Self-repairing hydrogel

Abstract

A hydrogel comprising guar gum, a (hetero) aryl diboronic acid, and an aqueous medium wherein the guar gum is crosslinked by the (hetero) aryl diboronic acid.

Description

Technical Field

[0001] The present invention relates to a hydrogel particularly suitable for 3D printing, especially in medical applications. The hydrogel can be used as an article, such as an implant, a tissue model and / or a drug delivery vehicle. Background Art

[0002] The lack of suitable donors for tissue or organ transplants prolongs suffering for many patients and can lead to death. Transplanting tissue or organs from one patient to another is also high risk due to the possibility of rejection and the side effects of immunosuppressive drugs. Transplants also generate long-term costs for the health system.

[0003] Three-dimensional (3D) bioprinting can be used to generate tissues for implantation. These tissues can be made using cells from the patient who will receive the tissue implant. This reduces the likelihood of rejection compared to transplantation and reduces the need for immunosuppressive drugs.

[0004] 3D bioprinting can also be used to provide in vitro 3D disease and / or tissue models with living human cells or tissues, thereby reducing the use of animals.

[0005] Materials used to produce engineered / artificial living tissue using 3D printing are called bioinks. Bioinks can encapsulate living cells, bioactive components, and / or growth factors during 3D bioprinting.

[0006] Tissues producible by bioprinting also find application in the field of tissue models. In this regard, it is important not to conduct safety and efficacy testing of compositions in animal models for ethical reasons. In fact, animal testing has been banned in the cosmetics industry. Therefore, providing tissue mimics for modeling diseases is highly desirable for research and development in various industries, including the cosmetics and pharmaceutical industries.

[0007] Additionally, it would be desirable to provide alternative vehicles for administering the therapeutic active ingredients.

[0008] An example of a widely used bio-ink is calcium alginate. However, bio-inks based on calcium alginate have variability between batches; reproducibility is poor. Bio-inks based on calcium alginate also suffer from the use of buffers containing calcium ions to achieve gelation, which can affect cell function and affect the activity of active agents present in the bio-ink. Summary of the invention

[0009] According to a first aspect, the present invention provides a hydrogel comprising guar gum, (hetero)aryl diboronic acid and an aqueous medium, wherein the guar gum is crosslinked by the (hetero)aryl diboronic acid. According to a second aspect, the present invention provides a composition comprising the hydrogel of the first aspect and an active agent. According to a third aspect, the present invention provides a pharmaceutical composition comprising the hydrogel of the first aspect or the composition of the second aspect.

[0010] It has surprisingly been found that the hydrogels of the claimed invention may be particularly suitable for forming predefined 3D shapes, especially by 3D printing, especially when the (hetero)aryldiboronic acid is 1,4-phenylenediboronic acid.

[0011] In addition, the concentration of the hydrogel can be changed to adjust its properties, especially its viscosity. A higher concentration will make the hydrogel more viscous. Therefore, by adjusting the concentration of the hydrogel, the hydrogel can be used for different applications, such as 3D printing or injection.

[0012] The combination of guar gum with (hetero)aryl diboronic acids, such as 1,4-phenylenediboronic acid, modifies the mechanical properties of guar gum and allows for rapid self-healing of the hydrogel.

[0013] Due to the combination of guar gum and (hetero)aryl diboronic acid, the hydrogels of the present invention contain dynamic borate crosslinks which are believed to be responsible for the self-healing ability of the hydrogels.

[0014] It has been determined that the hydrogels of the present invention can be used to enhance the mechanical properties of hydrogels made from proteins such as gelatin. For example, gelatin forms very weak hydrogels that tend to degrade at high temperatures. However, composite hydrogels that further include gelatin in addition to the materials required in the hydrogels of the present invention can have the benefits of gelatin hydrogels and enhanced mechanical properties.

[0015] It has been found that the hydrogels of the present invention form instantly upon 3D printing. Therefore, only a short delay is required before subsequent layers can be printed.

[0016] Furthermore, no additional steps need to be performed to solidify and bond the layers of the hydrogel together, such as by using temperature, photopolymerization, pH changes, or additional cross-linking agents (such as acrylamide) that may be toxic.

[0017] The hydrogel can also exhibit shear thinning (thixotropic) behavior, in which the viscosity of the hydrogel decreases with increasing shear rate. This indicates good injectability and thixotropy of the hydrogel.

[0018] Therefore, the specific components of the claimed hydrogel make the hydrogel particularly suitable for the manufacture of articles, such as by 3D printing, and for tissue engineering applications.

[0019] According to a fourth aspect, the present invention provides a method for manufacturing an article, comprising providing the hydrogel of the first aspect or the composition of the second aspect or the third aspect; and forming the hydrogel or the pharmaceutical composition into a three-dimensional (3D) shape.

[0020] According to a fifth aspect, the present invention provides an article comprising the hydrogel of the first aspect and / or the composition of the second aspect or the third aspect. The article may be an implant, a tissue scaffold, a disease and / or tissue model and / or a drug delivery vehicle. In a preferred embodiment, the article has been prepared by the method of the fourth aspect, in particular by 3D printing.

[0021] Hydrogels formed from polysaccharides and monoboric acid are not suitable for 3D printing because they do not provide suitable mechanical properties under essentially neutral conditions or the ability to self-heal quickly enough after printing to maintain their 3D structure.

[0022] Importantly, the use of other boronic acid-containing compounds in place of the (hetero)aryl diboronic acid required by the present invention cannot achieve the structural stability necessary for precise 3D printing in a short period of time.

[0023] In addition, borax cross-linked guar gum hydrogel has been reported (Sci. Rep. 2016, 6, No. 36497; ACS Biomater. Sci. Eng. 2018, 4, 3397-3404). However, this hydrogel is very brittle, has low flexibility, slow self-healing effect and poor low temperature resistance.

[0024] However, it has surprisingly been found that the storage modulus of the hydrogels of the present invention is about 10 times greater than that of comparable guar borate hydrogels.

[0025] By using a combination of (hetero)aryl diboronic acids and guar gum, the claimed hydrogel comprises chemically functionalized guar gum, containing borate groups, wherein double bond crosslinks are present both between different chains of guar gum and within the guar gum chains. This provides both increased mechanical stability and the ability to maintain the dynamic properties of the hydrogel, which makes the claimed hydrogel particularly suitable for the development of bio-inks.

[0026] US2010 / 0099586 A1 discusses a fracturing fluid composition and a method for fracturing a subterranean formation using a polyboron compound as a crosslinking agent. It discusses that the composition can be used to increase the viscosity of the fracturing fluid. Specifically, the composition and method allow for lower polymer loadings because a higher fracturing fluid viscosity can be achieved using less polymer than with conventional crosslinking systems.

[0027] US2010 / 0099586 A1 discusses very low guar gum concentrations of up to 25 ppt (0.3%) in fracturing fluids. The amount of polyboron compounds in the fracturing fluids is also very low, ranging from about 0.02% to about 0.5% by volume.

[0028] US 2010 / 0099586 A1 does not describe or provide any suggestion that the composition can be modified to prepare a hydrogel.

[0029] US2010 / 0099586 A1 does not provide any hint to explore the benefits that may be obtained at higher concentrations of guar gum and / or (hetero)aryl diboronic acid. 3D printing, medical injections, biopharmaceutical applications (e.g. controlled release drug delivery), self-healing or biocompatibility are not discussed in US2010 / 0099586 A1.

[0030] However, the claimed invention provides a hydrogel.The claimed invention can use higher amounts of the cross-linking composition (eg 3% w / v) and / or use specific ratios of guar gum to (hetero)aryl diboronic acid (eg 8:1).

[0031] The hydrogels of the present invention have been used to prepare highly stable articles, especially by 3D printing.

[0032] Further, the hydrogels of the present invention can be reliably and reproducibly manufactured on a large scale. The materials used to prepare the hydrogels of the present invention can be completely synthetic and free of animal-derived products. Therefore, the hydrogels of the present invention that comply with GLP and GMP can be easily prepared.

[0033] The hydrogels of the present invention may contain low concentrations of calcium ions or no calcium ions, thereby reducing the effects on cell function and on the activity of active agents present in the bio-ink compared to other hydrogels, and also increasing the biocompatibility of the claimed hydrogels.

[0034] The hydrogel of the present invention has been found to be about 7.4, making the hydrogel biocompatible and suitable for culturing living cells.

[0035] In addition, the claimed hydrogels and pharmaceutical compositions can also be used as drug delivery vehicles for administering one or more active agents, such as drugs, vitamins, or proteins, to subjects in need thereof. The claimed hydrogels and pharmaceutical compositions can provide controlled release of the active agent, for example, over an extended period of time or by adjusting the pH of the hydrogel (e.g., exposing the hydrogel to a more acidic environment).

[0036] Furthermore, and significantly, the hydrogels of the present invention have been demonstrated to support progressive cell growth during long-term cell culture.

[0037] Specifically, the hydrogel has been used to support human fibroblasts.

[0038] In contrast, the hydrogels of the prior art may lack biocompatibility. For example, Lu, X. et al., Colloids and Surfaces A: Physicochemical and Engineering Aspects, volume 561, 20 January 2019, pages 325-331 describe self-healing hydrogels containing hydroxypropyl guar gum, acrylamide and 3-acrylamide phenyl boronic acid. The document describes the use of acrylamide and 3-acrylamide phenyl boronic acid to form a copolymer of cross-linkable hydroxypropyl guar gum. However, acrylamide is carcinogenic and neurotoxic, and is therefore not suitable for use in bio-inks.

[0039] Further, the inventors of the present invention have also determined that the concentrations of the guar gum and (hetero)aryl diboronic acid components in the claimed hydrogels can be varied to control the viscosity and degradation rate of the hydrogels. As discussed in more detail by the examples, the hydrogels have been controlled to remain stable for up to about 50 days. Thus, the durability of the hydrogels can be modified to suit the desired application.

[0040] The claimed hydrogel may be made from a hydrogel precursor. Thus, according to a sixth aspect, the present invention provides a solid precursor of a hydrogel, wherein the precursor comprises guar gum crosslinked by a (hetero)aryl diboronic acid. Thus, guar gum and (hetero)aryl diboronic acid may be present in a solid unit. In other words, one solid unit may contain both guar gum and (hetero)aryl diboronic acid. The precursor may comprise a plurality of solid units. The solid unit may be, for example, a powder particle. The hydrogel of the first aspect may be made from the precursor of the sixth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0042] Figure 1Shows a diagram illustrating the cross-linking of guar gum (GG) by 1,4-benzenediboronic acid (BDBA) to form guar gum-1,4-benzenediboronic acid (GGBDBA) hydrogel;

[0043] Figure 2 A photograph of a GGBDBA hydrogel containing phosphate buffer is shown;

[0044] Figure 3 Shown are: a) SEM micrograph of GGBDBA hydrogel, and b) TEM micrograph of GGBDBA hydrogel;

[0045] FIG4 shows a graph illustrating the rheological properties of GGBDBA hydrogels at various concentrations;

[0046] FIG5 shows: a) a photograph of a GGBDBA hydrogel extruded from a needle, b) a photograph of a lattice pattern of a 2 wt % GGBDBA hydrogel, and c) a photograph of a lattice pattern of a 4 wt % GGBDBA hydrogel;

[0047] Figure 6 CAD images of the article are shown, as well as CAD images of the resulting 3D printed article formed from the GGBDBA hydrogel;

[0048] Figure 7 Shows photographs of tests used to demonstrate the self-healing properties of GGBDBA hydrogel;

[0049] Figure 8 shows a graph showing human fibroblast counts over time in the presence of GGBDBA hydrogel;

[0050] Fig. 9 Shown are: a) a graph showing the absorption of crystal violet by GGBDBA hydrogel over time, and b) an image showing crystal violet being absorbed by GGBDBA hydrogel;

[0051] Fig.10 Shows the encapsulation and release of drugs using GGBDBA hydrogel;

[0052] Fig.11 Photographs showing degradation of GGBDBA hydrogel in DMEM (low glucose) medium over up to 50 days are shown;

[0053] Fig.12 Photographs showing the degradation of GGBDBA hydrogel in medium over 21 days are shown;

[0054] Fig.13 showing a photograph of a composite hydrogel made of GGBDBA and gelatin; and

[0055] Fig.14 Shown is a graph showing the rheological properties of composite hydrogels made of GGBDBA and gelatin at various concentrations. DETAILED DESCRIPTION

[0056] The present invention provides self-repairing hydrogels. These self-repairing hydrogels can repair their structure and function after damage, similar to the repair of tissue in vivo. The hydrogels can also exhibit good shear-thinning behavior. Self-repairing hydrogels with good shear-thinning behavior are particularly useful in tissue engineering, 3D bioprinting and drug delivery applications.

[0057] The self-healing properties of the hydrogels are postulated to be due to electrostatic attractions within the hydrogels, which may affect the ability of the hydrogels to spontaneously create new bonds at damaged interfaces. This in turn depends on both guar gum and (hetero)aryl diboronic acid as required by the claimed invention. Thus, hydrogels containing dynamic chemical bonds and transient physical bonds generally exhibit self-healing properties.

[0058] Guar Gum

[0059] Guar gum (also known as guaran) is an edible, low-cost, and readily available polysaccharide. It is not animal-based, making it suitable for certain cultural requirements. Guar gum is a gel-forming galactomannan obtained by grinding the endosperm of guar beans (Cyamopsis tetragonolobus), a leguminous plant that has been grown primarily in India and Pakistan for centuries. Guar beans are an important crop that has long been used as food for humans and animals.

[0060] Guar is a plant cultivated primarily in certain tropical regions of the world. Guar is edible, and the gum extracted from it has a wide range of uses in industry, for example: in the textile industry, where the gum can be used to thicken dyes to allow for clear printing of patterns in carpets and textile prints; in food and beverages, where the gum is used as a thickener, stabilizer, suspending agent, and binder, and as a source of fiber; in medicine, where the gum is used as a laxative, and is used to treat diarrhea, irritable bowel syndrome, obesity, and diabetes; for lowering cholesterol; and for preventing atherosclerosis; in paper; in oils; and in cosmetics, where the gum is used as an emulsifier, stabilizer, and thickener in creams, lotions, and cosmetics. Guar gum is cheap, readily available, edible, and biocompatible.

[0061] It should be understood that guar gum is a polysaccharide composed of galactose and mannose. The backbone is a linear chain of β1,4-linked mannose residues to which almost every other mannose residue has a galactose residue linked 1,6- to form short side branches (although the exact substitution is random).

[0062] However, it has been recognized that it is difficult to control the viscosity or hydration of native guar gum, and that guar gum solutions may be unstable when left in solution for long periods of time. Therefore, there is a need to chemically modify guar gum and overcome these problems while also providing the specific benefits discussed in relation to the hydrogel itself when guar gum is combined with an aryl diboronic acid.

[0063] Mannose and galactose residues contain cis-1,2-diols. It is assumed that these groups can be combined with boronic acids (such as the aryl diboronic acids claimed in the claimed invention) to form boronates. This is shown in the accompanying drawings. Figure 1 Shown.

[0064] Therefore, the aryl diboronic acid used in the present invention is assumed to cross-link between the polysaccharide chains of guar gum.

[0065] The ability to form such crosslinks with diboric acid is believed to be a distinctive feature of guar gum that distinguishes the specific use of guar gum with diboric acid from other polysaccharides that do not contain cis-1,2-diol. This allows the hydrogels of the present invention to have the beneficial properties discussed herein.

[0066] Further, aqueous solutions of guar gum are believed to have a higher viscosity than any other natural polysaccharide.

[0067] It is assumed that the beneficial properties of the claimed hydrogels are due to this specific structure and functionality of guar gum compared to other polysaccharides.

[0068] The viscosity and hydration of the claimed hydrogels are not difficult to control, and the stability of the hydrogels does not pose a problem.

[0069] (Hetero)aryl diboronic acid

[0070] The hydrogel and the precursor comprise a (hetero)aryl diboronic acid. It should be understood that the term "(hetero)aryl" encompasses both aryl and heteroaryl groups. It should be understood that the term "diboronic acid" means that the (hetero)aryl group comprises two boronic acid groups bonded to the (hetero)aryl group.

[0071] The (hetero)aryl diboronic acid may contain 4 to 20 carbon atoms, such as 4 to 16 carbon atoms, or 4 to 10 carbon atoms. Preferably, the (hetero)aryl diboronic acid contains 4 to 8 carbon atoms, such as 4 to 6 carbon atoms, more preferably 5 to 8 carbon atoms, such as 5 or 6 carbon atoms. Most preferably, the (hetero)aryl diboronic acid contains 6 carbon atoms.

[0072] Preferably, the (hetero)aryl diboronic acid is an aryl diboronic acid. As used herein, the term "aryl" refers to a carbocyclic (i.e., heteroatom-free) aromatic group, including phenyl, naphthyl, indenyl and tetrahydronaphthyl groups. The aryl group can be monocyclic or polycyclic (e.g., bicyclic), as long as at least one ring is aromatic. The aryl group can be a five-membered or six-membered monocyclic ring, or a bicyclic structure formed by a fused five-membered ring and a six-membered ring or two fused six-membered rings. The aryl diboronic acid may contain 6 or 10 carbon atoms.

[0073] As used herein, the term "heteroaryl" refers to an aromatic ring system containing heteroatoms (e.g., N, O, S) in a ring structure. The heteroaryl group can be monocyclic or polycyclic (e.g., bicyclic), as long as at least one ring is aromatic. The heteroaryl group can be a five-membered or six-membered monocyclic ring, or a bicyclic structure formed by a fused five-membered ring and a six-membered ring or two fused six-membered rings. Each ring can contain up to five heteroatoms typically selected from N, S, and O. Typically, the heteroaryl ring will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more typically up to 2, such as a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom.

[0074] Examples of heteroaryl groups include the groups: pyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole, oxatriazole, isoxazole, thiazole, thiadiazole, isothiazole, pyrazole, triazole and tetrazole, pyridine, pyrazine, pyridazine, pyrimidine and triazine.

[0075] Examples of monocyclic groups are groups containing 4, 5, 6, 7 and 8 ring members, more typically 4 to 7 ring members, preferably 5, 6 or 7 ring members, more preferably 5 or 6 ring members. Examples of bicyclic groups are those containing 8, 9 and 10 ring members.

[0076] The (hetero)aryldiboronic acid may be substituted with one or more groups selected from the list consisting of cyano, halogen (e.g. F, Cl, Br and I), N3, -C(O)R Z 、-C(O)OR Z 、-OC(O)R Z 、-C(O)NHR Z 、-NHC(O)R Z 、-NHC(O)NHR Z 、-NHC(O)OR Z、-OC(O)NHR Z 、-OP(O)2OR Z 、-S(O)2NHR Z 、-NHS(O)2R Z 、-NR Z 2. -NHR Z AND-OR Z ; where each R Z independently represent H, C1-4 alkyl or C2-4 alkenyl. For example, the (hetero)aryl diboronic acid may be substituted by one or more groups selected from the list consisting of: cyano, halogen (e.g., F, Cl and Br), N3, -C(O)R Z 、-C(O)OR Z 、-OC(O)R Z 、-C(O)NHR Z 、-NHC(O)R Z 、-NHC(O)NHR Z 、-NHC(O)OR Z 、-OC(O)NHR Z AND-OR Z ; where each R Z and independently represent H, C1-4 alkyl or C2-4 alkenyl. Preferably, the (hetero)aryldiboronic acid is not substituted by any such groups.

[0077] Preferably, the (hetero)aryl diboronic acid is formed by a 6-membered (hetero)aromatic ring, for example wherein the (hetero)aryl group is benzene (i.e., phenyl), pyridine, pyrazine, pyrimidine, pyridazine or triazine. More preferably, the (hetero)aryl diboronic acid is phenyl diboronic acid, most preferably 1,4-phenyl diboronic acid:

[0078]

[0079] Hydrogel Precursor

[0080] The claimed invention defines a solid precursor of a hydrogel comprising both guar gum and a (hetero)aryldiboronic acid in a solid unit.

[0081] The hydrogel can be prepared via the precursor. The precursor can facilitate the production of the hydrogel.

[0082] The precursor can be prepared by a method comprising the following steps:

[0083] Providing a solution of guar gum and (hetero)aryl diboronic acid in an organic solvent;

[0084] adding an alkaline agent to the solution to form an alkaline solution; and

[0085] • Removing the organic solvent to provide the precursor.

[0086] In the precursor and / or hydrogel, guar gum and (hetero)aryl diboronic acid may be used in any suitable ratio. The exact ratio of guar gum to (hetero)aryl diboronic acid may be controlled to prepare a hydrogel with specific properties.

[0087] For example, the weight ratio of guar gum to (hetero)aryl diboronic acid may be 1:100 to 100:1, such as 1:100 to 50:1, or 1:100 to 20:1. The weight ratio of guar gum to (hetero)aryl diboronic acid may be 1:50 to 100:1, or 1:20 to 100: 1. Preferably, the weight ratio of guar gum to (hetero)aryl diboronic acid is 1:10 to 100:1, such as 1:5 to 100:1, or 1:2 to 100:1, such as 1:1 to 100:1.

[0088] More preferably, there is an amount of guar gum equal to or greater by weight than the amount of (hetero)aryl diboronic acid. For example, the weight ratio of guar gum to (hetero)aryl diboronic acid may be from 1:1 to 100:1, such as from 2:1 to 100:1 (e.g., from 2:1 to 40:1, or from 2:1 to 20:1), or from 4:1 to 100:1 (e.g., from 4:1 to 40:1, or from 4:1 to 20:1). Preferably, the weight ratio of guar gum to (hetero)aryl diboronic acid is greater than 5:1, such as from 6:1 or greater, or from 7:1 or greater, more preferably from 6:1 to 100:1, such as from 7:1 to 100:1, or from 8:1 to 100:1. The weight ratio of guar gum to (hetero)aryl diboronic acid may be 6: 1 to 60: 1, such as 6: 1 to 40: 1, or 6: 1 to 20: 1, such as 6: 1 to 15: 1, or 6: 1 to 10: 1. The weight ratio of guar gum to (hetero)aryl diboronic acid may be 8: 1 to 10: 1.

[0089] The organic solvent can be water-soluble. The organic solvent can be a protic solvent. The organic solvent can be an alcohol-containing solvent, such as methanol, ethanol, propanol or butanol. Relative to the total combined mass of guar gum and (hetero)aryl diboronic acid, the organic solvent can be provided in an amount of 0.1mL / g to 200mL / g, such as 0.1mL / g to 100mL / g or 0.1mL / g to 50mL / g. Relative to the total combined mass of guar gum and (hetero)aryl diboronic acid, the organic solvent can be provided in an amount of 0.1mL / g to 200mL / g, such as 1mL / g to 100mL / g or 10mL / g to 100mL / g.

[0090] Any suitable alkaline agent can be used to adjust the pH of the solution. The alkaline agent can be an aqueous solution of an alkali metal or alkaline earth metal hydroxide, a sulfate, a carbonate or a phosphate. In a preferred embodiment, the alkaline agent is an aqueous solution of an alkali metal or alkaline earth metal hydroxide. In a particularly preferred embodiment, the alkaline agent is an aqueous solution of sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, calcium hydroxide or strontium hydroxide; for example, it can be an aqueous solution of sodium hydroxide or potassium hydroxide. In a most preferred embodiment, the alkaline agent is an aqueous solution of sodium hydroxide. The concentration of the alkaline agent can be 0.001N to 19N, such as 0.01N to 10N, such as 0.01N to 5N or 0.02N to 4N, such as 0.05N to 2N, preferably 0.1N to 1N, such as 0.2N to 0.8N.

[0091] The pH of the alkaline solution may be between 7.5 and 14, such as between 8 and 13. Preferably, the pH of the alkaline composition is between 10 and 12, most preferably 11. Preferably, this pH is achieved due to the addition of the alkaline agent. The alkaline agent may be added to the solution in any suitable amount to make the pH of the solution alkaline, or one of the options discussed above. The pH may be determined by any method for determining pH, such as using a universal test paper, litmus (e.g., litmus paper), or a pH meter.

[0092] The alkaline solution can be heated before removing the solvent. Preferably, the alkaline solution can be heated at a temperature of 50°C or higher, such as 60°C or higher, or 70°C or higher, such as 50°C to 100°C or 70°C to 85°C. The time period that the heating step can be carried out is 30 minutes to 20 days, such as 1 hour to 10 days, or 1 hour to 6 days, such as 1 hour to 4 days. The time period is preferably 2 hours to 10 days, such as 6 hours to 6 days, or 12 hours to 6 days, such as 24 hours to 4 days. Preferably, the time period that the heating step is carried out at a temperature of 50°C or higher, such as 50°C to 100°C, is 30 minutes to 20 days, such as 6 hours to 6 days.

[0093] For example, the solvent may be removed in vacuo, or by evaporation at atmospheric pressure. Lyophilization or dehydration may be used to remove any residues of water present in the precursor.

[0094] The precursor may contain 10 wt % or less of organic solvent, such as 5 wt % or less, or 2 wt % or less. Preferably, the precursor contains 1 wt % or less of organic solvent, such as 0.5 wt % or less, or 0.1 wt % or less. The precursor may contain trace amounts of organic solvent, such as 0.0001 wt % or more, such as 0.0001 wt % to 10 wt %, or 0.0001 wt % to 1 wt %.

[0095] In this context, the term "solid" refers to the material phase of the precursor. The precursor may be in any solid form, for example as a powder, granules, flakes, agglomerates or blocks. Preferably, the precursor is a powder.

[0096] The precursor comprises both guar gum and (hetero)aryl diboronic acid in a solid unit. The solid unit may be a particle of a powder or flake, or a granule. It should be understood that this is different from a simple mixture of guar gum and (hetero)aryl diboronic acid, where each solid unit would contain either guar gum or (hetero)aryl diboronic acid, but not both components.

[0097] In the precursor, guar gum can be crosslinked by (hetero)aryldiboronic acids.

[0098] The hydrogel precursor may comprise an active agent, such as those discussed below, and / or may be a precursor to a (drug) composition.

[0099] Hydrogel

[0100] The hydrogel can be made from a hydrogel precursor. For example, the hydrogel can be made by a method comprising the following steps:

[0101] providing a hydrogel precursor as described above, and

[0102] • Contacting the hydrogel precursor with an aqueous medium.

[0103] Optionally, the hydrogel precursor is mixed with an aqueous medium.

[0104] The precursor may be added to an aqueous medium, or the aqueous medium may be added to the precursor. The precursor may be suspended and / or dispersed in the aqueous medium. Preferably, the precursor is mixed with the aqueous medium (e.g., stirred, vibrated and / or subjected to (super) sonication).

[0105] Once the precursors are contacted (eg, mixed) with the aqueous medium, the hydrogel typically forms almost immediately.

[0106] Organogels containing organic solvents are completely different from the hydrogels of the present invention. In addition, organogels are not suitable for biomedical applications due to their toxicity.

[0107] The aqueous medium comprises water. The aqueous medium can be water, such as deionized water and / or distilled water. It will be appreciated by those skilled in the art that a solution with a high concentration of salt can be used to precipitate protein, and that such a solution should not be used as an aqueous medium. Therefore, the aqueous medium can comprise 60% or more, such as 70% or more or 80% or more water by weight. Preferably, the aqueous medium comprises 90% or more, such as 95% or more, or 98% or more or 99% or more water by weight. The aqueous medium preferably does not substantially contain, or does not contain calcium ions. For example, the calcium ion concentration of the aqueous medium can be 10 mg / L or less, such as 1 mg / L or less or 0.1 mg / L or less. The aqueous medium (before contacting with the precursor) preferably does not substantially contain, or does not contain any organic solvent. By substantially free of, it means that the concentration of the substance in question of the solvent can be 0.05M or less, such as 0.01M or less, such as 5mM or less, or 1mM or less, such as 0.5mM or less, or 0.1mM or less. Substantially free may mean that the concentration of the substance is 0.05 mM or less, such as 0.01 mM or less, or 0.005 mM or less, such as 0.001 mM or less, or 0.01 μM or less.

[0108] The pH of the aqueous medium may be from 4 to 10, such as from 5 to 9, preferably from 6 to 8, such as from 7 to 8. The pH of the aqueous medium may be particularly suitable for biocompatibility.

[0109] Precursor can be suitably contacted with any amount of aqueous medium and precursor, for example, 10% or more by weight relative to the weight of the precursor, for example, 50% or more or 100% or more aqueous medium contact. Preferably, 200% or more by weight relative to the weight of the precursor, for example, 350% or more or 500% or more aqueous medium contact with the precursor. More preferably, 750% or more by weight relative to the weight of the precursor, for example, 1000% or more or 1500% or more, for example, 1800% aqueous medium contact with the precursor. The amount of the aqueous medium contacted with the precursor can be 100000% or less by weight relative to the weight of the precursor, for example, 50000% or less or 10000% or less. The amount of the aqueous medium contacted with the precursor can be 10% to 100000%, for example, 400% to 50000% by weight relative to the weight of the precursor.

[0110] The hydrogel may be separated from the excess aqueous medium. Separation of the hydrogel from the aqueous medium may be performed using techniques well known to those skilled in the art, such as filtration or centrifugation.

[0111] Hydrogels can be described as pasty masses, elastic solids, and / or gels.

[0112] It will be appreciated that the relative amounts (ratios) of guar gum to (hetero)aryldiboronic acid in the precursor (as described above) also apply to the hydrogel itself.

[0113] The hydrogel may contain any suitable amount of guar gum and (hetero)aryl diboronic acid. The inventors have determined that the concentrations of the guar gum and (hetero)aryl diboronic acid components of the claimed hydrogel can be varied to control the viscosity and degradation rate of the hydrogel. The hydrogel may typically contain a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1% or more, or 0.2% or more, such as 0.4% or more, preferably 0.5% or more, or 0.7% or more, or 0.8% or more, such as 1.0% or more, or 1.5% or more, or 2.0% or more, or 2.5% or more, and / or 50% or less, such as 20% or less, preferably 10% or less, such as 8% or less, or 6% or less by weight. The hydrogel may typically contain a combination of guar gum and (hetero)aryldiboronic acid (dry) in an amount of from 0.1% to 50%, such as from 0.5% to 10% by weight.

[0114] A hydrogel containing guar gum and (hetero)aryl diboronic acid at a concentration lower than 1.5 wt % (e.g., 1 wt % or lower) has a relatively low viscosity that allows free-flowing liquid behavior. Such a hydrogel may have a specific use as an injectable material and / or a drug delivery vehicle. However, 1.5 wt % and higher, such as 2 wt % and higher concentrations, will provide a more viscous / solid hydrogel that is not free-flowing. Such a hydrogel may have a specific use as a bio-ink, such as a tissue (e.g., disease) model and / or implant. Therefore, the viscosity of the hydrogel can be modified to adapt to the desired application.

[0115] Furthermore, a hydrogel containing guar gum and (hetero)aryl diboronic acid at a concentration of 6 wt% remained stable for about 50 days. However, reducing the concentration to 4 wt% kept the hydrogel stable for about 12 days. Thus, the durability of the hydrogel can be tailored to suit the desired application.

[0116] The hydrogel may contain a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1% or more by weight, such as 0.3% or more or 0.6% or more. Preferably, the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.8% or more by weight, or 1.0% or more, such as 1.4% or more. The hydrogel may contain this combination in an amount of 5% by weight or less, such as 4% by weight or less, or 3% by weight or less, preferably 2% by weight or less. For example, the hydrogel may contain a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1% to 5%, such as 0.6% to 3%, or 1% to 2% by weight.

[0117] The hydrogel may contain a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 2% or more by weight, such as 2.5% or more, or 3% or more, or 3.5% or more, such as 4% or more by weight. The hydrogel may contain a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 50% or less by weight, such as 20% or less, preferably 10% or less, such as 8% or less by weight, or 6% or less. For example, the hydrogel may contain a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 2% to 50% by weight, such as 2% to 10% by weight, or 2% to 6% by weight.

[0118] The aqueous medium may include a buffer, such as a phosphate-based buffer. Preferably, the buffer is a KH2PO4 and K2HPO4 buffer (i.e., a potassium phosphate buffer). The concentration of the buffer (before being added to the aqueous medium) may be 0.01M to 1.0M, or 0.05M to 0.5M, preferably 0.08M to 0.12M, such as about 0.1M.

[0119] The aqueous medium should be suitable for the survival of any cells with which it is contacted. It should be understood that the aqueous medium should not be toxic to such cells.

[0120] In one embodiment, the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1% or more, and the weight ratio of guar gum to (hetero)aryl diboronic acid is from 1:100 to 100:1. In one embodiment, the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.4% or more (e.g., 0.5% or more) by weight, and the weight ratio of guar gum to (hetero)aryl diboronic acid is from 1:100 to 100:1. In one embodiment, the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.4% or more (e.g., 0.5% or more) by weight, and the weight ratio of guar gum to (hetero)aryl diboronic acid is from 1:100 to 60:1, such as from 2:1 to 40:1. In one embodiment, the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1% to 50% by weight, and the weight ratio of guar gum to (hetero)aryl diboronic acid is 1:1 to 60:1.

[0121] Preferably, the (hetero)aryl diboronic acid is 1,4-phenylenediboronic acid, the weight ratio of guar gum to 1,4-phenylenediboronic acid is 1:1 to 60:1, and the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1% to 50% by weight.

[0122] Prior to use (e.g. for 3D printing), the hydrogel may be allowed to stand for a certain period of time to gel. The period of time may be 30 seconds or longer, or 2 minutes or longer, or 4 minutes or longer, or 6 minutes or longer, or 10 minutes or longer. The period of time may be 30 minutes or longer, such as 1 hour or longer, particularly in the case where the hydrogel contains a lower amount of (hetero)bisboronic acid. The period of time may be from 30 seconds to 48 hours, such as from 2 minutes to 6 hours.

[0123] The hydrogel may include proteins, such as gelatin. The resulting hydrogel may be referred to as a composite hydrogel. The hydrogel may include proteins, such as gelatin, in an amount of 0.1% by weight or more, such as 0.5% by weight or more, or 1% by weight or more, or 2% by weight or more, or 3% by weight or more, such as 3.5% by weight or more, or 4% by weight or more. The hydrogel may include proteins, such as gelatin, in an amount of 20% by weight or less, such as 10% by weight or less, or 8% by weight or less, or 6% by weight or less, such as 5% by weight or less. The hydrogel may include gelatin in an amount of 0.1% by weight to 20% by weight, such as 1% by weight to 10% by weight, or 2% by weight to 6% by weight. The hydrogel may comprise gelatin in an amount of 0.1 wt% or more and a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1 wt% or more, for example, the hydrogel may comprise gelatin in an amount of 0.1 to 20 wt% and a combination of guar gum and (hetero)aryl diboronic acid (dry) in an amount of 0.1 to 50 wt% (preferably 2 to 10 wt%, for example 2 to 6 wt%).

[0124] Composition

[0125] The present invention provides a composition comprising the hydrogel and an active agent. The present invention also provides a pharmaceutical composition comprising the hydrogel or the composition thereof.

[0126] The GGBDBA hydrogel can encapsulate active agents, such as drugs (medicaments), vitamins and / or proteins. The active agent can be dispersed in the hydrogel, such as dissolved or suspended in the hydrogel.

[0127] The hydrogel has wide applicability as a delivery vehicle because the physical presence of the hydrogel around the active agent can deliver the agent to, for example, an active site or tissue. Therefore, there is no particular restriction on the nature of the agent. The active agent can be easily encapsulated in the cross-linked fiber network of the hydrogel.

[0128] Furthermore, the examples show that the concentrations of guar gum and (hetero)aryl diboronic acid (dry) can be varied to control the degradation rate of the hydrogel. Thus, hydrogels containing such active agents can be modified release (eg, controlled release) vehicles.

[0129] The active agent may be selected from the group consisting of therapeutic agents, preventive agents, diagnostic agents and nutritional health agents, and the active agent is composed of a compound selected from the group consisting of small molecule active agents, proteins, polypeptides, polysaccharides and nucleic acids. The active agent may be selected from the group consisting of antibiotics, antivirals, antiparasitics, cytokines, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and biologically active fragments thereof, antigens and vaccine preparations, anti-inflammatory agents, immunomodulators and oligonucleotide drugs, paramagnetic molecules, fluorescent compounds, magnetic molecules and radionuclides, X-ray imaging agents and contrast agents.

[0130] The active agent may be an anti-cancer drug, such as doxorubicin (widely used in the treatment of breast, uterine, ovarian, lung and cervical cancer); or a phosphodiesterase inhibitor; or a drug used to treat COPD and / or asthma, such as theophylline; or a drug used to treat angina and / or hypertension; or a calcium channel blocker, such as nifedipine; or a drug used to treat epilepsy; or a sodium channel blocker, such as carbamazepine; or a drug used to treat diabetes, such as insulin; or a drug used to treat hyperparathyroidism, such as a vitamin D compound, such as calcifediol.

[0131] In one embodiment, the composition is a pharmaceutical composition, ie, the composition is pharmaceutically acceptable. The hydrogel may be non-toxic, eg, non-carcinogenic, and / or non-neurotoxic.

[0132] Preferably, the hydrogel is suitable for administration to a subject, such as an animal, such as a mammal or a human.

[0133] The hydrogels may be used in medical treatment methods comprising administering a pharmaceutical agent to a subject.

[0134] The pharmaceutical composition can be intended for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration or using a bioerodible / biodegradable insert.

[0135] In some embodiments, the composition is administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective to deliver the composition to target cells. Other possible routes include transdermal or oral administration.

[0136] The composition can be applied topically, for example, by direct injection to the position to be treated. The composition can be injected or otherwise directly applied to one or more tumors. Usually, local injection causes the local concentration of the composition to increase, and the local concentration is higher than the concentration achievable by systemic administration. The composition can be applied topically to appropriate cells by using a catheter or a syringe. Other means of locally delivering the composition to the cell include using an infusion pump or incorporating the composition into a polymer implant, which can affect the sustained release of the active agent to the immediate release region (immediatearea) of the implant.

[0137] The composition can be provided to the cell directly, such as by contacting the composition with the cell, or indirectly, such as by the action of any biological process. For example, the composition can be formulated in a physiologically acceptable carrier or vehicle and injected into the tissue or fluid surrounding the cell. The composition can cross the cell membrane by simple diffusion, endocytosis, or by any active or passive transport mechanism.

[0138] The selected dosage depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment. Typically, a dosage level of 0.001 to 10 mg / kg body weight is administered to a mammal daily. Generally, for intravenous injection or infusion, the dosage may be lower than that used for other modes of administration.

[0139] In a preferred embodiment, the composition is administered by parenteral injection. This is particularly because the hydrogel can exhibit shear thinning behavior, wherein the viscosity of the hydrogel decreases with increasing shear rate. This indicates good injectability and thixotropy of the hydrogel.

[0140] The composition may be in the form of a suspension or emulsion. Typically, pharmaceutical compositions are provided comprising an effective amount of one or more active agents, optionally including a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. Such compositions may include diluent sterile water, buffered saline with various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optional additives such as detergents and solubilizers (e.g., 20. 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite) and preservatives (e.g., thimerosal (Thimersol), benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation can be lyophilized and redissolved / resuspended just before use. The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0141] The composition can be applied topically. Topical application can include application to the lungs, nasal, oral (sublingual, buccal), vaginal or rectal mucosa. These methods of administration can be made effective by formulating the housing with a transdermal or mucosal transport element. For transdermal delivery, such elements can include chemical enhancers or physical enhancers, such as electroporation or microneedle delivery.

[0142] Transdermal formulations can also be prepared. These transdermal formulations are typically ointments, lotions, sprays or patches, all of which can be prepared using standard techniques. Transdermal formulations can contain penetration enhancers. Chemical enhancers and physical methods (including electroporation and microneedles) can work in conjunction with this method.

[0143] Compositions loaded with one or more active agents for delivering the one or more active agents to cells or the microenvironment of cells are generally used in treatment. The use of the composition generally includes contacting the composition loaded with the active agent with one or more cells. The contact can occur in vivo or in vitro. Therefore, it can be administered to a subject.

[0144] The composition can release the active agent when the pH changes, for example by raising or lowering the pH of the composition. Preferably, the pH is raised to release the active agent. The pH increase can occur by applying the hydrogel containing the active agent to the acidic conditions of the stomach, urine and / or vagina. The pH of the hydrogel environment can be controlled to 7.0 or lower, for example 6.5 or lower, or 6.0 or lower, for example 5.0 or lower, for example 7.0 to 0.0 or 6.0 to 2.0.

[0145] Implants and disease and / or tissue models

[0146] The hydrogels and products thereof of the present invention can be used as implants and / or tissue (eg, disease) models.

[0147] Implants or tissue (eg disease) models may be designed to support cell or tissue growth, such as in the development and / or repair of tissue, or to provide an analog of natural tissue. The tissue model may simulate diseased tissue.

[0148] Therefore, hydrogel and its products can be impregnated with cells and / or tissues. Cells and tissues can be selected from the following list: cardiac tissue, vascular implants, skin grafts, skeletal tissues, such as bone, cartilage, tendons, intervertebral discs (disc), related bones, cartilage precursor cells and bone grafts. Cells suitable for use in the hydrogel of the present invention and its products can be selected from the following group: embryonic stem cells, adult stem cells, osteoblasts, preosteoblasts, chondrocytes, nucleus pulposus cells, prechondrocytes, skeletal progenitor cells (including stem cells) derived from bone, bone marrow or blood, and combinations thereof. Relative to the recipient of the implant formed by the cell culture and tissue engineering functions of the present invention, the cell or tissue can be autologous, allogeneic or xenogeneic sources. It should also be understood that the term tissue as used herein includes connective tissue, cardiac tissue, vascular implants, skin grafts and bone grafts. The hydrogel and its products can provide an environment for at least one selected from the group consisting of: storage of tissue biopsies, digestion of tissue biopsies, cell sorting, cell washing, cell concentration, cell seeding, cell proliferation, cell differentiation, cell storage, cell transport, tissue formation, implant formation, storage of implantable tissues, and transport of implantable tissues.

[0149] The tissue (e.g., disease) model can be selected from the list consisting of: cardiac tissue, vascular tissue, skin tissue, and skeletal tissue (e.g., bone, cartilage, tendon, and intervertebral disc). The model can be used in methods including storage of tissue biopsies, digestion of tissue biopsies, cell sorting, cell washing, cell concentration, cell seeding, cell proliferation, cell differentiation, cell storage, cell transport, tissue formation, implant formation, storage of implantable tissues, and transport of implantable tissues.

[0150] Tissue (e.g., disease) models can be used in methods of determining the safety of a composition, wherein the method comprises exposing a model tissue to the composition and analyzing the tissue (e.g., visually) at a certain time point after the exposure and / or within a certain period of time after the exposure.

[0151] The implant may be selected from the list consisting of: cardiac tissue, vascular implants, skin grafts, skeletal tissue such as bone, cartilage, tendon, disc, associated bone, cartilage precursor cells and bone grafts.

[0152] Manufacturing of products

[0153] The hydrogel and / or pharmaceutical composition of the present invention can be used to prepare products. The products can include the above-mentioned tissue (eg, disease) models and implants.

[0154] It has been recognized that hydrogels exhibit shear thinning properties, allowing hydrogels to be injected and also formed into solid articles. In addition, hydrogels also exhibit self-healing properties, allowing layers of hydrogels to adhere to each other without the need for additional heat and / or adhesives.

[0155] A fourth aspect of the present invention provides a method for manufacturing an article, wherein the method comprises:

[0156] Providing the hydrogel of the first aspect or the composition of the second or third aspect; and

[0157] - forming the hydrogel or pharmaceutical composition into a three-dimensional (3D) shape, for example by 3D printing, CNC machining, casting, rotational molding, vacuum forming, injection molding, extrusion and / or blow molding the hydrogel to form the article.

[0158] Preferably, the hydrogel is formed using 3D printing.

[0159] In one embodiment, the method comprises forming a 3D shape into or onto a substrate and, after forming the 3D shape, peeling the article from the substrate.

[0160] This disclosure provides the subject matter of the following clauses:

[0161] 1. A hydrogel comprising guar gum, (hetero)aryl diboronic acid and an aqueous medium, wherein the guar gum is cross-linked by the (hetero)aryl diboronic acid.

[0162] 2. The hydrogel according to clause 1, wherein the (hetero)aryldiboronic acid comprises 4 to 10 carbon atoms.

[0163] 3. The hydrogel according to any one of clause 1 or clause 2, wherein the (hetero)aryldiboronic acid is 1,4-phenylenediboronic acid.

[0164] 4. The hydrogel according to any one of clauses 1 to 3, wherein the weight ratio of guar gum to (hetero)aryldiboronic acid is from 1:1 to 100:1.

[0165] 5. The hydrogel according to any one of clauses 1 to 4, wherein the hydrogel contains a combination of guar gum and a (hetero)aryldiboronic acid in an amount of 0.5% to 10% by weight.

[0166] 6. The hydrogel according to any one of clauses 1 to 5, wherein the aqueous medium is a phosphate-based buffer.

[0167] 7. The hydrogel according to any one of clauses 1 to 6, wherein the hydrogel further comprises gelatin.

[0168] 8. The hydrogel according to clause 7, wherein the hydrogel comprises gelatin in an amount of 2% to 10% by weight.

[0169] 9. A composition comprising the hydrogel of any one of clauses 1 to 8 and an active agent.

[0170] 10. The composition according to clause 9, wherein the composition is a pharmaceutical composition.

[0171] 11. A composition according to clause 9 or clause 10, wherein the active agent is a therapeutic agent, a prophylactic agent, a diagnostic agent and / or a nutritional agent, and the active agent comprises a compound selected from the group consisting of: small molecule active agents, proteins, polypeptides, polysaccharides and nucleic acids.

[0172] 12. A composition according to any one of clauses 9 to 11, wherein the active agent is selected from the group consisting of antibiotics, antivirals, antiparasitics, cytokines, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and biologically active fragments thereof, antigens and vaccine preparations, anti-inflammatory agents, immunomodulators and oligonucleotide drugs, paramagnetic molecules, fluorescent compounds, magnetic molecules and radionuclides, X-ray imaging agents and contrast agents.

[0173] 13. A method of manufacturing an article, wherein the method comprises:

[0174] • Providing a hydrogel according to any one of clauses 1 to 8 or a composition according to any one of clauses 9 to 12; and

[0175] • Forming the hydrogel or pharmaceutical composition into a three-dimensional (3D) shape.

[0176] 14. The method of clause 13, wherein the article is formed by 3D printing.

[0177] 15. An article comprising the hydrogel according to any one of clauses 1 to 8 or the composition according to any one of clauses 9 to 12.

[0178] 16. An article according to clause 15, wherein the article is manufactured by a method as defined by clause 13 or clause 14.

[0179] 17. The article according to clause 16, wherein the article is a tissue model or an implant.

[0180] 18. The article according to clause 17, wherein the tissue model is selected from the list consisting of: cardiac tissue, vascular tissue, skin tissue, and skeletal tissue, such as bone, cartilage, tendon, and intervertebral disc.

[0181] 19. The article according to clause 17, wherein the implant is selected from the list consisting of: cardiac tissue, vascular implants, skin grafts, skeletal tissue such as bone, cartilage, tendon, intervertebral disc, associated bone, cartilage precursor cells and bone grafts.

[0182] 20. A solid precursor of a hydrogel, wherein the precursor comprises guar gum cross-linked by (hetero)aryldiboronic acid.

[0183] Example

[0184] Guar gum, sodium hydroxide, 1,4-phenylenediboronic acid, and doxorubicin hydrochloride were all commercially available. Sodium phosphate buffer (PB) solution (0.1 M, pH 7.46) was prepared using Milli Q water.

[0185] The percentages given with respect to any hydrogel refer to the percentage of solids content (ie, guar gum and 1,4-phenylenediboronic acid) by weight relative to the total weight of the hydrogel.

[0186] Synthesis of GGBDBA hydrogel precursor

[0187] Guar gum (GG "Extra Pure", purchased from Sisco Research Laboratories Pvt Ltd, 1.6 g) and 1,4-phenylenediboronic acid (BDBA, 0.2 g) were mixed (weight ratio 8:1) and dissolved in ethanol (30 mL, approximately 20 ml / g, anhydrous). NaOH (0.5 N) was added dropwise to the mixture until the pH of the mixture became alkaline (pH about 11). The resulting mixture was heated under reflux (80° C.) for 48 h. After 48 h, the solvent was removed in vacuo and the light green residue / light yellow powder was dried in a desiccator (2 days) to provide a dry cross-linked powder that was ready for hydrogel preparation. The powder was optionally washed five times with ethanol to remove impurities. Ethanol (10 mL) was poured into the solid residue each time and the resulting suspension was scraped and sonicated for 10-15 minutes; the resulting suspension was allowed to settle and the supernatant was discarded. After washing, an off-white solid residue was obtained which was dried in vacuo and stored in a desiccator.

[0188] Synthesis of GGBDBA hydrogel

[0189] The dried cross-linked powder was used for hydrogelation. To prepare GGBDBA hydrogels of different concentrations (1% w / v, 2% w / v, 3% w / v, 4% w / v, 5% w / v, 6% w / v), an appropriate amount of the synthetic powder (e.g., 30 mg for 3% w / v) was first placed in a 5 mL glass vial. The required volume of phosphate buffer (pH 7.4; 0.1 M, 1 mL for 3% w / v example) was then added to the vial, and the solution was sonicated for 10-15 minutes and / or mixed using a vortex mixer for 5 minutes. At all mentioned concentrations, self-supporting hydrogels were immediately formed. The hydrogels were stored at 20°C for 6 h before conducting other experiments. Figure 2 The prepared hydrogel samples of different concentrations are shown. The samples are tilted or inverted to show their viscosity.

[0190] Tetrahydroxydiboron hydrogel (comparison)

[0191] In a comparative study, the same procedure was followed using guar gum and another diboric acid (tetrahydroxydiboron). However, tetrahydroxydiboron cross-linked guar gum failed to form a hydrogel under the same phosphate buffer conditions.

[0192] Gel time

[0193] The hydrogels of the claimed invention were synthesized using different ratios of guar gum and 1,4-phenylenediboronic acid as shown in the table below. The gelling time of these hydrogels was determined by mixing the precursors with phosphate buffer in a test tube, inverting the tube, and determining the time required for the mixture to reach a gel-like consistency.

[0194] The results are shown in the table below.

[0195] GG:BDBA ratio (w / w) Gel time 4:1 72h 5:1 48h 8:1 4-5min 10:1 4-5min

[0196] It can be seen that a ratio of guar gum to diboric acid greater than 5:1 provides a short gel time of 4-5 minutes. Lower guar gum ratios provide longer gel times, which may be due to the lower relative crosslinking due to the lower content of diboric acid.

[0197] Other experiments described herein were all conducted with hydrogels containing 8:1 by weight of GG:BDBA. It should be understood that the hydrogels need to be gelled before being used in 3D printing. Once the hydrogels have formed gels, any of the hydrogels tested are suitable for use in applications such as 3D printing.

[0198] FT-IR spectroscopy

[0199] FT-IR spectra of lyophilized GG-BDBA were recorded using a Bruker Tensor 27 spectrophotometer. These solid-state experiments were performed using a KBr pellet preparation technique with a scan range of 600 cm -1 With 4000cm -1 64 scans were performed with a resolution of 4 cm -1 , and the interval is 1cm -1 .

[0200] FT-IR spectroscopy showed the formation of boronate groups.

[0201] Morphological studies (electron microscopy)

[0202] Transmission electron microscopy (TEM) measurements were performed to evaluate the self-assembled morphology of the hydrogels. TEM images were taken using a transmission electron microscope (model: Jeol 100keV system) at an accelerating voltage of 300kV. The diluted hydrogel aqueous solution (5% V / V) was dried on a carbon-coated copper grid (300 mesh) by slow evaporation in air and then dried under reduced pressure at room temperature alone. Phosphotungstic acid (0.2%, w / v) was used as a negative stain.

[0203] Field emission gun scanning electron microscopy studies were performed by using a Carl Zeiss scanning electron microscope (FE-SEM Supra 55 Zeiss). The diluted hydrogel samples were dried on glass cover slips and coated with gold for SEM analysis with a working voltage of 5 kV.

[0204] SEM and TEM images are shown in the accompanying drawings. Figure 3 middle.

[0205] SEM image of GGBDBA ( Figure 3 a) shows distinct flaky and elongated surface morphology.

[0206] TEM image of GGBDBA ( Figure 3 b) shows the fiber network.

[0207] Rheological testing

[0208] Rheological analysis was performed using an Anton Paar Physica MCR 301 rheometer at 25 °C. The viscoelastic properties of the hydrogels were measured by measuring the storage modulus (G') and the loss modulus (G'). Freshly prepared hydrogels were placed on the rheometer plate and kept hydrated using a solvent trap. Parallel plates with a diameter of 25 mm were used with a Trugap of 1 mm. Dynamic strain sweep experiments were performed to determine the region of hydrogel deformation where the linear viscoelasticity was effective. The exact strain of the hydrogel was determined by measuring the linear viscoelastic regime at 10 rad s -1 To evaluate the mechanical strength of G-quadruplex hydrogels, the strain was 0.5% and the frequency was 1-600 rad s -1 The frequency sweep experiment was carried out in the frequency range of . The shear rate (0.1-1000s -1 ) to study the shear thinning behavior of the hydrogel.

[0209] The shear thinning behavior of the hydrogel was observed ( Figure 4a ), wherein the viscosity of the hydrogel decreases with increasing shear rate. This indicates the good injectability and thixotropy of the hydrogel.

[0210] Figure 4b An oscillatory strain sweep experiment at a constant frequency is shown in . These results show that the GGBDBA hydrogel maintains its viscoelastic properties at high strains up to 200%.

[0211] Figure 4c The dynamic frequency sweep experiment shown depicts that, when examined as a function of frequency, the storage modulus (G') remains greater than its loss modulus (G"), thereby exhibiting solid-like rheology.

[0212] Furthermore, it was found that the storage modulus of GGBDBA hydrogel was about 10 times that of comparable guar gum borate hydrogel (J. Agric. Food Chem. 2019, 67, 2, 746-752).

[0213] These properties are important for hydrogel applications in the form of materials injectable via syringes as well as extrusion-based 3D printing and bioprinting processes, and suggest that the resulting articles may be sufficiently stable for, e.g., implantation.

[0214] Injectability

[0215] The injectability of GGBDBA hydrogels (2%, 4%, and 6%) was tested by extruding the hydrogels through a 21-G needle using a syringe.

[0216] like Figure 5aAs shown, the hydrogel was successfully extruded through the needle. This demonstrates the potential of using hydrogels in 3D printing and as injectable drug delivery vehicles.

[0217] Printability

[0218] The ability to use the hydrogels (2%, 4%, and 6%) in printing was tested by extruding the hydrogels through a 21-G needle using a syringe to form patterns on a substrate.

[0219] like Figure 5b and Figure 5c As shown in the figure, the hydrogel successfully produced patterns on the substrate, which shows that the hydrogel can be used to print products.

[0220] 3D Printing

[0221] The scaffolds were designed using Solidworks (2016, Dassault Systemes) and the slicing software used was Slic3r (v.1.2.9). The hydrogels were prepared by mixing 2% of the preparation by weight / volume into 0.1 M, pH 7.4 sodium phosphate buffer. After vortexing, the formed hydrogels were directly used for 3D printing using an INKREDIBLE+ printer (CELLINK).

[0222] Figure 6 Shown are the CAD design to be printed (leftmost images; A, B, i, iii) and the resulting hydrogel implant that has been made from the hydrogel (C, E (both during production), D, F, ii, iv).

[0223] This suggests that the hydrogels could be used to produce articles via 3D printing.

[0224] Self-healing properties

[0225] The self-healing properties of GGBDBA gel (6%) were examined by preparing a two-part hydrogel in which a trace amount of rhodamine B (RdB) was added to one part to impart a color difference ( Figure 7 , above). The two parts of the hydrogel were each divided into two halves to provide four parts, which were then placed together alternately ( Figure 7 ,middle).

[0226] After standing for 2-3 minutes, the parts of the hydrogel had self-repaired. The resulting hydrogel was subjected to suspension and stretching tests. The tests confirmed that self-repair of the hydrogel had occurred. In addition, the tests confirmed that the boric acid crosslinks between the chains of guar gum are reversible and dynamic in nature. This is believed to be due to the rapid formation / hydrolysis of boric acid esters.

[0227] Cell compatibility

[0228] The 2% hydrogel was sterilized under ultraviolet (UV) light for 30 minutes and mixed with human fibroblasts in a cell culture hood. The mixture was placed in a cylinder and the cylinder was sealed.

[0229] During the 14-day cell culture in the presence of hydrogels, gradual cell growth was observed (e.g. Fig.12 shown).

[0230] This suggests that the hydrogel is nontoxic and an effective cell growth medium.

[0231] Nutritional intake

[0232] The ability of the hydrogel to absorb nutrients to support life within the hydrogel was examined by studying the absorbance of crystal violet from the solution over the course of 72 hours using ultraviolet-visible (UV-vis) spectroscopy.

[0233] The results are shown in the attached figure Fig. 9 It is shown in Fig. 9 a shows the UV-vis absorbance of crystal violet solution, and Fig. 9 b shows photographs of the test vials before and after the 72 hour test period.

[0234] The results showed that the amount of crystal violet in the solution was gradually taken up into the hydrogel.

[0235] Thus, the hydrogel may allow for the uptake of nutrients to support life within the hydrogel.

[0236] Drug delivery

[0237] Fig.10 It is shown how the hydrogels of the present invention can be used as delivery vehicles for active agents. For example, a drug (i.e., a therapeutic agent) can be encapsulated in the hydrogel. The drug may be released from the hydrogel, and / or the hydrogel may decompose / rupture, for example, in response to a pH change.

[0238] Controlled degradation

[0239] The hydrogel samples (0.4 mL, 4 wt% or 6 wt% solid content) were contacted with DMEM (Double Modified Eagle Medium, Low Glucose, 1 mL) in a vial. The samples were placed for a certain period of time (8 days, 12 days, 17 days, 50 days) and then inverted to visually determine whether the hydrogel had degraded.

[0240] The results are shown in the attached figure Fig.11Specifically, the 4 wt % hydrogel degraded after about 12 days in the medium, while the 6 wt % hydrogel degraded after about 50 days in the medium.

[0241] Another hydrogel sample (6 wt% solids content, 0.4 mL) was contacted with pH 7.4 buffer (1 mL) in the well plate. The samples were incubated in the buffer at 37°C and the supernatant buffer was replaced every 24 hours to simulate in vivo conditions.

[0242] Figure Fig.12 The results of this study are shown. After day 14, the hydrogel showed some degradation. At day 21, more degradation was visible.

[0243] Therefore, the concentration of the hydrogel can be changed to control the degradation rate of the hydrogel and the products made from it.

[0244] Enhancement of peptide hydrogels

[0245] Composite hydrogels containing GGBDBA and gelatin (GL) were prepared by mixing GGBDBA with GL. The concentration of GL was fixed at 4% w / v. Different concentrations of GGBDBA (0.5%, 1%, 1.5%, 2%, 3% and 4% w / v) were mixed with GL. The mixture was incubated at 37°C to form a gel. GL (4% w / v, without GGBDBA) was also prepared as a control.

[0246] Hydrogels were formed at each GGBDBA concentration. However, no hydrogels were formed with the control sample.

[0247] For the 4% GGBDBA-4% GL formulation, particularly stable hydrogels were observed, such as Fig.13 shown.

[0248] Rheological properties of composite hydrogels 3% GGBDBA-4% GL and 4% GGBDBA-4% GL are shown in Fig.14 shown. Fig.14 a shows an amplitude sweep experiment used to analyze LVE; Fig.14 b shows a frequency sweep experiment under 1% constant shear strain for analyzing G' and G" of the composite hydrogel, and Fig.14 c shows a viscosity versus shear rate study.

[0249] Amplitude strain sweep experiments were performed to measure the linear viscoelastic area (LVE). Results ( Fig.14a) shows that both formulations tested have LVE areas up to 20%. Frequency sweep experiments were used at 1% shear strain to determine the viscoelastic properties of the compositions. Both formulations exhibited a higher storage modulus (G') than the loss modulus (G"), which represents the viscoelasticity of the composite hydrogel. However, the hydrogel composed of 3% GGBDBA-4% GL exhibited a G' value of 60Pa, while the hydrogel composed of 4% GGBDBA-4% GL exhibited a G' value of 220Pa. Therefore, increasing the concentration of GGBDBA increases the viscoelastic properties of the solution. Therefore, as the GGBDBA concentration increases, the G' and G" of the hydrogels are enhanced ( Fig.14 b) Further, the 4% GGBDBA-4% GL formulation exhibited higher viscosity than the 3% GGBDBA-4% GL. However, upon increasing the concentration of GGBDBA in 4% GL, the viscosity of the solution increased.

[0250] Unlike some prior art composite hydrogels, the composite hydrogel of GGBDBA and GL does not require the addition of a crosslinker. However, the composite hydrogel can be tuned to optimize its functionality and viscosity.

Claims

1. A hydrogel comprising guar gum, (hetero)aryl diboronic acid and an aqueous medium, wherein the guar gum is cross-linked by the (hetero)aryl diboronic acid.

2. The hydrogel according to claim 1, wherein the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid in an amount of 0.5% by weight or more.

3. The hydrogel according to claim 2, wherein the hydrogel contains a combination of guar gum and (hetero)aryl diboronic acid in an amount of 0.5% by weight or more, and a weight ratio of guar gum to (hetero)aryl diboronic acid is 2:1 to 40:

1.

4. A hydrogel according to claim 2 or claim 3, wherein the combined amount of guar gum and (hetero)aryldiboronic acid is from 0.5% to 10% by weight.

5. The hydrogel according to any one of the preceding claims, wherein the (hetero)aryldiboronic acid is an aryldiboronic acid. The hydrogel according to claim 5 , wherein the (hetero)aryldiboronic acid is 1,4-phenylenediboronic acid.

7. The hydrogel according to any one of the preceding claims, wherein the aqueous medium is a phosphate-based buffer.

8. The hydrogel according to any one of the preceding claims, wherein the hydrogel further comprises gelatin.

9. The hydrogel according to claim 8, wherein the hydrogel comprises gelatin in an amount of 2 to 10% by weight.

10. A composition comprising the hydrogel according to any one of the preceding claims and an active agent.

11. A pharmaceutical composition comprising the hydrogel according to any one of claims 1 to 9 or the composition according to claim 10.

12. The composition of claim 10 or claim 11, wherein the active agent is a therapeutic, prophylactic, diagnostic and / or nutritional agent comprising a compound selected from the group consisting of small molecule active agents, proteins, polypeptides, polysaccharides and nucleic acids.

13. A composition according to any one of claims 10 to 12, wherein the active agent is selected from the group consisting of antibiotics, antivirals, antiparasitics, cytokines, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and biologically active fragments thereof, antigens and vaccine preparations, anti-inflammatory agents, immunomodulators and oligonucleotide drugs, paramagnetic molecules, fluorescent compounds, magnetic molecules and radionuclides, X-ray imaging agents and contrast agents.

14. A method of manufacturing an article, wherein the method comprises: Providing a hydrogel according to any one of claims 1 to 9 or a composition according to any one of claims 10 to 13; as well as - Forming the hydrogel or pharmaceutical composition into a three-dimensional (3D) shape.

15. The method of claim 14, wherein the object is formed by 3D printing.

16. An article comprising the hydrogel according to any one of claims 1 to 9 or the composition according to any one of claims 10 to 13.

17. An article according to claim 16, wherein the article is made by a method as defined by claim 14 or claim 15.

18. The article of claim 16 or 17, wherein the article is a tissue model or an implant.

19. The article of manufacture according to claim 18, wherein the article is a tissue model selected from the list consisting of: cardiac tissue, vascular tissue, skin tissue, and skeletal tissue, such as bone, cartilage, tendon, and intervertebral disc.

20. The article of manufacture according to claim 18, wherein the article is an implant selected from the list consisting of: cardiac tissue, vascular implants, skin grafts, skeletal tissue such as bone, cartilage, tendon, intervertebral disc, associated bone, cartilage precursor cells, and bone grafts.

21. A solid precursor of a hydrogel, wherein the precursor comprises guar gum cross-linked by (hetero)aryldiboronic acid.

Images