Dynamic covalent hydrogels, precursors thereof and their uses - Patent Application 20070122997

Abstract

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

Description

[Technical Field]

[0001] Related Applications This application claims priority to European Patent Application No. EP2016202.2, filed March 10, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Polymer networks are formed by physical or chemical crosslinking of functional monomers or polymer precursors. Physically crosslinked materials are held together by reversible, non-covalent 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 after shear is stopped) materials whose bonds can break and reform in response to external stimuli, including mechanical loading. However, the resulting structures are often unstable and lack robustness to small environmental perturbations. Chemically crosslinked networks, on the other hand, are held together by covalent bonds (Wichterle et al., Nature, 1960, 185: 117-118). This results in elastic gels that generally have better mechanical properties than physical networks. However, the irreversibility of chemical crosslinks limits their use in applications requiring shear-thinning and self-healing properties, such as 3D printing and minimally invasive drug delivery.

[0003] Recent research has introduced a new class of soft matter based on dynamic covalent chemistry, which 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 the network, which can be broken and reformed on experimental timescales. These dynamic covalent networks can then be reconfigured by bond exchange, allowing for stress relaxation and material flow. Crosslinking reactions that have been used to form dynamic covalent networks include transesterification, Diels-Alder cycloaddition, and boronate ester complexation. The reversible formation of boronate esters between boronic acids and cis-1,2- or cis-1,3-diol-containing molecules has emerged as a safe and synthetically easy-to-use dynamic covalent crosslinking motif for the design of stimuli-responsive biomedical materials. However, boronate ester complexation is generally supported at alkaline pH, and common diol coupling partners tend to yield relatively unstable condensation products and exhibit oxidation sensitivity. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] De Greef et al., Chem. Rev., 2009, 109: 5687-5754 [Non-patent document 2] Wichterle et al., Nature, 1960, 185: 117-118 [Non-patent document 3] Kloxin et al., Chem. Soc. Rev., 2013, 42: pp. 7161-7173 [Non-patent document 4] Wulff et al., Pure Appl. Chem., 1982, 54: 2093-2102 [Non-Patent Document 5] Wulff et al., Angew Chem., Int. Ed. Engl., 1984, 23: 741-742 [Non-patent document 6] Cromwell, J. Am. Chem. Soc., 2015, 137: 6492-6495 [Non-licensed Document 7] Piestら, Soft Matter, 2011, 7: 11111 pages [Non-licensed Document 8] Li ら, Chem. Commun., 2011, 47: 8169 pages [Non-licensed Document 9] Kim, J. Am. Chem. Soc., 2009, 131: 13908-13909 [Non-licensed Document 10] Berube, J. Org. Chem., 2008, 73: 6471~6479 pages [Non-licensed Document 11] Dowlut, J. Am. Chem. Soc., 2006, 128: 4226-4227 [Non-licensed Document 12] Curr. Med. Chem., 2018, doi: 10.2174 / 092986732566618100814443 [Non-licensed Document 13] Brooks and Sumerlin, Chem. Rev., 2016, 3: pages 1375~1397 [Non-licensed Document 14] Guan and Zhang, Chem. Soc. Rev., 2013, 42: 8106-8121 [Non-licensed Document 15] Tarus, Macromol. Rapid Commun., 2014, 35: pages 2089~2095 [Non-licensed Document 16] Tangら, Adv. Sci., 2018, 5(9): 1800638 [Non-licensed Document 17] Yesilyurt, Adv. Mater., 2015, 28(1): 86~91 pages [Non-licensed Document 18] Kolb, Angew. Chem., Int. Ed., 2001, 40: 2004~2021 pages [Non-licensed Document 19] "Bioconjugate Techniques", Greg T. Hermanson, 1996 [Non-licensed Document 20] Patterson, ACS Chemical Biology, 2014, 9: 592~605 pages [Non-licensed Document 21] Spicer, Nature Communications, 2014, 5: 4740 pages [Non-licensed Document 22] Madl and Heilshorn, Advanced Functional Materials, 2018, 28(11): 1706046 pages [Non-licensed Document 23] Brighid Pappinら、Boron-Carbohydrate Interactions [Non-licensed Document 24] Yannasら, Science, 1982, 215: pages 174~176 [Non-licensed Document 25] Lee and Mooney, Chem. Rev., 2001, 101: pages 1869~1880 [Non-licensed Document 26] Peppas, Adv.Mater., 2006, 18: 1345~1360 pages [Non-licensed Document 27] Jeongら, Cell, 2015, 162: pages 662~674 [Non-licensed Document 28] Parkら, Nature Biotechnology, 2015, 33: pages 1280~1286 [Non-licensed Document 29] Whitesides, Nature, 2006, 442: pages 386~373 [Non-licensed Document 30] Casavant, PNAS USA, 2013, 110: 10111~10116 pages [Non-licensed Document 31] Dong et al., Nature, 2006, 42: 551-554 [Non-Patent Document 32] Choi et al., Nature Photonics, 2013, 7: 987-994. [Non-Patent Document 33] Choi et al., Advanced Materials, 2015, 27: 4081-4086 [Non-Patent Document 34] Kim et al., Science, 2008, 320: 507-511 [Non-Patent Document 35] Rogers et al., Science, 2010, 327: 1603-1607 [Non-Patent Document 36] Xu et al., Science, 2014, 344: 70-74 [Non-Patent Document 37] Tee et al., Science, 2015, 350: 313-316 [Non-Patent Document 38] Shepherd et al., PNAS USA, 201, 108: 20400-20403 [Non-Patent Document 39] Morin et al., Science, 2012, 337: 828-832 [Non-Patent Document 40] "Remington's Pharmaceutical Sciences", EW Martin, 18th edition, 1990, Mack Publishing Co.: Easton, PA Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there remains a need in the art for improved boronic acid / diol couples suitable for producing dynamic covalent hydrogels that can be used for controlled drug release, cell culture, tissue engineering, 3D printing, and the like. [Means for solving the problem]

[0006] The present inventors have identified a new boronic acid / diol couple for the formation of dynamic covalent hydrogels. Compared to systems known in the art, the new boronic acid / diol couple of the present invention exhibits several advantages. In particular, hydrogel formation can be carried out in a single step at physiological pH and temperature. Furthermore, the crosslinking reaction is essentially instantaneous. The resulting hydrogels are highly stable over time, self-healing, minimally swellable, and possess viscoelastic properties similar to those of living tissue. Due to their advantageous shear-thinning properties, the polymer compositions are injectable. Furthermore, the composition of the hydrogels can be easily modified to adjust their biodegradability and / or mimic living tissue or environment.

[0007] As a result, the present invention relates to a crosslinked pair of hydrogel precursor polymers comprising: (1) a first hydrogel precursor polymer comprising a first polymer modified with phenylboronic acid or a phenylboronic acid derivative; and (2) a second hydrogel precursor polymer comprising a second polymer modified with glucamine.

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

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

[0010] In certain embodiments, at least one of the first and second polymers is selected from 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 benzoxaborole. 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 bioorthogonal functional moiety or a clickable moiety.

[0014] In certain embodiments, the crosslinked pair of hydrogel precursor polymers is such that a first hydrogel precursor polymer is in a first aqueous solution and a second hydrogel precursor polymer is in a second aqueous solution, the first and second aqueous solutions being separate aqueous solutions, and at least one of the first and second aqueous solutions may comprise a component selected from the group consisting of cells, a bioactive agent, a visualization agent, and any combination thereof.

[0015] The present invention also relates to a dynamic covalent hydrogel composed of a crosslinked pair of a first hydrogel precursor polymer and a second hydrogel precursor polymer, as defined herein, wherein the first and second hydrogel precursors are crosslinked by a dynamic covalent bond. The dynamic covalent hydrogel may further comprise a component selected from the group consisting of cells, bioactive agents, visualization agents, and any combination thereof.

[0016] The present invention further relates to a pharmaceutical composition comprising a crosslinked pair of hydrogel precursor polymers 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 precursor hydrogel polymer are contained in a multi-barrel syringe, preferably a double-barrel syringe.

[0018] The present invention also relates to a crosslinked pair of hydrogel precursor polymers 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 invention also relates to a crosslinked pair of hydrogel precursor polymers as defined herein, or a dynamic covalent hydrogel as defined herein, or a pharmaceutical composition as defined herein, for use as a therapeutic agent, e.g., in cell therapy, tissue engineering, regenerative medicine, viscosupplementation, delivery of cells and / or bioactive agents in vivo.

[0020] The present invention further relates to a kit comprising a crosslinked pair of hydrogel precursor polymers as defined herein, or a dynamic covalent hydrogel as defined herein, or a pharmaceutical composition as defined herein, and instructions for use of the crosslinked pair, the dynamic covalent hydrogel, or the pharmaceutical composition.

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

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

[0023] These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. [Brief explanation of the drawings]

[0024] [Figure 1]This is a diagram showing the viscoelastic behavior of a dynamic covalent hydrogel made from a mixture of Wulff-PBA modified and glucosamine modified polymer precursors. When Wulff-PBA modified hyaluronic acid and glucosamine modified hyaluronic acid are mixed at a volume ratio of 1:2, the storage modulus (G') and loss modulus (G") show a predicted crossover when measured over a range of frequencies. [Figure 2] This is a diagram showing the self-healing properties of a dynamic covalent hydrogel made from a mixture of Wulff-PBA modified and glucosamine modified polymer precursors. (A) Qualitatively, two hyaluronic acid (HA)-based dynamic covalent hydrogels form a single hydrogel within minutes, which can support its own mass. (B) Time sweep measurements for a typical HA-based dynamic covalent gel formulation (1% (w / v) HA-Wulff-PBA mixed with HA-glucosamine at a volume ratio of 1:1) at a constant frequency of 1 Hz under alternating low (1 Pa) and high (500 Pa) stresses, showing gel breakdown (G' < G") at high frequencies and recovery of initial mechanical properties (G' > G"). [Figure 3] This is a diagram showing a proof of concept for the tunability of the viscoelastic behavior of dynamic covalent gels by varying the molecular weight (100 kDa vs 500 kDa) and polymer content (1% vs 3% (w / v)) of hyaluronic acid-based dynamic covalent hydrogels. The storage modulus (G') and loss modulus (G") were measured as a function of frequency. [Figure 4] This is a diagram showing the swelling / stability study of hyaluronic acid (HA)-based hydrogels using HA-Wulff-PBA and HA-glucosamine at a volume ratio of 1:1 at 1% vs 2% (w / v) polymer concentrations. By adjusting the polymer concentration of these gels, it is possible to control their swelling and stability in an optimal formulation that shows minimal swelling and stabilization within 3 days, followed by long-term stability (at least 1 month). [Figure 5]Proof-of-concept diagram showing dynamic covalent hydrogels derived from various natural and synthetic polymers modified with either Wulff-PBA or glucamine. Storage modulus (G') and loss modulus (G") measured as a function of frequency for (A) 1% (w / v) poly(ethylene glycol) (PEG)-Wulff-PBA mixed with 1% (w / v) HA-glucamine at a 1:1 volume ratio, (B) 1% (w / v) alginate-Wulff-PBA mixed with 1% (w / v) alginate-glucamine at a 1:1 volume ratio, and (C) 1% (w / v) carboxymethylcellulose-Wulff-PBA mixed with 1% (w / v) carboxymethylcellulose-glucamine at a 1:1 volume ratio. [Figure 6] FIG. 1 shows the assessment of the viability of adipose-derived multipotent stromal cells encapsulated in a typical dynamic covalent hydrogel (1% (w / v) HA-Wulff-PBA mixed with 1% (w / v) HA-glucamine at a 1:1 volume ratio) using confocal microscopy and live / dead staining. [Figure 7] Figure 1 shows a comparison of the rheological properties (frequency sweep; G', shear modulus) of different boronic acid-diol couples immobilized on hyaluronic acid (HA) under physiological pH and temperature conditions (see Example 2, paragraph I). [Figure 8] Figure 1 shows the storage modulus (G') and loss modulus (G") of these specifically optimized formulations of dynamic covalent HA-Wulff-PBA and HA-glucamine hydrogels exhibiting various viscoelastic profiles. HA polymers with different molecular weights (300 kDa, 200 kDa, or 100 kDa), different total concentrations of HA (1% or 3% w / v), and different degrees of substitution for the two components (HA-wPBA = 26% or 40%; HA-glucamine - 52%) were used, while the molar ratio of wPBA:glucamine was kept constant at 1:1 (see Example 2, paragraph II). [Figure 9]Figure 1 shows the design of minimally to non-shrinking / non-swelling boronic acid-based hydrogels. The variation of hydrogel mass ratio as a function of time is reported for (A) a dynamic covalent hydrogel of HA-Wulff-PBA and HA-glucamine (300 kDa HA polymer was present at a total concentration of 1% w / v, the degree of substitution of HA-wPBA was 26%, the degree of substitution of HA-glucamine was 52%, and the molar ratio of wPBA:glucamine was 1:1); and (B) a dynamic covalent hydrogel of HA-Wulff-PBA and HA-glucamine, which was identical except that the HA polymer had a molecular weight of 200 kDa. The variation of hydrogel mass ratio as a function of time was studied in PBS (A and B), two different culture media (A), and in the presence of glucose, glucamine, and hyaluronidase (A) (see Example 2, paragraph II). [Figure 10] Figure 1 shows the cytocompatibility of boronate ester gels. Murine fibroblasts (L929 cell line) were encapsulated in boronate ester hydrogels according to the present invention (300 kDa HA; [HA] = 1% w / v; Wulff-PBA substitution = 26%; glucamine substitution = 52%; Wulff-PBA:glucamine ratio = 1:1), and cytocompatibility was assessed via cell viability (live / dead cell imaging), metabolic activity (CCK-8), and proliferation (PicoGreen) assays. Results are reported for days 0, 1, and 2 after encapsulation of cells in the hydrogels. The image on the right of the cell viability graph is representative of the high cell viability (≥ 98%) observed after 2 days of 3D cell culture (see Example 2, paragraph III). [Figure 11]Optimized dynamic covalent hydrogels are printable. The top three images show how a well-balanced extrusion pressure and rate enable the printing of continuous, well-degraded filaments of boronate ester hydrogels. The middle graph highlights in green the large range of extrusion / pressure couples that can be used to successfully print boronate ester hydrogels of the present invention, while showing in yellow conditions that lead to filament breakage and in red conditions that result in poorly degraded filaments. The bottom images illustrate geometries successfully printed from single-layer and multi-layer gels (see Example 2, paragraph IV). [Figure 12] Figure 1 shows the combination of the crosslinking mechanism of the present invention with "click" chemistry. (A) The scheme highlights the potential combination of the crosslinking mechanism with "click" chemistry to modulate the composition and physicochemical properties of the resulting gel. In the specific context of bioprinting, such a strategy can be used to mechanically strengthen the printed construct. (B) is a graph showing the successful mechanical strengthening of the printed construct upon immersion in a medium containing an appropriate "clickable" polymer. In this example, strain-promoted azide-alkyne cycloaddition (SPAAC) between bicyclonyne (BCN) and azide (N3) was used as a model for the "click" reaction. A boronate ester gel was chemically modified with BCN to enable post-printing modification with azide-modified hyaluronan (100 kDa) (see Example 2, paragraph V). DETAILED DESCRIPTION OF THE INVENTION

[0025] As mentioned above, the present invention provides hydrogel precursor polymers and dynamic covalent hydrogels, pharmaceutical compositions comprising such precursors or hydrogels, and their use in various applications.

[0026] I- Hydrogel precursor polymer pair 1. Hydrogel precursor polymer The present invention provides a cross-link couple or pair of hydrogel precursor polymers capable of cross-linking when mixed together. A cross-link couple or pair of hydrogel precursor polymers according to the present invention is composed of a first hydrogel precursor polymer modified with phenylboronic acid or a phenylboronic acid derivative and a second hydrogel precursor polymer modified with glucamine. The terms "couple," "pair," "cross-link couple," and "cross-link pair" are used interchangeably herein. They refer to the relationship or combination of two hydrogel precursor polymers, which are separate from each other but, when mixed, form a dynamic covalent hydrogel through cross-linking. In other words, the terms "couple," "pair," "cross-link couple," and "cross-link pair," as used herein, exclude mixtures, blends, and equivalents thereof. As used herein, the term "hydrogel precursor polymer" refers to a polymer that can play a role in a reaction to form a cross-linking molecule, e.g., a hydrogel network. Thus, although the hydrogel precursor polymers in a pair as defined herein are the same materials that form the dynamic covalent hydrogel according to the present invention, they do not contact each other and therefore do not undergo cross-linking as a pair.

[0027] A. Polymer In embodiments of the present invention in which 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, refers to a polymer that is not significantly toxic to cells and / or living tissues and does not elicit an immunopathogenic response in healthy individuals. The term "biodegradable," as used herein to characterize a polymer, refers to a polymer that degrades due to the activity of living organisms, light, air, water, or any combination thereof. Preferably, a biodegradable polymer is one that breaks down over time within a mammalian body into non-toxic molecules small enough to be metabolized or excreted under normal physiological conditions. As used herein, "hydrophilic polymer" refers to 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 mass in water within 24 hours and more than 4% at equilibrium, as determined by ASTM D570 testing.

[0028] The first and second hydrogel precursor polymers of a pair according to the present invention are independently selected from the group consisting of natural polymers, semi-synthetic polymers, and synthetic polymers. In embodiments of the present invention in which the polymer precursors are used in biological, biomedical, or medical applications, the first and / or second hydrogel precursor polymers of the pair are independently biocompatible, biodegradable, hydrophilic polymers selected from the group consisting of natural polymers, semi-synthetic polymers, and synthetic polymers.

[0029] In certain embodiments, at least one of the first and second hydrogel precursor polymers is selected from among natural polymers. The terms "natural polymer" and "biopolymer" are used interchangeably herein to refer to naturally occurring polymers (i.e., polymers that are found in nature but may have been obtained using methods involving human intervention, such as 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 invention include naturally occurring polysaccharides, collagen, and gelatin.

[0030] As used herein, the term "polysaccharide" has its art-recognized meaning and refers to a complex carbohydrate composed of 10 to up to several thousand monosaccharides arranged in a chain and linked via glycosidic bonds. The most common monosaccharides occurring as part of polysaccharides are glucose, fructose, galactose, and mannose. The molecular weights of naturally occurring polysaccharides vary from several hundred to several thousand daltons.

[0031] Exemplary polysaccharides include, but are not limited to, arabinan, fructan, fucan, galactan, galacturonan, glucan, mannan, xylan (e.g., inulin), levan, fucoidan, carrageenan, galactolose, pectic acid, pectin, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, 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 naturally occurring homopolymers or heteropolymers, such as those containing one or more of aldose, ketose, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, 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 naturally occurring derivatives thereof.

[0032] 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.

[0033] 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 the majority of the extracellular matrix and connective tissue, providing strength and flexibility to bodily tissues. Any type of collagen may be used in the practice of the present invention. Thus, the collagen may be selected from 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 a human or porcine source.

[0034] 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 thermal denaturation of collagen isolated from animal skin and bones with very dilute acid or extracted from fish skin. Gelatin is a heterogeneous mixture of single- or multi-chain polypeptides, each with an extended left-handed proline helix and containing between 50 and 1000 amino acids. Approximately half of gelatin's total amino acid content is glycine residues (approximately every third residue, spaced every third), proline residues, and 4-hydroxyproline residues.

[0035] In certain embodiments, at least one of the first and second hydrogel precursor polymers is selected from modified biopolymers. The terms "modified biopolymer" and "semi-synthetic polymer" are used interchangeably herein and refer to chemically modified naturally occurring polymers. Examples of modified biopolymers suitable for use in the present invention include derivatized cellulose (e.g., carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, methylcellulose, and methoxycellulose); derivatized hyaluronic acid (e.g., amine-modified hyaluronic acid and esterified hyaluronic acid); and derivatized collagen (e.g., amino-modified collagen and esterified collagen).

[0036] In certain preferred embodiments, at least one of the first and second hydrogel precursor polymers is a biocompatible, biodegradable, hydrophilic semi-synthetic polymer selected from the group consisting of carboxymethylcellulose, hydroxymethylcellulose, hydroxypropyl-cellulose, methylcellulose, and methoxycellulose.

[0037] In certain embodiments, at least one of the first and second hydrogel precursor polymers is selected from synthetic polymers. As used herein, the term "synthetic polymer" refers to a polymer that is neither naturally occurring nor semi-synthetic. Examples of synthetic polymers suitable for use in the context of the present invention 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, polycaprolactone or its copolymers. Other examples include polyacrylate derivatives, polymethacrylate derivatives (e.g., PEGMA), polyisoprene, polyamides, synthetic polypeptides (e.g., PBLG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL).

[0038] Those skilled in the art will appreciate that the list of polymers provided above is not exhaustive and can be extended to other similar polymers.

[0039] In the context of the present invention, the hydrogel precursor polymers may have any suitable molecular weight. For example, the polymers may have a molecular weight of about 5,000 g / mol to about 3,000,000 g / mol, preferably about 10,000 g / mol to about 700,000 g / mol. The terms "approximately" and "about," when used in reference to numbers, generally include numbers that are within a 10% range in either direction (greater or less than the number) unless otherwise stated or otherwise clear from the context (except where such number is considered to exceed 100% of the possible number).

[0040] B. Phenylboronic Acid and Phenylboronic Acid Derivatives The first hydrogel precursor polymer according to the present invention 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, refers to 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 may be covalently attached to the polymer through its ortho, meta, or para position (relative to the boronic acid group).

[0041] The term "phenylboronic acid," as used herein, refers to a molecule having the following chemical formula (I) and abbreviated as PhB(OH) or PBA:

[0042] [ka]

[0043] As used herein, the term "phenylboronic acid derivative" refers to a derivative of phenylboronic acid selected from the group consisting of ortho-, meta-, or para-monosubstituted phenylboronic acids, Wulff-type phenylboronic acids, and benzoxaboroles.

[0044] As used herein, the term "substituted" indicates that the particular group or compound being described has at least one hydrogen atom replaced by a non-hydrogen substituent. In the context of the present invention, the phenylboronic acid molecule is substituted to the extent that such substitution makes chemical sense. Examples of suitable substituents include, but are not limited to, alkyl, nitro (-NO), sulfo (-SO), and the like. - ), cyano (—CN), halogen (F, Br, Cl, or I), amine (—NRR′, where R and R′ are each independently H or alkyl), hydroxyl (—OH), sulfydryl (—SH), alkoxy (—OR, where R is alkyl), alkylthio (—SR, where R is alkyl), alkylsulfo (—O—SO—OR, 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 R and R′ are each independently H or alkyl), carboxylic acid (—COOH or —COOM, where M is a suitable cation, e.g., sodium or potassium), and sulfonic acid (—SOH or —R—SOH, where R is alkyl) groups. Those skilled in the art will know how to select a substituted phenylboronic acid such that the substituents are incompatible with grafting of the PBA onto the polymer.

[0045] As used herein, the term "alkyl" refers to a monovalent straight- or branched-chain group derived from an unsaturated hydrocarbon of 1 to 10 carbon atoms (C1-C10 alkyl), e.g., 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 5 carbon atoms (C1-C5 alkyl). Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Alkyl groups can be optionally substituted. For example, alkyl groups 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.

[0046] 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-sulfamoylbenzeneboronic 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, 2-(or 3- or 4-)carboxyphenylboronic acid, 2-(or 3- or 4-)(bromomethyl)phenylboronic acid, 2-(or 3- or 4-)aminocarbonylphenylboronic acid, o-tolylboric acid, m-tolylboronic acid, p-tolylboronic 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-)methylsulfonylphenylboronic 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-Trifluoroethoxy)phenylboronic acid, 4-(cyanomethyl)benzeneboronic 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-acetamidomethylphenyl) Boronic acids include 2-(dimethylaminocarbonyl)benzeneboronic acid, 4-(dimethylcarbamoyl)-phenylboronic acid, 2-(or 3- or 4-)isopropylphenylboronic acid, 4-propylphenylboronic acid, 3-propoxyphenylboronic acid, [3-(3-hydroxyl-propyl)phenyl]boronic acid, 4-(3-hydroxypropyl)benzeneboronic acid, 4-isopropoxyl-phenyl-boronic acid, 4-propoxyl-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.

[0047] The term "Wulff-type phenylboronic acid," as used herein, refers to a phenylboronic acid derivative containing an intermolecular tetracoordinate BN (formula (II)) bond that promotes the formation of a tetragonal boronate anion (sp3). The sp3 hybridization state remains stable even under neutral or slightly acidic conditions, promoting boronation with cis-diols.

[0048] [ka]

[0049] Wulff-type phenylboronic acids have been described (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, similar in structure to 2-dimethylaminomethylphenylboronic acid (DAPBA), are of great interest for 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).

[0050] In certain preferred embodiments, the Wulff-type phenylboronic acid has the formula (II): 20 Alkyl, substituted or unsubstituted C1-C 10 Alkenyl, substituted or unsubstituted C1-C 10 Alkynyl, acyl (-C(=O)R1, R1 is substituted or unsubstituted C1-C 20 alkyl group), and carboxy (—C(═O)OR, where R is a substituted or unsubstituted C-C 20 The alkyl, alkenyl, and alkynyl groups may be selected from one or more substituents, such as halogen (F, Br, I, Cl), hydroxy (—OH), amino (—NR2R3, where R2 and R3 are independently hydrogen and substituted or unsubstituted C1-C 20alkyl), alkoxy (-OR1, R1 is selected from substituted or unsubstituted C1-C 20 alkyl group), carboxy (-C(=O)OR, where R is a substituted or unsubstituted C-C 20 alkyl group), amide (-NR2C(=O)R3 or -C(=O)NR2R3, where R2 and R3 are independently hydrogen and substituted or unsubstituted C1-C 20 The group may be optionally substituted with a substituent selected from alkyl (--alkyl), nitro (--NO.sub.2), oxo (=O), and cyano (--CN).

[0051] The Wulff-type phenylboronic acid of formula (II) may also be such that one of R and R' is a linking moiety or spacer containing a free (non-bonded terminal) reactive group capable of binding 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 and refer to a moiety having a main chain length of at least 8 atoms, for example, more than 10, more than 20, more than 30, more than 35, more than 40, or more than 50 atoms. The linker may be linear, branched, cyclic, or monoatomic. In certain cases, the linker is substituted with sulfur, nitrogen, or oxygen heteroatoms. The bond between the main chain atoms may be saturated or unsaturated. The linker may contain one or more substituents.

[0052] 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 to enable grafting of the Wulff-type phenylboronic acid onto a first polymer. Alternatively, the substituent of the phenyl group is a linking moiety terminated with a functional group that can be used to graft the Wulff-type phenylboronic acid onto a first polymer.

[0053] The phenyl group of the Wulff-type phenylboronic acid of formula (II) may also be replaced with a heteroaryl group. As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic group of 5 to 10 ring atoms containing one, two, or three ring heteroatoms selected from N, O, and S, with at least one aromatic ring containing carbon atoms, the remaining ring atoms being carbon atoms, and it is understood that the point of attachment of the heteroaryl group is on the aromatic ring. In certain embodiments, the heteroaryl 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.

[0054] The term "benzoxaborole," as used herein, refers to a cyclic boronic acid half ester, particularly a bicyclic organic heterocycle having the chemical formula (III).

[0055] [ka]

[0056] Such compounds with intermolecular BO coordination (Berube et al., J. Org. Chem., 2008, 73: 6471-6479; Dowlut et al., J. Am. Chem. Soc., 2006, 128: 4226-4227) have been described.

[0057] In certain preferred embodiments, the benzoxaborole has formula (III) (wherein n is an integer equal to 1, 2, 3, 4, 5, 6, 7, or 8, and the phenyl 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 of the substituents is selected to enable grafting of the benzoxaborole onto the first polymer. Alternatively, the substituent on the phenyl group of formula (III) is a linking moiety terminated with a functional group that can be used to graft the benzoxaborole onto the first polymer. It is also envisioned that the phenyl group of the benzoxaborole of formula (III) can be replaced with a heteroaryl group, for example, a heterophenyl group.

[0058] C. Glucamine The second hydrogel precursor polymer according to the present invention is modified with glucamine. As used herein, the term "glucamine-modified" refers to a polymer grafted with glucamine (i.e., a polymer covalently linked to glucamine such that the polymer is functionalized with glucamine). As used herein, the term "glucamine" refers to a molecule having the following chemical formula (IV): Glucamine is also known as D-glucamine, and its IUPAC name is (2R,3R,4R,5S)-6-aminohexane-1,2,3,4,5-pentol.

[0059] [ka]

[0060] 2. Preparation of hydrogel precursor The first hydrogel precursor polymer modified with phenylboronic acid or a phenylboronic acid derivative and the second hydrogel precursor polymer modified with glucamine can be prepared using any suitable method known in the art, or using a procedure adapted from a method known in the art. Methods for grafting phenylboronic acid onto 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 onto 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).

[0061] In certain embodiments, grafting may be performed using the methods described in the Examples section below.

[0062] The reaction to obtain the glucamine-modified second hydrogel precursor polymer allows for 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., anywhere from about 1% to about 99%). In fact, because glucamine is completely soluble, it does not make the polymer more hydrophobic. Therefore, the molar ratio between the polymer and glucamine is not limited.

[0063] The hydrogel precursor polymers may be purified using any method known in the art, including, but not limited to, washing, filtration, precipitation, decantation, centrifugation, distillation, and the like, or any combination thereof.

[0064] If desired, after preparation, at least one of the first and second hydrogel precursors is placed in a solution prior to use or for storage. A preferred solution is an aqueous solution having a pH between about 6 and about 8. In certain preferred embodiments, the aqueous solution is a physiologically compatible solution, such as a buffered isotonic saline solution (see "Formulations" below).

[0065] Alternatively, if desired, after preparation, at least one of the first and second hydrogel precursors can be dried (e.g., freeze-dried or lyophilized) under vacuum to a substantially anhydrous form or powder, which can then be rehydrated prior to use.

[0066] If desired, after preparation, the hydrogel precursor polymers may be sterilized using any of a variety of sterilization techniques known in the art, for example, by bacteria-retaining filtration, by gamma irradiation, by electron beam irradiation, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0067] The hydrogel precursor polymers can be stored under appropriate conditions in either liquid or solid form. Suitable storage conditions for 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 appropriate conditions for periods of days, weeks, or months.

[0068] In certain embodiments, each of the first and second hydrogel precursors is placed in a recipient, such as a vial, flask, or other sealable container. When hydrogel precursor solutions are used, each solution may be placed in a syringe, such as a double-barrel syringe, or any other suitable syringe system in which the first and second hydrogel precursor polymers are physically separated, before co-extrusion, co-mixing, and co-injection of the combined first and second hydrogel precursors through a needle (cannula) into the body of a subject (see "Kits" below).

[0069] 3. Additional Ingredients Depending on the intended use of the crosslinked pair of hydrogel precursors, at least one of the first and second hydrogel precursor polymers or the hydrogel precursor solution may include cells, bioactive agents, visualization agents, or combinations thereof. Examples of such cells, bioactive agents, and visualization agents are provided below (see Pharmaceutical Compositions and Kits below).

[0070] The bioactive or visualization agent may be either covalently or ionically bound to at least one of the hydrogel precursor polymers, or the bioactive or visualization agent may be mixed with at least one of the hydrogel precursor polymers or the hydrogel precursor polymer solution.

[0071] To broaden the range of biomedical applications of the dynamic covalent hydrogels of the present invention, the first hydrogel precursor polymer or the second hydrogel precursor polymer of a crosslinked pair of hydrogel precursors may be modified to covalently link reactive chemical moieties, particularly bioorthogonal functional or clickable moieties. The presence of bioorthogonal functional or clickable moieties may be used to modulate the physicochemical properties of the hydrogel in time and space. For example, their presence can be used to stabilize and / or mechanically strengthen bioprinted constructs after printing. Alternatively, or in addition, the presence of bioorthogonal functional or clickable moieties may be used to facilitate post-gelation immobilization of molecules of interest (e.g., peptides, growth factors, drugs, fluorophores, etc.), development of evolving 3D culture systems in which cell-material interactions are tuned in time and space, etc.

[0072] Thus, in certain embodiments, the first hydrogel precursor polymer or the second hydrogel precursor polymer of a crosslinked pair of hydrogel precursors is directly or indirectly covalently linked to a bioorthogonal functional moiety or clickable moiety. As used herein, the term "bioorthogonal functional moiety or clickable moiety" refers to a reactive chemical group that can participate in a "bioorthogonal chemical reaction or click chemistry" and belongs to a pair of chemical reaction partners. Bioorthogonal chemical reactions and click chemistry reactions are conjugation reactions known in the art. The term "bioorthogonal chemical reaction" more specifically refers to a reaction that uses a chemical reaction pair that (1) does not naturally occur in biological systems; (2) does not cross-react with functional groups present in living organisms; and (3) does not require a cytotoxic catalyst or produce cytotoxic by-products. Examples of bioorthogonal chemical reactions include Staudinger ligation, which utilizes two reaction partners, an azide and a functionalized triarylphosphine, to form a stable amide bond; strain-promoted azide-alkyne cycloaddition (SPAAC, or copper-free click chemistry), which uses an azide and a strained cyclooctyne to obtain a stable triazole linkage via a 1,3-dipolar cycloaddition; 1,3-cycloaddition between a nitrone and a cyclooctyne, or between norbornene and a nitrile oxide (norbornene cycloaddition); oxime / hydrazine formation from aldehydes and ketones; tetrazine ligation, which involves an alkene (e.g., trans-cyclooctene, norbornene) and an s-tetrazine in a reverse-demand Diels-Alder reaction followed by nitrogen gas removal via a reverse Diels-Alder reaction; isocyanide-based click reactions; and quadricyclane ligation. The terms "click reaction," "click chemistry reaction," and "click chemistry" are used interchangeably herein. They more specifically refer to reactions that are "modular, versatile, give very good yields, produce only harmless by-products that can be removed by non-chromatographic methods, and are stereospecific" (Kolb et al., Angew. Chem., Int. Ed., 2001, 40: 2004-2021). Such reactions are characterized as "simple reaction conditions (ideally, the process is oxygen- and water-sensitive)." The reaction should require "non-reactive (should not be labile), readily available starting materials and reagents, no solvent or the 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 robust but not necessarily free of undesired effects in biological contexts, even though some click reactions are bioorthogonal chemical reactions. Click reactions include 1,4-conjugate additions (i.e., thiol-vinyl sulfone and thiol-maleimide reactions), aldehyde-nucleophile reactions (hydrazone and oxime ligation), Diels-Alder reactions, and photoactivated thiol-ene coupling.

[0073] The bioorthogonal functional moieties used in the practice of the present invention belong to a bioorthogonal partner pair, while the clickable moieties belong to a click reaction partner pair. The bioorthogonal functional moiety or clickable moiety may be any suitable chemical group that belongs to a bioorthogonal partner pair or a click reaction partner pair. Representative pairs of such 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 moiety or clickable moiety may be selected from the group consisting of cyclooctene; trans-cyclooctene; tetrazine; azide; alkynes, including strained alkynes, e.g., cyclooctene or cyclooctyne derivatives; amines; activated esters; isocyanates; isothiocyanates; thiols; aldehydes; amides; norbornenes; vinyl derivatives, including acrylates, methacrylates, etc.; phosphines, e.g., triarylphosphines; nitrones; quadricyclanes; maleimides; hydrazones, etc. Other bioorthogonal functional moieties and clickable moieties may also be used.

[0074] The bioorthogonal functional or clickable moiety may be covalently attached, directly or indirectly (e.g., via a linker), to the first hydrogel precursor polymer or the second hydrogel precursor polymer of a crosslinked pair of hydrogel precursors. It may be attached to the first polymer, phenylboronic acid or a phenylboronic acid derivative, the second polymer, or glucamine. In certain preferred embodiments, the bioorthogonal functional or clickable moiety is covalently attached, directly or indirectly (e.g., via a linker), to the first polymer or the second polymer. As will be appreciated by those skilled in the art, more than one bioorthogonal functional or clickable moiety may be attached to the first or second hydrogel precursor.

[0075] In certain embodiments, conjugation using bioorthogonal or click reactions typically involves a two-step strategy: first, introduction of a bioorthogonal functional moiety or click moiety into the first hydrogel precursor polymer or the second hydrogel precursor polymer of a crosslinked pair of hydrogel precursors, followed by introduction of a desired molecule or biomolecule via bioorthogonal or click conjugation after formation of the dynamic covalent hydrogel.

[0076] In other embodiments, conjugation using bioorthogonal or click reactions may be used to synthesize double lattice gels (i.e., a mixture of four polymers forming a 2:2 network), or for co-crosslinking (a mixture of three polymers with two network participating polymers). Co-crosslinking can be performed in one or two steps.

[0077] II- Dynamic Covalent Hydrogels The present invention also provides dynamic covalent hydrogels composed of two hydrogel precursor polymers of the crosslinked pair described herein. As used herein, the term "hydrogel" has its art-recognized meaning and refers to a three-dimensional polymeric structure that is insoluble in water or other aqueous media but can absorb and retain water to form a stable, often soft, flexible structure. In embodiments of the present invention in which the hydrogel is used in biological, biomedical, or medical applications, the hydrogel may be composed of at least one biocompatible, biodegradable, hydrophilic polymer. Crosslinked hydrogels can be considered solids because they do not flow or deform without the application of shear stress. However, crosslinked hydrogels with certain formulations may distort under the influence of gravity. "Hydrogel" may be used interchangeably with "hydrogel scaffold" or "scaffold" or "viscoelastic material." Hydrogels according to the present invention can be formed in vitro. Alternatively, they can be formed in situ, i.e., at a tissue site in a living animal or human body (see below). Hydrogels according to the present invention may further encapsulate or include any number of cells, biomolecules and / or bioactive agents (see below).

[0078] 1. Dynamic covalent hydrogel The hydrogel according to the present invention is composed of a first hydrogel precursor polymer modified with phenylboronic acid or a phenylboronic acid derivative as described above, and a second hydrogel precursor polymer modified with glucamine as described above, wherein the first and second polymers are crosslinked by a dynamic covalent bond.

[0079] As used herein, the term "dynamic covalent bond" refers to a covalent bond that can reversibly form and dissociate. For example, components of a dynamic system can respond to changes in the chemical environment (e.g., complex entities) or physical conditions (e.g., temperature, mechanical stress, electric field, irradiation, etc.). In the context of the present invention, first and second hydrogel precursor polymers are crosslinked by a dynamic covalent bond formed between the boronic acid functional group of the first polymer and the diol functional group of the second polymer. Boronic acids have the ability to form reversible covalent bonds with 1,2- and 1,3-diols, leading to the formation of boronate esters (Brighid Pappin et al., Boron-Carbohydrate Interactions). Thus, in the hydrogels according to the present invention, the first and second hydrogel precursor polymers are crosslinked by a boronic ester dynamic covalent bond.

[0080] The hydrogels of the present invention are "dynamic covalent hydrogels." The dynamic properties of crosslinking via boronic ester bonds result in hydrogels that exhibit 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 within a material when old bonds are broken. Thus, self-healing hydrogels of the present invention autonomously repair external stress damage and substantially recover their original elastic modulus and strength. The stress is typically applied via physical force and / or pressure. Thus, "self-healing" is the process by which a hydrogel that exhibits reduced flow resistance when subjected to external stress regains some or all of its stiffness and strength after the external stress is removed. As used herein, the term "shear thinning" refers to the effect in which the viscosity of a hydrogel (a measure of a fluid's resistance to flow) decreases with increasing shear rate or shear stress. Shear-thinning and self-healing hydrogels exhibit many unique and useful properties, including externally tunable strength, moldability, and low-energy synthesis / processing. In certain embodiments, upon removal of the mechanical shear force, the original hydrogel recovers within 30 minutes, preferably within about 20 minutes, about 10 minutes, about 5 minutes, or 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. For hydrogels prepared by the present inventors, recovery was on the order of a few seconds.

[0081] Preferably, the dynamic covalent hydrogels according to the present invention are biocompatible. The terms "biocompatible" and "medically acceptable" are used interchangeably herein. They refer to materials that are not significantly toxic to cells and / or living tissues. When used in medical applications, a hydrogel is considered biocompatible if, after placement in a physiological environment, it elicits minimal inflammatory responses, no anaphylactic reactions, and minimal unwanted cell growth on the surface of the biomaterial. Upon implantation into a host mammal, a biocompatible hydrogel does not elicit a host response that adversely affects the function of the hydrogel. Such a host response may include the formation of fibrous structures on or around the hydrogel, immune rejection of the hydrogel, or the release of toxic or pyrogenic compounds from the hydrogel into surrounding host tissues and / or body fluids.

[0082] Preferably, the dynamic covalent hydrogels according to the present invention are biodegradable. As used herein, the term "biodegradable" refers to the predictable breakdown of the hydrogel into molecules small enough to be metabolized or excreted under normal physiological conditions.

[0083] Dynamic covalent hydrogels according to the present invention prepared in vitro (or ex vivo) may be of any desired shape (geometry) and dimensions (size). As will be appreciated by those skilled in the art, the shape and dimensions will generally be dictated by the intended use of the hydrogel (e.g., 3D cell culture, tissue engineering, etc.). Shaping and sizing may include custom shaping and sizing to match the implantable device to a particular treatment site in a particular patient, as determined by imaging or other techniques known to those skilled in the art.

[0084] In certain embodiments, the hydrogels may be formed as microparticles or nanoparticles having sizes between about 50 nm and about 1000 nm, preferably between about 100 nm and 500 nm. These nanoparticles are suitable for administration by injection. However, due to the shear-thinning / shear-healing properties of the hydrogels described herein, the use of microparticles or nanoparticles is not necessary for easy injectability.

[0085] 2. Preparation of dynamic covalent hydrogels The dynamic covalent hydrogels of the present invention are formed by crosslinking a first hydrogel precursor polymer modified with phenylboronic acid or a phenylboronic acid derivative and a second hydrogel precursor polymer modified with glucamine. The reaction is carried out in a single step by mixing the first and second hydrogel precursor polymers, each contained in a solution.

[0086] The crosslinking reaction may occur in vitro (including ex vivo, i.e., in vitro prior to administration to a subject). Alternatively, the hydrogel may be formed in situ (e.g., directly at a given site in a living animal or human body), where hydrogel formation is initiated by mixing two hydrogel precursor polymers at the injection site.

[0087] The crosslinking reaction is carried out under physiological conditions in the absence of a catalyst. As used herein, the term "physiological conditions" refers to an artificial environment that mimics a natural environment compatible with living cells, e.g., primarily aqueous conditions of temperature, pH, osmolality, osmolality, oxidation, and electrolyte concentrations that are compatible with living cells and / or that are optionally considered to be within normal ranges at the site of administration or action in a subject. When the crosslinking reaction is carried out in vitro (or ex vivo), physiological conditions include an aqueous solution having a pH ranging from about 6 to about 8, preferably about 7.2 to 7.4, and a temperature ranging from about 4°C to about 42°C. When the crosslinking reaction is carried out in situ, the two hydrogel precursor polymers are placed in a buffered aqueous solution having a pH ranging from approximately 7.4 (e.g., between about 7 and 7.6) and a temperature ranging from about 30°C to about 42°C, preferably about 37°C.

[0088] The formation of dynamic networks at physiological pH is extraordinary compared to other boronate-cis-diol complexes, which can only be stable at alkaline pH (Springsteen et al., Tetrahedron, 2002, 58: 5291-5300; Peters, Coordination Chemistry Reviews, 2014, 268: 1-22).

[0089] 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 comprised between about 1:10 and about 10:1, e.g., between about 1:8 and about 8:1, or between about 1:6 and about 6:1, or between about 1:4 and about 4:1, or even between about 1:2 and about 2:1.

[0090] In the context of the present invention, the crosslinking reaction is substantially instantaneous. In certain embodiments, the crosslinking reaction can occur within a time frame of about 1 second to about 5 minutes, e.g., about 3 seconds to about 1 minute, about 10 seconds to about 2 minutes, and the gelation time can be less than about 30 seconds, or less than about 20 seconds, or even less than about 10 seconds. For example, the crosslinking reaction can occur within a time frame of about 10 seconds.

[0091] If desired, following in vitro preparation, the dynamic covalent hydrogels may be sterilized using any suitable method known in the art, such as gamma irradiation, autoclaving, ethylene oxide sterilization, infrared irradiation, and electron beam irradiation. A sterilization method is suitable for use if it does not induce a significant loss of the useful physical and / or mechanical properties of the hydrogel. Selecting a suitable sterilization method is within the skill of one of ordinary skill in the art.

[0092] Dynamic covalent hydrogels prepared in vitro may be stored under sterile conditions at low temperatures (eg, 4° C.) prior to use.

[0093] 3. Preparation of dynamic covalent hydrogels Dynamic covalent hydrogels according to the present invention can be characterized by their viscoelasticity. The term "viscoelasticity," as used herein to characterize a hydrogel, is meant to refer to a characteristic provided by the scaffold that can vary with the time and / or rate of loading. Accordingly, where appropriate, a viscoelastic hydrogel provides a loading time and / or rate characteristic that matches or approximates that observed in a given tissue or site (see below). This characteristic is responsible for the dissipation of energy, which can be provided by the scaffold itself and / or by the scaffold in complex with cells growing thereon. For example, it may be desirable to provide a scaffold that approximates the viscoelastic properties of the tissue that the hydrogel is intended to repair, restore, or replace (see below).

[0094] The dynamic covalent hydrogels according to the present invention may have a shear storage modulus (G') comprised between about 10 Pa and about 10,000 Pa, preferably between about 100 Pa and about 1000 kPa; and a shear loss modulus (G") comprised between about 0.01 Pa and about 1000 Pa, preferably between about 0.1 kPa and about 500 Pa. Measurement of the G' and G" values ​​of the dynamic gels is typically carried out in the frequency range comprised between 0.001 and 1000 Hz, using a stress constraint comprised between 0.1 and 10 Pa.

[0095] The dynamic covalent hydrogels according to the present invention undergo minimal swelling and exhibit high stability. As used herein, the term "swelling" is understood to refer to the property of a hydrogel that increases in mass and volume when it absorbs a fluid (here, water). As used herein, the term "swelling ratio" refers to the ratio of the mass of a hydrogel when substantially fully hydrated to its mass when unswollen (i.e., the mass before immersion in a fluid). The swelling ratio is generally provided as a percentage. Those skilled in the art are aware of suitable processes and measurement methods for determining the swelling ratio. As used herein, the term "stable," when referring to a hydrogel, means no change in the swelling ratio over time. The dynamic covalent hydrogels according to the present invention have a swelling ratio between about 100% and about 300%, for example, between about 100% and about 200%. In the experiments presented in the Examples section below (see Example 1), the stability of the prepared dynamic covalent hydrogels was observed over a 35-day period (for a 35-day experiment). There is no doubt that the dynamic covalent hydrogels according to the present invention are stable for weeks and up to months.

[0096] 4. Additional Ingredients Depending on the intended use of the dynamic covalent hydrogels according to the present invention, the hydrogels may comprise cells, bioactive agents, visualization agents, or any other additional components, or combinations thereof. Examples of such cells and additional components are provided below (see Pharmaceutical Compositions and Kits).

[0097] A bioactive agent, visualization agent, or any other additional component may be incorporated into the hydrogel before, during, or after crosslinking of the first and second hydrogel precursor polymers. For example, prior to the crosslinking step, any additional component may typically be added to at least one of the first and second hydrogel precursor solutions (as described above). During the crosslinking step, any additional component may be added to the reaction mixture comprised of the first and second hydrogel precursors. Alternatively, or in addition, a bioactive agent, visualization agent, or any other active component may be incorporated into the hydrogel after its formation. For example, a dynamic covalent hydrogel according to the present invention may be immersed in a solution containing any additional active component to allow the component to diffuse into the hydrogel, or any additional active component may be added to a solution containing the dynamic covalent hydrogel. Alternatively, dynamic covalent hydrogels (see above) prepared from crosslinked pairs of hydrogel precursors in which the first or second hydrogel precursor is directly or indirectly covalently linked to a bioorthogonal functional or clickable moiety may undergo bioorthogonal or click conjugation to introduce desired molecules or biomolecules (e.g., peptides, growth factors, drugs, fluorophores, etc.).

[0098] In embodiments in which the dynamic covalent hydrogels according to the present invention comprise cells, the cells may be encapsulated within the hydrogel. As used herein, the term "encapsulated" has its art-recognized meaning and refers to the containment, immobilization, and / or entrapment of one or more cells within a three-dimensional structure defined by a physical barrier (i.e., a barrier that reduces or controls the permeability of said structure).

[0099] III—Hydrogel Precursor Polymer Compositions and Uses of Dynamic Covalent Hydrogels 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 biointegrated electronics, and soft robotics (e.g., 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 invention may find application in a variety of fields, particularly in the treatment or prevention of diseases or medical conditions in a subject.

[0100] 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.) that may or may not have a disease or disorder. A non-human subject may also be a transgenic or otherwise modified animal. In many embodiments of the invention, a subject is often referred to as an "individual" or "patient." The terms "subject," "individual," and "patient" do not denote a particular age and, therefore, encompass newborns, children, teenagers, and adults. The term "patient" more specifically refers to an individual afflicted with a disease or disorder.

[0101] The term "treatment" is used herein to characterize a method or process intended to (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or halt the progression, worsening, or progression of a disease, disorder, or condition; (3) alleviate the symptoms of a disease, disorder, or condition; or (4) cure a disease, disorder, or condition. Treatment may be administered after the onset of a disease, disorder, or condition, for a therapeutic effect. Alternatively, treatment may be administered prior to the onset of a disease, disorder, or condition, for a prophylactic or preventative effect. In this instance, the term "prevention" is used.

[0102] 1. 3D Cell Culture and Cell Therapy The dynamic covalent hydrogels of the present invention are attractive 3D cell culture alternatives to natural animal-derived matrices such as MATRIGEL®. 3D cell culture is an artificially created environment that allows biological cells to grow and interact with their surroundings in all three dimensions, similar to how they would do in vivo. Compared to traditional culture, cells in 3D culture more closely resemble their in vivo environment in terms of cell shape and cellular environment. The diversity of structures and materials is much greater in 3D matrices than in 2D substrates. Thus, in certain embodiments, the dynamic covalent hydrogels of the present invention are used for 3D cell culture.

[0103] The term "cell culture" refers to the process of maintaining cells under conditions suitable for their maintenance and / or growth, such as temperature, availability of nutrients, atmospheric CO2 content, and the cell density at which the cells are maintained. In the hydrogel, cells can be cultured in vitro or in vivo. Culture conditions suitable for maintaining the proliferation, expansion, and differentiation of different cell types 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 invention include any cells that can grow in a 3D hydrogel matrix (see below).

[0104] Dynamic covalent hydrogels used for 3D cell culture may include any of a variety of biomolecules, the presence of which is desirable in the context of cell culture, including, but not limited to, proteoglycan or glycosaminoglycan chains, hormones, growth factors, chemoattractants, etc. (See Pharmaceutical Compositions and Kits below).

[0105] After being cultured in the dynamic covalent hydrogel according to the present invention, the cells may be harvested before use. The term "harvesting" as used herein refers to the procedure of removing or dissociating cells from the 3D hydrogel in which they were cultured. Cells may be separated from the hydrogel fragments obtained from enzymatic degradation of the hydrogel by filtration. Examples of enzymes that can be used to degrade hydrogel scaffolds include, but are not limited to, collagenase, elastase, trypsin, pullulanase, hyaluronidase, chondroitinase (ABC), cellulase, etc. Alternatively, dynamic gels have the great advantage of degrading in the presence of a large amount of one of the two reactive groups in the form of small free molecules. Typically, the addition of glucamine or any other diol dissolves the gel, allowing the cells to be recovered by centrifugation without the need for enzymatic reaction or trituration of the material.

[0106] After harvesting, cells cultured in dynamic covalent hydrogels may find many uses. Thus, for example, harvested cells can be used in cell therapy, where living cells are injected, transplanted, or implanted into a subject to treat or prevent a disease or medical condition in that subject. Thus, harvested cells may be used to treat human clinical conditions (e.g., cancer, infectious diseases, autoimmune diseases) or in regenerative medicine (e.g., restoration and repair of damaged, injured, or missing tissues, such as articular cartilage or the spinal cord). The availability of large quantities of functional stem cells, which have been proposed as promising candidates for therapy, may facilitate the development of autologous (patient-derived) or allogeneic (separate donor-derived) transplants. Similarly, using the dynamic covalent 3D hydrogel matrices of the present invention, large quantities of cells from genetically modified stem cell lines can be generated for cell-based therapy. The dynamic covalent 3D hydrogels described herein can also be used for the expansion of stem cells in therapeutic cloning (also known as somatic cell nuclear transfer).

[0107] Alternatively, the harvested cells may be used for tissue engineering applications. The availability of large quantities of functional cells is also advantageous in cell biology research. Other potential uses of the dynamic covalent hydrogels according to the present invention include, but are not limited to, the production of lineage-dependent viruses, e.g., viruses that require differentiated cells to produce sufficient particles for use as a vaccine; and protein manufacturing (cells on the hydrogel produce factors that can be isolated from the culture medium and / or from the cells and then purified). Examples of proteins that can be produced in this way include, but are not limited to, growth factors, hormones, signaling molecules, inhibitors of cell growth, and antibodies.

[0108] 2. Tissue Engineering, Organoid Formation and Applications The dynamic covalent hydrogels of the present invention may 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, viable tissue to repair or replace whole or parts of tissues (e.g., bone, cartilage, blood vessels, bladder, skin, muscle, etc.) damaged by disease, trauma, genetic or chromosomal abnormalities, or aging. Thus, in certain embodiments, the dynamic covalent hydrogels described herein are used in tissue engineering.

[0109] As is known in the art, tissue culture should be performed in a suitable culture medium, with or without stimuli such as stress or orientation. Additionally, the tissue culture medium or dynamic covalent hydrogel may contain any of a variety of biomolecules, the presence of which is desirable in such a context. Examples of such biomolecules include growth factors, nutrients and / or cell-binding domains, sugars, tissue adhesives, etc.

[0110] An example of a tissue equivalent is an organoid. 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 confined lineage commitment in a manner similar to that in vivo. Thus, organoids exhibit the native physiology of cells and have a cellular composition (including residual stem cells and specialized cell types) and anatomy that closely resembles the native situation. The cells that generate organoids differentiate to form organ-like tissues that exhibit multiple cell types that self-organize to form structures very similar to organs in vivo. Therefore, organoids are an excellent model for studying human organ development in a system that closely resembles human organs and in vivo development. Organoids may be used for drug response screening, toxicity assays, or regenerative medicine. Organoids can also be used to cultivate pathogens, such as norovirus, for which there are currently no suitable tissue culture or animal models.

[0111] 3. In vivo delivery of cells and / or bioactive agents In certain embodiments, the dynamic covalent hydrogels according to the present invention may be used as a system for the delivery of cells and / or bioactive agents in vivo (i.e., within the body of a subject in need thereof).

[0112] In certain embodiments, hydrogels according to the present invention may be used for cell delivery. Hydrogels according to the present invention may be used as raw materials for preparing cell delivery systems that can be administered to a subject for therapeutic or diagnostic purposes. In certain embodiments, hydrogels according to the present invention may be used to prepare patches, biofilms, or dressings that can be loaded with cells. For example, hydrogels according to the present invention may be used to prepare dressings that can be applied to the skin (e.g., damaged or injured skin) to rebuild or heal the skin. Alternatively, the dressing may be applied to a subject's heart to treat ischemia (myocardial infarction). In such embodiments, cells entrapped in the hydrogel may migrate to the target tissue or organ.

[0113] In other embodiments, crosslinked pairs of hydrogel precursor polymers may be used for cell delivery. Indeed, in situ gelling polymer matrices are of great interest in tissue regeneration because these materials can be used as injectable hydrogels. They can act as cell vehicles with the ability to adopt the shape of the corresponding tissue cavity. Furthermore, cells can be directly incorporated into the injectable solution, minimizing issues related to cell adhesion.

[0114] The dynamic covalent hydrogel and hydrogel precursor polymer pair according to the present invention can be used as a vehicle 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 substances that act locally or systemically in the body, such as therapeutic, prophylactic, and / or diagnostic agents, substances that affect the structure or function of the body (e.g., affecting or participating in tissue growth or cell differentiation), compounds that can initiate a biological effect such as an immune response or play any other role in one or more biological processes, prodrugs that become biologically active or more active after being placed in a given physiological environment, and compounds or agents that support or assist cell growth, cell differentiation, and / or cell implantation. Examples of bioactive agents are provided below (see Pharmaceutical Compositions and Kits).

[0115] The bioactive agent can be mixed with the hydrogel, covalently bonded to the hydrogel, and / or adsorbed in or onto the hydrogel. Alternatively, or in addition, the bioactive agent can be included in a solution of the first or second hydrogel precursor polymer of the cross-linked couple used to form the dynamic covalent hydrogel, and after cross-linking of the first and second polymers, the dynamic covalent hydrogel thus formed contains the bioactive agent.

[0116] 4. Viscosupplementation / artificial joint lubricants In certain embodiments, crosslinked pairs of hydrogel precursor polymers or dynamic covalent hydrogels according to the present invention may be used in viscosupplementation therapy. Viscosupplementation therapy is a procedure that involves injecting a gel-like substance (e.g., hyaluronate) into a joint to reinforce the viscosity of synovial fluid. Joint injections of viscoelastic fluids, most commonly crosslinked hyaluronic acid, have been used to treat osteoarthritis for over 20 years. The effectiveness of these viscosupplementation treatments is said to stem, in part, from the high viscosity of the injected polymer solution.

[0117] Polymer hydrogels that undergo crosslinking during or after administration into the joint space may show improvement because such hydrogels increase in viscosity within the joint space after administration. Thus, a crosslinked pair of hydrogel precursor polymers according to the present invention may be administered as a relatively low-viscosity liquid and then form a much more viscous or viscoelastic gel in the intra-articular space of the joint within a short time frame. Additionally, the higher viscosity imparted by in situ crosslinking may result in a longer residence time in the joint and therefore a longer duration of treatment than can be achieved with a single administration.

[0118] Thus, crosslinked pairs of hydrogel precursor polymers or dynamic covalently bonded hydrogels according to the present invention may be used in viscosupplementation therapy for the treatment of osteoarthritis or rheumatoid arthritis, or other inflammatory arthritis, such as gout or calcium pyrophosphate deposition disease (e.g., by injection into the intra-articular space of a joint), including the knee, shoulder, temporomandibular and carpometacarpal joints, elbow, hip, wrist, ankle, and lumbar intervertebral (facet) joints of the spine.

[0119] In certain embodiments, at least one of the hydrogel precursor polymer solutions contains a corticosteroid (e.g., triamcinolone acetonide, cortisone acetate), which is useful for providing relief from the pain and swelling caused by inflammation experienced by subjects suffering from osteoarthritis. Several advantages are associated with in situ entrapment / incorporation of the corticosteroid within the hydrogel: entrapment (1) is effective in preventing the majority of the steroid from coming into direct contact with joint tissue, (2) is effective in maximizing the local concentration of steroid in the joint while minimizing its systemic concentration, (3) is effective in preventing premature elimination of steroid from the joint, and (4) can achieve therapeutic efficacy at a lower total dose than would be achieved in the absence of hydrogel entrapment, while minimizing undesirable local and systemic side effects.

[0120] In certain embodiments, at least one of the hydrogel precursor polymer solutions contains a fibroblast growth medium that activates the cellular components of the artificial connective tissue, in particular synoviocytes and chondrocytes, thereby ensuring their cellular regeneration and stimulating their endogenous synthesis.

[0121] Viscosupplementation can be achieved via a single injection or multiple intra-articular injections administered over a period of several weeks into the affected joint. The goal is to provide the joint with the ability to absorb shock during movement and be lubricated at least at rest. In certain embodiments, viscosupplementation is administered with the goal of delaying total hip or knee replacement.

[0122] 5. 3D Printing and 3D Bioprinting In certain embodiments, the crosslinked pairs of hydrogel precursor polymers and dynamic covalent hydrogels described herein find application in 3D printing or 3D bioprinting. 3D printing builds three-dimensional objects, typically by layer-by-layer addition of material from a computer-aided design model. 3D bioprinting utilizes 3D printing technology to generate functional miniature tissue constructs from biocompatible materials, cells, and supporting components, such as cell culture media. Key applications include high-throughput in vitro tissue models, drug discovery and toxicology, and regenerative medicine / tissue engineering applications. It involves precise layer-by-layer positioning of biomaterials and biological cells with spatial control of the placement of functional components. This technology has made significant advances toward clinical reconstruction of tissues and organs, such as the ear, nose, bone, heart, liver, and skin.

[0123] 6. Soft Robotics The cross-linked pairs of hydrogel precursor polymers and dynamic covalent hydrogels described herein can be applied to soft robotics, a specific subfield of robotics that deals with building robots from highly compliant materials similar to those found in living organisms (Robosoft, first IEEE International Conference on Soft Robotics, April 24–28, 2018, Livorno, Italy). In contrast to robots constructed from rigid materials, soft robots allow for increased flexibility and adaptability to accomplish tasks, improving safety when operating around humans. These features enable their potential use in the fields of medicine and manufacturing. Thus, for example, soft robots could be implemented in medical practice, specifically in invasive surgery. Due to their shape-changing properties, soft robots can assist in surgery. Shape-changing is important because soft robots can adjust their morphology to navigate around different structures in the human body.

[0124] IV- Pharmaceutical Compositions and Kits To use the crosslinked pairs of hydrogel precursor polymers and dynamic covalent hydrogels described herein for therapeutic treatment of mammals, including humans, in some embodiments, the hydrogel precursor polymers and dynamic covalent hydrogels are formulated as pharmaceutical compositions in accordance with standard pharmaceutical practice. Accordingly, the present invention provides pharmaceutical compositions comprising the dynamic covalent hydrogels described herein and at least one pharmaceutically acceptable diluent or carrier. The present invention also provides pharmaceutical compositions in which a first hydrogel precursor polymer of a crosslinked pair described herein is formulated with at least one pharmaceutically acceptable diluent or carrier, and a second hydrogel precursor polymer of a crosslinked pair described herein is formulated with at least one pharmaceutically acceptable diluent or carrier.

[0125] Pharmaceutical compositions according to the present invention may be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as multiple single unit doses. As used herein, the term "unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of each hydrogel precursor polymer or a predetermined number of dynamic covalent hydrogels of a given shape and size.

[0126] Pharmaceutical compositions according to the invention are formulated, dosed and administered in a manner consistent with good medical practice, i.e., amounts, concentrations, schedules, courses, vehicles and routes of administration. Factors to be considered in this context include the particular disease or clinical condition being treated, the particular subject being treated (age, weight, etc.), the clinical condition (health) of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the schedule of administration, and other factors known to physicians.

[0127] 1. Formulation As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not excessively toxic to the host at the concentration at which it is administered. This term includes solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. The use of such media and agents for pharmaceutical 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, incorporated herein by reference).

[0128] Examples of suitable pharmaceutically acceptable carriers or excipients include, but are not limited to, saline and / or buffer solutions (e.g., citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, 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, disaccharides, and other carbohydrates. (e.g., glucose, mannose, or dextrin), 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), non-ionic surfactants (e.g., TWEEN®, PLURONICS®, polyethylene glycol), lubricants (e.g., magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, natural oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc.), and any combination thereof.

[0129] Suitable preservatives include antimicrobial preservatives (e.g., benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal), antifungal preservatives (e.g., butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, propionylparaben, propyl ... The preservative may be any of the preservatives known to be useful in pharmaceutical compositions (e.g., tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, 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.), and any combination thereof.

[0130] For example, injectable preparations can be formulated as known in the art using suitable vehicles and solvents, such as water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile, fixed oils are conveniently used as a solution or suspending medium. For this purpose, any bland fixed oil, including synthetic mono- or diglycerides, may be used. Fatty acids, such as oleic acid, may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for administration by injection. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. If necessary or desired, the injectable formulation may also contain a local anesthetic to ease pain at the injection site.

[0131] For topical application, the crosslinked pair of hydrogel precursor polymers and dynamic covalent hydrogels according to the present invention, e.g., containing cells and / or bioactive agents, are preferably combined with other components, e.g., carriers and / or adjuvants. The nature of such other components is not limited, except that they must be physiologically acceptable and effective for their intended administration and cannot degrade the activity of the active ingredients of the composition. In certain embodiments, pharmaceutical compositions according to the present invention may be impregnated into or deposited on articles, which may 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 invention may be used to prepare patches, biofilms, or dressings that can be loaded with cells and / or bioactive agents.

[0132] Although the description of compositions provided herein is primarily directed to compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications of compositions suitable for administration to humans to make them suitable for administration to various animals are well understood.

[0133] 2. Additional Ingredients The pharmaceutical composition according to the present invention may further comprise at least one additional component selected from cells, biologically active agents, visualization agents, or any combination thereof. As already mentioned above, the biologically active agents and visualization agents may be associated with at least one of the hydrogel precursor polymers or dynamic covalent hydrogels present in the pharmaceutical composition, either covalently or ionically. Alternatively, or in addition, the biologically active agents and visualization agents may be mixed with the hydrogel precursor solution or pharmaceutical composition according to the present invention and do not form an association with the hydrogel precursor polymers or hydrogel.

[0134] A. Cell In certain embodiments, the hydrogel precursor polymer solution or dynamic covalent hydrogel contains cells. As used in the context of the present invention, the term "cells" refers to various forms of cells, including, but not limited to, cell clusters (e.g., pancreatic islets or portions thereof) and individually isolated cells. In certain preferred embodiments, the cells used in association with the hydrogel precursor polymer solution or dynamic covalent hydrogel according to the present invention are of mammalian (animal or human) origin. Mammalian cells may be of any organ, body 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). Cells may be primary, secondary, or immortalized cells (i.e., established cell lines). They may be isolated or derived from ex vivo biological samples or obtained from volunteers or patients by techniques well known in the art. Cells used in regenerative medicine and tissue engineering may be derived from the patient to whom the cells are administered (autologous administration) or from another individual (allogeneic administration). Additionally, xenogeneic cells, such as those derived from animals, can also be adapted for regenerative medicine strategies. Alternatively, cells may be purchased from commercial sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, or in addition, cells may be genetically engineered to contain genes of interest, such as genes or receptors that express growth factors, or to contain defective genes, or even Oct3 / 4, Sox2, Klf4, and c-Myc genes to prepare human induced stem cells from adult somatic cells.

[0135] Cells that may be used in combination with the hydrogel precursor polymer solutions or dynamic covalent hydrogels described herein include differentiated cells, stem cells (including induced pluripotent stem cells), and progenitor cells.

[0136] As used herein, the term "differentiated cells" refers to cells that are specialized for a particular function and do not have the ability to generate other types 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, neurons, neuroblasts, glioblastomas, giant cells, dendritic cells, macrophages, blastomeres, endothelial cells, stromal cells, Kupffer cells, Langerhans cells, dyke cells, and tissue cells. Specific examples of differentiated cells include, but are not limited to, fibroblasts, chondrocytes, osteoblasts, osteoclasts, osteocytes, synoviocytes, bone marrow stromal cells, stem cells, fibrochondrocytes, endothelial cells, smooth muscle cells, adipocytes, cardiac myocytes, myocytes, keratinocytes, hepatocytes, leukocytes, macrophages, endocrine cells, urogenital cells, lymphatic cells, pancreatic islet cells, muscle cells, enterocytes, kidney cells, vascular cells, thyroid cells, parathyroid cells, cells of the adrenal-hypothalamic-pituitary axis, bile duct cells, ovarian or testicular cells, salivary secretory cells, kidney cells, epithelial cells, and neuronal cells.

[0137] As used herein, the term "stem cell" refers to a relatively undifferentiated cell that has the capacity for continuous self-renewal and the potential to give rise to 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 cells" and "ES cells" are used interchangeably herein. They refer to stem cells derived from a group of cells called the inner cell mass, which is part of an early (4-5 day old) embryo called a blastocyst. "Human embryonic stem cells" or "hES cells" are embryonic stem cells of human origin, generally derived from a fertilized egg less than one week old. In vitro, embryonic stem cells can proliferate indefinitely, a property not shared by adult stem cells. The term "adult stem cells" refers to stem cells that are not of embryonic origin or derived from embryonic or fetal tissue. The term "adult stem cells" also encompasses stem cells isolated from subjects of all ages (e.g., human infants and children). As used herein, the term "induced pluripotent stem cells" (or "iPS cells") refers to a type of pluripotent stem cell artificially induced 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 may be human induced pluripotent stem cells. The terms "human induced pluripotent stem cells" and "human iPS cells" are used interchangeably herein. They refer to induced pluripotent stem cells of human origin. Typically, human induced stem cells can be obtained by inducing the expression of Oct3 / 4, Sox2, Klf4, and c-Myc genes in any adult somatic cell (e.g., fibroblast). Essentially, somatic cells are transfected with a viral vector, such as a retrovirus, which contains the Oct3 / 4, Sox2, Klf4, and c-Myc genes.

[0138] The terms "progenitor cells" and "precursor cells" are used interchangeably herein. They refer to partially specialized cells that arise in fetal or adult tissues. 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. Compared to stem cells, they have limited capacity for self-renewal and differentiation. Such capacity for self-renewal (or proliferation) is demonstrated by the expression of proliferation markers, such as Ki-67 nuclear antigen. Furthermore, progenitor cells also express specific markers as they undertake specific differentiation processes. 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, etc.

[0139] The cells present in the hydrogel precursor polymer solution or dynamic covalent hydrogel can form a substantially homogeneous population or a heterogeneous cell population. The term "substantially homogeneous cell population," as used herein, 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 cell population," as used herein, refers to a population of cells comprising at least two cell types.

[0140] The cells may be present in the hydrogel precursor polymer solution or dynamic covalent hydrogel in any suitable amount, for example, the cells may be added to the hydrogel precursor polymer solution or dynamic covalent hydrogel at a density of about 500 to about 1000 cells / μL.

[0141] B. Bioactive Agents In certain embodiments, pharmaceutical compositions according to the present invention comprise at least one bioactive agent. As will be appreciated by those skilled in the art, the selection of one or more bioactive agents will be based on the intended purpose of the pharmaceutical composition (e.g., use in viscosupplementation, joint treatment, cell therapy, 3D cell culture, tissue engineering, etc.). Generally, the amount of bioactive agent present in an inventive pharmaceutical composition will be the usual dosage required to obtain the desired result via a given route of administration. Such dosages will be known to or readily determined by those skilled in the pharmaceutical and / or medical arts.

[0142] Suitable bioactive agents may belong to various classes 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 bioactive agent may be a single compound or multiple compounds, including, for example, a combination of two or more bioactive agents.

[0143] Examples of suitable therapeutic agents include, but are not limited to, analgesics, anesthetics, pain relievers, anti-cancer agents, antibacterial agents, antiviral agents, antifungal agents, antibiotics, anti-inflammatory agents, antioxidants, antiseptics, antipruritics, immunostimulants, angiogenesis inhibitors, antineoplastic agents, antiproliferative agents, antidiabetic agents, decongestants, antihypertensive agents, dermatological agents, anticholinergics, immunosuppressants, antidepressants, antipsychotics, beta-adrenergic blockers, cardiovascular active agents, vasoactive substances, nonsteroidal agents, sex hormones, steroidal agents, osteogenic agents, osteoconductive agents, osteoinductive agents, antirejection agents, antiarthritic agents, thrombolytic agents, antifibrinolytic agents, hemorheologic agents, antiplatelet agents, and the like.

[0144] Other examples of suitable bioactive agents include cytokines, growth factors, proteoglycans or portions thereof, adhesion molecules, and any combination thereof.

[0145] Cytokines and growth factors are polypeptide molecules that regulate the migration, proliferation, differentiation, and metabolism of mammalian cells. A diverse range of these biomolecules have been identified as potentially playing important roles in regulating healing. Cytokines may be lymphokines, monokines, or chemokines. Examples of cytokines include, but are not limited to, interleukins (IL) (e.g., IL-1, IL-2, IL-4, and IL-8), interferons (IFN) (e.g., IFN-α, IFN-β, and IFN-γ), and tumor necrosis factors (e.g., TNF-α), or any variants, synthetic analogs, active portions, or combinations thereof. Examples of growth factors include, but are not limited to, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), heparin-binding growth factor (HBGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), connective tissue activating peptide (CTAP), transforming growth factor alpha (TGF-α) and beta (TGF-β), nerve growth factor (NGF), colony-stimulating factors (G-CSF and GM-CSF), and the like, or any variant, synthetic analog, active portion, or combination thereof.

[0146] Proteoglycans are protein-carbohydrate complexes characterized by their glycosaminoglycan (GAG) components. GAGs are highly charged sulfated and carboxylated polyanionic polysaccharides. Examples of GAGs suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, hyaluronan, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate.

[0147] Adhesion molecules constitute a diverse family of extracellular and cell surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, recognition, activation, and migration. Adhesion molecules are essential for the structural integrity and homeostatic functioning of most tissues and are involved in a wide range of biological processes, including embryogenesis, inflammation, thrombosis, and tissue repair. Adhesion molecules include matricellular proteins (e.g., thrombospondin and tenascin) and cell surface adhesion molecules (e.g., integrins, selectins, cadherins, and immunoglobulins).

[0148] Other examples of bioactive agents include, but are not limited to, hormones and hormone analogs (e.g., growth hormone), morphogens (e.g., retinoic acid, arachidonic acid, etc.), extracellular matrix molecules (e.g., fibronectin, vitronectin, laminin, collagen, elastin, etc.), blood clotting / clotting factors (e.g., fibrinogen, prothrombin, hemophilia A, etc.); and the like.

[0149] The biologically active agent may be a bioactive peptide sequence that can be attached to the surface of the hydrogel to promote protein adsorption and subsequent cell and tissue adhesion. Examples include adhesive peptides derived from fibronectin, vitronectin, laminin, and collagen. The term "RGD" or "RGD sequence" refers to the minimally bioactive arginine-glycine-aspartic acid (RGD) sequence, which is the minimum (minimal) fibronectin-derived amino acid sequence sufficient to mimic cells that bind to fibronectin and / or promote adhesion of anchorage-dependent cells. Such short RGD bioactive peptides are known in the art.

[0150] Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, as well as radionuclides, x-ray imaging agents and contrast agents.

[0151] In certain embodiments, the bioactive agent is a particle of bioactive glass, soluble glass, resorbable calcium phosphate, hydroxyapatite, calcium carboxylate, calcium sulfate, glass ceramics, and the like.

[0152] In certain embodiments, the pharmaceutical compositions described herein contain less than about 80% by weight, less than about 75% by weight, less than about 70% by weight, less than about 60% by weight, less than about 50% by weight, less than about 40% by weight, less than about 30% by weight, less than about 20% by weight, less than about 15% by weight, less than about 10% by weight, less than about 5% by weight, less than about 1% by weight, less than about 0.5% by weight, or less than about 0.1% by weight of the bioactive agent.

[0153] C. Visualization Agents In certain embodiments, the hydrogel precursor polymer solution or dynamic covalent hydrogel may contain a visualization agent to improve visibility, e.g., to allow a surgeon to accurately and conveniently position the in situ forming hydrogel during a surgical procedure. The visualization agent may be selected from a variety of non-toxic coloring substances, e.g., dyes suitable for use in implantable medical devices. Suitable dyes include, for example, dyes for visualizing the thickness of the hydrogel as it forms in situ, e.g., 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 suitable dyes include fluorescent compounds (e.g., fluorescein or eosin), x-ray contrast agents (e.g., iodine 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, e.g., 11 C-DASB, 11 C-flumazenil, 11 Other visualization agents such as C-raclopride may also be used.

[0154] The visualization agent may be covalently bound to at least one of the hydrogel precursor polymers. However, in a preferred embodiment, the visualization agent is not covalently linked to the hydrogel precursor polymers. For example, the visualization agent may be present in the precursor hydrogel solution. The visualization agent may be used in a small amount, for example, at a concentration of less than 1% w / v, or less than 0.01% w / v, or even less than 0.001% w / v.

[0155] 3. Administration The pharmaceutical compositions according to the present invention may be administered using any suitable route of administration, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual, etc. In particular, a preferred route in the context of the present invention is direct administration to the affected site (e.g., in the intra-articular space of a joint, e.g., by transdermal injection, by topical placement at the site of injury or trauma to the skin, by placement in conjunction with a surgical procedure, etc.).

[0156] Dynamic covalent hydrogels that exhibit viscous flow under shear stress (shear thinning) and rapid recovery (self-healing) when the applied stress is relaxed enable minimally invasive implantation in vivo by direct injection or catheter-based delivery. Thus, in certain embodiments, dynamic covalent hydrogels according to the present invention can be compressed and loaded into a delivery device, e.g., a catheter, endoscope, syringe, etc. The delivery device is flexed 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 expands to approximately its original relaxed size and shape.

[0157] In other embodiments, the implantable hydrogel is inserted by an open surgical procedure.

[0158] As mentioned above, dynamic covalent hydrogels according to the present invention may be formed in situ (e.g., directly at or near a given site in the body of a living animal or human). In this case, hydrogel formation is initiated by mixing two hydrogel precursor polymers at an injection site. Thus, in certain embodiments, two hydrogel precursors of a crosslinked pair according to the present invention may be applied (e.g., injected) via a spray to tissue to form a coating or space-filling hydrogel in situ. Preferably, the two hydrogel precursors are contained in separate chambers of the spray. When the spray is activated, the emergent spray contacts the tissue, the two hydrogel precursor polymers mix and crosslink, gelation occurs, and the hydrogel is formed at the desired site in the body. The spray may be a multi-barrel system, preferably a double-barrel syringe system.

[0159] 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 may have two or more plungers. The term "double-barrel system" refers to any system or device, typically a syringe, that includes two separate barrels and may have one or two plungers. Furthermore, a multi-barrel (e.g., double-barrel) syringe system generally includes a needle or cannula, with or without a tip cap or needle shield for sealing the end of the syringe system. The barrel generally has a storage capacity for containing sufficient first and second hydrogel precursor solutions. The barrel may be made of glass, plastic, or any other material and may have various geometries, inner diameters, material compositions, transparencies, etc. Furthermore, a multi-barrel syringe system may be a double-barrel syringe system in the form of two integrated syringes, i.e., a syringe with two integrated barrels and a single or double plunger assembly for expelling the contents from the barrel. The syringe system may also include two removably connected barrels and two or more removably connected plungers. Furthermore, the syringe system may also include a means (e.g., an applicator tip) configured to thoroughly mix the contents contained in the barrels prior to ejection through the applicator tip. Thus, the barrels are generally connected, and the plunger assemblies are generally configured to eject the contents from the barrels simultaneously, so that the proper mixing ratio of the hydrogel precursor solutions is maintained.

[0160] Treatment according to the invention may be administered in a single dose or multiple doses (e.g., daily, weekly, monthly, every two months, every three months, every six months, every year, every two years, etc.). The dose and dosing regimen will be determined by a physician.

[0161] A dose may be any amount of pharmaceutical composition sufficient to achieve a desired biological or medical response. For example, a dose may correspond to between about 0.1 μg and about 1 μg of a crosslinked pair of hydrogel precursors or hydrogel, or between about 0.001 mg and about 0.01 mg, or between about 0.01 mg and about 0.1 mg, or between about 0.1 mg and about 1 mg, or between about 1 mg and about 3 mg, or between about 3 mg and about 10 mg, or between about 10 mg and about 30 mg, or between about 30 mg and about 100 mg, or between about 100 mg and about 300 g, or between about 300 mg and about 1,000 mg, or between 1 g and about 10 g of a crosslinked pair of hydrogel precursors or hydrogel.

[0162] 4. Kit In another aspect, the invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks, or bottles) containing one or more of the ingredients of the inventive pharmaceutical compositions described herein.

[0163] The various components of the pharmaceutical pack or kit may be supplied in solid (e.g., lyophilized) or liquid or semi-liquid form. Each component is generally suitable for delivery to its respective container or provided in concentrated form. A pack or kit according to the invention may also include a medium for reconstituting the lyophilized components. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

[0164] In certain embodiments, kits according to the invention comprise a first and second hydrogel precursor polymer of a crosslink pair as described herein, wherein the first and second hydrogel precursor polymers (either neat or individually formulated in solution) 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.

[0165] In certain embodiments, kits according to the present invention 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 medium and / or reagent include, but are not limited to, rehydration medium and / or reagents; antibiotics; biomolecules, biologically active agents as described herein, cell culture medium and / or reagents, cells, seeding means, harvest medium and / or reagents, filters, wash medium and / or reagents, etc.

[0166] In certain embodiments, the pack or kit includes one or more additional bioactive agents. Optionally, the container is provided with a notice or package insert in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting approval by the agency for manufacture, use, or sale for human administration. The package insert notice may contain instructions for use of the hydrogel precursor polymer pair or dynamic covalent hydrogel according to the methods of treatment disclosed herein.

[0167] An identifier, e.g., a bar code, radio frequency, ID tag, etc., may 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. [Example]

[0168] The following examples describe some preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the present invention. Furthermore, unless statements in the examples are presented in the past tense, as in the rest of the specification, such statements are not intended to imply that the experiments were actually performed or the data actually obtained.

[0169] Example 1 I. Synthesis and Polymer Modification - Synthesis of Aminated Wulff Type B 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 under argon was added 2-formylphenylboronic acid (1 eq; 2.66 mmol; 398 mg). After stirring at room temperature for 16 h, sodium borohydride (4.26 mmol; 161 mg) was slowly added to the yellowish 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. After removing 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 five times with dichloromethane (5 × 12 mL). The combined organic phases were dried over MgSO4 and concentrated in vacuo to give the expected compound as a white solid (0.76 g; yield: 93%).

[0170] 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 HCl gas. 1 The reaction was monitored by H NMR. After 30 min, the reaction mixture was concentrated in vacuo 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-evaporation with toluene. The solid residue was partitioned by the addition of dichloromethane. After drying over Na2SO4 and filtration, the organic phase was concentrated in vacuo to give the expected aminated Wulff-type phenylboronic acid derivative as a yellowish powder (427 mg; 72%).

[0171] Synthesis of Boronic Acid-Modified Polysaccharides. A typical synthesis was as follows: 500 kDa hyaluronic acid (HA) (100 mg) was dissolved in MES buffer, pH 5.5 (10 mL). DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) (69 mg; 1 eq) was added to the HA solution and reacted at room temperature for 30 minutes under stirring. Wulff-type phenylboronic acid (wPBA; 27.5 mg; 0.5 eq) was added to the activated HA solution and reacted at room temperature for 3 days under stirring. The solution was filter-sterilized and dialyzed against 1x PBS buffer (pH 7.4) for 1 day, 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: 1 The solubility of the boronic acid derivatives was determined by H NMR (400 MHz, DO, δ). A similar procedure was applied to graft various aminated boronic acid derivatives (i.e., 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, 5-aminobenzoxaborole) onto various polysaccharides (i.e., 20 / 100 / 500 kDa hyaluronic acid, carboxymethylcellulose, alginate), adjusting the equivalent weight based on the solubility of the reagents and the resulting modified polymer.

[0172] Synthesis of diol-modified polysaccharides. A typical synthesis was as follows: 500 kDa HA (100 mg) was dissolved in MES buffer, pH 5.5 (10 mL). DMT-MM (137 mg; 2 eq) was added to the HA solution and allowed to react at room temperature for 30 days under stirring. Glucamine (90 mg; 1 eq) was added to the activated HA solution and allowed to react at room temperature for 3 days under stirring. The solution was filter-sterilized and dialyzed against 1x 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 either by elemental analysis (nitrogen / carbon ratio) of the purified product using 2,4,6-trinitrobenzenesulfonic acid (TNBSA) titration or by quantification of unreacted aminated molecules in the crude mixture. A similar procedure was applied to graft various diol-containing aminated molecules (i.e., glucamine, isoserinol, glucosamine, galactosamine, fructosamine, dopamine, 1-amino-1-deoxy-D-galactitol, tris(hydroxyl-methyl)aminomethane) onto various polysaccharides (i.e., 20 / 100 / 500 kDa hyaluronic acid, carboxymethylcellulose, alginate), adjusting the equivalent amounts based on the solubility of the reagents and the resulting modified polymer.

[0173] Synthesis of Wulff-type boronic acid-modified PEG. Four-arm PEG-NH2 (MW = 2 kDa; 2.418 g) was dissolved in methanol (24 mL). 2-Formylarylboronic acid (943 mg; 1.3 eq) was added to the PEG solution and reacted overnight at room temperature under stirring. The solution was cooled on ice, and NaBH4 (274 mg; 1.5 eq) was added to it, followed by stirring at room temperature for 48 hours. 20 mL of deionized water was added, and liquid-liquid extraction was performed using dichloromethane (3 × 50 mL), after which the organic phase was dried over anhydrous Na2SO4. 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 as follows: 1 Determined by H NMR (400 MHz, D2O, δ).

[0174] Synthesis of Boronic Acid-Based Polysaccharide Hydrogels. A typical synthesis of a boronic acid hydrogel was as follows: In a 2 mL Eppendorf tube, 10 mg of HA-wPBA was dissolved in 1 mL of PBS for 1-2 hours at room temperature. Using a similar procedure, 10 mg of HA-glucamine was dissolved in 1 mL of PBS. In a 2 mL Eppendorf tube, the HA-wPBA and HA-glucamine solutions were quickly mixed together in a 1:1 volume ratio, and a pipettable dynamic hydrogel solution was used prior to gelation. A similar procedure was used to test various combinations of phenylboronic acid- and diol-modified polysaccharides. When necessary, a double-barrel syringe was used for convenience.

[0175] II. Physicochemical properties Swelling / Stability Test. Hydrogels were prepared as described above. Aliquots of hydrogel (3 x 100 μL) were transferred to pre-weighed 2 mL Eppendorf tubes and allowed to equilibrate at 37°C for 30 minutes. The gel-containing tubes were weighed, and 900 μL of warm PBS (37°C) was added to each. At the designated time points, the supernatant was removed, the gel surface was carefully dried with a Kimwipe, and the tubes were weighed. Swelling was determined as the ratio of the hydrogel mass at a given time point divided by its initial mass.

[0176] Rheological evaluation of dynamic hydrogels. Hydrogels were prepared as described above. Viscoelastic data were collected using a HAAKE MARS rheometer (Thermo Fisher 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. A solvent trap was used to minimize evaporation during measurements. To measure the shear storage modulus (G') and shear loss modulus (G") of the gels, frequency sweeps (0.01–10 Hz) were performed at 37°C under a constant shear stress of 1 Pa. Self-healing properties were evaluated by measuring G' and G" over time at 1 Hz and 7°C under alternating stresses of 1 Pa for 100 s (non-breaking stress) and 500 Pa for 50 s (breaking stress).

[0177] III. Biological properties Cytocompatibility of Hydrogel Components. Using L929 fibroblasts as a model cell line, HA-wPBA, PEG-wPBA, and HA-glucamine solutions were separately tested for cytocompatibility. Polymer solutions (1%) were prepared under sterile conditions using sterile polymer and cell culture medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). Cells were seeded at 15,000 cells / well (100 μL) in cell culture medium in standard polystyrene 96-well plates and incubated overnight (37°C, 5% CO2, 95% humidity). The medium was replaced with 100 μL of the 1% polymer solution before incubating the cells. After the indicated incubation times (0, 24, and 48 h), cells were tested for metabolic activity using a CCK-8 assay kit according to the supplier's protocol. The CCK-8 medium was then replaced with Tris / EDTA (TE) buffer, and the cells were frozen overnight (-80°C). Cell proliferation was then assessed using the Quant-iT™ PicoGreen™ Assay Kit for DNA quantification, according to the supplier's recommendations.

[0178] 3D cell viability assessment. 1% HA-wPBA and 2% HA-glucamine 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-glucamine solution was added to 8 × 10 6 Mix 4 x 10 cells / mL of adipose-derived multipotent stromal cells (MSCs) at a 1:1 ratio. 6 A 1% HA-glucamine solution containing 5 x 10 cells / mL was obtained. 0.5 mL of the 1% HA-wPBA solution was mixed with 0.5 mL of the 1% HA-glucamine / cell solution, and 100 μL of hydrogel (100K cells / well) was plated in a 96-well plate. The cell-containing hydrogel was incubated (37°C, 5% CO2, 95% humidity) on 150 μL of cell culture medium before analysis, and the medium was changed every other day. 2D controls were fed with 250 μL of cell culture medium every other day to culture 5 x 10 cells per well. 3 Cells were cultured at a density of 1000 cells / well. After the indicated incubation times (0, 1, and 7 days), cell viability was assessed by live / dead staining and confocal microscopy (Nikon A1) using Calcein AM (live cells, Sigma-Aldrich), ethidium homodimer (dead cells, Sigma-Aldrich), and Hoechst (all cell control, Invitrogen) according to the manufacturer's instructions. Average viability was obtained from three biological replicates.

[0179] IV. Results The results obtained are shown in FIGS.

[0180] As a proof-of-concept, we first demonstrated the successful synthesis of dynamic covalent hydrogels when Wulff-PBA-modified hyaluronic acid (HA-wPBA) was mixed with glucamine-modified hyaluronic acid (HA-glucamine). Using 1% (w / v) HA and a 1:2 wPBA:glucamine ratio, we observed by rheometry a frequency-dependent crossover of the storage modulus (G') and loss modulus (G"), a distinctive feature of dynamic covalent hydrogels (Figure 1).

[0181] The inventors further demonstrated the newly designed self-healing properties of hydrogels inherent to dynamic covalent polymer networks. When two separate qualitative HA-based dynamic hydrogels were brought into contact with each other, a single gel formed within minutes (Figure 2A). Time sweep measurements at a constant frequency of 1 Hz under low (1 Pa) and high (500 Pa) alternating stresses showed an effective recovery of their initial mechanical properties (G' > G") following repeated breakdown of the gel under high shear (G' < G"), further confirming self-healing (Figure 2B).

[0182] Next, the inventors examined the effect of their molecular weight and polymer content on the viscoelastic behavior of these HA-based hydrogels. By decreasing the molecular weight of the polymer precursor from 500 kDa to 100 kDa, it was possible to increase the polymer content from 1% to 3% (w / v), verifying the processable viscosity of the precursor solution and a wide range of crosslink densities for the designed hydrogels. This enabled the obtaining of dynamic covalent hydrogels with a shear storage modulus component (G') spanning several orders of magnitude (tens to thousands of Pa), reflecting the various viscoelastic behaviors and high tunability of the system (Figure 3).

[0183] Using the newly discovered crosslinking pair (i.e., wPBA and glucamine), the swelling and stability of HA-based dynamic covalent hydrogels were evaluated. By comparing 1% and 2% (w / v) polymer contents, the inventors showed that the optimal formulation of these new hydrogels results in minimal swelling and long-term stability (at least 1 month) despite the reversible crosslinking of the polymer network (Figure 4). This further enables the tuning of the swelling and stability properties of these dynamic hydrogels for various applications.

[0184] As a proof-of-concept for its versatility, we applied the new cross-linking pair to various polymers. Dynamic covalent hydrogels were successfully obtained when 1% (w / v) poly(ethylene glycol) (PEG)-wPBA was mixed with 1% (w / v) HA-glutamine at a 1:1 volume ratio (Figure 5A); 1% (w / v) alginate-wPBA was mixed with 1% alginate-glucamine at a 1:1 volume ratio (Figure 5B); and 1% (w / v) carboxymethylcellulose-wPBA was mixed with 1% (w / v) carboxymethylcellulose-glucamine at a 1:1 volume ratio (Figure 5C). Collectively, these results suggest that the new cross-linking pair can be applied to synthetic and natural polymers of various molecular weights, ionic strengths, and degradability properties.

[0185] Finally, we evaluated the cytocompatibility of the HA-HA and HA-PEG dynamic covalent gels by confocal microscopy to assess the survival (live / dead assay) of encapsulated adipose-derived multipotent stromal cells (A-MSCs) after 7 days of 3D culture. These results revealed excellent survival (>90%) of A-MSCs encapsulated in these gels.

[0186] Example 2 Example 2 reports data on the physicochemical and biological properties of various boronic acid-diol couples according to the present invention for dynamic hydrogel design.

[0187] I. Comparison of the rheological properties of various polymer-immobilized boronic acid-diol couples We sought to identify new boronic acid-diol couples with sufficiently high affinity to form dynamic covalent hydrogels when immobilized on polymers under physiological pH and temperature conditions.

[0188] Hyaluronic acid (HA) was chosen as the polymer backbone because it is often considered the polymer of choice for biomedical applications. Using DMT-MM as the activator (see Example 1), various aminated phenylboronic acid (PBA) derivatives were each immobilized onto HA via amidation. A series of diol-containing molecules were grafted onto HA following a similar procedure. To allow comparison of various boronic acid-diol couples, reaction conditions were adjusted to match the degree of substitution of various PBAs (≈20-25%) and diols (≈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 small amounts of DMSO to reach a degree of substitution of ≈20-25%; 3PBA-modified HA precipitated during synthesis. In contrast, Wulff-type phenylboronic acid (wPBA) could be immobilized under aqueous conditions (i.e., without the use of DMSO) up to a degree of substitution of at least 40% due to its positively charged nature.

[0189] All boronic acid-diol couples were then tested by mixing all PBA-modified and diol-modified HA components in pairs. The resulting rheological measurements (frequency sweeps; G', shear modulus) are reported in Figure 7. They revealed that a series of couples were capable of forming hydrogels under physiological conditions (e.g., HA-glucamine and HA-wPBA; HA-fructosamine and HA-wPBA; HA-dalcitramine and HA-wPBA; HA-glucamine and HA-2PBA; and HA-dalcitramine and HA-2PBA). In particular, the association of HA-glucamine and HA-wPBA resulted in the formation of viscoelastic materials with elasticity components one to several orders of magnitude higher than any of the other couples. Interestingly, the BX-modified polymer, which was reported to more readily support hydrogel formation in the presence of diols due to its chemical structure, did not result in any significant gelation properties.

[0190] II. Optimized formulation of HA-wPBA-HA-glucamine gel with improved physicochemical (viscoelastic) properties To tune the viscoelastic behavior of gels made with HA-wPBA and HA-glucamine, we screened a large panel of compositions that varied in the molecular weight of the polymeric hyaluronic acid (HA), the total concentration of HA, the degree of substitution of each of the two components (HA-wPBA and HA-glucamine), and the molar ratio of wPBA:glucamine.

[0191] Several compositions of interest were identified that exhibited specific viscoelastic behavior, as observed via rheological measurements (in frequency sweep tests, the G' / G" crossover of a dynamic gel is directly related to its relaxation time). Figure 8 shows three such specific optimized compositions: one with a total HA concentration of 1%, a HA molecular weight of 300 kDa, a 26% HA-wPBA substitution degree, and a 52% HA-glucamine substitution degree; one with a total HA concentration of 1%, a 200 kDa HA molecular weight, a 26% HA-wPBA substitution degree, and a 52% HA-glucamine substitution degree; and one with a total HA concentration of 3%, a 100 kDa HA molecular weight, a 40% HA-wPBA substitution degree, and a 52% HA-glucamine substitution degree. In each of the three specific compositions, the molar ratio of wPBA to glucamine is 1:1.

[0192] More importantly, while boronic acid-based hydrogels tend to shrink, we were able to design minimally to non-shrinking / non-swelling boronic acid-based hydrogels by carefully balancing the shrinking and swelling tendencies of the network of HA, a charged hydrophilic polymer (see Figure 9(B) and Figure 9(A), curves for PBS, culture medium 1, and culture medium 2). The optimal formulation was also stable for weeks to months under physiological pH and temperature conditions, which has not been reported for boronic acid-based hydrogels to date. Thanks to the dynamic characteristics of their networks, the new gels can be easily dissolved by simply adding competing free diol-containing molecules, such as glucose or glucamine (see Figure 9(A)). The HA-based gels can also be enzymatically degraded using hyaluronidase, making the dynamic covalent hydrogels of the present invention glycoresponsive and biodegradable (see Figure 9(A)).

[0193] III. Cytocompatibility of Boronic Ester Hydrogels To validate the use of the new gels for biomedical applications, the cytocompatibility of the cell-encapsulated boronate hydrogels of the present invention was evaluated using a model cell line (L929 murine fibroblasts). The gels used in the experiments reported in Figure 6 were HA-Wulff-PBA and HA-glucamine hydrogels, with a total concentration of 300 kDa HA polymer of 1% w / v, a boro:diol molar ratio of 1:1, and a degree of substitution of 26% for HA-Wulff-PBA and 52% for HA-glucamine. Cytocompatibility was assessed via cell viability (live / dead cell imaging), metabolic activity (CCK-8), and proliferation (PicoGreen) assays.

[0194] The results obtained are reported in Figure 10. After 2 days, the data collected showed high cell viability, accompanied by increased metabolic activity and cell number, which together indicate the excellent cytocompatibility of the test gel. Similar results were obtained with various other hydrogels according to the invention.

[0195] IV. Optimized Boronic Acid Hydrogels Are Printable Because our dynamic covalent hydrogels have the particularity of transiently flowing under shear (see Example 1), we envisioned the use of the new hydrogels for the development of innovative bioinks in the context of bioprinting. Preliminary data are shown in Figure 11. The following composition was used as a typical example: 200 kDa HA, total HA concentration = 1% w / v, degree of substitution of HA-Wulff-PBA: 26%, degree of substitution of HA-glucamine = 52%, molar ratio of Wulff-PBA:glucamine = 1:1. The obtained results show that, in conjunction with optimal printing conditions (e.g., head displacement, extrusion pressure), the optimized formulation of the new hydrogels can be easily printed with good shape fidelity.

[0196] V. Combination with other cross-linking mechanisms To expand the range of biomedical applications of the new hydrogel, we sought to demonstrate the feasibility of linking the discovered cross-linking mechanism with other chemical reactions, particularly "click" and bioorthogonal chemistries.

[0197] Preliminary data indicate that clickable moieties can be co-immobilized onto one of the two polymeric components of a hydrogel, allowing the physicochemical properties of the gel to be tuned in time and space (see Figure 12(A)). For example, co-crosslinking of boronic acid-based hydrogels can be used to stabilize and / or mechanically reinforce bioprinted constructs after printing. Strain-promoted azide-alkyne cycloaddition (SPAAC) between bicyclonyne (BCN) and azide (N3) was used as a model for the "click" reaction. A 200 kDa boronate gel (total HA concentration = 1%; Wulff-PBA substitution = 16%; glucamine substitution = 52%; Wulff-PBA:glucamine molar ratio = 1:1) was chemically modified with BCN (4% substitution as a co-substituent to HA-glucamine) to enable post-printing modification with azide-modified hyaluronan (100 kDa; HA-N3). Immersion of the bioprinted boronate gel in 0.5% HA-N3 containing PBS overnight increased the gel hardness from ≈600 Pa to ≈3000 Pa.

[0198] This strategy can be used to immobilize molecules of interest (e.g., peptides, growth factors, drugs, fluorophores), develop evolvable 3D culture systems with cell-material interactions tuned in time and space, and more.

Claims

1. (1) a first hydrogel precursor polymer comprising a first polymer modified with Wulff-type phenylboronic acid; and (2) a second hydrogel precursor polymer consisting of a second polymer modified with glucamine; A crosslinked pair of hydrogel precursor polymers comprising: the Wulff-type phenylboronic acid has the formula (II): 【Chemistry 1】 wherein R and R' are independently selected from hydrogen, substituted or unsubstituted C 1 -C 20 alkyl, substituted or unsubstituted C 1 -C 10 alkenyl, substituted or unsubstituted C 1 -C 10 alkynyl, acyl (-C(=O)R 1 , where R 1 is a substituted or unsubstituted C 1 -C 20 alkyl group), and carboxy (-C(=O)OR 1 , where R 1 is a substituted or unsubstituted C 1 -C 20 alkyl group); The substituted C 1 -C 20 alkyl, the substituted C 1 -C 10 alkenyl and the substituted C 1 -C 10 alkynyl groups may be selected from halogen (F, Br, I, Cl), hydroxy (—OH), amino (—NR 2 R 3 , R 2 and R 3 are independently selected from hydrogen and substituted or unsubstituted C 1 -C 20 alkyl), alkoxy (—OR 1 , R 1 is a substituted or unsubstituted C 1 -C 20 alkyl group), carboxy (—C(═O)OR 1 , R 1 is a substituted or unsubstituted C 1 -C 20 alkyl group), amido (—NR 2 C(═O)R 3 or —C(═O)NR 2 R 3 , R 2 and R 3 are independently selected from hydrogen and substituted or unsubstituted C 1 -C 20 alkyl), nitro (—NO 2 a crosslinked pair of hydrogel precursor polymers substituted with one or more substituents selected from aryl (=O), oxo (=O), and cyano (-CN).

2. 2. The crosslinked pair of hydrogel precursor polymers of claim 1, wherein the Wulff-type phenylboronic acid is 2-((dimethylamino)methyl)phenylboronic acid.

3. 2. The crosslinked pair of hydrogel precursor polymers of claim 1, wherein the Wulff-type phenylboronic acid is (2-(((4-aminobutyl)amino)methyl)phenyl)boronic acid.

4. 4. The crosslinked pair of hydrogel precursor polymers of any one of claims 1 to 3, wherein a first polymer is grafted with Wulff-type phenylboronic acid and a second polymer is grafted with glucamine.

5. 5. The crosslinked pair of hydrogel precursor polymers of any one of claims 1 to 4, wherein the first and second polymers are independently selected from natural polymers, semi-synthetic polymers, and synthetic polymers.

6. 6. The crosslinked pair of hydrogel precursor polymers of claim 5, wherein at least one of the first and second polymers is selected from biocompatible, biodegradable, hydrophilic natural polymers, semi-synthetic polymers, and synthetic polymers.

7. 7. The crosslinked pair of hydrogel precursor polymers of any one of claims 1 to 6, wherein the first hydrogel precursor polymer or the second hydrogel precursor polymer is directly or indirectly covalently bonded to a bioorthogonal functional moiety or a clickable moiety.

8. 8. The crosslinked pair of hydrogel precursor polymers of any one of claims 1 to 7, 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.

9. 9. The crosslinked pair of hydrogel precursor polymers of claim 8, wherein at least one of the first and second aqueous solutions comprises a component selected from the group consisting of cells, bioactive agents, visualization agents, and any combination thereof.

10. 7. A dynamic covalent hydrogel composed of a first hydrogel precursor polymer and a second hydrogel precursor polymer of a crosslinked pair according to any one of claims 1 to 6, wherein the first and second hydrogel precursors are crosslinked by a dynamic covalent bond.

11. 11. The dynamic covalent hydrogel of claim 10, further comprising a component selected from the group consisting of cells, bioactive agents, visualization agents, and any combination thereof.

12. 12. A pharmaceutical composition comprising a crosslinked pair of hydrogel precursor polymers according to any one of claims 1 to 9, or a dynamic covalent hydrogel according to claim 10 or claim 11, and at least one pharmaceutically acceptable carrier or excipient.

13. 13. The pharmaceutical composition of claim 12, wherein the first hydrogel precursor polymer and the second precursor hydrogel polymer are contained in a multi-barrel syringe.

14. 14. The pharmaceutical composition according to claim 13, wherein the multi-barrel syringe is a double-barrel syringe.

15. 14. A crosslinked pair of hydrogel precursor polymers according to any one of claims 1 to 9, or a dynamic covalent hydrogel according to claim 10 or claim 11, or a pharmaceutical composition according to claim 12 or 13, for use as a therapeutic agent in cell therapy, tissue engineering, regenerative medicine, viscosupplementation, delivery of cells and / or bioactive agents in vivo.

16. 14. A kit comprising a crosslinked pair of hydrogel precursor polymers according to any one of claims 1 to 9, or a dynamic covalent hydrogel according to claim 10 or claim 11, or a pharmaceutical composition according to claim 12 or 13, and instructions for use of the crosslinked pair, the dynamic covalent hydrogel or the pharmaceutical composition.

17. 10. A method for preparing a dynamic covalent hydrogel, comprising mixing a first hydrogel precursor polymer and a second hydrogel precursor polymer of a crosslinked pair according to any one of claims 1 to 9 to obtain a dynamic covalent hydrogel.

18. 18. The method of claim 17, carried out under physiological conditions.

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