NL2020453B1 - Tripodal squaramide-based monomers - Google Patents

Abstract

The present invention relates to a tripodal squaramide-based monomer according to formula (I) 5 for the formation of supramolecular polymers and hydrogel materials: NH R1 T n N O H 3 0 (I) wherein T represents a central atom; n is an integer of from 1 to 12; and R1 is a group according to Formula (||): O /R\N 2 JL O‚R 3 10 H wherein R2 is a hydrophobic group; and R3 is a hydrophilic group, to hydrogels produced by self-assembly of the monomer and to use of the hydrogels for cell culture, preferably pluripotent stem cells, advantageously human induced pluripotent stem cells and their derivatives. 15

Description

Field of the Invention

The present invention relates to the synthesis and self-assembly of squaramide-based tripodal monomers into supramolecular materials in water for biological application More specifically, the invention relates to their self-assembly in water based on non-covalent interactions to form supramolecular polymers that can eventually form hydrogel materials. This invention also encompasses their capacity to form hydrogels with self-recovering characteristics, making them particularly suitable to for cell culture applications with various cell types.

Background of the Invention

Recently, there is growing interest in developing supramolecular polymer materials, such as supramolecular hydrogels, for a broad range of applications, from biomedicine to electronics due to their unique properties that arise from their non-covalent character in comparison to their covalent counterparts. Consequently, these materials are endowed with characteristics such as facile preparation, responsiveness and self-healing. As biomaterials, their easy processing permits the mixing of numerous functionalized monomers with complex cargoes, such as peptides, and their responsiveness to stimuli such as temperature, pH, light and enzymes makes them candidates as designer materials that can deliver therapeutic cargoes or as scaffolds for 3D cell cultures.

One particular area where supramolecular hydrogels can be especially useful is in the cell culture, especially in the culture of human pluripotent stem cells (hPSCs) that are unique in their capacity to generate any body cell type. Human induced pluripotent stem cells (hiPSCs) have been shown to reproduce all properties of human embryonic stem cells (hESCs) derived from the pre-implantation stage of human embryos, but can be derived from somatic cells obtained in a non-invasive manner. Excitingly, hiPSCs have the potential for decreased immunogenicity because they can be derived from autologous sources, but they require specific culture conditions to maintain their pluripotent state. To enable their expansion and directed differentiation in 3D for applications such as drug screening, disease modelling and eventually regenerative medicine, inert synthetic scaffolds and gentle release methods have been required for optimal culture and recovery of the hiPSC cells for further downstream applications. However, to reach such end-stage applications in the biomedical area with supramolecular materials, structurally simple and biocompatible monomers with high synthetic accessibility that robustly self-assemble into polymeric architectures are necessary.

To promote supramolecular polymerization by self-assembly of a given monomer, a combination of non-covalent interactions, such as hydrogen-bonding, ttstacking, van der Waals and/or electrostatic interactions, need to be engineered into the monomer unit. Hydrogen bonds have often been employed because of their capacity to engender directional interactions between monomer units while providing a handle to tune the strength of their association by their type, number, arrangement and microenvironment. Commonly used hydrogen bonding synthons have included amides, thioamides, ureas and thioureas.

The self-assembly of squaramide monomers has been explored to some extent, see for instance Busschaert et al, Angew. Chem. Int. Ed. 2012, 51,4426, Schiller et al, D. D. Soft Matter 2016, 12, 4361 and Wu et al, Inorg. Chem. Front. 2016, 3, 1597. Examples of self-assembly of squaramide monomers has also been reported in water, as disclosed by Saez Talens et al, in Angew. Chem. 2015, 127, 10648, López et al, Chem. Eur. J. 2017, 23, 7590 and Noteborn et al, ChemBioChem. 2017, 18, 1. The use of squaramide-based tripodal monomers as ionophores has been described in Yueling et al, Analyst 2015, 15, 140, 2015, 5317 and Xiong-Jie et al, Bioorganic & Medicinal Chemistry Letters 2017, 27, 9, 1, 1999.

However, there is a need for monomers that are synthetically accessible and robustly self-assemble into polymer architectures in water and eventually hydrogel materials, for biological applications, such as in cell culture. More specifically, such materials would be an advantage for the culture of pluripotent stem cells (PSCs), more particularly for the culture of human pluripotent stem cells (hPSCs), human pluripotent stem cells (hiPSCs) and their derivatives.

Summary of the Invention

It is therefore an object of the present invention to provide a monomer that can be polymerized to produce a supramolecular polymer, advantageously in the form of fibers, that can result in hydrogel materials for the cell culture, particularly for the culture of an hPSC, more particularly for the culture of hiPSCs.

Accordingly, one aspect of the present invention relates to a tripodal squaramide-based supramolecular monomer according to formula (I):

NHR¹

(I) wherein T represents a central atom;

n is an integer of from 1 to 12; and

R¹ is a group according to Formula (Ila) or (lib):

wherein R² is a hydrophobic group; R³ is a hydrophilic group, and X and/or Y independently represent -NH-; -NR⁴-; O or C.

Brief Description of the Figures

Figure 1 shows an oscillatory rheology measurements of hydrogel 2 (5.6 mM) and hydrogel 3 (3.1 mM) in deionized water at 25 °C: Amplitude sweep (1 Hz) for hydrogels 2 (A) and 3 (B); Frequency sweep (0.05% strain) of hydrogels 2 (C) and 3 (D); and Step strain measurements (1 Hz) for hydrogels 2 (E) and 3 (F). The absence of data between the application of high strain is due to the acquisition of a frequency sweep (from 0.01 to 2 Hz, γ = 0.05%).

Figure 2 discloses oscillatory rheology measurements of the hydrogel 2 (5.6 mM) in PBS at 37 °C: (A) Amplitude sweep (f = 1 Hz); (B) Frequency sweep (γ = 0.05%); and (C) Step strain measurements (f = 1 Hz). The absence of data before the application of high strain is due to the acquisition of a frequency sweep (from 0.01 to 2 Hz, γ = 0.05%).

Figure 3 is a Cryo-TEM image of a hydrogel 2 (5.6 mM) with 20 minutes sonication in an ultrasonic bath that is left to stand overnight. Insert are histograms of the width distribution of the fibers for a sample size of N = 50.

Figure 4 is a Cryo-TEM image of hydrogel 3 (3.1 mM). Insert: Histograms of the width distribution of the fibres for a sample size of N = 50.

Figure 5 is a Cryo-electron tomography image of hydrogel 3 (3.1 mM).

Figure 6 is a Small-angle X-ray scattering profile of fibers of hydrogel 3, collected at a concentration of 2 mg mL'¹. Black dots represent experimental data; red line represents fit with a form factor for flexible cylinders.

Figure 7 shows an lcs(q) determination plot of the scattering profile in Figure 6. The lcs(q) plateau (0.0143 < q < 0.0383 A-1) is indicated by a red line.

Figure 8 shows a (A) UV-Vis spectra of monomers 1 -3 in deionized water (1.5x10-5 M). (B) Solutions of Nile Red (1.0 x 10 ^s M, Ex. 550 nm, Em. 560-750 nm) in deionized water and in the presence of 1-3 (1.5 χ 10'⁵ M) (C) FTIR spectra of lyophilized monomers 1-3 in the solid state (3.1 mM in deionized water, arrows highlight peaks).

Table 2 below shows infrared spectra assignments of vibrational modes from 3700-1500 cm¹ for the various peaks of the lyophilized monomers 1-3 indicated in Figure 8C.

Table 2: infrared spectra assignments

	Sample 1	Sample 2	Sample 3
v(N-H)	3317 cm1 (carbamate) 3164 erm1 (squaramide)	3317 cm’1 (carbamate) 3165 erm1 (squaramide)	3315 cm'1 (carbamate) 3167 cm-1 (squaramide)
v(C-H)	2931 cm’1 (antisym) 2861 cm'1 (sym)	2925 cm'1 (antisym) 2854 cm-1 (sym)	2918 cm1 (antisym) 2850 erm1 (sym)
v(C=O)	1719 and 1693 cm'1 (carbamate) 1654 and 1639 erm1 (squaramide)	1719 and 1692 cm*1 (carbamate) 1644 erm1 (squaramide)	1720 and 1689 cm*1 (carbamate) 1652cm-1 (squaramide)
Ring breathing	1799 erm1	1799 erm1	1799 cm1

Figure 9 shows Cryo-TEM images of (A) diluted solution of monomer 2 (1.5 x 10-5 M) and (B) diluted solution of monomer 3 (1.5 x 10-5 M) with 20 minutes sonication in an ultrasonic bath and left to stand overnight. Figure 10 shows concentration-dependent UV-Vis spectra of monomers 2 (A) and 3 (B) in deionized water.

Figure 11 shows UV-Vis spectra of monomers 2 and 3 (1.5 x 10-5 M) in HFIP/H2O (v/v, 1:1).

Figure 12 shows results of the MTT cytotoxicity test for the monomers 1-3 ranging in concentration (1-200 μΜ) on NIH 3T3 cells under various conditions (N = 3): (A) in deionized water after 24 h. (B) in deionized water after 72 h. (C) in PBS after 24 h. (D) in PBS after 72 h.

Figure 13 illustrates a supramolecular hydrogel preparation and 3D cell seeding strategy in water.

Figure 14 shows confocal microscopy images of NIH 3T3 cells encapsulated in a hydrogel of monomer 2 (5.6 mM) in 3D: after 2 h incubation (left); after 48 h incubation (right) (green: viable cells, red: dead cells). Scale bar 200 pm.

Figure 15 shows confocal microscopy images of NIH 3T3 cells encapsulated in a hydrogel of monomer 3 (3.1 mM): after 2 h incubation (left); after 48 h incubation (right) (green: viable cells, red: dead cells). Scale bar 200 pm.

Figure 16 shows a 3D culture of hiPSC-derived ECs encapsulated in the hydrogel of monomer 3 (3.1 mM): (A) Representative images of hiPSC-ECs seeded in the hydrogel at low density (5 x 10⁵ cells/ml_) and at (B) high density (2 x 10⁶ cells/ml_) and cultured for 24 h. Dead cells were detected using NucGreen® Dead reagent (ThermoFisher). Scale bar 200 pm.

Figure 17 shows a 3D cell culture of hiPSCs in hydrogel of monomer 3 (3.1 mM): Representative images at ~24 (A) and ~72 h (B). Dead cells were detected using the NucGreen® Dead reagent. Scale bar 100 pm; (C) hiPSC spheroid diameter distribution in a hydrogel of monomer 3 (3.1 mM) after 24 h, 48 h and 72 h, with approximately 135 spheroids per day; (D) FACS analysis of hiPSCs cells after 24 h under standard 2D culture conditions or after retrieval from hydrogel 3 (3.1 mM) after 24 h of culture; upper panel: side scatter (SSC-A) and forward scatter (FSC-A) showing live cell population (Black circles are the gated live cells); lower panel: expression level of TRA-1-60 and SSEA-4 pluripotent stem cell markers of the gated live cells.

Figure 18 shows representative images of hiPSC 3D spheroids in hydrogel of monomer 3 (3.1 mM) just after seeding, after 24 h, 48 h and 72 h of culture. Scale bar 100 pm.

Figure 19 finally shows a 3D cell culture of hiPSCs encapsulated in hydrogel of monomer 3 (3.1 mM): The values of mean fluorescence intensity (MFI) of the positive cell populations in the expression level of TRA-1-60 (A) and SSEA-4 (B).

Detailed Description of the Invention

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The present invention relates to a new class of soft materials, in the form of lowmolecular-weight gels (LMWGs), or supramolecular gels. Such gels typically arise from the self-assembly of small molecules into entangled fibers through a combination of non-covalent interactions, such as hydrogen-bonding, π-stacking, van der Waals and/or electrostatic interactions, and eventually hydrogel materials above a critical concentration. Due to their inherent non-covalent nature, supramolecular polymer materials may exhibit unique features in comparison to their covalent counterparts such as facile preparation, responsiveness and self-healing abilities. Based on specific features and properties, supramolecular materials are promising candidates for many biological applications, such as cell culture, tissue engineering, drug delivery, cancer therapy, imaging, and immunology. However, to reach such end-stage applications in the biomedical area with supramolecular materials, structurally simple and biocompatible monomers with high synthetic accessibility that robustly self-assemble into polymeric architectures are necessary.

Hydrogen-bonds are often employed in the design of the monomers because of their capacity to engender directional interactions between them while providing a handle to tune the strength of their association by their type, number, arrangement and microenvironment. Despite their extensive use in the areas of bioconjugation, medicinal chemistry, catalysis and anion recognition, ditopic hydrogen-bonding squaramide units have been explored to a far lesser extent in the materials domain especially with respect to selfassembly, with few examples reported in water, as set out above.

The presented monomers advantageously have the capacity to prepare noncovalent materials. Moreover, the present design allows the facile synthesis of a library of flexible tripodal squaramide-based supramolecular polymer monomers, and their eventual transformation into hydrogels with self-recovering character upon self-assembly. These supramolecular polymer hydrogels according to the invention were found to be highly cytocompatible with a range of cell types, including several considered sensitive such as hiPSCs and their differentiated derivatives.

Moreover, when pluripotent stem cells are encapsulated in these materials in 3D they can be can be collected from the soft gel upon dilution in a gentle manner. The simplicity of the seeding and release approach combined with the biocompatibility of these squaramide-based materials make this material particularly suitable for such applications.

In accordance with this invention, the tripodal squaramide-based monomers of formula (I) can each be made by reacting a oligomeric or polymeric multi-ethylene glycol alkyl ether, preferably tetraethylene glycol monomethyl ether, preferably with 1,1carbonyldiimidazole, and by reacting the resulting product can then be reacted with an excess of a carboxybenzyl (Cbz)-protected, linear 1 ,n’-alkyldiamine (wherein ri is 4-20, preferably 612, more preferably 8-10).

The amine moiety of the Cbz group can be protected during this step in a conventional manner, e.g., with a protective group and subsequently deprotected in a conventional manner, e.g., by a catalytic hydrogenation. The amine moiety of the Cbz group can then be reacted with 3,4-Dibutoxy-3-cyclobutene-1,2-dione (Dibutyl Squarate) and diisopropylethylamine (DIPEA). In a final step, the resulting squaramide monomer, also referred to as an amphiphile can be reacted with tris(2-aminoethyl)amine (TREN) and DIPEA to obtain one of the tripodal squaramide-based monomers of formula (I).

The tripodal squaramide-based monomers of formula (I) can self-assemble into supramolecular fibrous and eventually, hydrogel materials useful for applications in the 3D cell culture, namely as scaffolds for cell culture, or with hiPSCs and their derivatives. In this regard, the monomers, when dissolved in aqueous solutions, preferably water, can polymerize to form fibers and hydrogels. The monomers can be dissolved in a conventional manner, e.g., by sonication instead of heating.

The monomers of formula (I) feature a flexible tripodal core with squaramides that are attached to varied hydrophobic and hydrophilic domains. With increased concentrations of the monomers, self-recovering hydrogels can be formed even under physiological conditions at 37°C. Such hydrogels are composed of a network of entangled fibrils of a few nanometres in width and microns in length as seen in cryo-EM. Spectroscopic measurements of the self-assembly properties of the monomers in water indicate that hydrogen-bonding and hydrophobic interactions facilitate the polymerization and growth of the monomers into supramolecular polymer fibers. The resulting hydrogels are highly cytocompatible with a range of cell types, including several considered sensitive such as hiPSCs and their differentiated derivatives. Moreover, the single cell hiPSCs can form spheroids within the hydrogels, likely due to the hydrogels’ mechanically soft and supramolecular character, and can retain their pluripotent stem cell phenotype upon their gentle isolation by simple dilution. The simplicity of the seeding and release approach to forming hydrogels from the monomer of formula (I) as shown in Figure 14, combined with the biocompatibility of these squaramide-based materials make it possible to use the hydrogels for applications in 3D cell culture and delivery, namely with hiPSCs and cells that are derived from them.

The subject invention advantageously also relates to a family of synthetically accessible, squaramide-based tripodal monomers comprising a tris(2-aminoethyl)amine (TREN) core. These monomers can self-assemble into supramolecular polymers, and eventually also into hydrogels with self-recovering character depending on their hydrophilic/hydrophobic ratio.

The invention furthermore may be applied to prepare cytocompatible, selfrecovering supramolecular hydrogels for the encapsulation of several cell types, such as NIH

3T3 fibroblasts, human induced pluripotent stem cells (hiPSCs) and hiPSC-derived endothelial cells (ECs) in a 3D environment for a range of cell culture applications, including those where the subsequent release of the cells from the scaffold in a gentle manner is necessary.

In Formula (II), R² is advantageously an alkylene group, more advantageously a straight chain alkylene group, more advantageously a straight chain alkylene group of 4 to 20 carbon atoms, still more advantageously a straight chain alkylene group of 6 to 12 carbon atoms; yet more advantageously a straight chain alkylene group of 8 to 10 carbon atoms.

R³ is advantageously a group of the formula (III):

ⁿ (HI), wherein n is 2 to 10, preferably 3 to 6, more preferably 4 or 5. Preferably, R¹ is a group according to formula (III)

wherein a represents an optionally substituted alkyl, alkenyl and/or alkynyl moiety, wherein a is an integer ranging of from 1 to 15; and p is a polyethylene glycol group comprising of from 10 to 10.000 ethylene glycol units; and W represents a functional anchoring group.

Preferably, T is selected from nitrogen, boron, phosphorus, silicon, or carbon, preferably wherein T is boron, nitrogen or phosphorus, and wherein the core has a C3 or pseudo-C3 symmetry. Preferably, n represents of from 1 to 6 atom carbon chains linking T and the squaramide moiety. More preferably, n is 2 to 10, advantageously 3 to 6, more advantageously 4 or 5.

Preferably, the core is derived from any one of the following compounds T1 to

T5:

nh₂

T3 _T4 T5

A preferred monomer is defined as formula (IV)

UP (IV), wherein R is as defined as R¹ herein above.

Another aspect of the present invention relates to a supramolecular polymer, advantageously in the form of fibers, more advantageously in the form of a hydrogel, produced by self-assembly of the tripodal squaramide-based monomer of formula (I) in water over several length scales. The present invention relates to a process for polymerizing the tripodal squaramidebased monomer of formula (I) comprising the step of self-assembling the monomer in water.

Another aspect of the present invention relates to the use of a supramolecular polymer, advantageously in the form of fibers, more advantageously in the form of a hydrogel, produced by polymerization of the tripodal squaramide-based monomer of formula (I), for cell culture of pluripotent stem cells, preferably human pluripotent stem cells (hPSCs), advantageously for the culture of human induced pluripotent stem cells (hiPSCs).

For the purpose of the invention, the amount of the monomers in the supramolecular polymer or hydrogel is indicated in mM. In the present invention, the ditopic hydrogen bond moiety, the squaramide, 3Q, with two C=O hydrogen bond acceptors and two N-H hydrogen bond donors opposite one another on a conformationally rigid cyclobutenedione ring provide directional and intermolecular interactions between the monomer units during polymerization. And through the formation hydrogen bonds, increased aromatic character was reported, that significantly to the strength of the overall hydrogen bond interaction. The flexible tripodal core could advantageously be selected from commercially available tris(2-aminoethyl)amine (T1), tris(3aminopropyl)amine (T2), and synthetically accessible moieties T3, T4 and T5. The free amines in the tripodal core offer a simple way to synthetically couple the hydrogen bonding moiety 3Q.

A hydrophobic component a is preferably selected from the group comprising C2-50 alkyl, or C2-50 alkenyl, or C2-50 alkynyl, or C3-12 cycloalkyl.

Preferably, a is an integer from 2 to 50. In theory all alkyl moieties of varied length can be obtained from commercial sources or by synthetic means using metathesis with different unsaturated fatty acids. The alkyl chains can then be oxidized to a mixture of alkenes and alkynes.

The hydrophilic component p comprises an integer from 2 to 14. This may advantageously be obtained from commercially available oligo or polyfethylene glycol), or synthesized from commercially available starting compounds.

The component depicted in formula Ila and lib represents the linker between hydrophobic and hydrophilic group in this invention, which could be a -NH(C=O)O- (carbamate), -NH(C=O)NH(urea), and -NH(C=O)- (amide) moiety, which also offered the additional hydrogen bonding effect during the monomers aggregation. -O- (ether) and -(C=O)O- (ester) moiety could also be used as a linker in this invention.

The anchoring unit W may be neutral, cationic, anionic or zwitterionic, organic, bio-organic, inorganic a metal complex or a metallic nanoparticle, or a combination. W may be an acid anhydride, acrylic acid, acyl halide, aldehyde, alkenyl, alkoxy group, alkyl, alkynyl, amide, amine, anhydride, azide, borate, borinic acid, borinic ester, boronic acid, boronic ester, bromide group, carbamate, carboxylate, carboxylic acid, chloride group, cyanate, cyanide, diimide, diselenide, disulfide, dithiolane, epoxide, ester, ether, fluor group, hydrazine, hydroxyl, N-hydroxysuccinimide esters, imide, iodide group, isocyanate, isocyanide, isothiocyanate, ketone, maleimide, nitro group, phenyl, phosphate, phosphate amide, phosphate ester, phosphine, phosphine imide, phosphine oxide, phosphinic acid, phosphinic ester, phosphonic acid, phosponic ester, polysulfide, selenide, seleninic acid, selenium halide, selenoaldehyde, selenoketone, selenol, selenone, selenoxide, selenyl halide, silanol, silene, silole, siloxane, silyl ether, s-nitrosothiols, sulfide, sulfilimine, sulfinyl halide, sulfonate, sulfone, sulfonyl, sulfoximides, sulfur halide, thioaldehyde, thioamide, thiocarbamate, thiocyanate, thioester, thioether, thioketone, thiol, thiosulfinate.

Based on the non-covalent nature of the backbone and decorating with different functional W groups, these biofunctional materials in this invention have the potential for a broad range of applications that involve interaction with biological environments, such as in cell culture.

Supramolecular hydrogels, composed of physically crosslinked fibrillar networks possess high water contents and when combined with some bioactive moieties, demonstrate the potential to mimic extracellular matrix for 3D cell culture. Cellular response is known to be affected by cell biomaterial interactions, cell-cell interactions, and soluble factors, among other factors found in their microenvironment. For example, biophysical cues, such as the rigidity of biomaterial matrices, are critical in cell culture since different cell types encounter a range of rigidities in their microenvironment based on their tissue origin. Hence, materials that vary in elastic modulus, from soft to stiff, are required to regulate several cell behaviors from proliferation to differentiation. In this invention, the tripodal squaramide-based monomer may be self-assembled into hydrogel materials through physical non-covalent crosslinking, and the addition of monomers or covalent polymers that enable further crosslinking by through complementary reactive groups or permit the inclusion of bioactive functions (e.g peptides, sugars, nucleic acids) can further their use in this area.

Non-covalent crosslinking of the hydrogel network can be facilitated by several interactions such as hydrogen bonding, ττ-π stacking, ionic interactions (negative charge group with positive charge group) or metal coordination (for instance with metal cations, such as Ca²⁺ with negative charged groups, like carboxyl or sulfonate group). Based on the functionality of the W groups, crosslinking strategies such as thiol-ene, azide-alkyne cycloaddition azide-alkyne cycloaddition, chelating agents, oxime chemistry, Diels-Alder reactions, amide bond formation, maleimide coupling, chelating agents and thiol-based chemistry to trigger the hydrogel formation and modulate hydrogel stiffness. The synthetic molecule with functional W group could also be conjugated to many biologically active proteins, nucleobase, amino acids, saccharide, peptides, or DNA via radical polymerization, a range of synthetic and natural covalent polymers, or other conjugation strategies, such as “click” reactions.

Supramolecular hydrogels in this invention can be advantageously used as a promising material for drug delivery. For example, the drugs and bioactive molecules can physically encapsulated in hydrogels, e.g. by physical loading, or the drug could be covalently conjugated to the self-assembled supramolecular polymer, i.e. a chemical loading. The hydrogels could carry a controlled amount of bioactive small molecules or drugs by their non-covalent interaction. .Either of these modes of interactions can be applied for drug/prodrug like acetaminophen, ibuprofen, naproxen, folic acid, Taxol, lamivudine, rapamycin, curcumin, zidovudine, dexamethasone, betamethasone, hydrocortisone and triamcinolone acetonide. These drugs could even be directly conjugated to the W group with the appropriate chemistries.

W may also be a fluorophore, like aminocoumarin, benzoxadiazole, dansyl, eosin, fluorescein, hydroxycoumarin, indocarbocyanine, merocyanine, methoxycoumarin, nitrobenzoxadiazole, Oregon green, oxacarbocyanine, phthalocyanine, rhodamine, to permit visualization of the hydrogels on their own and in interacting with biological media.

W may be a MRI contrast agent such as gadobenate, gadobutrol, gadocoletic acid, gadodiamide, gadofosveset, gadomelitol, gadomer 17, gadopentetate, gadopentetic acid dimeglumine, gadoterate, gadoteridol, gadoversetamide to track the hydrogels if applied in vivo.

W may also include a peptide, such as for instance a therapeutic peptide, a cell targeting peptide, a cell entry peptide, or an extracellular matrix peptide; a targeting ligand; an aptamer, and/or an antibody. Suitable peptides comprise 3 to 100 amino acids, more preferably 3 to 90 amino acids, more preferably 3 to 80 amino acids and most preferable from 3 to 70 amino acids, and include cyclic peptides, polypeptides, oligopeptides and even proteins.

Preferably, the W group comprises a peptide having a function selected from the group consisting of targeting, therapeutic, cell-penetrating abilities and/or bioactive cues that interface with the cell surface for 3D cell culture, cell delivery and tissue engineering. W can also be a cell targeting peptide. Such peptides are able to bind to protein receptors present in specific tissues or on specific cells, usually they consist of short peptides (e.g. 3-70 amino acids long) that can target extra- or intracellular receptors. W can also be RGD, cRGD, a vascular endothelial growth factor receptor (VEGFR) binding peptide, angiopep-2, angiocept, nuclear localization signal (NLS) peptide, chlorotoxin peptide, plasma membrane targeting peptide, and ER targeting peptide.

W may advantageously comprise of one or more therapeutic peptide(s). Therapeutic peptides may be antibiofilm peptides, antifungal peptides, antiparasitic peptides, antibacterial peptides, antiprotozoa peptides, antimalarial peptides, antiprotozoa peptides, antiviral peptides, anti-HIV peptides, anticancer peptides, wound healing peptides, antioxidant peptides, chemotactic peptides, and insecticidal peptides,.

W may advantageously also be a cell-penetrating peptide (CPP). CPPs are short peptides that facilitate cellular uptake if the supramolecular polymers are used below the critical gelation concentration. CPPs are typically composed of numerous positively charged amino acids such as arginine or lysine with sequences that possess polar/charged amino acids and non-polar, hydrophobic amino acids in an alternating pattern. W may be a trans-activating transcriptional activator (Tat), Pep-1, penetratin, transportan, CADY, TP, ΤΡΙΟ, MPG, arginine octamer. polyarginine sequences, VP22, SAP Prolinerich motifs, Arg8, HSV-1 structural protein, Vectocell® peptides, PPTG 1, hCT (9-32), SynB, and Pvec.

W may advantageously also comprise of one or more peptides to synthesize materials that can serve as extracellular matrix mimics for 3D cell culture. Preferably, W comprises a peptide that has a function as a biophysical or biochemical cue from the natural extracellular matrix. Peptides serving as biophysical cues are sequences that could recognize cell membrane proteins extracellularly resulting in cellular attachment, proliferation or even differentiation. Examples serving as biophysical cues include RGD peptide, cyclic RGD peptide, RGD mimics, IKVAV peptide, DGEA peptide, fibronectin, vitronectin, laminin and collagen type-1.

Peptides that mimic biochemical cues may also comprise sequences that bind to transmembrane receptors, thereby inducing a biochemical cascade resulting in cell growth, 5 proliferation, differentiation or migration. Examples include growth factors, including insulin-like growth factor (IGF-1), transforming growth factor pl(TGF-pi), vascular endothelial growth factor (VEGF), hepatocyte growth factor (hGF), fibroblast growth factor-2 (FGF-2), nerve growth factor (NGF), epidermal growth factor (EGF), bone morphogenetic proteins (BMP-2, BMP-4), placental growth factor (PIGF), cytokines and chemokines.

Further examples are set out in Table 1:

Table 1: Peptides and associated origins and key sequences

Peptide	Origin	Sequence
RGD	Collagen I	Arg-Gly-Asp
cRGD	Collagen I	c(Arg-Gly-Asp-D-Phe-Lys)
DGEA	Collagen I	Asp-Glye-Glu-Ala
Angiopep-2	synthetic	TFFYGGSRGKRNNFKTEEY
Chlorotoxin	synthetic	MCMPCFTTDHQMARKCDDCCGGKGRGKCYG PQCLCR
NLS peptide	synthetic	P ro- Pro- Lys- Ly s- Ly s-A rg - Ly s-Va I
ER targeting peptide	synthetic	Lys-Asp-Glu-Leu
IKVAV	Laminin-1	Iso-lys-val-ala-val
KNRLTIELEVRT	Laminin-2	KNRLTIELEVRT
FN-C/H-I	Fibronectin	YEKPGSPPREVVPRPRPGV
FN-C/H-II	Fibronectin	KNNQKSEPLIGRKKT
CS1 (LDV)	Fibronectin	DELPLVTLPHPNLHGPELIDVPST
EDGIHEL	Fibronectin	EDGIHEL
VFDNFVLK	Tenascin-C	VFDNFVLK
EIDGIELT	Tenascin-C	EIDGIELT
peptide	vitronectin	MAPLRPLLILALLAWVALA
peptide	VEGF	KLTWQELYQLKYKGI
peptide	hGF	MWVTKLLPALLLQHVLLHLLLLPIAIPYAEG
peptide	EGF	MLLTLIILLPVVSKFSFVSLSA
peptide	TGF-βΙ	MPPSGLRLLLLLLPLLWLLVLTPGRPAAG
peptide	IGF-1	MGKISSLPTQLFKCCFCDFLK
peptide	VEGF	MNFLLSWVHWSLALLLYLHHAKWSQA
peptide	synthetic	KLTWQELYQLKYKGI
peptide	NGF	MSMLFYTLITAFLIGIQA
peptide	BMP-2	MVAGTRCLLALLLPQVLLGGAAG
peptide	BMP-4	MIPGNRMLMVVLLCQVLLG
Tat	HIV-Tat protein	PGRKKRRQRRPPQ
Penetratin	Homeodomain	RQIKIWFQNRRMKWKK

Transportan	Galaninmastoparan	GWTLNSAGYLLGKINLKALAALAKKIL
VP-22	HSV-1 structural protein	DAATATRG RSAAS R PTE R P RAPAR- SASRPRRPVD
MPG	HIV Gp41-SV40 NLS	GALFLGFLGAAGSTMGAWSQPKKKRKV
Pep-1	T rp-rich motif- SV40 NLS	KETWWETWWTEWSQPKKKRKV
MAP	chimeric	KA LA KA LA KA LA
SAP	Proline-rich motif	VRLPPPVRLPPPVRLPPP
PPTG1	Chimeric	GLFRALLRLLRSLWRLLLRA
hCT (9-32)	Human calcitonin	LGTYTQDFNKTFPQTAIGVGAP
SynB	Protegrin	RGGRLSYSRRRFSTSTGR
Pvec	Murine VE- cadherin	LLIILRRRIRKQAHAHSK

W may advantageously be comprised of targeting moieties to apply the supramolecular hydrogel materials for drug delivery, gene delivery, and tissue engineering. The targeting moiety can consist of a peptide, aptamers or monoclonal antibodies. W may also advantageously comprise 5 targeting ligands that interact with over-expressed cell surface receptors or intracellular proteins.

W may include CD13 receptors, CD44 receptors, folate receptors (FRs), integrins ανβ3, Integrin a-3, cholecystokinin receptors (CCKRs), integrin ανβ5, somatostatin receptors, endothelin receptors, epidermal growth factor receptors (EGFR), fibroblast growth factor receptors (FGFR), bombesin receptors,transferrin receptors, or prostate-specific membrane antigen (PSMA).

W may advantageously be comprised of aptamers. Aptamers are short nucleic acid sequences of RNA or DNA that can fold into various 3D conformations that show high binding affinity and specificity to various (bio)molecular targets. W may be an aptamer targeting tenascin C, PSMA (A9 and A10), TTA1, PTK7, sgc8c, nucleolin (AS1411), MUC1 (5TR1), Tn antigen (5TRG2), and N-acetylgalactosamine (GalNAc3).

W may advantageously be comprised of monoclonal antibodies. Monoclonal antibodies (mAb or moAb) are antibodies that have monovalent affinity to bind with antigens. They are made by identical immune cells that are all clones of a unique parent cell. W may advantageously comprise of alemtuzumab (CD52), bevacizumab (VEGF), acetuximab, panitumumab and gefinitib (EGFR), gemtuzumab (CD 33), ibritumomab tiuxetan (CD 20), ofatumumab (CD 20), rituximab and tostiumomab, and trastuzumab (HER 2).

Depending on the desired application, a mixture of different amount of different functional 5 monomers with distinct W’s can be self-assembled together to create tailored and functional particles or hydrogels directed towards a particular application as outlined above.

The following examples illustrate the invention:

Examples

Tripodal squaramide-based monomers of formula (I) wherein n in formula (III) is 3-6 were synthesized in the following Examples 1-4 by the following route:

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DIPEA; CHCh reflux evernight _u 5a(n«3) · π 5b

5c (n®5) 5d (n~6)

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-- 8a {n«3)

8b inM}

8c (n-5)

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/ >7 H	H
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Example 1 -- Synthesis of compounds 6a-d

To a solution of benzyl chloroformate (a: 2.94 g, 17.23 mmol; b: 1.89 g, 11.08 mmol; c: 1.98 g, 11.61 mmol; d: 1.11 g, 6.51 mmol) in CH2CI2 (100 ml.) a stirred solution of alkyldiamine (5a: 10.00 g, 86.14 mmol; 5b: 8.03 g, 55.66 mmol; 5c: 10.00 g, 58.05 mmol; 5d: 6.49 g, 32.39 mmol) in CH2CI2 (100 ml.) was added dropwise over 2 hours atO°C. The reaction mixture was allowed to stir overnight at room temperature. The solution was then concentrated by rotary evaporation and ethyl acetate was added. The mixture washed 3x with water and the aqueous layers were discarded. The organic layer was dried over MgSCU and the solvent was removed under vacuum to obtain a white solid (6a) without further purification. For 6b-d, when 1 M HCI was added, a white precipitate formed in the organic layer. The solid was collected by filtration, washed with ethyl acetate and used without further purification.

Compound 6a

Yield: 2.67 g, 62%. 1H-NMR (CD₃OD, 400 MHz): 7.34-7.28 (m, 5H), 5.06 (s, 2H),

3.12- 3.09 (m, 2H), 2.64-2.60 (m, 2H), 1.51-1.43 (m, 4H), 1.35-1.32 (m, 4H). ¹³C-NMR (CD3OD, 100 MHz): 158.85, 138.44, 129.21, 128.90, 128.71,67.24, 42.28, 41.66, 33.33, 30.81,27.56,

27.38. MALDI-TOF-MS: m/z calc: 250.17, found: 250.47 [M+H]⁺.

Compound 6b

Yield: 2.31 g, 75%. ¹H-NMR (CD₃OD, 400 MHz): 7.34-7.27 (m, 5H), 5.06 (s, 2H),

3.12- 3.07 (m, 2H), 2.94-2.89 (m, 2H), 1.69-1.63 (m, 2H), 1.53-1.46 (m, 2H), 1.41-1.31 (m, 8H). ¹³C-NMR (CD3OD, 100 MHz): 158.49, 138.07, 129.13, 128.60, 128.48, 66.82, 41.28, 40.32, 30.41, 29.64, 28.09, 27.20, 26.92, 26.90. MALDI-TOF-MS: m/z calc: 278.20, found: 278.59 [M+H]⁺.

Compound 6c

Yield: 2.77 g, 78%. ¹H-NMR (CD3OD, 400 MHz): 7.34-7.27 (m, 5H), 5.06 (s, 2H),

3.11- 3.08 (m, 2H), 2.93-2.89 (m, 2H), 1.67-1.62 (m, 2H), 1.50-1.46 (m, 2H), 1.40-1.32 (m, 12H). ¹³C-NMR (CD3OD, 100 MHz): 158.47, 138.07, 129.11, 128.56, 128.39, 66.81, 41.35, 40.34, 30.46, 30.08, 29.96, 29.90, 29.74, 28.13, 27.35, 27.00. MALDI-TOF-MS: m/z calc: 306.23, found: 306.65 [M+H]⁺.

Compound 6d

Yield: 1.39 g, 64%. ¹H-NMR (CD3OD, 400 MHz): 7.34-7.27 (m, 5H), 5.06 (s, 2H),

3.12- 3.07 (m, 2H), 2.92-2.89 (m, 2H), 1.67-1.61 (m, 2H), 1.49-1.46 (m, 2H), 1.41-1.30 (m, 16H). ¹³C-NMR (CD3OD, 100 MHz): 158.90, 138.50, 129.43, 128.75, 128.69, 67.24, 41.79,

40.77, 30.91, 30.66, 30.64, 30.60, 30.48, 30.39, 30.22, 28.58, 27.82, 27.45. MALDI-TOF-MS: m/z calc: 334.26, found: 334.67 [M+H]⁺.

Example 2 -- Synthesis of compounds 7a-d

Tetraethyleneglycol monomethyl ether (a: 1.03 g, 4.95 mmol; b: 1.30 g, 6.24 mmol; c: 1.50 g, 7.20 mmol; d: 1.30 g, 6.24 mmol) was first activated with 1,1’-carbonyldiimidazole (a: 0.88 g, 5.44 mmol; b: 1.11 g, 6.85 mmol; c: 1.28 g, 7.92 mmol; d: 1.11 g, 6.87 mmol) for 1 hour at room temperature. Subsequently, 6a-d (6a: 1.49 g, 5.94 mmol; 6b: 2.08 g, 7.49 mmol; 6c: 2.65 g, 8.64 mmol; 6d: 2.50 g, 7.49 mmol), di-isopropylethylamine (DIPEA -- a: 1.7 ml_, 9.90 mmol; b: 2.2 ml_, 12.48 mmol; c: 2.5 ml_, 14.41 mmol; d: 2.2 ml_, 12.48 mmol) and CHCI₃ (15 ml.) were added to the reaction mixture and refluxed overnight. Once the reaction was finished, DCM (15 ml.) was added and washed with H₂O (30 ml_). The combined aqueous fractions were then back-extracted 3x with DCM (3 x 30 ml_). The combined organic fractions were dried with MgSO4, prior to removal of the solvent in vacuo. The crude product was purified by silica column chromatography using a DCM/ethyl acetate gradient (20-50 vol% EtOAc). The product was evaporated to dryness by rotary evaporation to obtain a white solid 7a-d and placed in a vacuum oven overnight.

Compound 7a

Yield: 1.46 g, 61%. ¹H-NMR (CDCI3, 400 MHz): 7.29-7.24 (m, 5H), 5.20 (br s, 1H), 5.11 (brs, 1H), 5.03 (s, 2H), 4.15-4.13 (m, 2H), 3.76-3.58 (m, 12H), 3.50-3.48 (m, 2H), 3.32 (s, 3H), 3.13-3.05 (m, 4H), 1.45-1.38 (m, 4H), 1.27-1.21 (m, 4H). ¹³C-NMR (CDCI3, 100 MHz): 156.23, 136.42, 128.35, 128.01, 127.84, 71.69, 70.37, 70.31, 70.27, 69.43, 66.34, 63.60, 58.79, 40.65, 40.57, 29.62, 26.02. LC-MS: t = 6.66 min, m/z: 485.27 [M+H]⁺. MALDI-TOF-MS: m/z calc: 484.28, found: 506.81 [M+Na]⁺.

Compound 7b

Yield: 2.26 g, 71%. ¹H-NMR (CDCIs, 400 MHz): 7.33-7.25 (m, 5H), 5.06 (s, 2H), 4.98 (brs, 1H), 4.89 (brs, 1H), 4.18-4.16 (m, 2H), 3.65-3.60 (m, 12H), 3.53-3.51 (m, 2H), 3.34 (s, 3H), 3.17-3.08 (m, 4H), 1.47-1.42 (m, 4H), 1.25 (s, 8H). ¹³C-NMR (CDCIs, 100 MHz): 156.17,

136.38, 128.52, 128.17, 127.98, 71.59, 70.27, 70.21, 70.17, 69.35, 66.24, 63.46, 59.03, 40.76, 40.67, 29.59, 28.83, 26.32. LC-MS: t = 7.37 min, 513.33 m/z [M+H]⁺. MALDI-TOF-MS: m/z calc: 512.31, found: 534.88 [M+Na]⁺.

Compound 7c

Yield: 2.45 g, 63%. ¹H-NMR (CDCh, 400 MHz): 7.35-7.29 (m, 5H), 5.08 (s, 2H), 4.83 (brs, 1H), 4.77 (brs, 1H), 4.21-4.19 (m, 2H), 3.68-3.62 (m, 12H), 3.55-3.53 (m, 2H), 3.37 (s, 3H), 3.20-3.11 (m, 4H), 1.49-1.43 (m, 4H), 1.32-1.24 (m, 12H).¹³C-NMR (CDCIs, 100 MHz): 156.50, 136.75, 128.35, 128.20, 128.16, 71.99, 70.66, 70.61, 70.57, 69.75, 66.62, 63.86, 59.06, 41.18, 41.10, 30.00, 29.48, 29.28, 26.78. LC-MS: t = 8.09 min, m/z: 541.33 [M+H]⁺. MALDI-TOF-MS: m/z calc: 540.34, found: 562.82 [M+Na]⁺.

Compound 7d

Yield: 1.70 g, 48%. Ή-NMR (CDCIs, 400 MHz): 7.35-7.27 (m, 5H), 5.08 (s, 2H), 4.87 (brs, 1H), 4.77 (br s, 1H), 4.21-4.18 (m, 2H), 3.70-3.61 (m, 12H), 3.54-3.52 (m, 2H), 3.37 (s, 3H), 3.19-3.10 (m, 4H), 1.48-1.43 (m, 4H), 1.26-1.23 (m, 16H). ¹³C-NMR (CDCIs, 100 MHz): 156.53, 136.78, 129.01, 128.61, 128.18, 72.02, 70.70, 70.64, 70.60, 69.79, 66.66, 63.89, 59.13, 41.22, 41.14, 30.04, 29.60, 29.35, 26.83. LC-MS: t = 8.80 min, 569.40 m/z: [M+H]⁺. MALDI-TOF-MS: m/z calc: 568.37, found: 590.87 [M+Na]⁺.

Example 3 -- Synthesis of compounds 8a-d

Compound 7a-d (7a: 1.33 g, 2.75 mmol; 7b: 1.74 g, 3.40 mmol; 7c: 1.14 g, 2.11 mmol; 7d: 1.17 g, 2.05 mmol) was dissolved in anhydrous methanol (10 mL), and Pd/C (a: 29.80 mg, 0.28 mmol; b: 36.18 mg, 0.34 mmol; c: 22.35 mg, 0.21 mmol; d: 22.35 mg, 0.21 mmol) was added. The solution was degassed with nitrogen, prior to the dropwise addition of triethylsilane (a: 4.4 mL, 27.50 mmol; b: 5.4 mL, 34.00 mmol; c: 3.4 mL, 21.10 mmol; d: 3.3 mL, 20.50 mmol). The addition of triethylsilane resulted in the formation of an effervescent solution and once the reaction was complete (as demonstrated by TLC), the solution was filtered through Celite to remove the remaining Pd/C. The filtrate was concentrated by rotary evaporation and afterwards, a gentle stream of nitrogen gas. The dried product was redissolved in chloroform (15 mL). 3,4-Dibutoxy-3-cyclobutene-1,2-dione (Dibutyl Squarate -a: 0.65 mL, 3.03 mmol; b: 0.81 mL, 3.74 mmol; c: 0.50 mL, 2.32 mmol; d: 0.49 mL, 2.26 mmol) and DIPEA (a: 0.95 mL, 5.50 mmol; b: 1.1 mL, 6.80 mmol; c: 0.74 mL, 4.22 mmol; d: 0.71 mL, 4.10 mmol) were added to the reaction mixture and stirred at room temperature overnight. Subsequently, DCM (15 mL) was added and washed with H₂O (30 mL). The aqueous fractions were back-extracted 3x with DCM (3 x 30 mL). The organic fractions were combined and dried with MgSCu, prior to removing the solvent in vacuo. The crude product was further purified by silica gel column chromatography using a DCM/ethyl acetate gradient (10-50 vol% EtOAc). The productwas concentrated by rotary evaporation to provide an oil (8a-d) that was further dried in a vacuum oven overnight.

Compound 8a

Yield: 1.09 g, 73%. ¹H-NMR (CDCb, 400 MHz): 5.14 (brs, 1H), 4.71-4.68 (m, 2H), 4.17-4.15 (m, 2H), 3.64-3.59 (m, 12H), 3.52-3.50 (m, 2H), 3.38-3.35 (m, 2H), 3.33 (s, 3H), 3.14-3.09 (q, 2H), 1.76-1.71 (m, 2H), 1.59-1.56 (m, 2H), 1.47-1.30 (m, 8H), 0.95-0.89 (m, 3H). ¹³C-NMR (CDCb, 100 MHz): 189.71, 182.75, 177.52, 172.47, 156.64, 73.45, 71.87, 70.54, 70.49, 70.46, 70.43, 69.62, 63.82, 58.99, 44.65, 40.70, 32.03, 30.43, 29.77, 26.10, 25.90, 18.67, 13.70. LC-MS: t = 6.20 min, 503.20 m/z [M+H]⁺. MALDI-TOF-MS: m/z calc: 502.29, found: 524.81 [M+Na]⁺.

Compound 8b

Yield: 1.23 g, 68%. ¹H-NMR (CDCb, 400 MHz): 7.47 (br s, 1H), 5.10 (br s, 1H), 4.60-4.57 (m, 2H), 4.08-4.03 (m, 2H), 3.53-3.48 (m, 12H), 3.41-3.39 (m, 2H), 3.30-3.24 (m, 2H), 3.22 (s, 3H), 3.01-2.96 (q, 2H), 1.66-1.60 (m, 2H), 1.48-1.45 (m, 2H), 1.35-1.16 (m, 12H), 0.84-0.78 (m, 3H). ¹³C-NMR (CDCb, 100 MHz): 189.33, 182.88,176.97, 172.30, 156.27, 72.99, 72.84, 71.57, 70.23, 70.19, 70.16, 70.12, 69.30, 63.47, 58.66, 44.52, 40.64, 31.72, 30.29, 29.57, 28.81, 28.73, 26.32, 26.02, 18.37, 13.41. LC-MS: t = 6.83 min, m/z: 531.33 [M+H]⁺. MALDI-TOF-MS: m/z calc: 530.32, found: 552.87 [M+Na]⁺.

Compound 8c

Yield: 0.81 g, 69%. ¹H-NMR (CDCb, 400 MHz): 6.59 (br s, 1H), 4.91 (br s, 1H),

4.74- 4.71 (m, 2H), 4.20-4.18 (m, 2H), 3.67-3.62 (m, 12H), 3.55-3.52 (m, 2H), 3.41-3.36 (m, 2H), 3.36 (s, 3H), 3.16-3.11 (q, 2H), 1.79-1.75 (m, 2H), 1.61-1.57 (m, 2H), 1.48-1.26 (m, 16H), 0.97-0.94 (m, 3H). ¹³C-NMR (CDCb, 100 MHz): 189.77, 182.84,177.42, 172.66, 156.63, 73.42, 72.02, 70.68, 70.63, 70.61, 70.58, 69.75, 63.91, 59.11, 44.99, 41.13, 32.15, 30.77, 30.04, 29.53, 29.34, 29.23, 26.83, 26.50, 18.80, 13.83. LC-MS: t = 7.60 min, m/z: 559.27 [M+H]⁺. MALDI-TOF-MS: m/z calc: 558.35, found: 580.91 [M+Na]⁺.

Compound 8d

Yield: 0.62 g, 52%. Ή-NMR (CDCb, 400 MHz): 6.23 (brs, 1H), 4.87 (brs, 1H),

4.75- 4.67 (m, 2H), 4.21-4.19 (m, 2H), 3.68-3.63 (m, 12H), 3.56-3.53 (m, 2H), 3.44-3.41 (m, 2H), 3.37 (s, 3H), 3.17-3.12 (q, 2H), 1.62-1.56 (m, 2H), 1.49-1.25 (m, 22H), 0.98-0.95 (m, 3H). ¹³C-NMR (CDCb, 100 MHz): 189.66, 182.70, 177.40, 172.43, 156.40, 73.36, 71.89, 70.56, 70.51, 70.47, 69.66, 63.79, 59.00, 44.89, 41.02, 32.01, 30.66, 29.92, 29.49, 29.45, 29.23, 29.12, 26.72, 26.37, 18.66, 13.68. LC-MS: t = 8.32 min, m/z: 587.27 [M+H]⁺. MALDI-TOF-MS: m/z calc: 586.38, found: 608.55 [M+Na]⁺.

Example 4 -- Synthesis of tripodal squaramide-based monomers 1-4

Compound 8a-d (8a: 0.52 g, 1.04 mmol; 8b: 0.49 g, 0.92 mmol; 8c: 0.64 g, 1.15 mmol; 8d: 0.32 g, 0.54 mmol) was dissolved in chloroform (15 ml_). Tris(2-aminoethyl)amine (a: 46 μΙ_, 0.31 mmol; b: 40 pl_, 0.27 mmol; c: 52 μΙ_, 0.35 mmol; d: 24 μΙ_, 0.16 mmol) and DIPEA (a: 181 μΙ_, 1.04 mmol; b: 160μ!_, 0.92 mmol; c: 200μ!_, 1.15 mmol; d: 94μΙ_, 0.54 mmol) were added to the reaction mixture. The solution was refluxed overnight and purified by flash column chromatography on a C18 silica gel column using a gradient of 10-90% CH3CN/H2O over 35 minutes. The product was concentrated by rotary evaporation and lyophilized to provide a white solid (compounds 1-4).

Monomer 1

Yield: 0.22 g, 51%. Ή-NMR (DMSO-d₆, 400 MHz): 7.46 (br s, 3H), 7.27 (brs, 3H), 7.18 (brs, 3H), 4.03-4.01 (m, 6H), 3.55-3.40 (m, 54H), 3.23 (s, 9H), 2.96-2.93 (m, 6H), 2.702.68 (m, 6H), 1.49-1.26 (m, 24H). ¹³C-NMR (DMSO-d₆, 100 MHz): 182.65, 168.27, 167.77, 156.42, 71.54, 70.07, 70.03, 69.98, 69.84, 69.16, 63.27, 58.31, 54.96, 43.53, 41.83, 40.40, 30.96, 29.57, 26.13, 25.85. LC-MS: t = 5.14 min, m/z: 1431.87 [M+H]⁺. MALDI-TOF-MS: m/z calc: 1430.80, found: 1452.69 [M+Na]⁺.

Monomer 2

Yield: 0.22 g, 54%. Ή-NMR (DMSO-d₆, 400 MHz): 7.53 (br s, 3H), 7.34 (br s, 3H), 7.14 (br s, 3H), 4.04-4.01 (m, 6H), 3.58-3.40 (m, 54H), 3.23 (s, 9H), 2.96-2.91 (m, 6H), 2.73 (s, 6H), 1.53-1.21 (m, 36H). ¹³C-NMR (DMSO-d_s, 100 MHz): 182.63, 168.28, 167.71, 156.45, 71.61, 70.14, 70.11, 70.07, 69.91, 69.25, 63.32, 58.30, 55.05, 43.66, 41.60, 40.52, 31.05, 29.98, 29.72, 29.08, 29.02, 26.60, 26.21. LC-MS: t = 5.93 min, m/z: 1515.67 [M+H]⁺. MALDITOF-MS: m/z calc: 1514.81, found: 1536.80 [M+Na]⁺.

Monomer 3

Yield: 0.30 g, 52%. Ή-NMR (DMSO-d₆, 400 MHz): 7.49 (brs, 3H), 7.30 (brs, 3H), 7.18 (brs, 3H), 4.04-4.02 (m, 6H), 3.56-3.38 (m, 54H), 3.24 (s, 9H), 2.96-2.91 (q, 6H), 2.69 (s, 6H), 1.49-1.23 (m, 48H). ¹³C-NMR (DMSO-d₆, 100 MHz): 182.59, 168.23, 167.68, 156.36, 71.52, 70.05, 70.02, 69.96, 69.82, 69.15, 63.22, 58.29, 54.97, 43.55, 41.63, 40.44, 30.97, 29.66, 29.28, 29.25, 29.02, 28.94, 26.53, 26.15. LC-MS: t = 6.89 min, m/z: 1599.87 [M+H]⁺. MALDI-TOF-MS: m/z calc: 1598.99, found: 1622.42 [M+Na]⁺.

Monomer 4

Yield: 0.11 g, 42%. Ή-NMR (DMSO-d₆, 400 MHz): 7.61 (br s, 3H), 7.45 (brs, 3H), 7.16 (brs, 3H), 4.03-4.01 (m, 6H), 3.57-3.40 (m, 54), 3.23 (s, 9H), 2.95-2.90 (q, 6H), 2.64 (s, 6H), 1.51-1.21 (m, 60H). ¹³C-NMR (DMSO-d₆, 100 MHz): 182.40, 182.27, 168.03, 167.45, 156.17, 71.33, 69.86, 69.83, 69.78, 69.64, 68.96, 63.03, 58.09, 54.80, 43.38, 41.43, 40.25,

30.76, 29.47, 29.14, 29.12, 28.86, 28.77, 26.35, 25.96. LC-MS: t = 7.81 min, m/z: 1684.73 [M+H]⁺. MALDI-TOF-MS: m/z calc: 1683.08, found: 1706.45 [M+Na]⁺.

Example 5 -- Self-assembly of monomers 2 and 3

The sol-to-gel transition of monomers 2 and 3 in aqueous solution was tested by the gel inversion method. In this regard, 2.5 mg (V1, 3.0 mg (V2), 3.5 mg (V3), 4.0 mg (V4), 4.5 mg (V5) and 5.0 mg (V6) of each monomer was placed in 500 pl_ of deionized water in a glass vial (2 ml_). The vials were sonicated 20 minutes in an ultrasound bath at 298K. The two monomers were poorly soluble in the deionized water, even with heating, but their sonication at room temperature in an ultrasonic bath resulted in dissolution and formation of transparent hydrogels. Monomer 3 formed a hydrogel 3 directly after sonication when above its critical gelation concentration (CGC) of 1.3-1.9 mM, but monomer 2 required a significantly higher concentration (4.0-4.6 mM) and a longer period of time to form a hydrogel 2.

Example 6 - Analysis of hydrogels formed from monomers 1,2 and 3

The mechanical properties of the hydrogels 2 and 3 (formed from monomers 2 and 3, respectively) were quantified using oscillatory rheology measurements both in deionized water at 25 °C and also in phosphate buffered saline (PBS) at 37 °C and a pH of 7.4. See Figures 1 and 2. The linear viscoelastic regime (LVE) was first determined by an amplitude experiment. For hydrogel 3 in deionized water at 25 °C or in PBS at 37 °C, the storage (G’) and loss (G”) moduli remained constant until 3% strain at fixed frequency of 1 Hz, whereas for hydrogel 2, both variables remained constant until the application of 10% strain. In frequency sweep experiments of hydrogels 2 and 3, G’ was found to be greater than G” by nearly an order of magnitude and frequency independent over the measuring range from 0.01 to 2 Hz, synonymous with the formation of a viscoelastic material. The hydrogels 2 and 3 at 37 °C in PBS (G’ = 37 Pa for 2; G’ = 64 Pa for 3) showed similar mechanical proper-ties to those at 25 °C in deionized water (G’ = 46 Pa for 2; G’ = 67 Pa for 3). Moreover, step-strain experiments were performed to examine the potential of the supramolecular hydrogels 2 and 3 to self-recover after the application of large strain. When a large amplitude strain was applied (200% for hydrogel 2 and 100% for hydrogel 3 at frequency of 1 Hz, in deionized water at 25 °C or in PBS at 37 °C) for 120 seconds, the G’ value of both hydrogels 2 and 3 decreased and showed an inversion of modulus (G”>G’). However, when the large amplitude strain was removed, both hydrogels 2 and 3 quickly recovered back to the gel state (G’>G”). The recovery of the hydrogel materials after the application of a cyclic strain was demonstrated over 2 cycles.

The structure of the hydrogels 3 was analysed by transmission electron (cryoTEM) microscopy. In this regard, stock solutions of hydrogels 2 (5.6 mM) and 3 (3.1 mM) were prepared in deionized water (1.5 mM) and sonicated for 20 minutes sonication in an ultrasonic bath. Cryo-TEM samples were prepared by applying 3 pl_ of the hydrogel 2 (5.6 mM) and 3 (3.1 mM), or resulting solution (1.5 χ 10^-5 M) without further dilution to a freshly glow-discharged 300 mesh copper grid with a lacey-carbon support film (Supplier-Electron Microscopy Sciences, Pennsylvania, USA). Excess liquid was blotted away for 2 seconds (95% humidity, 21 °C, Whatman No.4 filter paper) and plunge-frozen in liquid ethane at 183 °C using a Leica EM GP (Leica Microsystems, Wetzlar, Germany) before imaging.

Probing of the network at the nanoscale by cryo-TEM revealed fibers greater than a micron in length with a width of 5.4±1.0 nm for hydrogel 2 (5.6 mM). See Figure 3. Probing of the network at the nanoscale by cryo-TEM revealed fibers greater than a micron in length with a width of 4.2±1.1 nm for hydrogel 3 (3.1 mM). See Figure 4. Cryo-TEM tomography was further executed on hydrogel 3, providing a view into the organization of the 3D fibrillar network. See Figure 5.

From this, it appears that the gel properties of the hydrogels of monomers of formula (I) arise from the entanglements of their self-assembled fibers of their squaramidebased monomers of formula (I). (Figure 5). These results were supported by small angle X-ray scattering experiments (SAXS) in the solution phase, where scattering profiles showed the formation of high-aspect ratio one-dimensional aggregates with a length beyond the resolution of the instrument. The data was best described with a form factor for flexible cylinders, which yielded to a cross-sectional radius (res) of 2.6 nm, a cross-sectional mass per unit length (ML) of 2.42 10-21 g nm^-1 and a Kuhn length of 6.6 nm, See Figure

6. By applying equations 1 and 2:

(1) (2) and by estimation of the L from Figure 7, 1 monomer/nm was determined along the fibrillar axes. These results indicate that transitioning from a bolaamphiphilic to a pseudo-C3 symmetric monomer geometry reduces lateral monomer aggregation and increases fibre length and flexibility. Bolaamphiphiles are amphiphilic molecules that have hydrophilic groups at both ends of a sufficiently long hydrophobic hydrocarbon chain. Compared to single-headed amphiphiles, the introduction of a second head-group generally induces a higher solubility in water, an increase in the critical micelle concentration, and a decrease in aggregation number. The aggregate morphologies of bolaamphiphiles include spheres, cylinders, disks, and vesicles. Bolaamphiphiles are also known to form helical structures, that can form monolayer microtubular self-assemblies.

Spectroscopic methods showed the effect of the hydrophobic-hydrophilic balance of the monomers 1 to 3 on squaramide self-assembly. Monomers 2 and 3 (1.5 x 10'⁵ M) showed two distinct absorption bands at 255 and 329 nm consistent with selfassembly of the squaramide moiety. (See Figure 8A). Their self-assembly were further supported by cryo-TEM performed on dilute solutions (1.5 x 10^-5 M) of monomers 2 and 3 in deionized water which showed the presence of long fibers. (See Figure 9.) Concentration-dependent measurements of monomers 2 and 3 displayed retention of these bands even at the lowest concentration: 3.75 x 10 ⁶ M for monomer 2 and 1.5 x 10^-6 M for monomer 3. (See Figure 10.) Conversely, compound 1 with the shortest alkyl spacer (n = 6) displayed only a single band. This result is on par with UV-Vis spectra collected upon the addition of hexafluoroisopropanol (HFIP), a hydrogen bond disrupting solvent, and was suggestive of depolymerization of monomers 2 and 3. (See Figure 11.) Fluorescence measurements using Nile Red as a probe of the hydrophobic environment support the results of the UV-Vis measurements. (See Figure 8B.) Monomer 1 has a similar fluorescence intensity and maximum wavelength as the Nile Red dye in water, while compound monomer 3 showed the greatest increase in fluorescence intensity and blueshifting (622 nm) of the Nile Red peak (659 nm) relative to 2 (633 nm). Fourier transform infrared (FTIR) spectroscopy on lyophilized monomers (3.1 mM) from gel inversion experiments provided additional evidence for the hydrogen-bonding and hydrophobic interactions between the tripodal squaramide-based supramolecular monomers. Monomers 1 to 3 displayed two distinct frequencies for the N-H stretch of the carbamate (3317 cnr¹-3315 cm'¹) and squaramide units (3167 cm'¹-3164 cm'¹). Additionally, the antisymmetric and symmetric C-H stretches were shifted to lower wavenumbers from 2931 cm'¹, 2861 cm'¹ to 2918 cm^-1, 2850 cm'¹, respectively, with increasing length of the alkyl chains (from n = 6 to n = 10); these are indicative of their close packing within the supramolecular polymer. (See Figure 8C, inset). All monomers showed a small broad band at 1799 cm^-1 associated with ring breathing of the squaramide unit and one or two C=O stretching modes of the carbamates and squaramides in the amide I region. (See Figures 9C and 13). The variable number of bands in the amide I region suggested that more than one packing mode exists for the various monomers in the self-assembled state.

From these analyses, it is believed that the squaramide unit can be employed to robustly self-assemble flexible amphiphilic monomers through hydrophobic and hydrogen-bonding interactions into long fibrillar aggregates and eventually gel phase materials in water. The mechanically soft and self-recovering character of these materials is believed to be advantageous for the culture of induced pluripotent stem cells due to their mechanical similarity with tissues found in the embryonic microenvironment and their potential for gentle encapsulation and release of various cell types, including human pluripotent stem cells.

Example 7 - Cytocompatibility of supramolecular polymers and hydrogels formed from monomers 1,2 and 3 and their application in 3D cell cultures

To apply the tripodal squaramide-based monomers of formula (I) as supramolecular polymers or hydrogel scaffolds in the biomedical area, the cytocompatibility of the monomers was first evaluated against NIH 3T3 cells. The cytotoxicity of monomers 13 with increasing concentration (1-200 μΜ) in both deionized water and PBS was evaluated by an MTT cytotoxicity assay. (See Figure 13.) The cell viability was approximately 95% for monomers 1-3 as the concentration was increased from 1 to 200 μΜ and applied for 24 h and 72 h, on par with the control sample. These results indicate that supramolecular polymers and hydrogels constructed from monomers 1-3 in the solution-phase are cytocompatible.

Hydrogels formed from cytocompatible monomers 2 and 3 were further examined for their capacity to encapsulate cells in a 3D environment using the self-recovery character of the material. For this purpose, supramolecular hydrogels shown schematically in Figure 14 were used for 3D cell seeding. A suspension of NIH 3T3 cells (5 χ 10⁶ cells/ml_, 20 μΙ_) were pipetted with preformed hydrogels of monomer 2 (6.2 mM, 180 μΙ_) or of monomer 3 (3.4 mM, 180 μΙ_) to provide final gel concentrations above the CGC of the squaramide-based hydrogelator (5.6 mM for hydrogel of monomer 2 and 3.1 mM for hydrogel of monomer 3). For both hydrogels, 99% and 98% of the cells, respectively, were viable and homogeneously dispersed in 3D 2h after seeding process as calculated by counting of viable cells in z-stack images collected by confocal microscopy (Figure 15 and Figure S17). Moreover, after 48 h of seeding, 74% of cells seeded in 3D in hydrogel 2 and 77% of cells in hydrogel 3 remained viable, as evidenced by the numerous Calcein AM positive cells and very few propidium iodide stained cells.

These tripodal squaramide-based supramolecular materials were further examined for their suitability for the 3D cell culture of hiPSC and hiPSC-derived endothelial cells (hiPSC-ECs). First, hiPSC-ECs were encapsulated in a hydrogel of monomer 3 (with a final concentration of 3.1 mM) using the same technique of Figure 14 as for the NIH 3T3s. The majority of hiPSC-ECs were well-dispersed throughout the gel and remained viable after 24 h, as observed by the virtual absence of NucGreen® Dead positive cells. (See Figure 17.) Despite being viable, no cell proliferation or cell attachment/spreading of the hiPSC-ECs was observed in the hydrogel. This result could be expected based on the lack of adherent cues (e.g. RGD) within the supramolecular matrix. Surprisingly, the encapsulation of undifferentiated hiPSCs as single cells in the supramolecular hydrogel of monomer 3 resulted in the formation of compact spheroids of increasing diameter after 24 h when seeded in 3D. Their formation was followed by time-lapse microscopy over a period of 72 h. Importantly, the hiPSC spheroids remained viable as observed by their morphology and the largely absent staining of the dead cells in the spheroid cluster with the NucGreen® Dead reagent after 24 h and 72 h. (See Figures 19A and 19B.) The evolution of the hiPSC spheroid diameter after encapsulation in the hydrogel of monomer 3 (3.1 mM) with time (after 24 h, 48 h and 72 h culture) trended to larger sizes with longer culture periods (after 72 h, spheroid diameters ranged from 26-143 pm from single cells on Day 0) (representative images of hiPSC 3D spheroids in hydrogel are shown in Figure 18C and Figure 19). These results show the potential of these supramolecular materials for application in the culture of hPSCs and their derivatives.

A major difficulty in the 3D culture of hiPSCs is their release from the 3D cultures for further processing or movement since even enzymatic methods may not be effective. This challenge has prompted the development of materials that enable cell release in response to temperature change or UV light. Therefore, cells were tested to determine whether they could be easily released from the hydrogels, formed from monomers of formula (I), without affecting viability, for example by simple mechanical disruption of the hydrogels by pipetting and dilution the hydrogels. To release the cells, hiPSC spheroid-laden hydrogels were diluted into a 50x greater volume of culture medium.

Cells after 24 h of culture in 2D with conventional cell culture conditions or after 24 h in 3D in the supramolecular hydrogels were released and collected. Cell surface expression of TRA-1-60 and SSEA-4, pluripotent stem cell markers associated with the undifferentiated state, were analysed by fluorescent activated cell sorting (FACS). hiPSCs cultured ether in 5 2D or 3D were positive for TRA-1 -60 and SSEA-4 pluripotent stem cell surface markers, as shown using histograms of fluorescence intensity of the markers in gated live cell population. (See Figure 18D.) The values of mean fluorescence intensity (MFI) also increased in TRA-1-60 and SSEA-4 level with the longer culture time in hydrogel. See

Figure 20. Moreover, the percentage of live hiPSCs cells, as determined by side scatter 10 (SSC-A) and forward scatter (FSC-A) events, after 24 h were comparable for either 2D or

3D culture. Thus, the squaramide-based supramolecular hydrogels, formed from monomers of formula (I), proved cytocompatible towards any of the cell types tested including sensitive cells such as hiPSCs and their derivatives. Importantly, the pluripotent stem cells showed spheroid formation from single cells and retained their undifferentiated 15 state within the supramolecular polymer materials.

Claims (19)

CONCLUSIONS A supramolecular tripodal squaramide-based monomer according to formula (I): wherein T represents a central atom; n is an integer from 1 to 12; and R ^{1 is} a group according to formula (Ba) or (Ilb): wherein R ^{2 is} a hydrophobic group; R 'is a hydrophilic group, and X and / or Y 20 independently represent -NH-; -NR ⁴ -; O or 0, or The monomer of claim 1, wherein R ^{3 comprises} a polyethylene glycol chain. A monomer according to claim 1 or claim 2, wherein R ^{2 is} an alkylene group, preferably a straight-chain alkylene group. A monomer according to claim 3, wherein R ^{2 is} a straight-chain alkylene group with 4-20 carbon atoms, preferably with 6-12 carbon atoms, and more preferably with 8-10 carbon atoms. A monomer according to any one of claims 1 to 4, wherein R ^{3 is} a group of formula (III): n (lil), wherein n is 2-10, preferably 3-6, and even better 4 or 5. The monomer of claim 1, wherein R ^{1 is} a group of formula (III) wherein a represents an optionally substituted alkyl, alkenyl, and / or alkinyl group, wherein a is an integer comprised between 1 and 15; and p is between 10 and 10,000; and W stands for a functional anchor group. The monomer of claim 1, wherein T is selected from nitrogen, boron, phosphorus, silicon, or carbon, wherein T is preferably boron, nitrogen, phosphorus, and wherein the core has a C3 symmetry. The monomer of any one of claims 1 to 4, wherein n represents 1 to 6 carbon atom chains connecting T and the squaramide group. A monomer according to any one of the preceding claims, wherein the core is derived from any of the following compounds T1 to T5: NH ₂ T5 T3 T4 10, Monomer according to one of the claims 1 to 9, represented by a structural formula (IV): (IV), wherein R is as defined above. A supramolecular polymer produced by an auto-assembly of the tripodal squaramide-based monomer according to any of claims 1 to 10 in an aqueous solvent, preferably in water. A supramolecular polymer according to claim 11, in the form of fibers, which can preferably form a hydrogel. A method of polymerizing the monomer according to any of claims 1 to 10, comprising: a) providing the monomer, and b) subjecting the monomer to an auto assembly in the presence of water A supramolecular polymer obtained from a polymerization of the monomer according to any of claims 1 to 10 for culturing cells, including pluripotent stem cells, preferably human pluripotent stem cells (hPSCs), even better for the culturing human-induced pluripotent stem cells (hiPSCs). Use of a monomer according to any of claims 1 to 10 for the preparation of a supramolecular polymer. 1/19 G (Pa) θ '- ^G ( ^Pa ) θ' · ^G < ^Pa ) * 100 100 Strain (%) Frequency (Hz) Frequency (Hz) Strain Strain (%) Strain (%) Time (sh Figure 1 Is 2/19 0.1 4 «τÖ.01 oS 10 100 Strain (%) 5 10-a. G j G © 1 ;; © i 1 y ^{0 1} --οϊ ^r - ^^ 0J ——r Frequency (Hz) Time (s) Figure 2 3/19 Figure 3 4/19 (; i Figure 4 5/19 Figure 5 6/19 Figure 6 7/19 Figure 7 8/19 Figure 8 9/19 Figure 9 10/19 Figure 10 Absorbance (A.U.) 0.8 η Wavelength (nm) 11/19 Wavelength (nm) Figure 11 12/19 Ceii Viability {%} Ceii Viability (%) Figure 12 13/19 2 (π = 8) 3 (π = 40) 4 (π ~ 12) PBS (οΗ-7.4}:> sonication 20 msnut.es and overnight equilibration supramolecular hydrogel 3D cehscÈClen supramolecular hydrogel Figure 13 14/19 Figure 14 15/19 Calcein AM PI • SS: Figure 15 16/19 Figure 16 17/19 SFL86 (2D) SFL86 (30: .. 24h Figure 17 5SC-A 'Jf.W ·. J; vK your: W1X a ’: vK FSC-A Control SSEA-4 18/19 Figure 18 19/19 TRA-1-60 Figure 19