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WO2025221145A1 - Cryogels de poly(isocyanopeptide) - Google Patents

Cryogels de poly(isocyanopeptide)

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Publication number
WO2025221145A1
WO2025221145A1 PCT/NL2025/050182 NL2025050182W WO2025221145A1 WO 2025221145 A1 WO2025221145 A1 WO 2025221145A1 NL 2025050182 W NL2025050182 W NL 2025050182W WO 2025221145 A1 WO2025221145 A1 WO 2025221145A1
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Prior art keywords
cryogels
poly
isocyanide
isocyanopeptide
cryogel
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Inventor
Lotte GERRITS
Roelof HAMMINK
Paul Herco Jaroslav KOUWER
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Radboud Universiteit Nijmegen
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Radboud Universiteit Nijmegen
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Publication of WO2025221145A1 publication Critical patent/WO2025221145A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L65/00—Compositions of macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain; Compositions of derivatives of such polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G61/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G61/02—Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
    • C08G61/04—Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/10—Definition of the polymer structure
    • C08G2261/12—Copolymers
    • C08G2261/122—Copolymers statistical
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/10—Definition of the polymer structure
    • C08G2261/14—Side-groups
    • C08G2261/142—Side-chains containing oxygen
    • C08G2261/1424—Side-chains containing oxygen containing ether groups, including alkoxy
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/10—Definition of the polymer structure
    • C08G2261/14—Side-groups
    • C08G2261/142—Side-chains containing oxygen
    • C08G2261/1426—Side-chains containing oxygen containing carboxy groups (COOH) and/or -C(=O)O-moieties
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/10—Definition of the polymer structure
    • C08G2261/14—Side-groups
    • C08G2261/143—Side-chains containing nitrogen
    • C08G2261/1432—Side-chains containing nitrogen containing amide groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/10—Definition of the polymer structure
    • C08G2261/20—Definition of the polymer structure non-conjugated
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/30—Monomer units or repeat units incorporating structural elements in the main chain
    • C08G2261/33—Monomer units or repeat units incorporating structural elements in the main chain incorporating non-aromatic structural elements in the main chain
    • C08G2261/332—Monomer units or repeat units incorporating structural elements in the main chain incorporating non-aromatic structural elements in the main chain containing only carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/40—Polymerisation processes
    • C08G2261/42—Non-organometallic coupling reactions, e.g. Gilch-type or Wessling-Zimmermann type
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/70—Post-treatment
    • C08G2261/76—Post-treatment crosslinking

Definitions

  • 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).
  • cryogels suitable for bio-medical applications such as in the fields of tissue engineering and in vitro cell culture.
  • 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.
  • 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.
  • trans-cyclooctene e.g. DBCO
  • a background disclosure on such azide-modified poly(isocyanopeptides) is EP 2454302.
  • 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.
  • the PIC hydrogels have excellent properties, improvement is desired.
  • 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.
  • 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) 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 C1 to C 3 alkoxy, more preferably methoxy.
  • 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.
  • the invention presents a poly(isocyanopeptide) obtainable by the aforementioned process.
  • 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.
  • 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.
  • 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.
  • 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).
  • polyisocyanides thus comprise an all-carbon backbone, whereby each carbon atom carries a substituent.
  • substituents are referred to with the term “side-chain.”
  • 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
  • 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.
  • 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 C 1 to C 3 alkoxy, more preferably methoxy, are inert for the coupling to biomolecules, as well as for
  • 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) 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.
  • 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).
  • 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.
  • 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.
  • the isocyanopeptides are functionalized with at least 3 ethylene glycol units to lead to water soluble materials after polymerization.
  • alkylene glycol units 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.
  • suitable alkylene glycols are ethylene-, propylene-, butylene- or pentylene glycol.
  • 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.
  • 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.
  • 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.
  • 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.
  • a mixture of the comonomers, (A), (B), and (C) is subjected to random copolymerization using a suitable catalyst, preferably a Ni 2+ catalyst.
  • a suitable catalyst preferably a Ni 2+ 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.
  • cryopolymerization 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.
  • crosslinking agents suitable for crosslinking acrylic functionalized monomers Generally, this will involve crosslinking agents suitable for crosslinking acrylic functionalized monomers.
  • 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.
  • an alternative to APS is potassium persulfate, which is preferably applied in conjunction with TEMED, riboflavin or riboflavin phosphate.
  • TEMED riboflavin
  • 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.”
  • 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).
  • the main parameters that the skilled person will be able to take into account are: - the length of the polyisocyanides backbone; generally this will vary 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, if so, the amount thereof; - the polymer concentration in the mixture from which a cryogel is 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.
  • 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.
  • higher swelling ratios are associated with cryogels with lower degree of crosslinking and lower density of polymer walls.
  • the observed increase in swelling ratio for PIC cryogels with HEMA could not be attributed to the cryogels’ polymer wall thickness.
  • the inventors attribute the increased swelling ratio to the introduction of hydrophobic groups in the cryogel network by addition of HEMA.
  • 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.
  • cryogelation temperature has a substantial impact on cryogel features such as pore size, structural homogeneity and polymer wall thickness.
  • compositional changes i.e., the PIC molecular weight and the PIC concentration
  • 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.
  • poly(isocyanopeptide) cryogels of the invention can be broadly applied inter alia, in in vitro or ex vivo systems for cell culturing.
  • acrylic functionalized 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.
  • 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 (0 o C ) 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).
  • 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.
  • DMAP 4-dimethylaminopyridine
  • DCC N,N'-dicyclohexylcarbodi- imide
  • Boc-deprotection using HCl (4M in Dioxane) a similar condensation reaction with Boc-D-Alanine was performed using DMAP, DCC and N-hydroxybenzotriazole (HOBt).
  • 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 %).
  • cryogels 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.
  • 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.
  • the axial force and displacement data were used to obtain stress-strain curves.
  • the compressive stress ( ⁇ ) was calculated from the recorded axial force (F) per cross sectional area (A) of the un-deformed sample.
  • the strain ( ⁇ ) was determined by calculating the ratio between the deformed (dl) and initial (l) lengths.
  • the ice crystals were thawed and the cryogel was thoroughly washed with water to remove unreacted residual ingredients.
  • cryogel 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.
  • HEMA 2- hydroxylethyl methacrylate
  • 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
  • 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.
  • 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.%.
  • 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).
  • cryogels C-P1, C-P2 and C-P3, respectively.
  • 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 ⁇ m, 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.
  • 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.

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Abstract

L'invention concerne des poly (isocyanopeptides) fonctionnalisés par des groupes acryliques. Ces poly(isocyanopeptides) peuvent être soumis à une réticulation. Ceci peut être accompli dans une solution aqueuse, laquelle peut être soumise à une cryopolymérisation à l'aide d'un agent de réticulation approprié. On obtient ainsi des cryogels présentant des propriétés intéressantes. En particulier, ces cryogels permettent d'ajuster les propriétés mécaniques sans pour autant affecter nécessairement l'architecture de cryogel, telle que la taille des pores et la distribution de la taille des pores.
PCT/NL2025/050182 2024-04-18 2025-04-17 Cryogels de poly(isocyanopeptide) Pending WO2025221145A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2454302A1 (fr) 2009-07-16 2012-05-23 Stichting Katholieke Universiteit Meer In Het Bijzonder Radboud Universiteit Nijmegen Procédé de préparation de polyisocyanopeptides fonctionnalisés par des oligo(alkylène glycol) de poids moléculaire élevé
WO2015007771A1 (fr) * 2013-07-18 2015-01-22 Stichting Katholieke Universiteit, More Particularly Radboud Universiteit Nijmegen Polymère adapté à l'utilisation en culture cellulaire
WO2018104324A1 (fr) * 2016-12-05 2018-06-14 Stichting Katholieke Universiteit Réseaux biomimétiques comprenant des hydrogels de polyisocyanopeptide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2454302A1 (fr) 2009-07-16 2012-05-23 Stichting Katholieke Universiteit Meer In Het Bijzonder Radboud Universiteit Nijmegen Procédé de préparation de polyisocyanopeptides fonctionnalisés par des oligo(alkylène glycol) de poids moléculaire élevé
WO2015007771A1 (fr) * 2013-07-18 2015-01-22 Stichting Katholieke Universiteit, More Particularly Radboud Universiteit Nijmegen Polymère adapté à l'utilisation en culture cellulaire
WO2018104324A1 (fr) * 2016-12-05 2018-06-14 Stichting Katholieke Universiteit Réseaux biomimétiques comprenant des hydrogels de polyisocyanopeptide

Non-Patent Citations (3)

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Title
ELIZABETH A PUMFORD ET AL.: "Nontoxic Initiator Alternatives to TEMED for Redox Hydrogel Polymerization", ACS APPL BIO MATER, 14 March 2024 (2024-03-14)
MANDAL, S. ET AL., CHEM. SCI., vol. 4, 2013, pages 4168 - 4174
TOM�S SEDLA�K ET AL: "Macroporous Biodegradable Cryogels of Synthetic Poly([alpha]-amino acids)", BIOMACROMOLECULES, vol. 16, no. 11, 16 October 2015 (2015-10-16), US, pages 3455 - 3465, XP055305982, ISSN: 1525-7797, DOI: 10.1021/acs.biomac.5b01224 *

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