH), 73.23 (Ar-CH2), 70.63 (OCH2), 70.55 (OCH2), 70.53 (OCH2), 70.51 (OCH2), 70.48 (OCH2), 69.39 (OCH2), 68.94 (OCH2), 64.41 (OCH2), 48.35 (CHCH3), 47.12 (CHCH3), 17.90 (CH3), 17.56 (CH3). Synthesis of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl formyl-D-alanyl-L-alaninate (3). Palladium catalyst (60.6 mg, 10% Pd / C) was added to a solution of 2 (600 mg, 1.32 mmol) in ethanol (25.0 mL). The resulting mixture was stirred under H2flow at rt for 18h. The mixture was filtered over celite, washed with ethanol and concentrated in vacuo to yield 3 as a yellow oil (450 mg, 1.23 mmol). TLC (MeOH: DCM, 1:20, v / v): Rf = 0.23.1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H, HC=ONH), 7.14 (d, J = 7.8 Hz, 1H, NH), 6.79 (s, 1H, NH), 4.69 – 4.54 (m, 2H, 2 x CHCH3), 4.29 (ddd, J = 5.9, 3.4, 1.3 Hz, 2H, OCH2), 3.77 – 3.59 (m, 14H, OCH2), 1.42 (dd, J = 11.5, 7.1 Hz, 6H, 2 x CH3).13C-NMR (101 MHz, CDCl3): δ 172.35 (C=OCHCH3), 171.49 (C=OCHCH3), 161.25 (HC=ONH), 72.49 (OCH2), 70.45 (OCH2), 70.44 (OCH2), 70.42 (OCH2), 70.10 (OCH2), 69.00 (OCH2), 64.25 (OCH2), 61.57 (OCH2), 48.32 (CHCH3), 47.16 (CHCH3), 18.04 (CH3), 17.92 (CH3). Synthesis of (3R,6S)-3,6-dimethyl-1,4,7-trioxo-8,11,14,17- tetraoxa-2,5-diazanonadecan-19-yl methacrylate (4). To a solution of 3 (260 mg, 0.71 mmol) in dry dichloromethane (DCM, 18.0 mL) sodium methacrylate (386 mg, 3.60 mmol), Di-isopropylethylamine (DIPEA) (184 mg, 1.40 mmol) and DMAP (43.6 mg, 0.360 mmol) were added and the resulting mixture was cooled to 0 °C. A solution of N-(3- Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC-HCl, 684 mg, 3.60 mmol) in DCM (12.0 mL) was added dropwise to the mixture and allowed to warm to rt. After stirring for 18h, the reaction mixture was washed with an acidic brine solution (1 M HCl, 3 x 50 mL) and sat. aq. NaHCO3 (3 x 50 mL). The combined organic layers were dried with Na2SO4, filtered and concentrated in vacuo to afford 4 as a yellow oil (174 mg, 0.40 mmol). TLC: (MeOH: DCM, 1:10 v / v): Rf = 0.22.1H-NMR (400 Hz, CDCl3): δ 8.18 (s, 1H, HC=ONH), 6.93 (d, J = 7.7 Hz, 1H, NH), 6.70 (d, J = 7.8 Hz, 1H, NH), 6.13 (s, 1H, C=CH2), 5.61 – 5.54 (m, 1H, C=CH2), 4.68 – 4.51 (m, 2H, 2 x CHCH3), 4.36 – 4.20 (m, 4H, OCH2), 3.74 (ddd, J = 5.8, 3.7, 0.9 Hz, 2H, OCH2), 3.66 (dddt, J = 10.2, 6.4, 2.4, 1.4 Hz, 10H, OCH2), 1.98 – 1.92 (m, 3H, CCH2CH3), 1.41 (ddd, J = 11.6, 7.1, 0.9 Hz, 6H, 2 x CH3).13C NMR (101 MHz, CDCl3): δ 172.46 (C=OCHCH3), 171.36 (C=OCHCH3), 167.33 (C=OC=CH2), 161.20 (HC=ONH), 136.09 (C=CH2), 125.77 (C=CH2), 70.61 (OCH2), 70.52 (OCH2), 70.49 (OCH2), 70.47 (OCH2), 69.08 (OCH2), 68.92 (OCH2), 64.37 (OCH2), 63.75 (OCH2), 48.29 (CHCH3), 47.13 (CHCH3), 18.27 (C=CH2CH3), 17.89 (CH3), 17.75 (CH3). Synthesis of (14S,17R)-17-isocyano-14-methyl-13,16-dioxo- 3,6,9,12-tetraoxa-15-azaoctadecyl methacrylate (5). A solution of Burgess reagent (121 mg, 0.510 mmol) in dry DCM (15.0 mL) was added dropwise to a solution of 4 (169 mg, 0.390 mmol) in dry DCM (20.0 mL) under nitrogen flow. The resulting reaction mixture was stirred at rt for 6h and concentrated in The crude oil was purified using column chromatography (SiO2, 1 ^ 20% MeOH in DCM, v / v) to afford 4 as a yellow oil (95 mg, 0.23 mmol). TLC (MeOH: DCM, 1:10, v / v): Rf = 0.46.1H-NMR (400 Hz, CDCl3): δ 6.98 (d, J = 7.5 Hz, 1H, NH), 6.13 (dt, J = 2.0, 1.0 Hz, 1H, C=CH2), 5.58 (p, J = 1.6 Hz, 1H, C=CH2), 4.59 (p, J = 7.2 Hz, 1H, CHCH3), 4.38 – 4.22 (m, 5H, CHCH3, 2 x OCH2), 3.78 – 3.70 (m, 4H, OCH2), 3.69 – 3.61 (m, 8H, OCH2), 1.95 (dd, J = 1.6, 1.0 Hz, 3H, CCH2CH3), 1.65 (d, J = 3.1 Hz, 3H, CH3), 1.48 (d, J = 7.2 Hz, 3H, CH3).13C NMR (101 MHz, CDCl3): δ 171.98 (C=OCHCH3), 167.35 (C=OC=CH2), 165.71 (C=OCHCH3), 136.16 (C=CH2), 125.73 (C=CH2), 70.63 (OCH2), 69.14 (OCH2), 68.82 (OCH2), 64.69 (OCH2), 63.82 (OCH2), 53.40 (CHCH3), 48.56 (CHCH3), 19.66 (C=CH2CH3), 18.30 (CH3), 18.03 (CH3). MS (ESI) m / z: found 437.10 (M + Na+), calcd 437.20 Example 2 Synthesis of methacrylate functionalized PIC. In short, PICs comprising azide-, methacrylate-, and methoxy groups were synthesized. To this end, the azide-terminated isocyanide monomers were prepared as described in literature (Mandal, S. et al.; Chem.Sci. 2013, 4, 4168-4174).Commercially available methoxy-terminated monomers (Chiralix) were purified via column chromatography (SiO2, MeOH:DCM, 5:95, v / v) before use. Stock solutions of azide-terminated (1.29 mL, 10.3 mM), methacrylate-terminated (0.693 mL, 40.1 mM) and methoxy terminated monomer (7.35 mL, 189 mM) in dry toluene, MBraun SPS 800 Solvent system) were combined. Dry toluene was added to reach a final concentration of 55 mg monomers per mL. To obtain polymers with various lengths, three polymerization reactions were carried out with catalyst to monomer ratios of 1:1000 (P1), 1:3000 (P2) and 1:10000 (P3), respectively. A solution of Ni(ClO4)2· 6 H2O (1:9, EtOH : toluene, v / v) was added to the reaction mixtures (584 µL, 2.45 mM for P1, 167 µL, 2.84 mM for P2 and 130 µL, 1.10 mM for P3). The resulting mixtures were reacted for 18h at rt. Isocyanide consumption was confirmed by the disappearance of the characteristic FT-IR peak at 2140 cm-1. The polymers were precipitated three times in cold (0 °C) diisopropylether and dried overnight to yield P1 as an off-white solid (1.35 g, 87 %), P2 as an off-white solid (1.16 g, 78 %) and P3 as an off-white solid (404 mg, 77 %). Average polymer lengths were determined using atomic force microscopy (AFM, Nanoscope IV Bruker, NSG-10 tapping mode tips, NT-MDT) and were found to be 210 ± 138 nm (P1), 346 ± 231 nm (P2) and 562 ± 390 nm (P3) (Mean ± SD) The characteristic helical backbone of the P1, P2 and P3 was confirmed by Circular Dichroism spectroscopy of polymer solutions in Milli-Q (0.2 mg / mL). Example 3 Preparation of PIC cryogels. Unless specified otherwise, cryogels were made via the following protocol. Stock solutions of PIC (P1, 833 μL, 11.0 mg / ml), 2-hydroxylethyl methacrylate (HEMA, Sigma-Aldrich) (97 μL, 7.33 mg / ml) and N,N,N′,N′- Tetramethyl ethylenediamine (TEMED, Sigma-Aldrich) (20.0 μL, 58.0 mg / ml) in Milli-Q were combined and cooled on ice (0 °C) for 1h prior to use. A cooled (0 °C) solution of Ammonium persulfate (APS, Sigma-Aldrich) (50 µL, 100 mg / mL in Milli-Q) was added, the reaction mixture was mixed by gently pipetting up and down and transferred to a custom-made Teflon mold (200 μL per well) that was cooled on ice (0 °C) for 1h before use. After filling of each well, the mold was placed in a cryostat (-20 °C, SLEE Medical, MEV) for 18h. The mold was then removed from the cryostat and Milli-Q was added to thaw the cryogels. The cryogels were washed with Milli-Q and stored in the fridge (5 °C) until further use. For synthesis of cryogels with varying polymer concentrations, stock solutions of P1 (4.50, 8.00 and 11.0 mg / mL) in Milli-Q were used, and the amount of HEMA added was adjusted accordingly by using HEMA stock solutions of 3.00, 5.33 and 7.33 mg / mL, respectively. TEMED and APS were added following the general protocol. For synthesis of cryogels consisting of polymers with different lengths, stock solutions of P1, P2 or P3 (all 4.5 mg / mL) in Milli-Q were made, and the amount of HEMA added was adjusted accordingly (3.00 mg / mL). TEMED and APS were added following the general protocol. Cryogel pore size analysis. Cryogels were labeled with AzDye 647 through addition of dibenzocyclooctyne (DBCO)-functionalized AzDye 647 (Click Chemistry Tools) as follows: cryogels were dehydrated using a medical gauze (kliniray® gauze compress x-ray) and submerged in a solution of AzDye 647 DBCO (0.25 eq r.t. azides in cryogel, 0.250 mL, 0.051 mM) in Milli-Q. After incubation for 1h at rt, gels were washed three times with 0.05% PBS Tween, three times with PBS and submerged in PBS. Confocal Microscopy was performed on hydrated cryogels using a Leica SP8x AOBS-WLL microscope. Per cryogel, 3 z-stacks (30 slices) were recorded. Pore sizes were determined from z-stack analysis in Fiji. Z-stacks were segmented using the Trainable Weka Segmentation plugin, followed by determination of the pore sizes using the BoneJ plugin. Determination of mechanical properties of the cryogels. Hydrated cryogels were subjected to uniaxial compression tests on a Discovery HR-2 (TA Instruments), using a 20 mm steel Peltier plate at 20 °C. Cryogels were compressed at constant linear rate of 10 μm / s. The axial force and displacement data were used to obtain stress-strain curves. The compressive stress (σ) was calculated from the recorded axial force (F) per cross sectional area (A) of the un-deformed sample. The strain (ε) was determined by calculating the ratio between the deformed (dl) and initial (l) lengths. The Young’s modulus (also known as compression modulus, E) was calculated from the slope of the stress-strain curves at 80% strain via equation (1): ^^^^⁄ ^^^^ == ^^^ ⁄(1) ^^^ ^^Determination of cryogel swelling ratio. To analyse the swelling behaviour of the prepared cryogels, lyophilized cryogels (mdry) were weighed before immersion in Milli-Q. After 5 min incubation to allow the cryogels to swell, excess water was removed and the cryogel weight (mwet) was (2). ^^^^^^^^Preparation and analysis of PIC cryogels. After successful synthesis of methacrylate polymers, we set out to develop PIC based cryogels via a cryopolymerization process using free-radical polymerization. In a typical experiment, an aqueous reaction mixture containing methacrylate-functionalized polymers and crosslinking agents (ammonium persulfate, APS, and tetramethylethylenediamine, TEMED) was cooled to the polymerization temperature (e.g. –20 °C) and the crosslinking reactions was allowed to take place overnight. Then, the ice crystals were thawed and the cryogel was thoroughly washed with water to remove unreacted residual ingredients. The mechanical properties of the cryogels were studied in compression mode, where a freshly fully hydrated gel was subjected to uniaxial compression up to 90% (without breaking), which resulted stress-strain curves that demonstrates the elastic and ductile nature of the cryogel. From the stress-strain curves, we determine the Young’s modulus E′ at a fixed strain (ε = 80%). PIC cryogels can undergo multiple rounds of compression without losing their mechanical properties. Because of its interconnected and macroporous structure, the PIC cryogel is sponge-like and exhibits shape memory behavior. After lyophilization, that shrinks the gel, the cryogel regains its original shape when rehydrated. The architecture of the cryogels was studied by determining the cryogel swelling ratio, which is a measure for porosity, and by confocal fluorescence microscopy after labeling the cryogels with a fluorescent dye. From the latter experiment, average pore sizes and pore size distributions were calculated. Initiator and comonomer concentrations. Before setting out to investigate which parameters in the cryogelation process could be employed to tune the properties of PIC cryogels, we determined the optimal concentration initiators required for cryogel formation. At APS and TEMED concentrations below 0.02 mM and 0.01 mM, respectively, the formed PIC cryogels were weak and disintegrated upon handling, indicating that the degree of polymer crosslinking was insufficient for cryogel formation. Beyond these concentrations, however, the formed cryogels were mechanically stable. Based on these findings, we continued cryopolymerization with 0.02 mM APS and 0.01 mM TEMED. To further establish the optimal composition of PIC cryogels, we investigated the influence of addition of an acrylate chain extender on the architectural and mechanical cryogel properties. It is well-known that the ratio between chain extender and polymer within a cryogel affects properties such as pore size and swelling ratio. Here, we added 2- hydroxylethyl methacrylate (HEMA) as a chain extender to the PIC cryogel reaction mixture. Cryogels that were prepared with HEMA had a smaller average pore size (27 μm) than cryogels that were prepared without HEMA (58 μm). The distribution of the pore sizes was narrower for cryogels that contained HEMA than for cryogels without HEMA (Fig.3), which suggests that the addition of HEMA results in cryogels with a more homogenous structure. Uniaxial compression tests showed that cryogels prepared with HEMA display a slightly higher Young’s modulus (5.3 kPa) than those prepared without HEMA (3.9 kPa). The graphs of Fig.3 present the pore size and pore size distribution of cryogels made (a) with HEMA, and (b) without HEMA, as well as the swelling ratio of these cryogels (c). Polymer concentration influences the mechanical properties of PIC cryogels To determine how the properties of PIC cryogels can be tuned, we investigated the influence of polymer concentration. In general, an increase in polymer concentration results in cryogels with smaller pores due to the decreased amount of free water available for ice crystallization. We prepared cryogels with low, medium and high polymer concentrations (respectively 3.75, 6.67 and 9.17 mg / mL), resulting in cryogels C-low, C- med and C-high, respectively. Here, we observed that polymer concentration had no significant influence on pore size and pore size distribution. This is shown in Fig.4, showing pore sizes and pore size distribution for (a) C-low, (b) C-med and (c) C-high. The average pore size is shown in Fig.4(d). All cryogels have highly interconnected macroporous structures, narrow pore size distributions and similar pore sizes: 14.6, 17.9 and 16.8 μm for C-low, C-med and C-high, respectively. Without wishing to be bound by theory, the inventors believe that the polymer concentration does not affect ice crystal formation rates, which yields in similar cryogel architectures, albeit with higher densities of polymer walls for the higher concentration cryogels. Surprisingly, in deviation from, generally, other cryogels, the polymer concentration is considerably lower for PIC cryogels: for PIC cryogels of the invention the highest polymer concentration is typically 1 wt.%, whilst the polymer concentrations for other cryogels typically vary from 2 to 8 wt.%. Next, we examined the influence of polymer concentration on their mechanical properties by subjecting cryogels C-low, C-med and C-high to uniaxial compression tests. C-low cryogels have a Young’s modulus of 1.1 kPa, C-med cryogels of 3.4 kPa and C-high cryogels of 6.9 kPa, as depicted in Fig,.4(e). There is a linear correlation between polymer concentration and the Young’s modulus. The prepared cryogels displayed similar swelling behavior. C-low cryogels had a slightly higher swelling ratio (5450%) than C-med (4320%) and C-high (4137%), as shown in Fig.4(f). A decreased polymer concentration advantageously affords cryogels with a lower density polymer walls, which results in a higher swelling ratio due to the increased flexibility of the structure. The absence of significant differences for swelling ratio and pore size between the cryogels further underlines the previously described argument that the swelling ratio of PIC cryogels mostly correlates to the porous structure of the cryogels. Based on these results, we note that we can employ polymer concentration to tune the mechanical properties of PIC cryogels without affecting pore size and porosity. Polymer molecular weight influences the mechanical properties of PIC cryogels Besides cryogelation temperature and polymer concentration, we find that the molecular weight of the polymers influences cryogel properties. Usually, at a fixed polymer concentrations, the use of polymers with lower molecular weights results in cryogels with larger pores. This phenomenon can be explained by the Mark-Kuhn-Houwink equation, which states that an increase in molecular weight results in a decrease in free water content in the polymer solution that is available for crystallization. As a result, cryogels with smaller pores and thicker polymer walls are generated. We used PIC scaffolds of increasing length (and molecular weight), P1, P2 and P3, at a constant concentration, to prepare cryogels C-P1, C-P2 and C-P3, respectively. We observed that polymer molecular weight had no significant influence on pore size and pore size distribution. All cryogels have a highly interconnected macroporous structure and similar pore size distributions. Additionally, all cryogels displayed a similar pore size of 14.6, 15.7 and 17.4 μm, as shown in Fig.5 for (a) C-P1, (b) C-P2, and (c) C-P3, respectively, with average pore size presented in Fig.5(d).These results deviate from the common trend for cryogels. Without wishing to be bound by theory, the inventors believe that in the PIC cryogels of the invention, the ice crystal formation does not depend on the polymer molecular weight and thus gives cryogels with similar architectures in which polymers walls have higher density for the cryogels with higher molecular weight polymer scaffolds. The Young’s modulus of cryogels C-P1, CP-2 and CP-3 was determined from the uniaxial compressions tests and found that the molecular weight of the PIC scaffolds slightly influences the mechanical properties of the formed cryogels. C-P1 cryogels shows a Young’s modulus of 1.1 kPa, C-P2 cryogels of 1.7 kPa and C-P3 cryogels of 2.4 kPa, as depicted in Fig.5(e). The molecular weight between crosslinks of a cryogel plays an important role in determining the mechanical strength. Cryogel networks with larger molecular weight between crosslinks usually have a lower compressive strength than cryogels with smaller molecular weight between crosslinks. We hypothesize that for C-P3 the molecular weight between the crosslinks is smaller than for C-P1 and C-P2. Because of the larger polymer scaffold used, it could be that more crosslinks are made in close proximity, resulting in a lower molecular weight between crosslinks. Additionally, the observed increase in compressive strength underlines the hypothesis that PIC scaffolds with high molecular weight are more tightly packed within the non-frozen microphase than scaffolds with low molecular weight. All prepared cryogels displayed similar swelling behavior. As shown in Fig.5(f), C-P1 and CP-2 cryogels had a slightly higher swelling ratio than C-P3, which can be explained by their somewhat lower mechanical strength. Cryogels with less stiff networks are capable of taking up more water than cryogels with a more compact, dense network. Together, we conclude that while the polymer molecular weight has some effect on cryogel features such as compressive strength and swelling ratio, the influence is moderate compared to the influence of cryogelation temperature or polymer concentration, which makes it a less effective parameter.

Claims

Claims 1. A poly(isocyanopeptide) polymer comprising side chains provided with an oligo (alkylene glycol) group positioned between an oligopeptide moiety and a terminal functional group, wherein the terminal functional groups in the polymer comprise azide groups and acrylic groups.

2. A poly(isocyanopeptide) according to claim 1, further comprising side chains having alkoxy terminal groups.

3. A poly(isocyanopeptide) obtainable by subjecting isocyanide comonomers to Ni(II) catalyzed random copolymerization, wherein the isocyanide monomers comprise: (a) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an azide terminal functional group; (b) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an acrylic terminal group; (c) isocyanide substituted with an oligo(alkylene glycol) functionalizedpeptide side chain having an alkoxy terminal group, preferably methoxy.

4. A poly(isocyanopeptide) according to claim 3, wherein the isocyanide monomers are consisting of the monomers defined as (a), (b), and (c).

5. A poly(isocyanopeptide) according to claim 3 or 4, wherein the relative amounts of the comonomers are 1-3 mole % of (a), 2-5 mole % of (b), and 92-97 mole % of (c).

6. A poly(isocyanopeptide) according to any one of the preceding claims, wherein the oligopeptide is dialanine or trialanine.

7. A poly(isocyanopeptide) according to any one of the preceding claims, wherein the acrylic groups are methacrylate groups.

8. A cryogel mixture comprising an aqueous solution of a poly(isocyanopeptide) according to any one of the preceding claims, acrosslinking agent for crosslinking acrylic functional group, and an acrylic crosslinking catalyst.

9. A cryogel mixture according to claim 8, further comprising an acrylic chain extender, preferably hydroxyethyl methacrylate.

10. A cryogel obtainable by subjecting a cryogel mixture according to claim 8 or 9 to cooling to a temperature below 0°C, preferably in a range of from -80°C to -10°C.

11. A process for the preparation of poly(isocyanopeptide), the process comprising subjecting isocyanide comonomers to Ni(II) catalyzed random copolymerization, wherein the isocyanide monomers comprise: (a) isocyanide substituted with an oligo(alkylene glycol) functionalized peptide side chain having an azide terminal functional group; (b) isocyanide substituted with an oligo(alkylene glycol) functionalized peptide side chain having an acrylic terminal group; (c) isocyanide substituted with an oligo(alkylene glycol) functionalized peptide side chain having an alkoxy terminal group, preferably methoxy.

12. A process for preparing a cryogel, the process comprising subjecting a cryogel mixture according to claim 8 or 9 to cooling to a temperature below 0°C, preferably in a range of from -80°C to -10°C.

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