Dynamic covalent hydrogels, precursors thereof and uses thereof

Abstract

The present invention provides cross-linking pairs of hydrogel precursor polymers, dynamic covalent hydrogels prepared from the cross-linking pairs of hydrogel precursors, pharmaceutical compositions comprising the precursors or hydrogels, and uses thereof in various applications.

Description

[0001] Related Applications

[0002] This application claims priority to European Patent Application No. EP 2016202.2, filed March 10, 2020, which is incorporated herein by reference in its entirety. BACKGROUND

[0003] Polymeric networks are formed through physical or chemical crosslinking of functional monomers or polymer precursors. Physically crosslinked materials are bound together through non-covalent, reversible interactions (De Greef et al., Chem. Rev., 2009, 109: 5687-5754). This generally results in shear-thinning (viscous flow upon increasing shear) and self-healing (reformation of gel properties upon cessation of shear) materials, as the bonds can break and reform in response to external stimuli, including mechanical loading. However, the structures thus obtained are generally unstable to small environmental perturbations and lack strength. Chemically crosslinked networks, on the other hand, are bound together through covalent bonds (Wichterle et al., Nature, 1960, 185: 117-118). The elastic gels that result generally have better mechanical properties than physical networks. However, the irreversibility of chemical crosslinking limits their use in application scenarios that require shear-thinning and self-healing properties, such as 3D printing and minimally invasive drug delivery.

[0004] Recent research has introduced a new class of soft materials based on dynamic covalent chemistry that combines the advantages of both physically and chemically crosslinked materials (Kloxin et al., Chem. Soc. Rev., 2013, 42: 7161-7173). In this approach, reversible covalent bonds are formed in a network that can break and reform on the experimental timescale. Thus, these dynamic covalent networks rearrange due to exchange of bonds, enabling stress relaxation and material flow. Crosslinking reactions that have been used for dynamic covalent network formation include ester exchange, Diels-Alder cycloaddition, and boronate complexation. The reversible formation of boronate esters between boronic acids and molecules containing cis-1,2 or cis-1,3 diols has emerged as a safe and synthetically tractable dynamic covalent crosslinking motif for the design of stimuli-responsive biomedical materials. However, boronate complexation is generally preferred at basic pH, common diol coupling partners tend to yield relatively unstable condensation products, and exhibit sensitivity to oxidation.

[0005] Accordingly, there remains a need in the art for improved boronic acid / diol pairs suitable for producing dynamic covalent hydrogels useful in drug controlled release, cell culture, tissue engineering, 3D printing, and the like. SUMMARY

[0006] The present inventors have identified a new boronic acid / diol pair for the formation of dynamic covalent hydrogels. The new boronic acid / diol pair of the present invention has multiple advantages compared to the systems known in the art. In particular, the formation of the hydrogels can be carried out in one step at physiological conditions of pH and temperature. Furthermore, the cross-linking reaction is essentially instantaneous. The hydrogels thus obtained have a high temporal stability, self-healing properties and a minimal swellability, and have viscoelastic properties similar to living tissues. Due to their advantageous shear-thinning properties, the polymer compositions are injectable. Moreover, the composition of the hydrogels can be easily varied, thereby adjusting their biodegradability and / or mimicking living tissues or environments.

[0007] Thus, the present invention relates to a cross-linking pair of hydrogel precursor polymers comprising: (1) a first hydrogel precursor polymer consisting of a first polymer modified with a phenylboronic acid or a phenylboronic acid derivative, and (2) a second hydrogel precursor polymer consisting of a second polymer modified with glucosamine.

[0008] In certain embodiments, the first polymer is grafted with a phenylboronic acid or a phenylboronic acid derivative, and the second polymer is grafted with glucosamine.

[0009] In certain embodiments, the first and second polymers are independently selected from the group consisting of natural polymers, semi-synthetic polymers and synthetic polymers.

[0010] In certain embodiments, at least one of the first and second polymers is selected from the group consisting of biocompatible, biodegradable, hydrophilic natural polymers, semi-synthetic polymers and synthetic polymers.

[0011] In certain embodiments, the first and second polymers are the same. In other embodiments, the first and second polymers are different.

[0012] In certain embodiments, the phenylboronic acid derivative is an ortho-, meta- or para-monosubstituted phenylboronic acid, a Wulff-type phenylboronic acid or a benzoboroxole. In certain preferred embodiments, the phenylboronic acid derivative is a Wulff-type phenylboronic acid.

[0013] In certain embodiments, the first hydrogel precursor polymer or the second hydrogel precursor polymer is directly or indirectly covalently linked to a bio-orthogonal functional moiety or a clickable moiety.

[0014] In certain embodiments, the cross-linking pair of hydrogel precursor polymers is such that the first hydrogel precursor polymer is in a first aqueous solution; and the second hydrogel precursor polymer is in a second aqueous solution, the first and second aqueous solutions being separate aqueous solutions. At least one of the first and second aqueous solutions can comprise an ingredient selected from the group consisting of cells, bioactive agents, visualizing agents and any combination thereof.

[0015] The present application also relates to a dynamic covalent hydrogel consisting of a first hydrogel precursor polymer and a second hydrogel precursor polymer of a cross-linking pair as defined herein, wherein the first and second hydrogel precursors are cross-linked via dynamic covalent bonds. The dynamic covalent hydrogel can further comprise an ingredient selected from the group consisting of cells, bioactive agents, visualizing agents, and any combination thereof.

[0016] The present application also relates to a pharmaceutical composition comprising a cross-linking pair of a hydrogel precursor polymer as defined herein or a dynamic covalent hydrogel as defined herein, and at least one pharmaceutically acceptable carrier or excipient.

[0017] In certain embodiments, the pharmaceutical composition is such that the first hydrogel precursor polymer and the second hydrogel precursor polymer are contained in a multi-barrelled syringe, preferably a double-barrelled syringe.

[0018] The present application also relates to a cross-linking pair of a hydrogel precursor polymer as defined herein, or a dynamic covalent hydrogel as defined herein, or a pharmaceutical composition as defined herein, for use as a therapeutic agent.

[0019] The present application also relates to a cross-linking pair of a hydrogel precursor polymer as defined herein, or a dynamic covalent hydrogel as defined herein, or a pharmaceutical composition as defined herein, for use as a therapeutic agent, for example in cell therapy, tissue engineering, regenerative medicine, viscoelastic supplementation therapy, delivery of cells and / or bioactive agents in vivo.

[0020] The present application also relates to a kit comprising a cross-linking pair of a hydrogel precursor polymer as defined herein, or a dynamic covalent hydrogel as defined herein, or a pharmaceutical composition as defined herein, and instructions for use of the cross-linking pair, dynamic covalent hydrogel or pharmaceutical composition.

[0021] The present application also relates to a method for preparing a dynamic covalent hydrogel comprising the step of mixing a first hydrogel precursor polymer and a second hydrogel precursor polymer of a cross-linking pair as defined herein to obtain a dynamic covalent hydrogel.

[0022] In certain embodiments, the method for preparing a dynamic covalent hydrogel is performed under physiological conditions.

[0023] These and other objects, advantages, and features of the application will become apparent to those persons of ordinary skill in the art upon reading the following detailed description of preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1Schematic of the viscoelastic behavior of a dynamic covalent hydrogel made from a mixture of Wulff-PBA-modified polymer precursors and glucosamine-modified polymer precursors. Wulff-PBA-modified hyaluronic acid and glucosamine-modified hyaluronic acid were mixed at a 1 :2 volume ratio, and the storage modulus (G') and loss modulus (G") show the expected cross-over when measured over a range of frequencies.

[0025] Figure 2 Schematic of the self-healing properties of a dynamic covalent hydrogel made from a mixture of Wulff-PBA-modified polymer precursors and glucosamine-modified polymer precursors. (A) Qualitatively, two dynamic covalent hydrogels based on hyaluronic acid (HA) form a single hydrogel within minutes that can support its own weight. (B) Time sweep measurements on a typical HA-based dynamic covalent gel formulation (1% w / v) (HA-Wulff-PBA mixed with HA-glucosamine at a 1 : 1 volume ratio) under alternating low (1 Pa) and high (500 Pa) stresses at a constant frequency of 1 Hz show repeated breakage of the gel at high frequencies (G' < G") and recovery of the initial mechanical properties (G' > G").

[0026] Figure 3 Proof of concept of the tunability of the viscoelastic behavior of dynamic covalent gels, varying the molecular weight (100 kDa vs 500 kDa) and polymer content (1% vs 3% (w / v)) of the HA-based dynamic covalent hydrogels. The storage modulus (G') and loss modulus (G") were measured as a function of frequency.

[0027] Figure 4 Swelling / stability study of HA-based hydrogels using HA-Wulff-PBA and HA-glucosamine at a 1 : 1 volume ratio and polymer concentrations of 1% vs 2% (w / v). Tuning the polymer concentration of these gels allows control of their swelling and stability, with the optimal formulation showing minimal swelling and stability over 3 days and long-term stability (at least one month).

[0028] Figure 5Concept testing of dynamic covalent hydrogels obtained from various natural and synthetic polymers modified with Wulff-PBA or glucosamine. (A) 1% (w / v) poly(ethylene glycol) (PEG)-Wulff-PBA mixed with 1% (w / v) HA-glucosamine at a 1:1 volume ratio, (B) 1% (w / v) alginate-Wulff-PBA mixed with 1% (w / v) alginate-glucosamine at a 1:1 volume ratio, and (C) 1% (w / v) carboxymethylcellulose-Wulff-PBA mixed with 1% (w / v) carboxymethylcellulose-glucosamine at a 1:1 volume ratio, storage modulus (G') and loss modulus (G") measured as a function of frequency.

[0029] Figure 6 Evaluation of adipose-derived multipotent stromal cells viability encapsulated in a typical dynamic covalent hydrogel (1% (w / v) HA-Wulff-PBA mixed with 1% (w / v) HA-glucosamine at a 1:1 volume ratio) using confocal microscopy and live / dead staining.

[0030] Figure 7 Comparison of the rheological properties of different boronic acid-diol pairs immobilized on hyaluronic acid (HA) at physiological conditions of pH and temperature (frequency sweep; G', shear elastic modulus) (see Example 2, paragraph I).

[0031] Figure 8 Storage modulus (G') and loss modulus (G") of three specific optimized formulations of dynamic covalent hydrogels of HA-Wulff-PBA and HA-glucosamine showing different viscoelastic profiles. Different molecular weight (300 kDa, 200 kDa or 100 kA) of HA polymers, different total concentration of HA (1% or 3% w / v), different degree of substitution of both components (HA-wPBA - 26% or 40%; HA-glucosamine - 52%) were used, while the molar ratio of wPBA:glucosamine was kept constant at 1:1. (see Example 2, paragraph II).

[0032] Figure 9. Design of boronic acid-based minimal shrinkage / minimal swelling to no shrinkage / no swelling hydrogels. Hydrogel mass ratio as a function of time is reported for: (A) a dynamic covalent hydrogel of HA-Wulff-PBA and HA-glucosamine, where 300 kDa HA polymers are present at a total concentration of 1% w / v, the degree of substitution of HA-wPBA is 26%, the degree of substitution of HA-glucosamine is 52%, and the molar ratio of wPBA:glucosamine is 1:1; and (B) the same dynamic covalent hydrogel of HA-Wulff-PBA and HA-glucosamine except that the molecular weight of the HA polymers is 200 kDa. Hydrogel mass ratio as a function of time is investigated in PBS (A and B), in two different culture media (A), and in the presence of glucose, glucosamine, and hyaluronidase (A). (See Example 2, paragraph II).

[0033] Figure 10 . Cell compatibility of boronate gels. Murine fibroblasts (L929 cell line) are encapsulated in boronate hydrogels according to the application (300 kDa HA; [HA] = 1% w / v; Wulff-PBA degree of substitution = 26%; glucosamine - degree of substitution = 52%: Wulff-PBA:glucosamine ratio = 1:1) and cell compatibility is assessed by cell viability (live / dead cell imaging), metabolic activity (CCK-8), and proliferation (PicoGreen) assays. Results are reported at 0, 1, and 2 days after encapsulation of cells in the hydrogels. The image to the right of the cell viability plot is a representative image of high cell viability (>98%) observed after 2 days of 3D cell culture. (See Example 2, paragraph III).

[0034] Figure 11 . Optimized dynamic covalent hydrogels are printable. The top three images show how a good balance of extrusion pressure and speed can enable the printing of continuous and well-resolved boronate hydrogel filaments. The middle plot highlights in green the large range of extrusion / pressure pairs that can be used to successfully print the boronate hydrogels of the application, while conditions that lead to filament breakage are shown in yellow and conditions that lead to poor resolution of the filament are shown in red. The lower plots are examples of shapes that are successfully printed, either from one layer of gel or from multiple layers of gel. (See Example 2, paragraph IV).

[0035] Figure 12The cross-linking mechanism of the present invention is combined with "Click" chemistry reactions. (A) This scheme highlights the potential combination of cross-linking mechanisms with "Click" chemistry reactions to tune the composition and physico-chemical properties of the resulting gel. In the specific context of bioprinting, this strategy can be used to mechanically reinforce the post-printed construct. (B) is a graph showing the successful mechanical reinforcement of a post-printed structure upon immersion in a medium containing a suitable "clickable" polymer. In this example, strain-promoted azide-yne cycloaddition (SPAAC) between bis-cyclooctynl (BCN) and azide (N3) is used as a typical "click" reaction. This borate gel is chemically modified with BCN, enabling post-printing modification with azide-modified hyaluronic acid (100 kDa). (See Example 2, paragraph V). DETAILED DESCRIPTION

[0036] As mentioned above, the present invention provides hydrogel precursor polymers and dynamic covalent hydrogels, pharmaceutical compositions comprising the precursors or hydrogels and uses thereof in various applications.

[0037] I- Pairing of hydrogel precursor polymers

[0038] 1. Hydrogel precursor polymers

[0039] The present invention provides a cross-linking pair or pair of hydrogel precursor polymers that are capable of cross-linking when mixed together. The cross-linking pair or pair of hydrogel precursor polymers contemplated by the present invention consists of a first hydrogel precursor polymer modified with phenylboronic acid or a phenylboronic acid derivative and a second hydrogel precursor polymer modified with glucosamine. The terms "pairing", "pair", "cross-linking pair" and "cross-linking pair" are used interchangeably herein. They refer to the association or combination of two hydrogel precursor polymers, wherein the polymers are separate from each other, but when mixed, form a dynamic covalent hydrogel through cross-linking. In other words, the terms "pairing", "pair", "cross-linking pair" and "cross-linking pair" as used herein do not encompass mixtures, blends and synonyms thereof. As used herein, the term "hydrogel precursor polymer" refers to a polymer that is capable of participating in a reaction to form a cross-linked molecular network, e.g. a hydrogel. Thus, the paired hydrogel precursor polymers as defined herein are the same substances as the substances that form the dynamic covalent hydrogels according to the present invention, but they are not in contact with each other and thus, as a pair, do not undergo cross-linking.

[0040] A. Polymers

[0041] In embodiments of the present application, when the polymer precursors are used in biological, biomedical, or medical applications, at least one of the first and second polymers is a biocompatible, biodegradable, hydrophilic polymer. The term "biocompatible" as used herein to characterize a polymer means a polymer that is not significantly toxic to cells and / or living tissue, and does not elicit an immunopathogenic response in a healthy individual. The term "biodegradable" as used herein to characterize a polymer means a polymer that is degraded by the action of living organisms, light, air, water, or any combination thereof. Preferably, a biodegradable polymer is one that breaks down into sufficiently small, non-toxic molecules over time in the body of a mammal so as to be metabolized or excreted under normal physiological conditions. As used herein, "hydrophilic polymer" means a polymer (or copolymer) having groups with an affinity for water. The term "hydrophilic polymer" encompasses polymers that absorb more than 0.5% of their weight in water in 24 hours, and more than 4% of their weight in water at equilibrium, as determined by ASTM D570 testing.

[0042] The first and second hydrogel precursor polymers of the pair according to the present application are independently selected from the group of natural polymers, semi-synthetic polymers, and synthetic polymers. In embodiments of the present application, when the polymer precursors are used in biological, biomedical, or medical applications, the first and / or second hydrogel precursor polymers of the pair are biocompatible, biodegradable, hydrophilic polymers independently selected from the group of natural polymers, semi-synthetic polymers, and synthetic polymers.

[0043] In certain embodiments, at least one of the first and second hydrogel precursor polymers is selected from natural polymers. The terms "natural polymer" and "biopolymer" are used interchangeably herein and refer to a polymer that occurs naturally (i.e., a polymer found in nature, but which can be obtained using methods involving artificial intervention, e.g., isolation, purification, synthetic preparation, recombinant preparation, etc.). In certain preferred embodiments, both the first and second hydrogel precursor polymers are natural polymers. Examples of natural polymers suitable for use in the present application include naturally occurring polysaccharides, collagen, and gelatin.

[0044] As used herein, the term "polysaccharide" has its art-recognized meaning and refers to complex carbohydrates composed of 10 to as many as thousands of monosaccharides arranged in chains and linked by glycosidic bonds. The most common monosaccharides that occur as part of a polysaccharide are glucose, fructose, galactose, and mannose. Naturally occurring polysaccharides vary in molecular weight between a few hundred to several thousand Daltons.

[0045] Exemplary polysaccharides include, but are not limited to, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (e.g., inulin), levan, fucan, fucoidan, carrageenan, galactomannan, pectic acid, pectin, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glycose, polydextrose, polydextrose, pachyman, chitosan, chitin, agarose, keratin, chondroitin sulfate, heparan sulfate, dermatan, hyaluronic acid, alginic acid (alginate), xanthan gum, and starch. Other examples of naturally occurring polysaccharides include other natural homopolymers or heteropolymers containing one or more of the following: aldoses, ketoses, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and natural derivatives thereof.

[0046] In certain preferred embodiments, at least one of the first and second hydrogel- precursor polymers is a naturally occurring polysaccharide selected from the group consisting of hyaluronic acid, alginate, cellulose, heparan sulfate, chondroitin sulfate, chitosan, and chitin.

[0047] In certain embodiments, at least one of the first and second hydrogel- precursor polymers is collagen. As used herein, the term "collagen" has its art-recognized meaning and refers to the most ubiquitous protein in the mammalian proteome. Collagen forms a large part of the extracellular matrix and connective tissue, providing strength and flexibility to tissues in the body. Any type of collagen can be used in the practice of the present application. Thus, the collagen can be selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type VI collagen, and any combination thereof. Preferably, the collagen is derived from human or porcine sources.

[0048] In certain embodiments, at least one of the first and second hydrogel precursor polymers is gelatin. As used herein, the term "gelatin" has its art-recognized meaning and refers to an animal protein prepared by heat denaturation of collagen isolated from animal skin and bone with very dilute acid, or an animal protein extracted from fish skin. Gelatin is a heterogeneous mixture of single- or multiple-chain polypeptides, each having an extended left-handed polyproline helical conformation and containing 50 to 1000 amino acids. About half of the total amino acid content of gelatin is glycine residues (almost one-third of the residues, arranged every third residue), proline residues, and 4-hydroxyproline residues.

[0049] In certain embodiments, at least one of the first and second hydrogel precursor polymers is selected from modified biopolymers. The terms "modified biopolymer" and "semisynthetic polymer" are used interchangeably herein and refer to a natural polymer that has been chemically modified. Examples of modified biopolymers suitable for use in the present application include derivatized celluloses (e.g., carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and methoxy cellulose); derivatized hyaluronic acid (e.g., amine-modified hyaluronic acid and esterified hyaluronic acid); and derivatized collagen (e.g., amine-modified collagen and esterified collagen).

[0050] In certain preferred embodiments, at least one of the first and second hydrogel precursor polymers is a biocompatible, biodegradable, hydrophilic semisynthetic polymer selected from the group consisting of carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, and methoxy cellulose.

[0051] In certain embodiments, at least one of the first and second hydrogel precursor polymers is a selected synthetic polymer. As used herein, the term "synthetic polymer" refers to a polymer that is neither a natural polymer nor a semisynthetic polymer. Examples of synthetic polymers suitable for use in the present application include, but are not limited to, poly(acrylic acid) and its derivatives, poly(ethylene glycol) and its copolymers, poly(vinyl alcohol), poly(2-hydroxyethyl methacrylate), polyphosphazene, poly(caprolactone) or its copolymers. Other examples include polyacrylate derivatives, polymethacrylate derivatives (e.g., PEGMAs), polyisoprene, polyamides, synthetic polypeptides (e.g., PBLG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL).

[0052] It will be appreciated by those skilled in the art that the list of polymers provided above is not exhaustive and can be extended to other similar polymers.

[0053] In the context of the present invention, the hydrogel precursor polymer can have any suitable molecular weight. For example, the polymer can have a molecular weight of about 5,000 grams / mole to about 3,000,000 grams / mole, preferably about 10,000 grams / mole to about 700,000 grams / mole. As used in this document, the terms "about" and "approximately," when used in reference to a number, generally mean that the number includes ±10% of the number (greater or less than the number), unless otherwise stated or otherwise evident from the context (unless the number exceeds 100% as a possible value).

[0054] B. Phenylboronic acid and phenylboronic acid derivatives

[0055] The first hydrogel precursor polymer to which the present invention relates is modified with phenylboronic acid or a phenylboronic acid derivative. The term "modified with phenylboronic acid or a phenylboronic acid derivative," as used herein to characterize a polymer, means a polymer grafted with phenylboronic acid or a phenylboronic acid derivative (i.e., a polymer covalently linked to phenylboronic acid or a phenylboronic acid derivative such that the polymer is functionalized with phenylboronic acid or a phenylboronic acid derivative). The phenylboronic acid (or phenylboronic acid derivative) molecule can be covalently linked to the polymer through its ortho, meta, or para position (relative to the boronic acid group).

[0056] As used herein, the term "phenylboronic acid" refers to a molecule having the following chemical formula (I), abbreviated as PhB(OH)2or PBA.

[0057]

[0058] As used herein, the term "phenylboronic acid derivative" refers to a phenylboronic acid derivative selected from the group consisting of ortho, meta, or para monosubstituted phenylboronic acids, Wulff-type phenylboronic acids, and benzoboroxoles.

[0059] As used herein, the term "substituted" means that the particular group or compound described has at least one hydrogen atom replaced by a non-hydrogen substituent. In the context of the present application, the phenylboronic acid molecule is substituted to the extent that the substitution makes chemical sense. Examples of suitable substituents include, but are not limited to, alkyl, nitro (-NO2), sulfo (-SO4"), cyano (-CN), halogen (F, Br, Cl, or I), amine (-NRR', where each R and R' is independently H or alkyl), hydroxyl (-OH), thiol (-SH), alkoxy (-OR, where R is alkyl), alkylthio (-SR, where R is alkyl), alkylsulfo (-O-SO2-O-R, where R is alkyl), aldehyde (-CHO), ketone (-CO-R, where R is alkyl), ester (-COO-R or -OCO-R, where R is alkyl), amide (-CO-NRR', where each R and R' is independently H or alkyl), carboxylic acid (-COOH or -COOM, where M is a suitable cation, such as sodium or potassium), and sulfonic acid (-SO3H or -R-SO3H, where R is alkyl) groups. One skilled in the art knows how to select a substituted phenylboronic acid such that the substitution is not incompatible with grafting the PBA onto a polymer.

[0060] The term "alkyl" as used herein refers to a monovalent straight or branched chain radical of unsaturated hydrocarbon derived from 1 to 10 carbon atoms (Ci-Cio alkyl), such as from 1 to 8 carbon atoms (Ci-C8alkyl) or from 1 to 5 carbon atoms (Ci-C5alkyl). Representative saturated straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl; while saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, t-butyl, isopentyl, and the like. The alkyl group can be optionally substituted. For example, the alkyl group can be substituted with at least one substituent selected from the group consisting of nitro, sulfo, cyano, halogen, amine, hydroxyl, aldehyde, ketone, ester, amide, carboxylic acid, and sulfonic acid groups.

[0061] Specific examples of monosubstituted phenylboronic acids include, but are not limited to, 2-(or 3- or 4-)bromophenylboronic acid, 2-(or 3- or 4-)chlorophenylboronic acid, 2-(or 3- or 4-)fluorophenylboronic acid, 2-(or 3- or 4-)iodophenylboronic acid, 2-(or 3- or 4-)nitrophenylboronic acid, 2-(or 3- or 4-)mercaptophenylboronic acid, 2-(or 3- or 4-)hydroxyphenylboronic acid, 3-aminosulfonylphenylboronic acid, 2-(or 3- or 4-)aminophenylboronic acid, 2-(or 3- or 4-)(trifluoromethyl)phenylboronic acid, 2-(or 3- or 4-)(trifluoromethoxy)phenylboronic acid, 2-(or 3- or 4-)cyanophenylboronic acid, 2-(3- or 4-)formylphenylboronic acid, and 2-(or 3- or 4-)carboxyphenylboronic acid. 2-(or 3- or 4-)(bromomethyl)phenylboronic acid, 2-(or 3- or 4-)aminocarbonylphenylboronic acid, o-tolueneboronic acid, m-tolueneboronic acid, p-tolueneboronic acid, 2-(or 3- or 4-)(methylthio)phenylboronic acid, 2-methoxy(or 3- or 4-)phenylboronic acid, (or 3- or 4-)(hydroxymethyl)phenylboronic acid, 2-(or 3- or 4-)methylsulfinylphenylboronic acid, 2-(or 3- or 4-)[methylsulfonylphenyl]boronic acid, (2-(or 3- or 4-)[(methylamino)sulfonyl]-phenyl)boronic acid, (2-(or 3- or 4-)[(methylsulfonyl)-amino]phenyl)boronic acid, (2-(or 3- or 4-)amino-methylphenyl)boronic acid, 3-(2,2,2-trifluoroethyl 4-(cyanomethyl)phenylboronic acid, 4-cyano-methoxyphenylboronic acid, 4-(2-nitrovinyl)phenylboronic acid, 2-(or 3- or 4-)vinyl-phenylboronic acid, 2-(or 3- or 4-)acetylphenylboronic acid, 2-(or 3- or 4-)methoxycarbonylphenylboronic acid, 2-(or 3- or 4-)acetamidophenylboronic acid, 2-(or 3- or 4-)ethylphenylboronic acid, 2-(or 3- or 4-)ethoxyphenylboronic acid, 3-(ethylthio)phenylboronic acid, 3-ethylsulfinylphenylboronic acid, 4-ethylsulfinyl-phenylboronic acid, 3-(N,N-dimethylamino)phenylboronic acid, 4-(methylsulfonyl-aminomethylphenyl)boronic acid, 2-(or 3- or 4-)ethoxycarbonylphenylboronic acid, ( 4-Acetaminomethylphenyl)boronic acid, 2-(dimethylaminocarbonyl)phenylboronic acid, 4-(dimethylcarbamoyl)-phenylboronic acid, 2-(or 3- or 4-)isopropylphenylboronic acid, 4-propylphenylboronic acid, 3-propoxyphenylboronic acid, [3-(3-hydroxy-propyl)phenyl]boronic acid, 4-(3-hydroxypropyl)phenylboronic acid, 4-isopropoxy-phenylboronic acid, 4-propoxy-phenylboronic acid, 3-tert-butylphenylboronic acid, 4-tert-butylphenylboronic acid, 4-butylphenylboronic acid, 2-(3- or 4-)butoxy-phenylboronic acid, 2-isobutoxy-phenylboronic acid, 3-isobutoxyphenylboronic acid, 4-(dimethyl-amino)phenylboronic acid, 3-(isobutylaminocarbonyl)phenylboronic acid, and 4-(isobutylaminocarbonyl)phenylboronic acid.

[0062] As used herein, the term "Wulff-type phenylboronic acid" refers to a phenylboronic acid derivative containing an intermolecular tetra-coordinated B-N (chemical formula (II)) bond that facilitates the formation of tetrahedral boronate anion (sp3). The sp3 hybridization state remains stable even under neutral or mildly acidic conditions, facilitating boronate esterification with cis-diol.

[0063]

[0064] Wulff-type phenylboronic acids have been described in detail (Wulff et al., Pure Appl. Chem., 1982, 54: 2093-2102; Wulff et al., Angew Chem., Int. Ed. Engl., 1984, 23: 741-742). Examples of suitable Wulff-type phenylboronic acids include, but are not limited to, 2-((dimethylamino)methyl)phenylboronic acid (DAPBA). Wulff-type boronic acids (structures analogous to 2-dimethylaminomethylphenylboronic acid (DAPBA)) have important implications in biomedical applications (Cromwell et al., J. Am. Chem. Soc., 2015, 137: 6492-6495; Piest et al., Soft Matter, 2011, 7: 11111; Li et al., Chem. Commun., 2011, 47: 8169; Kim et al., J. Am. Chem. Soc., 2009, 131: 13908-13909).

[0065] In certain preferred embodiments, the Wulff-type phenylboronic acid has the chemical formula (II), wherein R and R' are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C10 alkenyl, substituted or unsubstituted C1-C10 alkynyl, acyl (-C(=0)R1, wherein R1 is substituted or unsubstituted C1-C20 alkyl), and carboxyl (-C(=0)OR1, wherein R1 is substituted or unsubstituted C1-C20 alkyl). The alkyl, alkenyl, and alkynyl groups can be substituted with one or more substituents, for example, selected from the group consisting of halogen (F, Br, I, Cl), hydroxyl (-OH), amino (-NR2R3, wherein R2 and R3 are independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-C20 alkyl), alkoxy (-OR1, wherein R1 is substituted or unsubstituted C1-C20 alkyl), carboxyl (-C(=0)OR1, wherein R1 is substituted or unsubstituted C1-C20 alkyl), amido (-NR2C(=0)R3 or -C(=0)NR2R3, wherein R2 and R3 are independently selected from the group consisting of hydrogen and substituted or unsubstituted C1-C20 alkyl), nitro (-N02), oxo (=0), and cyano (-CN)

[0066] The Wulff-type phenylboronic acid of formula (II) can also be: wherein one of R and R' is a linking moiety or spacer that comprises, at the free end (non-linking end), a reactive group capable of attaching to or reacting with a polymer to which the Wulff-type phenylboronic acid is to be grafted. The terms "linking moiety," "linker," and "spacer" are used interchangeably herein to refer to a backbone moiety having a length of at least 8 atoms, such as greater than 10, greater than 20, greater than 30, greater than 35, greater than 40, or greater than 50 atoms. The linker can be linear, branched, cyclic, or a single atom. In some cases, the linker is substituted with a sulfur, nitrogen, or oxygen heteroatom. The bonds between the backbone atoms can be saturated or unsaturated. The linker can include one or more substituents.

[0067] In certain embodiments, the phenyl group of the Wulff-type phenylboronic acid of formula (II) is substituted with at least one substituent (see definition above). In certain embodiments, the substituent is selected such that it allows grafting of the Wulff-type phenylboronic acid to the first polymer. Alternatively, the substituent on the phenyl group is a linking moiety terminated in a functional group that can be used to graft the Wulff-type phenylboronic acid to the first polymer.

[0068] The phenyl group in the Wulff-type phenylboronic acid of formula (II) can also be substituted with a heteroaryl group. As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic group of 5 to 10 ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon atoms, with the understanding that the point of attachment of the heteroaryl group is on the aromatic ring. In certain embodiments, the heteroaryl group is a heterophenyl group. As used herein, the term "heterophenyl" refers to a phenyl group in which at least one carbon atom is replaced with a heteroatom selected from N, O, and S.

[0069] The term "benzoborazole" as used herein refers to a cyclic boronic acid hemiester, in particular a bicyclic organic heterocycle having the chemical formula (III).

[0070]

[0071] Such compounds with intramolecular B-O coordination have been described (Berubee et al., J. Org. Chem., 2008, 73: 6471-6479; Dowlut et al., J. Am. Chem. Soc, 2006, 128: 4226-4227).

[0072] In certain preferred embodiments, the benzoboroxole has the formula (III), wherein n is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8, and wherein the benzene ring is substituted with at least one substituent (see definition above). Thus, in formula (III), R is one or more (i.e. 1, 2, 3 or 4) substituents. At least one substituent is selected such that it allows grafting of the benzoboroxole to the first polymer. Alternatively, the substituent on the phenyl group of formula (III) is a linking moiety ending in a functional group which can be used to graft the benzoboroxole to the first polymer. The phenyl group in the benzoboroxole of formula (III) can also be substituted with a heteroaryl group, such as a heterophenyl group.

[0073] C. Glucosamine

[0074] The second hydrogel precursor polymer according to the present application is modified with glucosamine. As used herein, the term “modified with glucosamine” refers to a polymer that is grafted with glucosamine (i.e. covalently linked to glucosamine such that the polymer is functionalized with glucosamine). As used herein, the term “glucosamine” refers to a molecule having the following chemical formula (IV). Glucosamine is also known as D-glucosamine, and its IUPAC name is (2R,3R,4R,5S)-6-aminohexan-1,2,3,4,5-pentol.

[0075]

[0076] 2. Preparation of the hydrogel precursor

[0077] The first hydrogel precursor polymer modified with phenylboronic acid or a phenylboronic acid derivative and the second hydrogel precursor polymer modified with glucosamine can be prepared using any suitable method known in the art or using steps that modify methods known in the art. Methods for grafting phenylboronic acid on polymers are known in the art (Ryu et al., Curr. Med. Chem., 2018, doi: 10.2174 / 092986732566618100814443, Brooks and Sumerlin, Chem. Rev., 2016, 3: 1375-1397; Guan and Zhang, Chem. Soc. Rev., 2013, 42: 8106-8121). Similarly, methods for grafting diols on polymers are known in the art (Tarus et al., Macromol. Rapid Commun., 2014, 35: 2089-2095; Figueiredo et al., Tang et al., Adv. Sci., 2018, 5(9): 1800638; Yesilyurt et al., Adv. Mater., 2015, 28(1 ): 86-91).

[0078] In certain embodiments, grafting can be performed using the methods described in the Examples section below.

[0079] The reaction to obtain the second hydrogel precursor polymer modified with glucosamine allows the diol substitution level to be varied in a controlled manner over a wide range (e.g., from about 0.5% to about 100%, e.g., from about 1% to about 99%). In fact, since glucosamine is completely soluble, it does not make the polymer more hydrophobic. Thus, there is no limitation on the molar ratio between the polymer and glucosamine.

[0080] The hydrogel precursor polymers can be purified using any method known in the art. Examples of suitable purification methods include, but are not limited to, washing, filtering, precipitating, decanting, centrifuging, distilling, and the like, or any combination thereof.

[0081] If desired, at least one of the first and second hydrogel precursor polymers can be placed in solution after preparation, prior to use or storage. A preferred solution is an aqueous solution having a pH of between about 6 and about 8. In certain preferred embodiments, the aqueous solution is a physiologically compatible solution, such as a buffered isotonic saline (see "Formulations" below).

[0082] Alternatively, if desired, at least one of the first and second hydrogel precursor polymers can be dried (e.g., freeze-dried or lyophilized) under vacuum after preparation into a substantially anhydrous form or powder. Thereafter, the powder can be rehydrated prior to use.

[0083] If desired, the hydrogel precursor polymers can be sterilized after preparation. Sterilization can be performed using any of a variety of sterilization techniques known in the art, such as by filtration through a bacteria-retaining filter, by gamma irradiation, by electron beam irradiation, or by addition of a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0084] The hydrogel precursor polymers can be stored in liquid form or in solid form under suitable conditions. Suitable storage conditions for the polymers are known in the art. For example, lyophilized / anhydrous hydrogel precursor polymers can be stored in a sealed container at -20°C. The hydrogel precursor polymers can be stored under suitable conditions for several days, weeks, or months.

[0085] In certain embodiments, each of the first and second hydrogel precursors are each placed in a receptacle, such as a vial, flask, or other sealed container. When using hydrogel precursor solutions, each solution can be placed in a syringe, such as a dual barrel syringe or any other suitable syringe system, where the first and second hydrogel precursor polymers are physically separated prior to simultaneous extrusion through a needle (cannula) in the body of a subject with concomitant mixing and co-injection of the mixed first and second hydrogel precursors (see below "kits").

[0086] 3. Additional ingredients

[0087] Depending on the intended use of the cross-linking pair of the hydrogel precursors, at least one of the first and second hydrogel precursor polymers or hydrogel precursor solutions can comprise cells, bioactive agents, visualizing agents, or combinations thereof. Examples of such cells, bioactive agents, and visualizing agents are provided below (see below "pharmaceutical compositions and kits").

[0088] The bioactive agents or visualizing agents can be covalently or ionically attached to the at least one hydrogel precursor polymer. Alternatively, the bioactive agents or visualizing agents can be mixed with the at least one hydrogel precursor polymer or hydrogel precursor polymer solution.

[0089] To broaden the biomedical application scope of the dynamic covalent hydrogels of the present application, the first or second hydrogel precursor polymer of the cross-linking pair of the hydrogel precursors can be modified to be covalently linked to a reactive chemical moiety, in particular a bio-orthogonal functional moiety or to a clickable moiety. The presence of the bio-orthogonal functional moiety or the clickable moiety can be used to temporally and spatially modulate the physico-chemical properties of the hydrogel. For example, it can be used to stabilize and / or mechanically reinforce the bioprinted structure after printing. Alternatively, or in addition, the presence of the bio-orthogonal functional moiety or the clickable moiety can be used to facilitate the immobilization of target molecules (e.g., peptides, growth factors, drugs, fluorophores, etc.) after gelation, to develop evolving 3D culture systems with temporally and spatially modulated cell-material interactions, etc.

[0090] Accordingly, in certain embodiments, the first hydrogel precursor polymer or the second hydrogel precursor polymer of the hydrogel precursor crosslinking pair is directly or indirectly covalently linked to a bio-orthogonal functional moiety or a clickable moiety. As used herein, the term "bio-orthogonal functional moiety or clickable moiety" refers to a reactive chemical group that can participate in a "bio-orthogonal chemical reaction or click chemical reaction" and belongs to a pair of chemical reaction partners. Bio-orthogonal chemical reactions and click chemical reactions are conjugation reactions known in the art. The term "bio-orthogonal chemical reaction" more specifically refers to a reaction that utilizes a pair of chemical reactions that (1) do not occur naturally in biological systems; (2) do not cross-react with functional groups found in biology; and (3) do not require a cytotoxic catalyst or produce cytotoxic byproducts. Examples of bio-orthogonal chemical reactions include Staudinger ligation, which utilizes a pair of reaction partners of an azide and a functionalized triaryl phosphine to form a stable amide bond; strain-promoted azide-alkyne cycloaddition (SPAAC or copper-free click chemistry), which utilizes an azide and a strained cyclooctyne to produce a stable triazole bond via 1,3-dipolar cycloaddition; 1,3-cycloaddition between a nitrone and a cyclooctene or between a norbornene and a nitrile oxide (norbornene cycloaddition); formation of an oxime / hydrazine from an aldehyde and a ketone; tetrazine ligation, which includes an olefin (e.g., trans-cyclooctene, norbornene) and s-tetrazine in a reverse demand Diels Alder reaction, followed by a reverse Diels Alder reaction to eliminate nitrogen, an isonitrile-based click reaction, and a tetraloop alkane ligation. The terms "click reaction," "click chemical reaction," and "click chemistry" are used interchangeably herein. More particularly, they refer to "modular, global, very high yielding, harmless side products that can be removed by non-chromatographic methods, and stereospecific" reactions (Kolb et al., Angew. Chem., Int. Ed., 2001, 40:2004-2021). Such reactions should require "simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, no use of solvents or use of benign (e.g., water) or easily removed solvents, and simple product isolation" (Kolb et al., Angew. Chem., Int. Ed., 2001, 40:2004-2021). Thus, the term "click chemistry" encompasses a broader range of reactions that are stable in a biological context, but not necessarily without off-target effects, even though some click reactions are bio-orthogonal chemical reactions. Click reactions include 1,4-conjugate addition (i.e., thiol-vinyl sulfone and thiol-maleimide reactions), aldehyde-nucleophile reactions (hydrazone and oxime coordination), Diels-Alder reactions, and photoactivated thiol-alkene coupling.

[0091] Bioorthogonal functional moieties used to practice the present application belong to a pair of bioorthogonal coupling partners, while click moieties belong to a pair of click reaction partners. The bioorthogonal functional moieties or click moieties can be any suitable chemical group belonging to a pair of bioorthogonal coupling partners or a pair of click reaction partners. Representative examples of such representative reaction partners are described, for example, in "Bioconjugate Techniques", Greg T. Hermanson, 1996 and in Patterson et al., ACS Chemical Biology, 2014, 9:592-605; Spicer et al., Nature Communications, 2014, 5:4740; Madl and Heilshorn, Advanced Functional Materials, 2018, 28(11): 1706046. Thus, for example, the bioorthogonal functional moieties or click moieties can be selected from the group consisting of cyclooctenes; trans-cyclooctenes; tetrazines; azides; alkynes, including strained alkynes, such as cyclooctenes or cyclooctene derivatives; amines; active esters; isocyanates; isothiocyanates; thiols; aldehydes; amides; norbornenes; vinyl derivatives, including acrylates, methacrylates, and the like; phosphines, such as triaryl phosphines; nitrone; tetraloop alkanes; maleimides; hydrazones; and the like. Other bioorthogonal functional moieties and click moieties can also be used.

[0092] The bioorthogonal functional moiety or click moiety can be covalently attached, directly or indirectly (e.g., through a linker), to the first hydrogel precursor polymer or the second hydrogel precursor polymer of the hydrogel precursor cross-linking pair. It can be attached to the first polymer, the phenylboronic acid or phenylboronic acid derivative, the second polymer, or the glucosamine. In certain preferred embodiments, the bioorthogonal functional moiety or click moiety is covalently attached, directly or indirectly (e.g., through a linker), to the first polymer or the second polymer. As will be appreciated by those skilled in the art, there can be more than one bioorthogonal functional moiety or click moiety attached to the first or second hydrogel precursor.

[0093] In certain embodiments, conjugation employing bioorthogonal or click reactions typically involves a two-step strategy: first, introducing a bioorthogonal functional moiety or click moiety into the first hydrogel precursor polymer or the second hydrogel precursor polymer of the hydrogel precursor cross-linking pair, and subsequently, introducing a desired molecule or biomolecule through bioorthogonal or click conjugation after the formation of the dynamic covalent hydrogel.

[0094] In other embodiments, conjugation employing bioorthogonal or click reactions can be used for the synthesis of a bimodal gel (i.e., formation of a mixture of 2 to 2 of the 4 polymers) or co-crosslinking (a mixture of 3 polymers with a polymer participating in 2 networks). Co-crosslinking can be performed in one or two steps.

[0095] II - Dynamic covalent hydrogels

[0096] The present application also provides a dynamic covalent hydrogel composed of two hydrogel precursor polymers of a cross-linking pair as described herein. The term "hydrogel" as used herein has its meaning as understood in the art and refers to a three-dimensional polymeric structure that is insoluble in water or other aqueous medium, but is able to absorb and retain water to form a stable, usually soft and pliable structure. In embodiments of the present application, the hydrogel is used for biological, biomedical or medical applications, and the hydrogel can be composed of at least one biocompatible, biodegradable, hydrophilic polymer. Cross-linked hydrogels can be considered as solids, as they do not flow or deform without the application of a shear stress. However, cross-linked hydrogels with certain formulations can deform under the influence of gravity. "Hydrogel" can be interchanged with "hydrogel scaffold" or "scaffold" or "viscoelastic material". The hydrogel according to the present application can be formed in vitro. Alternatively, the hydrogel can be formed in situ, i.e. a hydrogel that can be formed at a tissue site in a living animal or human body (see below). The hydrogel according to the present application can further encapsulate or comprise any number of cells, biomolecules and / or bioactive agents (see below).

[0097] 1. Dynamic covalent hydrogel

[0098] The hydrogel according to the present application is composed of a first hydrogel precursor polymer modified with a phenylboronic acid or a phenylboronic acid derivative as described above and a second hydrogel precursor polymer modified with glucosamine as described above, wherein the first and second polymers are cross-linked via a dynamic covalent bond.

[0099] As used herein, the term "dynamic covalent bond" refers to a covalent bond that can be formed and dissociated reversibly. For example, the constitution of the dynamic system can respond to changes in the chemical environment (complexing entities etc.) or physical conditions (temperature, mechanical stress, electric field, radiation etc.). In the case of the present application, the first and second hydrogel precursor polymers are cross-linked via a dynamic covalent bond formed between the boronic acid function on the first polymer and the diol function on the second polymer. Boronic acids have the ability to form reversible covalent bonds with 1,2- and 1,3-diols (Brighid Pappin et al., Boron-Carbohydrate Interactions), such that boronate esters are formed. Thus, in the hydrogel according to the present application, the first and second hydrogel precursor polymers are cross-linked via a dynamic covalent boronate ester bond.

[0100] The hydrogels according to the present application are "dynamic covalent hydrogels". The dynamic nature of crosslinking through boronate ester bonds provides a hydrogel that exhibits viscous flow under shear stress (shear-thinning) and rapid recovery (self-healing) when the applied stress is relaxed. As used herein, the term "self-healing" refers to the spontaneous formation of new bonds when old bonds in the material are broken. Thus, the self-healing hydrogels according to the present application autonomously repair external stress damage and substantially recover their original modulus and strength. Stress is typically applied by physical force and / or pressure. Thus, "self-healing" is a process in which the hydrogel has reduced resistance to flow when subjected to external stress, and recovers some or all of its rigidity and strength after removal of the external stress. As used herein, the term "shear-thinning" refers to the effect that the viscosity of a hydrogel (a measure of resistance to flow of a fluid) decreases with increasing shear rate or increasing shear stress. Shear-thinning and self-repairing hydrogels exhibit a number of unique and useful properties, including externally tunable strength, moldability, and low energy synthesis / processing. In certain embodiments, the original hydrogel recovers within 30 minutes, preferably within about 20 minutes, or within about 10 minutes, or within about 5 minutes or within about 1 minute, or within about 60 seconds, about 45 seconds, about 30 seconds, about 15 seconds, about 10 seconds, about 5 seconds, or about 1 second after removal of the mechanical shear force. In the case of the hydrogels prepared by the present inventors, recovery is on the order of seconds.

[0101] Preferably, the dynamic covalent hydrogels according to the present application are biocompatible. The terms "biocompatible" and "medically acceptable" are used interchangeably herein. They refer to a material that is not significantly toxic to cells and / or living tissue. A hydrogel is considered biocompatible if, when placed in a physiological environment, there is minimal inflammatory response, no signs of allergic reaction, and minimal unwanted cell growth on the surface of the biomaterial. A biocompatible hydrogel does not elicit a host response when implanted in a host mammal that is sufficient to adversely affect the function of the hydrogel; the host response includes the formation of a fibrotic structure on or around the hydrogel, immune rejection of the hydrogel, or release of toxic or pyrogenic compounds from the hydrogel into the surrounding host tissue and / or fluids.

[0102] Preferably, the dynamic covalent hydrogels according to the present application are biodegradable. As used herein, the term "biodegradable" refers to a hydrogel that predictably breaks down into molecules small enough to be metabolized or excreted under normal physiological conditions.

[0103] The dynamic covalent hydrogels prepared in vitro (or ex vivo) according to the present application can have any desired shape (geometry) and size (dimension). As understood by one skilled in the art, the shape and size are generally determined by the intended use of the hydrogel (e.g., 3D cell culture, tissue engineering, etc.). Shaping and sizing can include custom shaping and sizing in a manner such that the implantable device matches a particular treatment site of a particular patient, as determined by imaging or other techniques known to one skilled in the art.

[0104] In certain embodiments, the hydrogels can be formed as microparticles or nanoparticles having a size of about 50 nanometers to about 1000 nanometers, preferably about 100 nanometers to about 500 nanometers. These nanoparticles are suitable for injection administration. However, due to the shear-thinning / shear-healing properties of the hydrogels described herein, microparticles or nanoparticles are not required for ease of injection.

[0105] 2. Preparation of dynamic covalent hydrogels

[0106] The dynamic covalent hydrogels according to the present application are formed by crosslinking between a first hydrogel precursor polymer modified by phenylboronic acid or a phenylboronic acid derivative and a second hydrogel precursor polymer modified by glucosamine. The reaction occurs in a single step of mixing the first and second hydrogel precursor polymers, each polymer being comprised in solution.

[0107] The crosslinking reaction can occur in vitro (including ex vivo, i.e., in vitro prior to administration to a subject). Alternatively, the hydrogel can be formed in situ (e.g., directly at a given site in a living animal or human body). In this case, the formation of the hydrogel is initiated by mixing the two hydrogel precursor polymers at the injection site.

[0108] The crosslinking reaction is carried out in the absence of any catalyst under physiological conditions. As used herein, the term "physiological conditions" refers to artificial conditions that mimic the natural environment compatible with living cells, e.g., the prevailing aqueous conditions of temperature, pH, osmotic pressure, tonicity, oxidation, and electrolyte concentration that are compatible with living cells and / or are optionally considered to be within the normal range at the site of administration or action of a subject. When the crosslinking reaction occurs in vitro (or ex vivo), the physiological conditions include an aqueous solution having a pH ranging from about 6 to about 8, preferably about 7.2-7.4, and a temperature ranging from about 4°C to about 42°C. When the crosslinking reaction occurs in situ, the two hydrogel precursor polymers are placed in a buffered aqueous solution having a pH of about 7.4 (e.g., about 7-7.6), a temperature of about 30-42°C, preferably about 37°C.

[0109] The formation of a dynamic network at physiological pH is unusual compared to other boronate-cisdiol complexes that are only stable at basic pH (Springsteen et al., Tetrahedron, 2002, 58: 5291-5300; Peters, Coordination Chemistry Reviews, 2014, 268: 1-22).

[0110] The molar ratio between the first hydrogel precursor polymer and the second polymer precursor polymer in the reaction mixture can be any value that results in a dynamic covalent hydrogel. In certain embodiments, the molar ratio between the first hydrogel precursor polymer and the second polymer precursor polymer is about 1 : 10 to about 10: 1, for example about 1 :8 to about 8: 1, or about 1 :6 to about 6: 1, or about 1 :4 to about 4: 1, or about 1 :2 to about 2: 1.

[0111] In the case of the present application, the cross-linking reaction is substantially instantaneous. In certain embodiments, the cross-linking reaction can occur in the time range of about 1 second to about 5 minutes, for example about 3 seconds to about 1 minute, about 10 seconds to about 2 minutes, for example the gelation time can be less than about 30 seconds, or less than about 20 seconds, or less than about 10 seconds. For example, the cross-linking reaction can occur in the time range of about 10 seconds.

[0112] If desired, following in vitro preparation, the dynamic covalent hydrogel can be sterilized using any suitable method known in the art, for example gamma irradiation, autoclaving, ethylene oxide sterilization, infrared radiation and electron beam radiation. A sterilization method is suitable for use if it does not result in a significant loss of useful physical and / or mechanical properties of the hydrogel. The person skilled in the art is capable of selecting a suitable sterilization method.

[0113] The dynamic covalent hydrogel prepared in vitro can be stored at low temperature (for example 4°C) under sterile conditions prior to use.

[0114] 3. Properties of the dynamic covalent hydrogel

[0115] The dynamic covalent hydrogel according to the present application is characterized by its viscoelasticity. The term "viscoelasticity" used herein to characterize the hydrogel refers to the property that can vary with time and / or loading rate provided by the scaffold. Thus, it is envisaged that a suitable viscoelastic hydrogel provides time and / or rate loading properties that match or approximate those observed in a predetermined tissue or site (see below). This property is related to energy dissipation, which can be provided by the scaffold itself and / or the scaffold as a composite in which cells are grown. For example, it is possible to provide a scaffold that approximates the viscoelasticity of the tissue that the hydrogel aims to repair, restore or replace (see below).

[0116] The dynamic covalent hydrogels according to the present application can have a shear storage modulus (G') of between about 10 Pa and about 10,000 Pa, preferably between about 100 Pa and about 1000 kPa; and a shear loss modulus (G") of between about 0.01 Pa and about 1000 Pa, preferably between about 0.1 kPa and about 500 Pa. The measurement of the G' and G" values of the dynamic gels is typically performed in a frequency range of 0.001-1000 Hz, and using a stress constraint of 0.1-10 pa.

[0117] The dynamic covalent hydrogels according to the present application undergo minimal swelling and exhibit high stability. As used herein, the term "swelling" is to be understood as the property of a hydrogel to increase in weight and volume upon absorption of a fluid, here water. As used herein, the term "swelling ratio" refers to the ratio of the weight of a hydrogel when substantially fully hydrated to the weight when not swollen, i.e. the weight before immersion in the fluid. The swelling ratio is typically provided in percent. The skilled person is aware of suitable procedures and measurement methods for determining the swelling ratio. As used herein, the term "stability" when referring to a hydrogel refers to the fact that the swelling ratio does not change over time. The dynamic covalent hydrogels according to the present application have a swelling ratio of between about 100% and about 300%, for example between about 100% and about 200%. In experiments given in the example section below (see Example 1), a stability of the prepared dynamic covalent hydrogels was observed for 35 days (for the 35 day experiment). It is very likely that the dynamic covalent hydrogels according to the present application are stable for several weeks up to several months.

[0118] 4. Additional ingredients

[0119] Depending on the intended use of the dynamic covalent hydrogels according to the present application, the hydrogels can comprise cells, bioactive agents, visualizing agents or any other additional ingredients, or a combination thereof. Examples of such cells and additional ingredients are provided below (see Pharmaceutical compositions and kits).

[0120] The bioactive agent, visualizing agent, or any other additional ingredient can be incorporated into the hydrogel before, during, or after crosslinking of the first and second hydrogel precursor polymers. For example, typically, any additional ingredient can be added to at least one of the first and second hydrogel precursor solutions (as described above) prior to the crosslinking step. During the crosslinking step, any additional ingredient can be added to the reaction mixture composed of the first and second hydrogel precursors. Alternatively or additionally, the bioactive agent, visualizing agent, or any other active ingredient can be incorporated into the hydrogel after the hydrogel has been formed. For example, the dynamic covalent hydrogel according to the present application can be immersed in a solution containing any additional active ingredient to allow the ingredient to diffuse into the hydrogel, or any additional active ingredient can be added to a solution containing the dynamic covalent hydrogel. Alternatively, it is also possible to prepare a dynamic covalent hydrogel from the crosslinking of hydrogel precursors, wherein the first hydrogel precursor or the second hydrogel precursor is directly or indirectly covalently linked to a bioorthogonal functional moiety or a clickable moiety (see above), which can be subjected to bioorthogonal or click conjugation to introduce a desired molecule or biomolecule (e.g., a peptide, a growth factor, a drug, a fluorophore, etc.).

[0121] In embodiments where the dynamic covalent hydrogel according to the present application comprises cells, the cells can be encapsulated within the hydrogel. As used herein, the term "encapsulated" has its meaning as understood in the art and refers to the inclusion, immobilization, and / or entrapment of one or more cells in a three-dimensional structure defined by a physical barrier (i.e., a barrier that reduces or controls the permeability of the structure).

[0122] III - Use of hydrogel precursor polymer compositions and dynamic covalent hydrogels

[0123] Soft materials such as injectable hydrogels have enabled a variety of modern technologies, including tissue engineering, cell therapy, drug delivery, biomedical devices, microfluidics, optics, stretchable and bio-integrated electronics, and soft robotics (see, for example, Yannas et al., Science, 1982, 215: 174-176; Lee and Mooney, Chem. Rev., 2001, 101: 1869-1880; Peppas et al., Adv. Mater., 2006, 18: 1345-1360; Jeong et al., Cell, 2015, 162: 662-674; Park et al., Nature Biotechnology, 2015, 33: 1280-1286; Whitesides, Nature, 2006, 442: 386-373; Casavant et al., PNAS USA, 2013, 110: 10111-10116; Dong et al., Nature, 2006, 42: 551-554; Choi et al., Nature Photonics, 2013, 7: 987-994; Choi et al., Advanced Materials, 2015, 27: 4081-4086; Kim et al., Science, 2008, 320: 507-511; Rogers et al., Science, 2010, 327: 1603-1607; Xu et al., Science, 2014, 344: 70-74; Tee et al., Science, 2015, 350: 313-316; Shepherd et al., PNAS USA, 201, 108: 20400-20403; and Morin et al., Science, 2012, 337: 828-832). Thus, the polymer compositions and dynamic covalent hydrogels according to the present application can find application scenarios in a wide range of fields, particularly in the treatment or prevention of a disease or medical condition in a subject.

[0124] As used herein, the term "subject" refers to a human or another mammal (e.g., a primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, etc.) who does or does not have a disease or disorder. The non-human subject can be a transgenic or otherwise modified animal. In many embodiments of the application, the subject is commonly referred to as an "individual" or "patient." The terms "subject," "individual," and "patient" do not connote a particular age, thus encompassing neonates, children, adolescents, and adults. The term "patient" more specifically refers to an individual who has a disease or disorder.

[0125] The term "treatment" is used herein to characterize a method or process that is directed to (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing or stopping the progression, worsening, or deterioration of a disease, disorder, or condition; (3) ameliorating symptoms of a disease, disorder, or condition; or (4) curing a disease, disorder, or condition. For therapeutic effects, treatment can be performed after the onset of the disease, disorder, or condition. Alternatively, treatment can be performed prior to the onset of the disease, disorder, or condition, to act prophylactically or preventively. In this case, the word "prevent" is used.

[0126] 1.3D Cell Culture and Cell Therapy

[0127] The dynamic covalent hydrogels of the present application are attractive 3D cell culture alternatives to natural, animal-derived matrices such as 3D cell culture is an artificially created environment in which biological cells are allowed to grow in all three spatial dimensions or to interact with their surroundings, similar to their in vivo situation. Cells in 3D culture are closer to the in vivo situation in terms of cell shape and cell environment compared to conventional culture. The diversity of architectures and materials is much greater on 3D matrices than on 2D matrices. Thus, in certain embodiments, the dynamic covalent hydrogels of the present application are used for 3D cell culture.

[0128] The term "cell culture" refers to the process of maintaining cells under conditions suitable for maintenance and / or growth, where the conditions refer to, for example, the temperature at which the cells are kept, the availability of nutrients, the atmospheric CO2 content, and the cell density. In a hydrogel, cells can be cultured in vitro or in vivo. Suitable culture conditions for maintaining proliferation, expansion, and differentiation of different types of cells are well known and documented in the literature. Cells that can be cultured using the dynamic covalent hydrogels according to the methods of the present application include any cell that can grow in a 3D hydrogel matrix (see below).

[0129] The dynamic covalent hydrogels for 3D cell culture can comprise any of a variety of biomolecules that are desirable to be present in a cell culture environment. Examples of such biomolecules include, but are not limited to, proteoglycan or glycosaminoglycan chains, hormones, growth factors, chemoattractants, and the like (see below for pharmaceutical compositions and kits).

[0130] Following culture in the dynamic covalent hydrogels according to the present application, the cells can be harvested prior to use. The term "harvesting" as used herein refers to the act of isolating or dissociating the cells from the 3D hydrogel in which they were cultured. The cells can be isolated from the hydrogel fragments produced by enzymatic degradation of the hydrogel by filtration. Examples of enzymes that can be used to degrade the hydrogel scaffold include, but are not limited to, collagenase, elastase, trypsin, pullulanase, hyaluronidase, chondroitinase (ABC), cellulase, and the like. Alternatively, the dynamic gels have the great advantage of disintegrating in the presence of one of the two reactive groups in the form of a large number of small free molecules. Typically, glucosamine or any other diol is added to dissolve the gel, and the cells can be recovered by centrifugation without the need to use enzymatic reactions or to grind the material.

[0131] Following harvesting, the cells cultured in the dynamic covalent hydrogels can have a number of applications. Thus, for example, the harvested cells can be used in cell therapy, in which live cells are injected, transplanted, or implanted into a subject in order to treat or prevent a disease or medical condition in the subject. Thus, the harvested cells can be used to treat human clinical conditions (e.g., cancer, infectious diseases, autoimmune diseases) or for regenerative medicine (e.g., for repairing damaged, injured, or defective tissues, such as articular cartilage, spinal cord, etc.). Functional stem cells, which are considered promising therapeutic candidates, can be used in large quantities to facilitate the development of autologous (from the patient) or allogeneic (from another donor) transplants. Similarly, using the dynamic covalent 3D hydrogel matrices of the present application, large numbers of cells from genetically tailored stem cell lines can be generated for cell-based therapies. The dynamic covalent 3D hydrogels described herein can also be used for stem cell proliferation in therapeutic cloning (also known as somatic cell nuclear transfer).

[0132] Alternatively, the harvested cells can be used in tissue engineering applications. Functional cells, which can be used in large quantities, are also an advantage in cell biology research. Other potential uses of the dynamic covalent hydrogels according to the present application include, but are not limited to, the production of lineage-dependent viruses, e.g., for the production of viruses that require differentiated cells to produce enough particles to be used as a vaccine; and protein production (in which cells on the hydrogels produce factors that can be isolated from the culture medium and / or the cells, which are then purified). Examples of proteins that can be produced in this manner include, but are not limited to, growth factors, hormones, signaling molecules, cell growth inhibitors, and antibodies.

[0133] 2. Tissue engineering, organoid formation, and applications

[0134] The dynamic covalent hydrogels of the present application can be used in tissue engineering. Tissue engineering is generally defined as the creation of tissue or organ equivalents by seeding cells onto or into a scaffold suitable for implantation. Tissue engineering involves the use of tissue scaffolds to form new living tissue to repair or replace entire tissues or portions of tissues (e.g., bone, cartilage, blood vessels, bladder, skin, muscle, etc.) that have been damaged by disease, trauma, genetic or chromosomal disorders, or aging. Thus, in certain embodiments, the dynamic covalent hydrogels described herein are used in tissue engineering.

[0135] As is known in the art, tissue culture requires being in a suitable medium with or without stimulation, such as pressure or orientation. In addition, the tissue medium or dynamic covalent hydrogel can comprise any of a variety of biomolecules, the presence of which is desirable in this context. Examples of such biomolecules include growth factors, nutrients, and / or cell binding domains, sugars, tissue adhesives, and the like.

[0136] Examples of tissue equivalents include organoids. As used herein, the term "organoid" refers to a three-dimensional culture system of organ-specific cell types that develop from stem cells and self-organize (or self-pattern) through cell sorting and spatially restricted lineage commitment in a manner analogous to in vivo conditions. Thus, organoids represent the natural physiology of cells and have a cellular composition (including remaining stem cells and specialized cell types) and anatomical structure that mimic the natural conditions. The cells that generate organoids differentiate to form organ-like tissues that exhibit multiple cell types that self-organize to form structures that closely resemble in vivo organs. Thus, organoids are an excellent model for studying human organs and the development of human organs in a system that closely resembles in vivo development. Organoids can be used for drug response screening, toxicity analysis, or regenerative medicine. Organoids can also be used to culture pathogens, such as noroviruses for which there are currently no suitable tissue cultures or animal models.

[0137] 3. In vivo delivery of cells and / or bioactive agents

[0138] In certain embodiments, the dynamic covalent hydrogels according to the present application can be used as a system for the in vivo (i.e., in a subject in need thereof) delivery of cells and / or bioactive agents.

[0139] In certain embodiments, the hydrogels according to the present application can be used for cell delivery. The hydrogels of the present application can be used as a raw material for the preparation of cell delivery systems that can be administered to a subject for therapeutic or diagnostic purposes. In certain embodiments, the hydrogels of the present application can be used for the preparation of patches, biomembranes or dressings that can be loaded with cells. For example, the hydrogels according to the present application can be used for the preparation of dressings that can be applied to the skin for the reconstruction or healing of the skin (e.g. at the site of a damaged or wounded skin). Alternatively, the dressings can be applied on the heart of a subject for the treatment of ischemia (myocardial infarction). In this embodiment, the cells entrapped in the hydrogels can migrate into the target tissue or organ.

[0140] In other embodiments, the cross-linking of the hydrogel precursor polymers can be used for cell delivery. In fact, in situ gelling polymer matrices are of great interest in tissue regeneration, as these materials can be used as injectable hydrogels. They can act as cell carriers with the ability to form the shape of the corresponding tissue cavity. Moreover, since cells can be directly incorporated into the injectable solution, problems related to cell adhesion can be minimized.

[0141] The dynamic covalent hydrogels and hydrogel precursor polymers according to the present application can be used as carriers for the delivery and / or controlled release of at least one bioactive agent. The terms "bioactive agent" and "biologically active agent" are used interchangeably herein; and include, but are not limited to, physiologically or pharmacologically active agents that act locally or systemically in the body, such as therapeutic, prophylactic and / or diagnostic agents, agents that affect body structure or function (e.g. agents that affect or participate in tissue growth or cell differentiation), compounds capable of eliciting a biological effect (such as an immune response) or playing any other role in one or more biological processes, prodrugs that become biologically active or more active upon being placed in a predetermined physiological environment, and compounds or agents that support or promote cell growth, cell differentiation and / or cell engraftment. Examples of bioactive agents are provided below (see Pharmaceutical Compositions and Kits).

[0142] The bioactive agent can be mixed with the hydrogel, covalently bonded to the hydrogel and / or adsorbed in or on the hydrogel. Alternatively, or additionally, the bioactive agent is contained in the solution of the first hydrogel precursor polymer or the second hydrogel precursor polymer of the cross-linking pair used to form the dynamic covalent hydrogel, such that upon cross-linking of the first and second polymers, the dynamic covalent hydrogel thus formed comprises the bioactive agent.

[0143] 4. Viscoelastic supplementation therapy / artificial joint lubricant

[0144] In certain embodiments, the cross-linking of the hydrogel precursor polymers or dynamic covalent hydrogels according to the present application can be used in visco-supplementation therapy. Visco-supplementation therapy is a therapy involving the injection of a gel-like substance (e.g. hyaluronate) into a joint to supplement the viscosity of synovial fluid. Therapy for the treatment of osteoarthritis by injection of a viscoelastic fluid (most commonly cross-linked hyaluronic acid) into a joint has been performed for over 20 years. The effectiveness of these visco-supplementation therapy treatments is thought to come in part from the high viscosity of the injected polymer solution.

[0145] A polymer hydrogel that undergoes cross-linking during or after administration into a joint space can bring an improvement, as such a hydrogel experiences an increase in viscosity within the joint space after administration. Thus, the cross-linking of the hydrogel precursor polymers according to the present application can be dosed as a liquid of relatively small viscosity, and then form a gel of greater viscosity or stronger viscoelasticity within the intra-articular space of a joint in a short time. Furthermore, the higher viscosity imparted by in situ cross-linking can allow for an extended residence time in the joint, so that a single dose can give rise to a longer duration of treatment.

[0146] Thus, the cross-linking of the hydrogel precursor polymers or dynamic covalent hydrogels according to the present application can be used in visco-supplementation therapy for the treatment of osteoarthritis or rheumatoid arthritis, or other inflammatory arthritic conditions (such as gout) or calcium pyrophosphate deposition disease (e.g. by injection into the intra-articular space of a joint). Joints include the knee joint, shoulder joint, temporomandibular joint and carpometacarpal joint, elbow joint, hip joint, wrist joint, ankle joint and lumbar facet (plane) joints in the spine.

[0147] In certain embodiments, the at least one hydrogel precursor polymer solution comprises a corticosteroid (such as triamcinolone acetonide, cortisone acetate) that can be used to relieve pain and swelling due to inflammation in a subject suffering from osteoarthritis. The following advantages are associated with the in situ capture / incorporation of corticosteroids in a hydrogel: (1) effective prevention of direct contact of the bulk of the steroid with joint tissue; (2) effective maximization of the local concentration of the steroid in the joint while minimizing its systemic concentration, (3) effective prevention of premature clearance of the steroid from the joint, and (4) allow for a therapeutic effect at a lower total dose than without hydrogel capture, while minimizing unwanted local and systemic side effects.

[0148] In certain embodiments, the at least one hydrogel precursor polymer solution comprises fibroblast growth medium to revitalize the cellular component of the joint connective tissue, in particular synoviocytes and chondrocytes, and thereby ensure their cell regeneration and stimulate their endogenous synthesis.

[0149] The viscoelastic supplementation therapy can be accomplished by a single injection or multiple intra-articular injections over weeks to the affected joint. The goal is to provide a joint that has the ability to absorb shock during motion and at least be lubricated when at rest. In certain embodiments, the viscoelastic supplementation is administered with the goal of delaying total hip arthroplasty or total knee arthroplasty.

[0150] 5. 3D printing and 3D bioprinting

[0151] In certain embodiments, the crosslinking of the hydrogel precursor polymers and dynamic covalent hydrogels described herein can be applied to 3D printing or 3D bioprinting. 3D printing builds three-dimensional objects from computer-aided design models, usually by successively adding material layer by layer. 3D bioprinting utilizes 3D printing technology to produce functional miniaturized tissue constructs from biocompatible materials, cells, and supporting ingredients such as cell media. Major applications include high-throughput in vitro tissue models, drug development and toxicology, regenerative medicine / tissue engineering applications. It involves the precise layer-by-layer positioning of biomaterials and living cells, and spatial control over the placement of functional ingredients. The technology has made significant progress in the clinical repair of tissues and organs such as ear, nose, bone, heart, liver, and skin.

[0152] 6. Soft robotics

[0153] The crosslinking of the hydrogel precursor polymers and dynamic covalent hydrogels described herein can be applied to soft robotics. Soft robotics is a specific subfield of robotics that involves building robots from highly compliant materials that are similar to those found in living organisms (Robosoft, first IEEE International Conference on Soft Robotics, April 24-28, 2018, Livorno, Italy). Compared to robots made from rigid materials, soft robots can improve flexibility and adaptability for completing tasks, and improve safety for working around humans. These characteristics make it potentially useful in the fields of medicine and manufacturing. Thus, for example, soft robots can be implemented in the medical industry, particularly for invasive surgery. Due to their shape-changing properties, soft robots can be used to assist in surgical procedures. Because a soft robot can navigate through different structures of the human body by adjusting its shape, shape-changing is important.

[0154] IV. Pharmaceutical compositions and kits

[0155] For use in therapeutic treatment of mammals, including humans, of the hydrogel precursor polymers and dynamic covalent hydrogels described herein, in some embodiments, the hydrogel precursor polymers and dynamic covalent hydrogels are formulated into pharmaceutical compositions in accordance with standard pharmaceutical practice. Accordingly, the present application provides pharmaceutical compositions comprising a dynamic covalent hydrogel described herein and at least one pharmaceutically acceptable diluent or carrier. The present application also provides pharmaceutical compositions wherein the first hydrogel precursor polymer of a cross-linking pair described herein is formulated with at least one pharmaceutically acceptable diluent or carrier and the second hydrogel precursor polymer of a cross-linking pair described herein is formulated with at least one pharmaceutically acceptable diluent or carrier.

[0156] Pharmaceutical compositions according to the present application can be prepared, packaged, and / or sold in bulk, as single unit dosages, and / or as multiple unit dosages. As used herein, the term "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined quantity of each hydrogel precursor polymer or a predetermined quantity of a dynamic covalent hydrogel of a given shape and size.

[0157] Pharmaceutical compositions according to the present application are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject (age, body weight, etc.) the clinical condition of the individual patient (fitness), the cause of the disorder, the delivery site, the method of administration, the scheduling of administration, the scheduling of treatment with other medications, and other factors known to medical practitioners. The

[0158] 1. Formulation

[0159] As used herein, the term "pharmaceutically acceptable carrier or excipient" means a carrier medium that does not interfere with the biological activity of the active ingredient and that is nontoxic to the host in which it is administered at the dosages and concentrations employed. This term includes solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see, e.g., "Remington's Pharmaceutical Sciences", E. W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

[0160] Examples of suitable pharmaceutically acceptable carriers or excipients include, but are not limited to, saline and / or buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, etc.), antioxidants (e.g., ascorbic acid, alpha-tocopherol, ascorbyl palmitate, methionine, etc.), preservatives, low molecular weight (less than 10 residues) peptides, proteins (e.g., serum albumin, gelatin or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine or lysine), monosaccharides and disaccharides and other carbohydrates (e.g., dextrose, mannose or dextrins), chelating agents (e.g., EDTA, citric acid and its salts and hydrates, fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, etc.), sugars (e.g., sucrose, mannitol, trehalose, sorbitol), salt-forming counterions (e.g., sodium), nonionic surfactants (e.g., TWEEN TM , PLURONICS TM , polyethylene glycol), lubricants (e.g., magnesium stearate, calcium stearate, stearic acid, silica, talc, mallet, glycerol esters, natural oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc.), or any combination thereof.

[0161] Suitable preservatives can be antimicrobial preservatives (e.g., benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexylene glycol, imidurea, phenol, phenoxyethanol, phenylethanol, phenylmercuric nitrate, propylene glycol, and thimerosal), antifungal preservatives (e.g., butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid), antiprotozoal preservatives, alcoholic preservatives (e.g., ethanol, polyethylene glycol, phenol, phenolics, bisphenols, chlorobutanol, hydroxybenzoate esters, and phenylethanol), acidic preservatives (e.g., vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid), any other preservative known to be useful in pharmaceutical compositions (e.g., tocopherol, tocopherol acetate, methionine, sulfoximine, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, etc.), or any combination thereof.

[0162] For example, injectable formulations can be prepared according to known techniques using suitable carriers and solvents such as water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland, fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid find use in the preparation of injectables. Sterile liquid compositions can be used for parenteral administration by injection. Injectables can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. To the extent necessary or desirable, injectable formulations can include local anesthetics to lessen pain at the site of the injection.

[0163] When topically applied, the crosslinked pair of hydrogel precursor polymers and dynamic covalent hydrogels according to the present application, for example comprising cells and / or bioactive agents, are suitably combined with other ingredients such as carriers and / or adjuvants. There are no particular restrictions as to the nature of these other ingredients, except that they must be physiologically acceptable and effective for the intended administration thereof, and do not reduce the activity of the active ingredients in the composition. In certain embodiments, the pharmaceutical compositions according to the present application can be impregnated into or deposited onto articles, which can include, for example, transdermal patches, plasters, and bandages. In other embodiments, the crosslinked pair of hydrogel precursor polymers and dynamic covalent hydrogels according to the present application can be used to prepare patches, biofilms, or dressings that can be loaded with cells and / or bioactive agents.

[0164] While the description of compositions provided herein is primarily directed to compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to animals of all sorts. In order for a composition to be suitable for administration to various animals, modifications can be made to the composition that are well known.

[0165] 2. Additional ingredients

[0166] The pharmaceutical compositions according to the present application can further comprise at least one additional ingredient selected from the group consisting of cells, bioactive substances, visualizing agents, or any combination thereof. As described above, the bioactive ingredients and visualizing agents can be bound to at least one of the hydrogel precursor polymers or dynamic covalent hydrogels present in the pharmaceutical composition by covalent or ionic means. Alternatively or additionally, the bioactive substances and visualizing agents can be mixed with the hydrogel precursor solution or pharmaceutical composition according to the present application and do not form any association with the hydrogel precursor polymers or hydrogels.

[0167] A. Cells

[0168] In certain embodiments, the hydrogel precursor polymer solution or dynamic covalent hydrogel contains cells. In the context of the present application, the term "cell" refers to various forms of cells, including but not limited to cell colonies (such as pancreatic islets or portions thereof) and individually isolated cells. In certain preferred embodiments, the cells used in conjunction with the hydrogel precursor polymer solution or dynamic covalent hydrogel according to the present application are of mammalian (animal or human) origin. The mammalian cells can be of any organ, fluid or tissue origin (e.g., brain, liver, skin, lung, kidney, heart, muscle, bone, bone marrow, blood, amniotic fluid, umbilical cord blood, etc.) and of any cell type (see below). The cells can be primary cells, secondary cells or immortalized cells (i.e., established cell lines). They can be isolated or derived from an in vitro biological sample, or obtained from a volunteer or patient by techniques well known in the art. Cells for regenerative medicine and tissue engineering can be from the patient to whom the cells are to be administered (autologous administration) or from other individuals (allogeneic administration). Furthermore, xenogeneic cells such as from animals can also be used in regenerative medicine strategies. Alternatively, or additionally, cells can be purchased from commercial sources (e.g., from the American Type Culture Collection, Manassas, VA). Alternatively, or additionally, the cells can be genetically engineered to contain a gene of interest, such as a gene expressing a growth factor or receptor, or containing a defective gene, or further containing Oct3 / 4, Sox2, Klf4 and c-Myc genes, so as to make human induced stem cells from adult human somatic cells.

[0169] Cells that can be used in conjunction with the hydrogel precursor polymer solution or dynamic covalent hydrogel described herein include differentiated cells, stem cells (including induced pluripotent stem cells) and progenitor cells.

[0170] As used herein, the term "differentiated cell" refers to a cell that is specialized for a particular function and does not have the ability to give rise to other kinds of cells. Examples of differentiated cells include, but are not limited to, basal cells, epithelial cells, platelets, lymphocytes, T cells, B cells, natural killer cells, reticulocytes, granulocytes, monocytes, mast cells, neural cells, neuroblasts, glioblasts, macrophages, dendritic cells, Kupffer cells, Langerhans cells, lining cells, and histiocytes. Specific examples of differentiated cells include, but are not limited to, fibroblasts, chondrocytes, osteoblasts, osteoclasts, osteocytes, synoviocytes, bone marrow stromal cells, stem cells, fibrocartilage cells, endothelial cells, smooth muscle cells, adipocytes, cardiomyocytes, muscle cells, keratinocytes, hepatocytes, leukocytes, macrophages, endocrine cells, urogenital cells, lymphatic cells, pancreatic islet cells, muscle cells, intestinal cells, kidney cells, vascular cells, thyroid cells, parathyroid cells, adrenal-pituitary axis cells, bile duct cells, ovarian or testicular cells, salivary secretory cells, renal cells, epithelial cells, and neural cells.

[0171] As used herein, the term "stem cell" refers to a relatively undifferentiated cell that has the capacity for sustained self-renewal and the potential to generate differentiated progeny (i.e., specialized cells of different types). Examples of stem cells include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The terms "embryonic stem cell" and "ES cell" are used interchangeably herein. It refers to stem cells derived from a group of cells called the inner cell mass, which is a part of an early (4- to 5-day) embryo called a blastocyst. "Human embryonic stem cells" or "hES cells" are stem cells of human origin, typically derived from a fertilized embryo less than one week old. In vitro, embryonic stem cells can be propagated indefinitely, a property that adult stem cells do not share. The term "adult stem cell" refers to stem cells that are not of embryonic origin, nor are they derived from embryonic or fetal tissue. The term "adult stem cell" also encompasses stem cells isolated from subjects of all ages (e.g., human infants and children). As used herein, the term "induced pluripotent stem cell" (or "iPS cell") refers to a pluripotent stem cell artificially derived from a non-pluripotent cell (e.g., an adult somatic cell). Induced pluripotent stem cells are identical to embryonic stem cells in their ability to form any differentiated cell, but are not derived from an embryo. Induced pluripotent stem cells can be human induced pluripotent stem cells. The terms "human induced pluripotent stem cell" and "human iPS cell" are used interchangeably herein. They refer to induced pluripotent stem cells of human origin. Typically, human induced stem cells can be obtained by inducing expression of Oct3 / 4, Sox2, Klf4, and c-Myc genes in any adult somatic cell (e.g., fibroblast). Essentially, the somatic cell is transfected with a viral vector, such as a retrovirus, that contains the Oct3 / 4, Sox2, Klf4, and c-Myc genes.

[0172] The terms "progenitor cell" and "precursor cell" are used interchangeably herein. They refer to cells that occur in fetal or adult tissues and are partially specialized. These cells divide and give rise to differentiated cells. Progenitor or precursor cells belong to a transiently expanding population of cells derived from stem cells. In comparison to stem cells, they have a limited capacity for self-renewal and differentiation. This capacity for self-renewal (or proliferation) is evidenced by expression of proliferation markers (e.g., Ki-67 nuclear antigen). In addition, because progenitor cells undergo a specific differentiation process, progenitor cells also express specific markers. Examples of progenitor cells include, but are not limited to, hematopoietic progenitor cells, endothelial progenitor cells, neural progenitor cells, mesenchymal progenitor cells, osteogenic progenitor cells, stromal progenitor cells, and the like.

[0173] The cells present in the hydrogel precursor polymer solution or the dynamic covalent hydrogel can form a substantially homogenous population or a heterogeneous population of cells. As used herein, the term "substantially homogenous population of cells" refers to a population of cells in which a majority (e.g., at least about 90%, preferably at least about 95%, more preferably at least about 99%) of the total number of cells belong to a single cell type. The term "heterogeneous population of cells" as used herein refers to a population of cells comprising at least two cell types.

[0174] The cells can be present in the hydrogel precursor polymer solution or the dynamic covalent hydrogel in any suitable amount. For example, the cells can be added to the hydrogel precursor polymer solution or the dynamic covalent hydrogel at a cell density of about 500 to about 1000 cells / L.

[0175] B. Biologically Active Agents

[0176] In certain embodiments, the pharmaceutical compositions according to the present application comprise at least one biologically active agent. The choice of one or more biologically active agents should be based on the intended purpose of the pharmaceutical composition (e.g., for viscoelastic replenishment therapy, joint therapy, cell therapy, 3D cell culture, tissue engineering, etc.) as understood by one of skill in the art. Generally, the amount of biologically active agent present in the pharmaceutical compositions of the present application is the usual dosage required to achieve the desired result by a given route of administration. Such dosages are known, or are readily determined by those of skill in the pharmaceutical and / or medical arts.

[0177] Suitable biologically active agents can belong to a variety of types of molecules, including but not limited to small molecule drugs, peptides, polypeptides, proteins, antibodies, genes and gene products, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, glycoproteins, oligonucleotides, steroids, nucleic acids, DNA, RNA, aptamers, nucleodides, nucleosides, oligonucleotides, antisense oligonucleotides, polynucleotides, siRNA, lipids, hormones, vitamins, and combinations thereof. The biologically active agent can be a single compound or a plurality of compounds, including for example a combination of two or more biologically active agents.

[0178] Examples of suitable therapeutic agents include, but are not limited to, analgesics, anesthetics, analgesics, anticancer agents, antimicrobial agents, antibacterial agents, antiviral agents, antifungal agents, antibiotics, anti-inflammatory agents, antioxidants, antiseptics, antipruritics, immunostimulants, anti-angiogenic agents, antitumor agents, antiproliferative agents, antidiabetic agents, decongestants, antihypertensive agents, dermatological agents, anticholinergics, immunosuppressants, antidepressants, antipsychotics, beta-adrenergic blockers, cardiovascular active agents, vasoactive agents, non-steroidal agents, sex hormones, steroid agents, osteogenic agents, osteoconductive agents, osteoinductive agents, anti-rejection agents, anti-arthritic agents, thrombolytic agents, anti-fibrinolytic agents, hemorheologic agents, anti-platelet agents, and the like.

[0179] Other examples of suitable bioactive agents include cytokines, growth factors, proteoglycans or portions thereof, adhesion molecules, and any combination thereof.

[0180] Cytokines and growth factors are polypeptide molecules that regulate mammalian cell migration, proliferation, differentiation, and metabolism. Different ranges of these biological molecules have been identified to play potentially important roles in the regulation of healing. Cytokines can be lymphokines, monokines, or chemokines. Examples of cytokines include, but are not limited to, interleukins (ILs) (e.g., IL-1, IL-2, IL-4, and IL-8), interferons (IFNs) (e.g., IFN-a, IFN-b, and IFN-g), and tumor necrosis factors (e.g., TNF-a), or any variant, synthetic analog, active portion, or combination thereof. Examples of growth factors include, but are not limited to, epidermal growth factors (EGFs), platelet-derived growth factors (PDGFs), heparin-binding growth factors (HBGFs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), insulin-like growth factors (IGFs), connective tissue activating peptides (CTAPs), transforming growth factors a (TGF-a) and b (TGF-b), nerve growth factors (NGFs), colony stimulating factors (G-CSF and GM-CSF), and the like, or any variant, synthetic analog, active portion, or combination thereof.

[0181] Proteoglycans are protein-carbohydrate complexes characterized by their glycosaminoglycan (GAG) component. GAGs are highly charged, sulfated and carboxylated polyanionic polysaccharides. Examples of GAGs suitable for use in the pharmaceutical compositions of the present application include, but are not limited to, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate.

[0182] Adhesion molecules constitute a diverse family of extracellular and cell surface glycoproteins that participate in cell-cell and cell-extracellular matrix adhesion, recognition, activation, and migration. Adhesion molecules are essential for the structural integrity and homeostatic function of most tissues and are involved in a wide range of biological processes, including embryogenesis, inflammation, thrombosis, and tissue repair. Adhesion molecules include matrix cell proteins (such as thrombospondin and tenascin) and cell surface adhesion molecules (such as integrins, selectins, cadherins, and immunoglobulins).

[0183] Other examples of bioactive agents include, but are not limited to, hormones and hormone analogs (e.g., growth hormone), morphogenetic agents (e.g., retinoic acid, arachidonic acid, etc.), extracellular matrix molecules (e.g., fibronectin, vitronectin, laminin, collagen, elastin, etc.), coagulation / fibrinolysis factors (e.g., fibrinogen, prothrombin, hemophilia A, etc.); and the like.

[0184] The bioactive agent can be a bioactive peptide sequence that can be attached to the hydrogel surface to promote protein adsorption and subsequent cell tissue attachment. Examples include adhesion peptides derived from fibronectin, vitronectin, laminin, and collagen. The term "RGD" or "RGD sequence" refers to the minimal bioactive RGD sequence, which is the arginine-glycine-aspartic acid (RGD) sequence, and which is the minimal fibronectin-derived amino acid sequence sufficient to mimic cell binding to fibronectin and / or to promote adhesion of anchorage-dependent cells. Such short chain RGD bioactive peptides are known in the art.

[0185] Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents.

[0186] In certain embodiments, the bioactive agent is a particulate, such as a bioactive glass, a water glass, a resorbable calcium phosphate, hydroxyapatite, calcium carbonate, calcium sulfate, glass-ceramic, and the like.

[0187] In certain embodiments, the pharmaceutical composition described herein contains less than about 80%, less than about 75%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% by weight of the bioactive agent.

[0188] C. visualization agents

[0189] In certain embodiments, the hydrogel precursor polymer solution or dynamic covalent hydrogel can contain a visualization agent to improve visibility, for example to enable a surgeon to accurately and conveniently place the in situ forming hydrogel during a surgical procedure. The visualization agent can be selected from a variety of non-toxic colored substances, such as dyes suitable for use in implantable medical devices. For example, suitable dyes can include dyes for showing the thickness of the hydrogel when it is formed in situ, such as FD&C Blue #1, FD&C Blue #2, FD&C Blue #3, D&C Green #6, methylene blue, indocyanine green, other colored dyes, and combinations thereof. Other visualization agents can be used, such as fluorescent compounds (e.g., fluorescein or eosin), x-ray contrast agents (e.g., iodinated compounds), ultrasound contrast agents, MRI contrast agents (e.g., gadolinium-containing compounds), PET agents (e.g., fluorodeoxyglucose or FDG). PS or SPECT agents (e.g., radioligands such as 11 C-DASB, 11 C-flumazenil, 11 C-raclopride, and the like).

[0190] The visualization agent can be covalently attached to at least one of the hydrogel precursor polymers. However, in preferred embodiments, the visualization agent is not covalently attached to the hydrogel precursor polymers. For example, the visualization agent can be present in the precursor hydrogel solution. The visualization agent can be used in small amounts, for example, at a concentration of less than 1% weight / volume, or less than 0.01% weight / volume, or even less than 0.001% weight / volume.

[0191] 3. Administration

[0192] The pharmaceutical compositions according to the present application can be administered using any suitable route of administration. Routes of administration include enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual, and the like. In particular, in the context of the present application, the preferred route is direct administration to the affected site (e.g., by percutaneous injection, e.g., into the intra-articular space of a joint, by topical placement at a damaged or injured skin site, by placement in conjunction with a surgical procedure, and the like).

[0193] Dynamic covalent hydrogels exhibit viscous flow under shear stress (shear thinning) and rapidly recover (self-heal) when the applied stress is relaxed, enabling their minimally invasive implantation in vivo by direct injection or catheter-based delivery. Thus, in certain embodiments, the dynamic covalent hydrogels according to the present application can be compressed and loaded into a delivery device, such as a catheter, endoscope, syringe, or the like. The delivery device is navigated through the vasculature or other vascular system of the intended patient host, and the hydrogel is released from the delivery device and optionally anchored (e.g., sutured to the target repair or regeneration site). Once released at the site, the hydrogel elastically swells to approximately its original relaxed size and shape.

[0194] In other embodiments, the implantable hydrogel is inserted through open surgical procedures.

[0195] As noted above, the dynamic covalent hydrogels according to the present application can be formed in situ (e.g., directly at or near a given site in a living animal or human body). In this case, the formation of the hydrogel is initiated by mixing the two hydrogel precursor polymers at the injection site. Thus, in certain embodiments, the two hydrogel precursors of a cross-linking pair according to the present application can be applied (e.g., injected) onto a tissue via a sprayer to form a coating or space-filling hydrogel in situ. Preferably, the two hydrogel precursors are placed in separate chambers of the sprayer. When the sprayer is activated, the resulting spray contacts the tissue such that the two hydrogel precursor polymers mix and cross-link, gelation occurs and the hydrogel forms at the predetermined site in the body. The sprayer can be a multi-barrel system, preferably a dual-barrel syringe system.

[0196] As used herein, the term "multi-barrel system" refers to any system or device, typically a syringe, that includes at least two separate barrels and can have two or more plungers. As used herein, the term "dual barrel syringe system" refers to any system or device, typically a syringe, that includes two separate barrels and can have one or two plungers. Further, the multi-barrel (e.g., dual barrel) syringe system typically includes a tip cap, or a needle or cannula with or without a needle shield, in order to seal the end of the syringe system. The barrels typically have a storage capacity that contains sufficient first and second hydrogel precursor solutions. The barrels can be made of glass, plastic, or any other suitable material, and can have different geometries, internal diameters, material compositions, transparency, etc. Further, the multi-barrel syringe system can be a dual barrel syringe system in the form of a syringe having two integrally connected syringes, i.e., two integrally connected barrels, and a single or dual plunger assembly for dispensing the contents from the barrels. Further, the syringe system can include two detachably connected barrels and two or one detachably connected plungers. Further, the syringe system can also include a device (e.g., an applicator tip) configured for thorough mixing of the components contained in the barrels prior to dispensing through the applicator tip. Thus, the barrels are typically connected and the plunger assembly is typically configured to dispense the contents from the barrels simultaneously in a manner such that the hydrogel precursor solutions are held in the proper mixing ratio.

[0197] The treatment according to the present application can be administered in a single dose or in multiple doses (e.g., daily, weekly, monthly, every two months, every three months, every half year, every year, every two years, etc.). The dose and dosage regimen will be determined by the physician.

[0198] The dose can be any amount of the pharmaceutical composition sufficient to achieve the desired biological or medical response. For example, the dose can correspond to about 0.1 pg to about 1 pg of the cross-linking pair of hydrogel precursors or hydrogels, or about 0.001 mg to about 0.01 mg, or about 0.01 mg to about 0.1 mg, or about 0.1 mg to about 1 mg, or about 1 mg to about 3 mg, or about 3 mg to about 10 mg, or about 10 mg to about 30 mg, or about 30 mg to about 100 mg, or about 100 mg to about 300 g, or about 300 mg to about 1000 mg, or about 1 g to about 10 g of the cross-linking pair of hydrogel precursors or hydrogels.

[0199] 4. Kit

[0200] In another aspect, the present application provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampules, test tubes, flasks, or bottles) filled with one or more of the ingredients of the pharmaceutical compositions of the application as described herein.

[0201] The different components of the pharmaceutical packs or kits can be provided in solid (e.g., lyophilized) or liquid or semi-liquid form. Each component is typically suitable for dispensing in a separate container or provided in concentrated form. The packs or kits according to the application can include media for reconstitution of the lyophilized components. The individual containers of the kit will preferably be kept in a closed state for commercial sale.

[0202] In certain embodiments, the kits according to the application comprise a first hydrogel precursor polymer and a second hydrogel precursor polymer of a cross-linking pair as described herein, wherein the first and second hydrogel precursor polymers (as such or formulated in solution, respectively) are contained in different containers. In other embodiments, the first and second hydrogel precursor polymers are contained in a multi-barrel syringe system, preferably a double-barrel system.

[0203] In certain embodiments, the kits according to the application comprise at least one dynamic covalent hydrogel contained in a first container and at least one medium and / or reagent for use with the hydrogel. Examples of such media and / or reagents include, but are not limited to, rehydration media and / or reagents; antibiotics; biomolecules, bioactive agents as described herein, cell culture media and / or reagents, cells, inoculation tools, harvesting media and / or reagents, filters, washing media and / or reagents, and the like.

[0204] In certain embodiments, the packs or kits include one or more additional bioactive agents. Optionally, the containers can be accompanied by written or printed instructions or package inserts in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the package for human administration. The package insert can contain instructions for use of the hydrogel precursor polymer pair or dynamic covalent hydrogel according to the therapeutic methods disclosed herein.

[0205] An identifier, such as a bar code, radio frequency, ID tag, etc., can be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit, for purposes of quality control, inventory control, tracking movement between workstations, etc.

[0206] Examples

[0207] The following examples describe some preferred modes of making and practicing the application. However, it is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, unless the description in the examples is presented in the past tense, the text is not intended to suggest that experiments actually were performed, or that data actually were obtained, in the past.

[0208] Example 1

[0209] I. Synthesis and Polymer Modification - Synthesis of Amineated Wulff-B Type

[0210] Synthesis of (2-(((4-((tert-butoxycarbonyl)amino)butyl)amino)methyl)phenyl)boronic acid. To a solution of BOC-protected diaminobutane (BOC-DAB; 2.66 mmol; 500 mg) in 3 mL of methanol was added 2-formylphenylboronic acid (1 equiv; 2.66 mmol; 398 mg) under argon. After stirring at room temperature for more than 16 hours, sodium borohydride (4.26 mmol; 161 mg) was slowly added to the light yellow solution at 0 °C. The reaction mixture was then stirred at room temperature and monitored by TLC (dichloromethane / methanol (7 / 3, vol / vol)) until completion of the reaction. After removal of the solvent under vacuum, the crude product was dissolved in a mixture of water (12 mL) and dichloromethane (25 mL). The aqueous phase was extracted 5 times with dichloromethane (5 x 12 mL). The resulting combined organic phase was dried over MgS04and concentrated under vacuum to give the expected compound as a white solid (0.76 g; yield: 93%).

[0211] Synthesis of (2-(((4-aminobutyl)amino)methyl)phenyl)boronic acid. The above product was dissolved in 3 mL of methanol and subjected to slow bubbling of HC1 gas. The reaction was monitored by TLC (dichloromethane / methanol (7 / 3, vol / vol)). After 30 minutes, the reaction mixture was concentrated under vacuum to give the corresponding ammonium salt. The resulting solid was dissolved in pure water (10 mL) and the pH was adjusted to 11 by dropwise addition of NaOH (1 M). The reaction mixture was concentrated by co-solvent evaporation using toluene. The solid residue was isolated by addition of dichloromethane. After drying over Na2S04and filtration, the organic phase was concentrated under vacuum to give the expected amine Wulff-type phenylboronic acid derivative as a light yellow powder (427 mg; 72%). 1 H NMR was used to monitor the reaction. After 30 minutes, the reaction mixture was concentrated under vacuum to give the corresponding ammonium salt. The resulting solid was dissolved in pure water (10 mL) and the pH was adjusted to 11 by dropwise addition of NaOH (1 M). The reaction mixture was concentrated by co-solvent evaporation using toluene. The solid residue was isolated by addition of dichloromethane. After drying over Na2S04and filtration, the organic phase was concentrated under vacuum to give the expected amine Wulff-type phenylboronic acid derivative as a light yellow powder (427 mg; 72%).

[0212] Synthesis of boronic acid-modified polysaccharides. A typical synthesis is as follows: 500 kDa hyaluronic acid (HA) (100 mg) was dissolved in MES buffer (10 mL) at pH 5.5. DMT-MM (4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride) (69 mg; 1 equiv) was added to the HA solution and reacted for 30 minutes under stirring and room temperature. Wulff-type phenylboronic acid (wPBA; 27.5 mg; 0.5 equiv) was added to the activated HA solution and reacted for 3 days under stirring and room temperature. The solution was filtered sterilized and dialyzed against lx PBS buffer (pH 7.4) for 1 day and then against deionized water for 2 days (MWCO 12-14 kDa, Spectrum Labs). The solution was lyophilized and stored at 4 °C. The degree of substitution was determined by 1H NMR (400 MHz, D20, δ) determination. Similar procedures were used to graft various aminated boronic acid derivatives (i.e. 2-amino phenyl boronic acid, 3-amino phenyl boronic acid, 4-amino phenyl boronic acid, 5-amino benzene boronic acid) to various polysaccharides (i.e. 20 / 100 / 500 kDa hyaluronic acid, carboxymethyl cellulose, alginate) with the equivalents adjusted based on the reagents and solubility of the resulting modified polymer.

[0213] Synthesis of diol-modified polysaccharides. A typical synthesis is as follows: 500 kDa HA (100 mg) was dissolved in MES buffer (10 mL) at pH 5.5. DMT-MM (137 mg; 2 equivalents) was added to the HA solution and reacted for 30 minutes with stirring at room temperature. Glucosamine (90 mg); 1 equivalent) was added to the activated HA solution and reacted for 3 days with stirring at room temperature. The solution was filter sterilized, dialyzed against lx PBS buffer (pH 7.4) for 1 day, and then against deionized water for 2 days (MWCO 12-14 kDa, Spectrum Labs). The solution was lyophilized and stored at 4 °C. The degree of substitution was determined by elemental analysis (nitrogen / carbon ratio) of the purified product or by quantifying unreacted aminated molecules in the crude mixture using 2,4,6-trinitrobenzenesulfonic acid (TNBSA) titration. Similar procedures were used to graft various diol-containing aminated molecules (i.e. glucosamine, isosorbide, glucosamine, galactosamine, fructosamine, dopamine, 1-amino-1-deoxy-D-galactitol, tris(hydroxymethyl)aminomethane) to various polysaccharides (i.e. 20 / 100 / 500 kDa hyaluronic acid, carboxymethyl cellulose, alginate) with the equivalents adjusted based on the reagents and solubility of the resulting modified polymer.

[0214] Synthesis of Wulff-type boronic acid-modified PEG. 4-arm PEG-NH2(MW = 2 kDa; 2.418 g) was dissolved in methanol (24 mL). 2-Formyl aryl boronic acid (943 mg; 1.3 equivalents) was added to the PEG solution and reacted overnight with stirring at room temperature. The solution was cooled on ice, NaBH4(274 mg; 1.5 equivalents) was added, and then stirred at room temperature for 48 hours. 20 mL of deionized water was added, liquid-liquid extraction was performed with dichloromethane (3 x 50 mL), and then the organic phase was dried over anhydrous Na2S04. The solution was filtered and dialyzed against deionized water for 1 day (MWCO 1 kDa, Spectrum Labs). The solution was lyophilized and stored at 4 °C. The degree of substitution was determined by 1 H NMR (400 MHz, D20, δ) determination.

[0215] Synthesis of boronic acid-based polysaccharide hydrogels. A typical synthesis of a boronic acid hydrogel is as follows: 10 mg of HA-wPBA was dissolved in PBS (1 mL) using a 1-2 hour incubation at room temperature in a 2-mL Eppendorf tube. A similar procedure was used to dissolve 10 mg of HA-glucosamine in PBS (1 mL). In a 2-mL Eppendorf tube, the HA-wPBA and HA-glucosamine solutions were mixed together rapidly in a 1 : 1 volume ratio, and the pipettable dynamic hydrogel solution was used before it gelled. Similar procedures were used to test various combinations of benzene boronic acid-modified polysaccharides and diol-modified polysaccharides. If necessary, a double-barreled syringe was used for convenience.

[0216] II. Physical-chemical properties

[0217] Swelling / stability test. Hydrogels were prepared as described above. Aliquots (3 x 100 pL) of the hydrogels were transferred to pre-weighed 2-mL Eppendorf tubes and left at 37 °C for 30 min to reach equilibrium. The tubes containing the gels were weighed, and 900 pL of warm PBS (37 °C) was added to each tube. At specific time points, the supernatant was removed, the gel surface was carefully dried with Kimwipes, and the tubes were weighed. Swelling was determined by the ratio of the mass of the hydrogel at a given time point divided by its initial mass.

[0218] Rheological evaluation of dynamic hydrogels. Hydrogels were prepared as described above. Viscoelastic data were collected using a HAAKE MARS rheometer (ThermoFisher Scientific, Germany) equipped with a 20 mm titanium cone (Ti 20l; Thermo Fisher Scientific, Germany) for parallel-plate measurements, and a Peltier plate for temperature control. During the measurements, a solvent trap was used to minimize evaporation. To measure the shear storage modulus (G’) and shear loss modulus (G”) of the gels, frequency sweeps (0.01-10 Hz) were performed at a constant shear stress of 1 Pa and 37 °C. Self-healing properties were evaluated by measuring G’ and G” as a function of time at 1 Hz and 7 °C under an alternating stress of 100 s at 1 Pa (non-destructive stress) and 50 s at 500 Pa (destructive stress).

[0219] III. Biological properties

[0220] Hydrogel cytocompatibility. L929 fibroblasts were used as a model cell line to test the cytocompatibility of HA-wPBA, PEG-wPBA and HA-glucosamine solutions, respectively. Polymer solutions (1%) were prepared under sterile conditions using sterile polymers and cell culture medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). Cells were seeded at a density of 15,000 cells / well (100 pL) in cell culture medium in standard polystyrene 96-well plates and incubated (37°C, 5% C02, 95% humidity) overnight. Prior to cell culturing, the medium was replaced with 100 pL of 1% polymer solution. At specific incubation time points (0, 24 hours and 48 hours), the metabolic activity of the cells was tested using a CCK-8 assay kit and following the supplier's protocol. Subsequently, the CCK-8 medium was replaced with Tris / EDTA (TE) buffer prior to freezing the cells (-80°C) overnight. Then, DNA quantification was performed using the Quant-iT PicoGreen assay kit and following the supplier's recommendations, thus allowing the evaluation of cell proliferation. TM PicoGreen TM assay kit and following the supplier's recommendations, thus allowing the evaluation of cell proliferation.

[0221] 3D cell survival evaluation. 1% HA-wPBA and 2% HA-glucosamine solutions were prepared under sterile conditions using cell culture medium (DMEM supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin and 1% amphotericin B). The 2% HA-glucosamine solution was mixed with a solution of 8x10 6 cells / mL of adipose-derived multipotent stromal cells (MSCs) at a 1:1 ratio, resulting in a 1% HA-glucosamine solution containing 4x10 6 cells / mL. 0.5 mL of 1% HA-wPBA solution was mixed with 0.5 mL of 1% HA-glucosamine / cell solution and 100 pL of hydrogel (100K cells / well) was plated in a 96-well plate. Prior to analysis, the cell-containing hydrogels were incubated with 150 pL of cell culture medium (37°C, 5% C02, 95% humidity) and the cell culture medium was changed every other day. 2D controls were performed in 250 pL of cell culture medium at a density of 5x10 3 cells per well, supplemented every other day. At specific incubation time points (0, 1 day and 7 days), cell viability was evaluated by live / dead staining and confocal microscopy (Nikon Al) using Calcein AM (live cells, Sigma-Aldrich), Ethidium homodimer (dead cells, Sigma-Aldrich) and Hoechst (total cell control, Invitrogen) following the manufacturer's instructions. The average viability was obtained from 3 biological replicates.

[0222] IV. Results

[0223] The results obtained are shown in Figures 1 to 6 .

[0224] As a proof of concept, the inventors first demonstrated that the Wulff-PBA modified hyaluronan (HA-wPBA) successfully synthesized a dynamic covalent hydrogel upon mixing with a glucosamine-modified hyaluronan (HA-glucosamine). Using 1% (w / v) HA and a 1 :2 wPBA:glucosamine ratio, they observed a frequency-dependent cross-over of the storage (G’) and loss (G”) moduli by rheological measurements, which is characteristic of dynamic covalent hydrogels Figure 1 ).

[0225] The inventors further demonstrated the self-healing property of the newly designed hydrogels, which is inherent to dynamic covalent polymer networks. Qualitatively, two independent HA-based dynamic hydrogels were brought into contact with each other, forming a single gel within a few minutes Figure 2 A). Time sweep measurements at a constant frequency of 1 Hz under alternating low (1 Pa) and high (500 Pa) stresses showed repeated breakage of the gel under high shear (G’ < G”), followed by an effective recovery of its initial mechanical properties (G’ > G”), further confirming the self-healing Figure 2 B).

[0226] The inventors then investigated the influence of the molecular weight and polymer content of these HA-based hydrogels on their viscoelastic behavior. Reducing the molecular weight of the polymer precursor from 500 kDa to 100 kDa allowed increasing the polymer content from 1% to 3% (w / v), ensuring a wide range of cross-linking densities for the designed hydrogels and a usable viscosity of the precursor solution. This way, the following dynamic covalent hydrogels were obtained: their shear storage modulus component (G’) spanned over an order of magnitude (from tens to thousands of Pa), reflecting the various viscoelastic behaviors of the system and the high tunability Figure 3 ).

[0227] Using the newly discovered cross-linking pair (i.e., wPBA and glucosamine), the inventors evaluated the swelling and stability of the HA-based dynamic covalent hydrogels. Comparing 1% and 2% (w / v) polymer content, the inventors found that the optimal formulation of these new hydrogels could lead to minimal swelling and long-term stability (at least one month) despite the reversible cross-linking of the polymer network Figure 4 ). This would further allow tuning the swelling and stability of these dynamic hydrogels for various applications.

[0228] As a proof of concept of their versatility, the new cross-linking pairs were applied to various polymers. Dynamic covalent hydrogels were successfully obtained by mixing: 1% (w / v) polyethylene glycol (PEG)-wPBA and 1% (w / v) HA-glutamine (A) in a 1:1 volume ratio; 1% (w / v) alginate-wPBA and 1% alginate-glutamine (B) in a 1:1 volume ratio; and 1% (w / v) carboxymethylcellulose-wPBA and 1% (w / v) carboxymethylcellulose-glutamine (C) in a 1:1 volume ratio. Altogether, these results show that the new cross-linking pairs can be applied to synthetic and natural polymers with different molecular weights, ionic strengths, and degradation properties. Figure 5 Figure 5 Figure 5

[0229] Finally, the inventors evaluated the cell compatibility of HA-HA and HA-PEG dynamic covalent gels by assessing the viability of encapsulated adipose-derived multipotent stromal cells (A-MSCs) after 7 days of 3D culture by confocal microscopy (live / dead assay). These results revealed excellent viability (>90%) of A-MSCs encapsulated in these gels.

[0230] Example 2

[0231] Example 2 reports data on the physicochemical and biological properties of different boronic acid-diol pairs used for the design of dynamic hydrogels according to the present application.

[0232] I. Rheological properties comparison of different boronic acid-diol pairs immobilized on polymers

[0233] The inventors sought to identify new boronic acid-diol pairs that, when immobilized on polymers, have a high enough affinity to form dynamic covalent hydrogels under physiological conditions of pH and temperature.

[0234] ​​​Hyaluronic acid (HA) was chosen as the polymer backbone because it is generally considered a polymer of choice for biomedical applications. Using DMT-MM as the activating agent (see Example 1), different aminated phenylboronic acid (PBA) derivatives were each immobilized onto HA via amidation. Following a similar procedure, a series of diol-containing molecules were grafted onto HA. To be able to compare various boronic acid-diol pairs, the reaction conditions were adjusted to match the degree of substitution of the various PBAs (about 20-25%) and diols (about 30-35%). Interestingly, 2-amino-phenylboronic acid (2PBA), 3-amino-phenylboronic acid (3PBA), 4-amino-phenylboronic acid (4PBA), and amino-benzoxaborole (BX) all required the use of a small amount of DMSO to reach a degree of substitution of about 20-25%; and 3PBA-modified HA precipitated during the synthesis. In contrast, Wulff-type phenylboronic acid (wPBA), due to its positively charged nature, could be immobilized under aqueous conditions (i.e., without the use of DMSO) with a degree of substitution of up to at least 40%.

[0235] All boronic acid-diol pairs were then tested by mixing all PBA-modified and diol-modified HA components two by two. The rheological measurements obtained (frequency sweep; G', shear elastic modulus) are shown in Figure 7 They revealed a series of pairs capable of forming hydrogels under physiological conditions (e.g., HA-glucosamine and HA-wPBA; HA-fructosamine and HA-wPBA; HA-dulcitolamine and HA-wPBA; HA-glucosamine and HA-2PBA; and HA-dulcitolamine and HA-2PBA). In particular, the combination of HA-glucosamine and HA-wPBA resulted in the formation of a viscoelastic material with an elastic component several orders of magnitude higher than any other pair. Interestingly, the BX-modified polymer, which was reported to form hydrogels more easily in the presence of diols due to its chemical structure, did not obtain any significant gelation properties.

[0236] II. Optimized formulation of HA-wPBA-HA-glucosamine gels with improved physicochemical (viscoelastic) properties

[0237] To tune the viscoelastic behavior of the gels made from HA-wPBA and HA-glucosamine, the inventors screened a large set of compositions varying the molecular weight of the polymer hyaluronic acid (HA), the total concentration of HA, the degree of substitution of each of the two components (HA-wPBA and HA-glucosamine), and the molar ratio of wPBA:glucosamine.

[0238] Several target compositions were identified that showed specific viscoelastic behavior as observed by rheological measurements (in frequency sweep tests, the G' / G" crossover of a dynamic gel is directly related to its relaxation time).Figure 8 Three such specific optimized compositions are shown: in a first composition, the total concentration of HA is 1%, the molecular weight of HA is 300 kDA, the degree of substitution of HA-wPBA is 26%, and the degree of substitution of HA-glucosamine is 52%; in a second case, the total concentration of HA is 1%, the molecular weight of HA is 200 kDa, the degree of substitution of HA-wPBA is 26%, and the degree of substitution of HA-glucosamine is 52%; and in a third case, the total concentration of HA is 3%, the molecular weight of HA is 100 kDA, the degree of substitution of HA-wPBA is 40%, and the degree of substitution of HA-glucosamine is 52%. In each of the three specific compositions, the molar ratio of wPBA:glucosamine is 1:1.

[0239] More importantly, while boronic acid-based hydrogels tend to shrink, the present inventors were able to design boronic acid-based hydrogels that shrink minimally to not at all by carefully balancing the network’s tendency to shrink and the swelling tendency of HA as a charged, hydrophilic polymer (see PBS, Media 1, and Media 2 in Figure 9 (B) and Figure 9 (A) curves). The optimal formulations are also stable for weeks to months under physiological conditions of pH and temperature, which has never been reported for boronic acid-based hydrogels to date. Due to the dynamic nature of its network, the new gel can be easily dissolved by simply adding competing, free diol-containing molecules such as glucose or glucosamine (see Figure 9 (A)). Enzymatic degradation of the HA-based gel can also be used using hyaluronidase, while making the dynamic covalent hydrogel of the present invention sugar-responsive and biodegradable (see Figure 9 (A)).

[0240] III. Cell compatibility of boronate hydrogels

[0241] To validate the use of the new gel in biomedical applications, the cell compatibility of the boronate hydrogel of the present invention with encapsulated cells was evaluated using a model cell line (L929 murine fibroblasts). Figure 6 The gel used in the experiments reported in Example 2 was a hydrogel of HA-Wulff-PBA and HA-glucosamine with a total concentration of 300 KDa HA polymer of 1% w / v, a molar ratio of boron:diol of 1:1, a degree of substitution of HA-Wulff-PBA of 26%, and a degree of substitution of HA-glucosamine of 52%. Cell compatibility was evaluated by cell viability (live / dead cell imaging), metabolic activity (CCK-8), and proliferation (PicoGreen) assays.

[0242] The results obtained are shown in Figure 10As shown. Two days later, the collected data showed high cell viability, accompanied by increased metabolic activity and cell number, collectively indicating the excellent cell compatibility of the test gel. Similar results were obtained with other hydrogels according to the invention.

[0243] IV. The optimized boric acid hydrogel is printable.

[0244] Because the dynamic covalent hydrogel according to the present invention exhibits the property of temporary flow under shear (see Example 1), the inventors envision using this novel hydrogel to develop innovative biolinks in the context of bioprinting. Preliminary data are as follows: Figure 11 As shown. The following composition was used as a typical example: 200 kDa HA, total HA concentration = 1% w / v, HA-Wulff-PBA substitution degree: 26%, HA-glucosamine substitution degree = 52%, Wulff-PBA:glucosamine molar ratio = 1:1. The results obtained indicate that the optimal formulation of the novel hydrogel associated with optimal printing conditions (e.g., head displacement, extrusion pressure) can be easily printed with good shape fidelity.

[0245] V. Combination with other cross-linking mechanisms

[0246] In order to expand the biomedical applications of novel hydrogels, the inventors sought to demonstrate the feasibility of associating the discovered crosslinking mechanism with other chemical reactions, particularly "click" and bioorthogonal chemical reactions.

[0247] As preliminary data, it is shown that the clickable portion can be co-immobilized onto one of the two polymer components of the hydrogel, thereby allowing for temporal and spatial tuning of the gel's physicochemical properties (see [link]). Figure 12 (A)). For example, co-crosslinking of boric acid-based hydrogels can be used to stabilize and / or mechanically enhance the printed bioprinted structures. Bicycloalkyne (BCN) and azide (N... 3) Strain-promoted azide-alkyne cycloaddition (SPAAC) was used as a "click" reaction model. A 200 kDa borate gel (total HA concentration = 1%; Wulff-PBA degree of substitution = 16%; glucosamine degree of substitution = 52%; Wulff-PBA:glucosamine molar ratio = 1:1) was chemically modified with BCN (4% as a co-substituted HA-glucosamine) to allow for post-printing modification with azide-modified hyaluronic acid (100 kDa HA-N3). The gel stiffness increased from approximately 600 Pa to approximately 3000 Pa by soaking the bioprinted borate gel overnight in PBS containing 0.5% HA-N3.

[0248] This strategy can be used to immobilize target molecules (e.g., peptides, growth factors, drugs, fluorophores), to develop evolving 3D culture systems that modulate cell-material interactions in time and space, and so on.

Claims

1. A cross-linking pair of hydrogel precursor polymers comprising: (1) a first hydrogel precursor polymer consisting of a first polymer modified with a Wulff-type benzenaboronic acid, and (2) a second hydrogel precursor polymer consisting of a second polymer modified with glucosamine, wherein the Wulff-type benzenaboronic acid is 2-((dimethylamino)methyl)benzenboronic acid, or is (2-(((4-aminobutyl)amino)methyl)phenyl)boronic acid.

2. The cross-linking pair of hydrogel precursor polymers according to claim 1, wherein the first polymer is grafted with the Wulff-type benzenaboronic acid and the second polymer is grafted with glucosamine.

3. The cross-linking pair of hydrogel precursor polymers according to claim 1 or 2, wherein the first and second polymers are independently selected from the group consisting of natural polymers, semi-synthetic polymers, and synthetic polymers.

4. The cross-linking pair of hydrogel precursor polymers according to claim 3, wherein at least one of the first and second polymers is selected from the group consisting of biocompatible, biodegradable, hydrophilic natural polymers, semi-synthetic polymers, and synthetic polymers.

5. The cross-linking pair of hydrogel precursor polymers according to claim 1 or 2, wherein the first hydrogel precursor polymer or the second hydrogel precursor polymer is directly or indirectly covalently linked to a bio-orthogonal functional moiety or a clickable moiety.

6. The cross-linking pair of hydrogel precursor polymers according to claim 1 or 2, wherein the first hydrogel precursor polymer is in a first aqueous solution; and the second hydrogel precursor polymer is in a second aqueous solution, the first and second aqueous solutions being separate aqueous solutions.

7. The cross-linking pair of hydrogel precursor polymers according to claim 6, wherein at least one of the first and second aqueous solutions comprises an ingredient selected from the group consisting of cells, a biologically active agent, a visualizing agent, or any combination thereof.

8. A dynamic covalent hydrogel consisting of a first hydrogel precursor polymer and a second hydrogel precursor polymer of a cross-linking pair as defined in any one of claims 1-4, wherein the first and second hydrogel precursors are cross-linked by a dynamic covalent bond.

9. The dynamic covalent hydrogel according to claim 8, further comprising an ingredient selected from the group consisting of cells, a biologically active agent, a visualizing agent, or any combination thereof.

10. A pharmaceutical composition comprising: a cross-linking pair of hydrogel precursor polymers as defined in any one of claims 1-7 or a dynamic covalent hydrogel according to claim 8 or 9, and at least one pharmaceutically acceptable carrier or excipient.

11. The pharmaceutical composition according to claim 10, wherein the first hydrogel precursor polymer and the second hydrogel precursor polymer are contained in a multi-barrel syringe, preferably a dual-barrel syringe.

12. The pharmaceutical composition according to claim 11, wherein the multi-barrel syringe is a dual-barrel syringe.

13. The cross-linking pair of hydrogel precursor polymers according to any one of claims 1 to 7, or the dynamic covalent hydrogel according to claim 8 or claim 9, or the pharmaceutical composition according to any one of claims 10 to 12, for use as a therapeutic agent.

14. The cross-linking pair of hydrogel precursor polymers, or the dynamic covalent hydrogel, or the pharmaceutical composition for use according to claim 13, wherein the therapeutic agent is for cell therapy, tissue engineering, regenerative medicine, viscoelastic supplementation therapy, or in vivo delivery of cells and / or bioactive agents.

15. A kit comprising the cross-linking pair of hydrogel precursor polymers according to any one of claims 1 to 7, or the dynamic covalent hydrogel according to claim 8 or claim 9, or the pharmaceutical composition according to any one of claims 10 to 12, and instructions for use of the cross-linking pair, the dynamic covalent hydrogel, or the pharmaceutical composition.

16. A method for preparing a dynamic covalent hydrogel, comprising the step of mixing a first hydrogel precursor polymer and a second hydrogel precursor polymer of a cross-linking pair as defined in any one of claims 1 to 7 to obtain a dynamic covalent hydrogel.

17. The method according to claim 16, wherein the method is carried out under physiological conditions.

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