Carbohydrate Crosslinkers

Abstract

To provide a hydrogel having glycosaminoglycan (GAG) as a swellable polymer, a method for crosslinking GAG molecules which gives less affects to the natural properties of GAG molecules, and a method for preparing hydrogel of GAG molecules by a mild and efficient route.SOLUTION: The present invention relates to a hydrogel product containing glycosaminoglycan molecules as a swellable polymer, where glycosaminoglycan molecules are crosslinked in covalent bond via crosslinkage including a spacer group selected from the group consisting of disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of hydrogels containing cross-linked polysaccharides and the use of such hydrogels in medical and / or cosmetic applications. More particularly, the present invention relates to hydrogels made of cross-linked glycosaminoglycans, in particular cross-linked hyaluronic acid, cross-linked chondroitin, or cross-linked chondroitin sulfate. [Background technology]

[0002] Water-absorbing gels, or hydrogels, are widely used in the biomedical field. They are generally prepared by chemically cross-linking polymers into an infinite network. While many polysaccharides will absorb water until they are completely dissolved, cross-linked gels of the same polysaccharides can usually absorb a certain amount of water until they are saturated, i.e., they have a finite liquid-holding capacity or swelling degree.

[0003] Hyaluronic acid, chondroitin, and chondroitin sulfate are well-known biocompatible polymers. They are natural polysaccharides that belong to the glycosaminoglycan (GAG) group. All GAGs are negatively charged heteropolysaccharide chains that have the capacity to absorb large amounts of water.

[0004] Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic applications. HA is a natural polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example, in viscosurgery and as dermal fillers.

[0005] Chondroitin sulfate (CS) is an abundant GAG found in mammalian connective tissues, where it is bound to proteins, in part as proteoglycans, along with other sulfated GAGs. Hydrogels containing CS have previously been shown to be successful in biomedical applications due to their similarity to the natural extracellular matrix (Lauder, RM, Complement Ther Med 17: 56-62, 2009). Chondroitin sulfate has also been used, for example, as a dietary supplement in the treatment of osteoarthritis.

[0006] Cross-linking of glycosaminoglycans extends the durability of the degradable polymers that make up the network, which may be useful in many applications. However, cross-linking may also reduce the native properties of glycosaminoglycans. Therefore, it is typically desirable to keep the degree of modification by effective cross-linking low to preserve the native properties and effects of the glycosaminoglycans themselves. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Lauder, RM, Complement Ther Med 17: pp. 56-62, 2009 [Non-patent document 2] "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties"; Keisuke Kurita, *Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, pp. 7544-7549 [Non-patent document 3] "Library of mild and economic protocols for the selective derivatization of sucrose under microwave irradiation"; M. Teresa Barros, Krasimira T. Petrova, Paula Correia-da-Silva and Taterao M. Potewar; Green Chem., 2011, 13, pp. 1897-1906 Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to provide hydrogels having glycosaminoglycans (GAGs) as the swellable polymer.

[0009] It is a further object of the present invention to provide a method for cross-linking GAG molecules which has minimal impact on the native properties of the GAG molecules.

[0010] It is also an object of the present invention to provide a method for preparing hydrogels of GAG molecules by a mild and efficient route. [Means for solving the problem]

[0011] Towards these and other objectives that will become apparent from the present disclosure, the present invention provides, according to a first aspect, a hydrogel product comprising glycosaminoglycan molecules as swellable polymers, wherein the glycosaminoglycan molecules are covalently crosslinked via bridges comprising spacer groups selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0012] With respect to the inventive methods of preparing the hydrogel products described herein, the term "crosslinker" refers to a molecule having two or more functional groups, particularly nucleophilic functional groups, attached to non-reactive spacer groups, particularly disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0013] Each of the two or more functional groups is capable of reacting with a carboxylic acid group on a GAG molecule to form a stable covalent bond. Preferably, the cross-linker consists of two or more functional groups and a spacer.

[0014] With respect to the hydrogel products of the invention described herein, the term "crosslink" refers to the portion or residue of the crosslinker by which the GAG molecules are covalently linked after crosslinking. The crosslink typically consists of i) a spacer group and ii) a linking group formed when the functional group of the crosslinker reacts with a carboxylic acid group on the GAG. The spacer group can be composed of, for example, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residues.

[0015] Crosslinking with a crosslinker containing a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides provides hydrogel products based entirely on carbohydrate-based structures or their derivatives, minimizing the disruption of the native properties of glycosaminoglycans by crosslinking. The disaccharides, trisaccharides, tetrasaccharides, or oligosaccharides are preferably well-defined in terms of structure and molecular weight. Preferably, the spacer group is selected from one specific disaccharide, trisaccharide, tetrasaccharide, or oligosaccharide structure. Preferably, the disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides are monodisperse or have a narrow molecular weight distribution. The use of well-defined disaccharide, trisaccharide, tetrasaccharide, or oligosaccharide crosslinkers in conjunction with efficient condensation reactions allows for the assembly of products in a controlled manner. The crosslinker itself can also help maintain or enhance the properties of the hydrogel, for example, when crosslinking with structures related to hyaluronic acid (e.g., diaminohyaluronic acid tetrasaccharide) or with structures with enhanced water retention properties (e.g., trehalose).

[0016] The GAG may be, for example, a sulfated or non-sulfated glycosaminoglycan, such as hyaluronan, chondroitin sulfate, heparan sulfate, heparosan, heparin, dermatan sulfate, and keratan sulfate. In some embodiments, the GAG is hyaluronic acid, chondroitin, or chondroitin sulfate. In a preferred embodiment, the GAG is hyaluronic acid.

[0017] In a preferred embodiment, the GAG is a natural GAG. Preferably, the GAG used in connection with the present invention is a natural GAG. Preferably, the GAG is used in its natural state. That is, the chemical structure of the GAG is preferably not altered or modified by the addition of functional groups, etc. Using the GAG in its natural state is preferred because it results in a cross-linked structure that more closely resembles the natural molecule, while preserving the natural properties and effects of the GAG itself and minimizing the immune response when the cross-linked GAG is introduced into the body.

[0018] Covalently cross-linked GAG molecules preferably consist of or essentially consist of carbohydrate-type structures or their derivatives. This means that the cross-linked GAG molecules preferably do not contain or essentially do not contain synthetic non-carbohydrate structures or linkers. This can be achieved by using GAGs in their native state with a cross-linking agent that consists of or essentially consists of carbohydrate-type structures or their derivatives. The functional groups of the cross-linking agent are then covalently bonded directly to the carboxyl groups of the GAGs. Thus, the cross-links of covalently cross-linked GAGs preferably consist of or essentially consist of disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide spacer groups.

[0019] According to a second aspect, the present invention provides a method for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, comprising the steps of: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a bi- or poly-nucleophilic functional cross-linker containing a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to obtain cross-linked glycosaminoglycan molecules. The present invention provides a method comprising:

[0020] The present invention involves the cross-linking of glycosaminoglycan molecules by covalent bonds, preferably amide bonds, typically using a bi- or poly-nucleophilic functional cross-linker comprising an activator for the carboxyl groups on the glycosaminoglycan molecular backbone and a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. Cross-linking by the methods of the present invention can be achieved by a mild and efficient route that results in high yields with minimal degradation of the GAG molecules.

[0021] The di- or poly-nucleophilic functional crosslinker contains a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, which remains in the crosslink between GAG molecules. The di- or poly-nucleophilic functional disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides contain at least two nucleophilic functional groups attached thereto. Preferably, the at least two nucleophilic functional groups are separated by a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0022] The di- or poly-nucleophilic functional crosslinker contains two or more functional groups capable of reacting with the functional carboxyl groups of GAGs, resulting in the formation of a covalent bond, preferably an amide bond. Preferably, the nucleophilic functional group is capable of reacting with a carboxyl group on a glycosaminoglycan molecule to form an amide bond. In some embodiments, the nucleophilic functional group of the disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide is selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semicarbazide, thiosemicarbazide, thiocarbazate, and aminoxy.

[0023] The di- or poly-nucleophilic functional disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides may be derived from nucleophilic functional polysaccharides, such as chitobiose, which is derived from chitin. The di- or poly-nucleophilic functional disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides may also be disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides that have been modified by the introduction of two or more nucleophilic functional groups.

[0024] A preferred group of di- or polynucleophilic functional crosslinkers includes homobifunctional or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates, and aminoxies.

[0025] In certain embodiments, the activation step (b) and the cross-linking step (c) are performed simultaneously. In other embodiments, the activation step (b) is performed prior to and separate from the cross-linking step (c).

[0026] In a preferred embodiment, step (c) further comprises providing particles of cross-linked GAG molecules having an average size in the range of 0.01 to 5 mm, preferably 0.1 to 0.8 mm.

[0027] In a preferred embodiment, the coupling agent in step (b) is a peptide coupling reagent. The peptide coupling reagent may be selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma, and COMU. A preferred peptide coupling reagent is a triazine-based coupling reagent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), with DMTMM being preferred. Another preferred peptide coupling reagent is a carbodiimide coupling reagent, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) mixed with N-hydroxysuccinimide (NHS).

[0028] According to a related aspect, the present invention also provides for the use of the hydrogel product as a medicament, such as in the treatment of soft tissue disorders. Methods are provided for treating a patient suffering from a soft tissue disorder by administering a therapeutically effective amount of the hydrogel product to the patient. Methods are also provided for providing corrective or cosmetic treatment to a patient by administering a therapeutically effective amount of the hydrogel product to the patient.

[0029] Other aspects and preferred embodiments of the present invention will become apparent from the following detailed disclosure of the invention and the appended claims. DETAILED DESCRIPTION OF THE INVENTION

[0030] Itemized List of Preferred Embodiments 1. A hydrogel product comprising glycosaminoglycan molecules as swellable polymers, wherein the glycosaminoglycan molecules are covalently crosslinked via bridges comprising spacer groups selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0031] 2. The hydrogel product of embodiment 1, wherein the glycosaminoglycan molecule is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

[0032] 3. The hydrogel product of embodiment 2, wherein the glycosaminoglycan molecule is hyaluronic acid.

[0033] 4. The hydrogel product of any one of embodiments 1 to 3, wherein at least 75% of the crosslinks comprise a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0034] 5. The hydrogel product of embodiment 4, wherein at least 90% of the crosslinks comprise a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0035] 6. The hydrogel product of embodiment 5, wherein at least 95% of the crosslinks comprise a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0036] 7. The hydrogel product of any one of embodiments 1 to 6, wherein the spacer group is a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose.

[0037] 8. The hydrogel product of embodiment 7, wherein the spacer group is a residue of a hyaluronic acid tetrasaccharide or hyaluronic acid hexasaccharide.

[0038] 9. The hydrogel product of embodiment 7, wherein the spacer group is a residue of trehalose, lactose, maltose, sucrose, cellobiose, or raffinose.

[0039] 10. The hydrogel product of any one of embodiments 1 to 9, wherein the spacer group is selected from the group consisting of disaccharides, trisaccharides, and tetrasaccharides.

[0040] 11. The hydrogel product of any one of embodiments 1 to 10, wherein the crosslinks are attached to the glycosaminoglycan molecules by amide bonds.

[0041] 12. The hydrogel product of any one of embodiments 1 to 11, wherein at least 75% of the bonds between the glycosaminoglycan molecules and the crosslinks are amide bonds.

[0042] 13. The hydrogel product of embodiment 12, wherein at least 90% of the bonds between the glycosaminoglycan molecules and the crosslinks are amide bonds.

[0043] 14. The hydrogel product of embodiment 13, wherein at least 95% of the bonds between the glycosaminoglycan molecules and the crosslinks are amide bonds.

[0044] 15. The hydrogel product of any one of embodiments 1 to 14, wherein less than 5% of the bonds between glycosaminoglycan molecules and crosslinks are ester bonds.

[0045] 16. The hydrogel product of embodiment 15, wherein less than 1% of the bonds between the glycosaminoglycan molecules and the crosslinks are ester bonds.

[0046] 17. The hydrogel product of any one of embodiments 1 to 16, wherein the cross-linked glycosaminoglycan molecules are in the form of gel particles having an average size in the range of 0.01 to 5 mm, preferably 0.1 to 0.8 mm.

[0047] 18. The hydrogel product of any one of embodiments 1 to 17, in the form of an injectable formulation.

[0048] 19. A method for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, comprising: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a bi- or poly-nucleophilic functional cross-linker containing a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to obtain cross-linked glycosaminoglycan molecules. A method comprising:

[0049] 20. The method of embodiment 19, wherein the cross-linking in step (c) results in an amide bond between the glycosaminoglycan molecule and the cross-linking agent.

[0050] 21. The method of embodiment 19 or 20, wherein the activation step (b) and the cross-linking step (c) are carried out simultaneously.

[0051] 22. The method of any one of embodiments 19-21, wherein the coupling agent and the cross-linking agent are added to the glycosaminoglycan simultaneously.

[0052] 23. The method of embodiment 19 or 20, wherein the activation step (b) is carried out prior to and separate from the cross-linking step (c).

[0053] 24. The method of any one of embodiments 19 to 23, wherein step (c) further comprises providing particles of cross-linked glycosaminoglycan having an average size in the range of 0.01 to 5 mm, preferably 0.1 to 0.8 mm.

[0054] 25. The method of any one of embodiments 19 to 24, wherein the glycosaminoglycan molecule is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

[0055] 26. The method of any one of embodiments 19 to 25, wherein the glycosaminoglycan molecule is hyaluronic acid.

[0056] 27. The method of any one of embodiments 19 to 26, wherein the coupling agent in step (b) is a peptide coupling reagent.

[0057] 28. The method of embodiment 27, wherein the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, oxyma and COMU.

[0058] 29. The method of embodiment 28, wherein the peptide coupling reagent is a triazine-based coupling reagent.

[0059] 30. The method of embodiment 29, wherein the triazine coupling reagent is selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT).

[0060] 31. The method of embodiment 30, wherein the triazine-based coupling reagent is DMTMM.

[0061] 32. The method of embodiment 28, wherein the peptide coupling reagent is a carbodiimide coupling reagent.

[0062] 33. The method of embodiment 32, wherein the carbodiimide coupling reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) mixed with N-hydroxysuccinimide (NHS).

[0063] 34. The method of any one of embodiments 19 to 33, wherein the spacer group is a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose.

[0064] 35. The method of any one of embodiments 19 to 33, wherein the spacer group is a residue of a hyaluronic acid tetrasaccharide or hyaluronic acid hexasaccharide.

[0065] 36. The method of any one of embodiments 19-33, wherein the spacer group is a residue of trehalose, lactose, maltose, sucrose, cellobiose, or raffinose.

[0066] 37. The method of any one of embodiments 19 to 36, wherein the spacer group is selected from the group consisting of disaccharides, trisaccharides, and tetrasaccharides.

[0067] 38. The method of any one of embodiments 19 to 37, wherein the nucleophilic groups of the crosslinker are selected from the group consisting of primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates, and aminoxy.

[0068] 39. The method of embodiment 38, wherein the nucleophilic groups of the disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides are primary amines.

[0069] 40. The method of embodiment 39, wherein the crosslinking agent is a dinucleophilic functional crosslinking agent.

[0070] 41. The method of embodiment 40, wherein the cross-linking agent is selected from the group consisting of diaminohyaluronic acid tetrasaccharide, diaminohyaluronic acid hexasaccharide, diaminotrehalose, diaminolactose, diaminomaltose, diaminosucrose, chitobiose, or diaminoraffinose.

[0071] 42. 42. The method of any one of embodiments 19 to 41, further comprising the step of: (d) subjecting the cross-linked glycosaminoglycan molecules obtained in step (c) to alkaline treatment.

[0072] 43. A product obtained by the method according to any one of embodiments 19 to 42.

[0073] 44. The hydrogel product of any one of embodiments 1 to 18 and 43, for use as a medicament.

[0074] 45. The hydrogel product of embodiment 44, for use in treating a soft tissue disorder.

[0075] 46. Use of a hydrogel product according to any one of embodiments 1 to 18 and 43 for the manufacture of a medicament for the treatment of soft tissue disorders.

[0076] 47. A method of treating a patient suffering from a soft tissue disorder by administering to the patient a therapeutically effective amount of a hydrogel product according to any one of embodiments 1 to 18 and 43.

[0077] 48. A method of providing corrective or cosmetic treatment to a patient by administering to the patient a therapeutically effective amount of a hydrogel product according to any one of embodiments 1 to 18 and 43.

[0078] 49. A method of cosmetically treating skin, comprising administering to the skin a hydrogel product according to any one of embodiments 1 to 18 and 43.

[0079] Detailed Description of the Invention The present invention provides advantageous methods for preparing hydrogels made from cross-linked glycosaminoglycan (GAG) molecules, the resulting hydrogel products, and their uses. GAGs are negatively charged heteropolysaccharide chains that have the capacity to absorb large amounts of water. In hydrogel products according to the present invention, the cross-linked GAG molecules are swellable polymers that provide gel properties. The preparation methods described herein provide gentle yet efficient cross-linking of the GAG molecules.

[0080] Thus, the present invention provides a GAG molecule hydrogel by crosslinking in an aqueous medium using a bi- or poly-nucleophilic functional crosslinker that can form a covalent bond directly with the carboxylic acid groups of the GAG molecule by a reaction involving the use of a coupling agent.

[0081] The GAG according to the present invention is preferably selected from the group consisting of hyaluronic acid, chondroitin, and chondroitin sulfate. In a preferred embodiment, the GAG molecule is hyaluronic acid. Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic applications. HA is a natural polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example, in viscoelastic surgery and as dermal fillers.

[0082] Unless otherwise specified, the term "hyaluronic acid" encompasses all variants and combinations of variants of hyaluronic acid, hyaluronate, or hyaluronan, with various chain lengths and charge states, as well as various chemical modifications. That is, the term also encompasses various hyaluronate salts of hyaluronic acid, such as sodium hyaluronate, with various counterions. Hyaluronic acid can be obtained from a variety of sources, both animal and non-animal. Non-animal sources include yeast and, preferably, bacteria. The molecular weight of a simple hyaluronic acid molecule typically ranges from 0.1 to 10 MDa, although other molecular weights are possible.

[0083] The term "chondroitin" refers to a GAG having a repeating disaccharide unit consisting of alternating non-sulfated D-glucuronic acid and N-acetyl-D-galactosamine moieties. For the avoidance of doubt, the term "chondroitin" does not include any form of chondroitin sulfate.

[0084] The term "chondroitin sulfate" refers to a GAG having a repeating disaccharide unit consisting of alternating D-glucuronic acid and N-acetyl-D-galactosamine moieties. The sulfate moieties can be located at a variety of different positions. Preferred chondroitin sulfate molecules are chondroitin-4-sulfate and chondroitin-6-sulfate.

[0085] Chondroitin molecules can be obtained from a variety of sources, both animal and non-animal. Non-animal sources include yeast and, preferably, bacteria. The molecular weight of simple chondroitin molecules typically ranges from 1 to 500 kDa, although other molecular weights are possible.

[0086] Cross-linked GAGs contain cross-links between GAG molecular chains, thereby creating a continuous network of GAG molecules held together by covalent cross-links.

[0087] Preferably, the GAG molecular chains are cross-linked to each other via a cross-linking agent comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0088] Preferably, the cross-linking agent is attached to the glycosaminoglycan molecule by an amide bond.

[0089] Preferably, the crosslinked GAG product is biocompatible, meaning that it will not provoke any or only a mild immune response in the treated individual, i.e., it will not provoke any or only mild undesirable local or systemic effects in the treated individual.

[0090] The crosslinked product according to the present invention is a gel or hydrogel, i.e., it can be considered a crosslinked system of GAG molecules that is water-insoluble but substantially dilute when subjected to a liquid, typically an aqueous liquid.

[0091] Gels contain mostly liquid by mass, e.g., 90-99.9% water, but behave like solids due to the three-dimensional cross-linked GAG molecular network within the liquid. Because of their significant liquid content, gels are structurally flexible and resemble natural tissue, making them highly useful as scaffolds in tissue engineering and for tissue augmentation. Gels are also useful for the treatment of soft tissue disorders and for corrective or cosmetic treatments. They are preferably used as injectable formulations.

[0092] Crosslinking of GAG molecules can be achieved by activating them with a coupling agent and then reacting them with a crosslinker. The concentration and degree of crosslinking of GAG molecules affect the mechanical properties of the gel, such as the elastic modulus G' and stability characteristics. Crosslinked GAG molecule gels can be characterized in terms of their "degree of modification." The degree of modification of GAG molecule gels generally ranges between 0.01 and 15 mol %. The degree of modification (mol %) describes the amount of crosslinker bound to the GAG molecule, i.e., the molar amount of bound crosslinker relative to the total molar amount of repeating disaccharide units. The degree of modification represents the extent to which the GAG molecule has been chemically modified by the crosslinker. Reaction conditions for activation and crosslinking, as well as appropriate analytical techniques for determining the degree of modification, are all well known to those skilled in the art. These and other related factors can be easily adjusted by those skilled in the art to provide appropriate conditions for obtaining the desired degree of modification and to verify the characteristics of the resulting product in relation to the degree of modification.

[0093] The hydrogel product may also contain a portion of GAG molecules that are not crosslinked, i.e., not bound to a three-dimensional network of crosslinked GAG molecules, but it is preferred that at least 50%, preferably at least 60%, more preferably at least 70%, and most preferably at least 80% by weight of the GAG molecules in the gel composition form part of a network of crosslinked GAG molecules.

[0094] The cross-linked GAG molecules are preferably present in the form of gel particles, which preferably have an average size in the range of 0.01 to 5 mm, preferably 0.1 to 0.8 mm, for example 0.2 to 0.5 mm or 0.5 to 0.8 mm.

[0095] The hydrogel product may be in aqueous solution, but may also be in dry form or precipitated, for example in ethanol. The hydrogel product is preferably injectable.

[0096] Hydrogel products include: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a bi- or poly-nucleophilic functional cross-linker containing a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to obtain cross-linked glycosaminoglycan molecules. It can be prepared by a method comprising:

[0097] Preferably, the GAG according to the present invention is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In one preferred embodiment, the GAG molecule is hyaluronic acid.

[0098] In the activation step (b), the carboxyl groups on the GAG molecules are activated with a coupling agent to form activated GAG molecules.

[0099] In a preferred embodiment, the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, oxyma and COMU.

[0100] The peptide coupling reagent is preferably a triazine-based coupling reagent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT). A preferred triazine-based peptide coupling reagent is DMTMM.

[0101] Another preferred peptide coupling reagent is a carbodiimide coupling reagent, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) mixed with N-hydroxysuccinimide (NHS).

[0102] In the cross-linking step (c), the activated GAG molecules are cross-linked via their carboxyl groups using a cross-linking agent. The cross-linking agent is a bi- or poly-nucleophilic functional cross-linking agent containing a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. The cross-linking agent links the GAG chains to each other via the carboxyl groups on the GAG backbone. The spacer group can be, for example, a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose. The term "residue" herein means that the structure of the compound is similar but not identical to the parent compound hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose, respectively. The structure of the residue may differ from that of the parent compound in that it contains two or more nucleophilic functional groups and, optionally, is covalently linked to carboxyl groups on the GAG backbone via said nucleophilic functional groups.

[0103] A di- or poly-nucleophilic functional crosslinker contains two or more functional groups capable of reacting with the functional carboxyl groups of a GAG, resulting in the formation of a covalent bond, preferably an amide bond.

[0104] A preferred group of bi- or polynucleophilic functional crosslinkers includes homo- or hetero-bifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates, and aminoxies. Non-limiting examples of such hetero-bifunctional crosslinkers useful in the present invention include:

[0105] [ka]

[0106] Diaminotrehalose (6,6'-diamino-6,6'-dideoxytrehalose),

[0107] [ka]

[0108] Diaminosucrose (6,6'-diamino-6,6'-dideoxysucrose),

[0109] [ka]

[0110] Chitobiose (2,2'-diamino-2,2'-deoxycellobiose),

[0111] [ka]

[0112] Diaminolactose (6,6'-diamino-6,6'-dideoxylactose),

[0113] [ka]

[0114] "Reduced N-deacetylated hyaluronic acid tetrasaccharide" or "reduced diaminohyaluronic acid tetrasaccharide", and

[0115] [ka]

[0116] Diaminoraffinose (6,6"-diamino-6,6"-dideoxyraffinose)

[0117] Examples of coupling with heterobifunctional primary amines (1a), aminoxy (1b), carbazate (1c), semicarbazide (1d), thiosemicarbazide (1e), thiocarbazate (1f), hydrazine (1g), and hydrazide (1h) are shown in Reaction Schemes 1a-1h.

[0118] [ka]

[0119] [ka]

[0120] [ka]

[0121] [ka]

[0122] This is illustrated schematically in FIG.

[0123] The di- or poly-nucleophilic functional crosslinker contains a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, which remains in the crosslink between the GAG molecules.

[0124] The method can be carried out in a one-pot manner in aqueous media, involving the direct covalent coupling of a bi- or poly-nucleophilic functional crosslinker to unique carboxylic acid groups on the native GAG using an appropriate coupling agent. In a preferred embodiment, the activation step (b) and the crosslinking step (c) are carried out simultaneously.

[0125] In another embodiment, the activation step (b) is carried out prior to and separate from the cross-linking step (c).

[0126] Methods for making crosslinked hydrogels typically involve preparing a mixture of GAG molecules, such as hyaluronic acid, with a crosslinker, such as diaminotrehalose, DATH (0.001 to 10 molar equivalents of amine relative to carboxylic acid groups, or preferably 0.001 to 1 molar equivalents), and a coupling agent, such as DMTMM (0.01 to 10 molar equivalents relative to carboxylic acid groups, or preferably 0.05 to 1 molar equivalents). The mixture is incubated at 5 to 50°C, preferably 10 to 40°C, or more preferably 20 to 35°C, for 2 to 120 hours, preferably 4 to 48 hours, followed by alkali treatment, neutralization, precipitation, washing, and drying in vacuo to obtain the crosslinked polysaccharide as a solid. The precipitate is preferably swollen in a phosphate buffer containing NaCl to form a hydrogel, and the hydrogel is then micronized into hydrogel particles with a size of 0.01 to 5 mm, preferably 0.1 to 1 mm.

[0127] Typical uses for the resulting hydrogel products include, but are not limited to, the preparation of injectable formulations for the treatment of soft tissue disorders, including corrective and cosmetic treatments.

[0128] In a more specific embodiment, cross-linking of chondroitin sulfate with DATH can be carried out as follows: Diaminotrehalose (DATH) is synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties"; Keisuke Kurita, *Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

[0129] Chondroitin sulfate (CS) (10-200 kDa) is weighed into a Falcon tube. A stock solution of diaminotrehalose (DATH) is prepared by dissolving DATH in phosphate buffer, pH 7.4. DMTMM is weighed into a container, and the DATH solution is added to the DMTMM. The pH of the DMTMM-DATH solution is adjusted to approximately 7 by adding 1.2 M HCl or 0.25 M NaOH, and then the mixture is added to the CS. The contents are thoroughly homogenized and then incubated at 15-55°C for 2-48 hours. The resulting material is pressed twice through a 1 mm steel mesh and swollen in NaOH. The gel is neutralized to pH 7 with 1.2 M HCl and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed three times through a filter mesh. The cross-linked CS gel is loaded into a syringe and sterilized.

[0130] In another more specific embodiment, cross-linking of HA with diaminosucrose can be carried out as follows: Diaminosucrose is prepared as described in "Library of mild and economic protocols for the selective derivatization of sucrose under microwave irradiation"; M. Teresa Barros, Krasimira T. Petrova, Paula Correia-da-Silva and Taterao M. Potewar; Green Chem., 2011, 13, pp. 1897-1906.

[0131] Hyaluronic acid (HA) (10-1000 kDa) is weighed into a container. A diaminosucrose stock solution is prepared by dissolving diaminosucrose in phosphate buffer pH 7.4. DMTMM is weighed into a container and the diaminosucrose solution is added to the DMTMM. The pH of the DMTMM-diaminosucrose solution is adjusted to approximately 7 by adding 1.2 M HCl or 0.25 M NaOH, and then the mixture is added to the HA. The contents are thoroughly homogenized and then incubated at 15-55°C for 2-48 hours. The resulting material is pressed twice through a 1 mm steel mesh and swollen in NaOH. The gel is neutralized to pH 7 with 1.2 M HCl and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed three times through a filter mesh. The cross-linked HA gel is loaded into a syringe and sterilized.

[0132] In another more specific embodiment, cross-linking of HA with chitobiose can be carried out as follows: Hyaluronic acid (HA) (10-1000 kDa) is weighed into a container. A chitobiose stock solution is prepared by dissolving chitobiose (purchased from Carbosynth Ltd., UK) in phosphate buffer, pH 7.4. DMTMM is weighed into a container, and the chitobiose solution is added to the DMTMM. The pH of the DMTMM-chitobiose solution is adjusted to approximately 7 by adding 1.2 M HCl or 0.25 M NaOH, and then the mixture is added to the HA. The contents are thoroughly homogenized and then incubated at 15-55°C for 2-48 hours. The resulting material is pressed twice through a 1 mm steel mesh and swollen in NaOH. The gel is neutralized to pH 7 with 1.2 M HCl and then precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuum at 25°C. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The cross-linked HA gel is filled into a syringe and sterilized.

[0133] In another more specific embodiment, cross-linking of HA with reduced diamino HA tetrasaccharide can be carried out as follows: Hyaluronic acid (HA) (10-1000 kDa) is weighed into a container. A stock solution of reduced diamino HA tetrasaccharide is prepared by dissolving the reduced diamino HA tetrasaccharide in phosphate buffer, pH 7.4. DMTMM is weighed into a container, and the reduced diamino HA tetrasaccharide solution is added to the DMTMM. The pH of the DMTMM and reduced diamino HA tetrasaccharide solution is adjusted to approximately 7 by adding 1.2 M HCl or 0.25 M NaOH, and then the mixture is added to the HA. The contents are thoroughly homogenized and incubated at 15-55°C for 2-48 hours. The resulting material is pressed twice through a 1 mm steel mesh and swollen in NaOH. The gel is neutralized to pH 7 with 1.2 M HCl and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed three times through a filter mesh. The cross-linked HA gel is loaded into a syringe and sterilized.

[0134] In another more specific embodiment, cross-linking of HA with dicarbazate trehalose can be carried out as follows: α,α-D-Trehalose (1 eq.) (anhydrous) (Carbosynth Ltd., UK) is dissolved in dry dimethylformamide (DMF), followed by the addition of triethylamine (2–6 eq.). The flask is cooled to 0°C (ice / water) and N2 atmosphere. 4-Nitrophenyl chloroformate (2–6 eq.) is added dropwise to the flask. The resulting mixture is stirred at room temperature for 2–48 h, then concentrated, purified by FC, and dried in vacuo. The product is dissolved in DMF, and hydrazine monohydrate (2–20 eq.) is added to the solution, followed by stirring at 0–50°C for 4–48 h. The reaction mixture is then concentrated, purified by FC, and dried in vacuo to give α,α-D-6,6'-dideoxy-6,6'-dicarbazate trehalose (dicarbazatotrehalose, DCT).

[0135] Hyaluronic acid (HA) (10-1000 kDa) is weighed into a container. A stock solution of dicarbazate trehalose (DCT) is prepared by dissolving DCT in phosphate buffer pH 7.4. DMTMM is weighed into a container and the DCT solution is added to the DMTMM. The pH of the DMTMM-DCT solution is adjusted to approximately 7 by adding 1.2 M HCl or 0.25 M NaOH, and then the mixture is added to the HA. The contents are thoroughly homogenized and incubated at 15-55°C for 2-48 hours. The resulting material is pressed twice through a 1 mm steel mesh and swollen in NaOH. The gel is neutralized to pH 7 with 1.2 M HCl and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed three times through a filter mesh. The cross-linked HA gel is loaded into a syringe and sterilized.

[0136] In another more specific embodiment, cross-linking of HA with diaminooxytrehalose can be carried out as follows: To a stirred suspension of α,α-D-trehalose (1 equivalent) (anhydrous) (Carbosynth Ltd., UK) in anhydrous THF, N-hydroxyphthalimide (2–10 equivalents) and triphenylphosphine (2–10 equivalents) are added, and the mixture is stirred for 5–60 minutes. Diisopropyl azodicarboxylate (DIAD, 2–10 equivalents) is then added dropwise at 0–40°C, and the mixture is stirred for 2–48 hours at 0–40°C. The solvent is removed in vacuo, and the crude product is purified by FC and dried in vacuo. A suspension of the product in MeOH and CHCl is treated with hydrazine monohydrate (2–20 equivalents), and the mixture is stirred for 2–24 hours at 0–40°C. Diaminoxytrehalose is then obtained by concentration, purification by FC, and drying in vacuo.

[0137] Hyaluronic acid (HA) (10-1000 kDa) is weighed into a container. A stock solution of diaminoxytrehalose (DAOT) is prepared by dissolving DAOT in phosphate buffer, pH 7.4. DMTMM is weighed into a container, and the DAOT solution is added to the DMTMM. The pH of the DMTMM-DAOT solution is adjusted to approximately 7 by adding 1.2 M HCl or 0.25 M NaOH, and then the mixture is added to the HA. The contents are thoroughly homogenized and incubated at 15-55°C for 2-48 hours. The resulting material is pressed twice through a 1 mm steel mesh and swollen in NaOH. The gel is neutralized to pH 7 with 1.2 M HCl and precipitated with ethanol. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed three times through a filter mesh. The cross-linked HA gel is loaded into a syringe and sterilized. [Example]

[0138] By way of example, and without wishing to be limited thereto, the invention is illustrated below.

[0139] Definition and Analysis SwF - Swelling factor analysis was performed in saline. [PS] - polysaccharide concentration, e.g., HA concentration. PS concentration was measured by LC-SEC-UV or NIR. GelP - Gel fraction (sometimes called gel content or GelC) describes the percentage of polysaccharides that are part of the gel network. A figure of 90% means that only 10% of the polysaccharides are not part of the network. The amount of free polysaccharides in the gel was measured by LC-SEC-UV. SwC - swelling capacity is the total liquid uptake of 1 gram of polysaccharide, not corrected for gel fraction.

[0140]

number

[0141] SwCC - Corrected Swelling Capacity (sometimes called SwDC) is the total liquid uptake of one gram of polysaccharide corrected for gel fraction.

[0142]

number

[0143] CrR - effective cross-linking rate was analyzed by LC-SEC-MS.

[0144]

number

[0145] A CrR of 1.0 means that all of the cross-linking agent is cross-linked.

[0146] Alkaline or thermal hydrolysis In some of the examples below, the products were subjected to alkaline or thermal hydrolysis to hydrolyze the ester bonds formed during the cross-linking process. Alkaline / thermal hydrolysis results in only amide cross-links in the final product. Alkaline / thermal hydrolysis was performed as follows:

[0147] Alkaline hydrolysis The material was allowed to swell in 0.25 M NaOH (1 g material: 9 g 0.25 M NaOH, resulting in a pH of 13) at room temperature for at least 1 hour. The gel was neutralized to pH 7 with 1.2 M HCl and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagent, then with 70% ethanol to remove salts, and finally with ethanol to remove water. The ethanol was removed overnight in a vacuum oven.

[0148] The precipitate was swelled in 0.7% NaCl phosphate buffer, pH 7.4, and then pressed through a fine filter mesh three times. The gel was filled into syringes and sterilized. In some cases, some syringes were not sterilized to check the effectiveness of sterilization.

[0149] thermal hydrolysis The material was swollen at room temperature in 0.7% NaCl phosphate buffer, pH 7.4. If necessary, the pH was adjusted to 7.2-7.5. The gel was left at 70°C for 20-24 hours, and then the particle size was reduced by passing it through a fine filter mesh three times. The gel was filled into syringes and sterilized. In some cases, some syringes were not sterilized to observe the effectiveness of sterilization.

[0150] Synthesis of hyaluronic acid diaminotrehalose Diaminotrehalose (DATH) was synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties"; Keisuke Kurita, *Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

[0151] Example 1 Cross-linking of hyaluronic acid with diaminotrehalose (DATH) A series of experiments (Examples 1-1 to 1-5) was conducted involving crosslinking hyaluronic acid (HA) of various molecular weights with various molar ratios of DATH using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as the coupling agent. The ratios between HA, DATH, and DMTMM are listed in Table 1 below.

[0152] Hyaluronan (molecular weight (M) ranging from approximately 100 kDa to approximately 1000 kDa) w ) was weighed into a Falcon tube. A stock solution of diaminotrehalose (DATH) was prepared by dissolving DATH (0.001-0.005 equivalents) in phosphate buffer, pH 7.4. DMTMM (0.05 equivalents) was weighed into a PTFE container, and the DATH solution was added to the DMTMM until dissolved. The pH of the DMTMM-DATH solution was adjusted to 6-7 with 1.2 M HCl or 0.25 M NaOH and then added to the HA. The contents were thoroughly homogenized and then incubated at 35°C for 24 hours.

[0153] The resulting material was pressed twice through a 1 mm steel mesh and then treated with NaOH solution. The gel was neutralized to pH 7 with 1.2 M HCl and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagent, then with 70% ethanol to remove salts, and finally with ethanol to remove water. The ethanol was removed overnight in a vacuum oven.

[0154] The precipitate was swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The gel was filled into a syringe and sterilized.

[0155] [Table 1]

[0156] Example 2 Cross-linking of hyaluronic acid with diaminotrehalose (DATH) A series of experiments (Examples 2-1 to 2-11) was conducted involving crosslinking hyaluronic acid (HA) of various molecular weights with various molar ratios of DATH using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as the coupling agent. The ratios between HA, DATH, and DMTMM are listed in Table 2 below.

[0157] Hyaluronic acid was weighed into a reaction vessel. A stock solution of the crosslinker (DATH) was prepared by dissolving it in phosphate buffer, pH 7.4. DMTMM was weighed into a PTFE container, and the crosslinker solution was added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinker solution was adjusted to 6-7 with 1.2 M HCl or 0.25 M NaOH and then added to the hyaluronic acid. The contents were thoroughly homogenized and then incubated at 35°C for 24 hours. The resulting material was pressed twice through a 1 mm steel mesh and then treated with either heat or alkali. The results are shown in Table 2.

[0158] [Table 2]

[0159] Example 3 Cross-linking of hyaluronic acid with chitobiose (CB) Hyaluronic acid was weighed into a reaction vessel. A stock solution of the crosslinker (chitobiose) was prepared by dissolving it in phosphate buffer (pH 7.4). DMTMM was weighed into a PTFE container, and the crosslinker solution was added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinker solution was adjusted to 6-7 with 1.2 M HCl and then added to the HA. The contents were thoroughly homogenized and then incubated at 35°C for 24 hours. The resulting material was pressed twice through a 1 mm steel mesh and then treated with either heat or alkali according to the general procedure. The ratios between HA, chitobiose, and DMTMM are listed in Table 3 below (Examples 3-1 and 3-2).

[0160] Example 4 Cross-linking of hyaluronic acid with diaminotetra-HA (DA-4HA) Diaminotetra-HA (DA-4HA) was synthesized according to the scheme below.

[0161] [ka]

[0162] Process 1 A solution of HA-4 (500 mg, 0.61 mmol) in water (5 mL) at room temperature was treated with sodium borohydride (23.05 mg, 0.61 mmol), and the resulting solution was stirred for 3 h and concentrated to dryness to give the reduced product 1 (532 mg, assumed 100%) as a white foam. LCMS(t r =0.28min., ES+=779.4(M-2 Na+2H)

[0163] Process 2 The reduced product 1 (532 mg) was dissolved in aqueous NHOH (5 mL, 50% v / v), and solid NHI (100 mg) was added. The resulting suspension was heated at 70 °C for 48 h, cooled to room temperature, and concentrated to dryness to give a residue. The residue was precipitated in neat EtOH, and the resulting precipitate was collected by filtration and dried to constant weight to give a 1:1 mixture of diamine 2 and monoamine 3 in quantitative yield. The crude reaction product was used without further purification. 2: LCMS(t r =0.16min., ES+=695.36(M-2 Na+2H) 3: LCMS(t r =0.19min., ES+=737.47(M-2 Na+2H)

[0164] Hyaluronic acid was weighed into a reaction vessel. Stock solutions of the crosslinker (diaminotetra-HA) synthesized as described above and DMTMM were prepared by dissolving them in phosphate buffer pH 7.4, respectively. The pH of both solutions was adjusted to 7 and then added to the HA. The contents were thoroughly homogenized and then incubated at 23°C for 24 hours. The resulting material was pressed twice through a 1 mm steel mesh and then heat-treated according to the general procedure. The ratios between HA, diaminotetra-HA, and DMTMM are listed in Table 3 below (Example 4).

[0165] Example 5 Cross-linking of heparosan (HEP) with diaminotrehalose (DATH) The coupling agent DMTMM and the cross-linking agent DATH were weighed into separate reaction vessels and dissolved in phosphate buffer (pH 7.4). The pH of the solution was adjusted to pH 7-7.5 with 1.2 M HCl or 0.25 M NaOH. The DMTMM and DATH solutions were then added sequentially to the heparosan weighed into the reaction vessel. The contents were thoroughly homogenized and then incubated at 35°C for 24 hours. The resulting material was pressed twice through a 1 mm steel mesh and then heat-treated according to the general procedure. The ratios between heparosan, DATH, and DMTMM are listed in Table 3 below (Examples 5-1 and 5-2).

[0166] Example 6 Cross-linking of chondroitin sulfate (CS) with diaminotrehalose (DATH) The coupling agent DMTMM and the cross-linking agent DATH were weighed into separate reaction vessels and dissolved in phosphate buffer (pH 7.4). The pH of the solution was adjusted to pH 7-7.5 with 1.2 M HCl or 0.25 M NaOH. The DMTMM solution and DATH solution were then added sequentially to the weighed chondroitin sulfate in the reaction vessel. The contents were thoroughly homogenized and then incubated at 35°C for 24 hours. The resulting material was pressed twice through a 1 mm steel mesh and then heat-treated according to the general procedure. The ratios of chondroitin sulfate, DATH, and DMTMM are listed below in Table 3 (Examples 6-1 and 6-2).

[0167] [Table 3]

Claims

1. 1. A method for preparing a hydrogel product comprising cross-linked hyaluronic acid (HA) molecules, comprising: carrying out alkaline hydrolysis to hydrolyze the ester bonds between the HA molecules and the crosslinker to obtain a hydrogel product; the HA molecules are crosslinked via amide bonds formed between the crosslinker and activated carboxyl groups on the HA; the cross-linking agent is a di-, tri-, tetra-, or oligosaccharide with two or more nucleophilic functionalities; the ester bond is formed between HA and the crosslinker during the crosslinking process that forms the amide bond; The method, wherein the hydrogel product has less than 50% by weight of non-crosslinked HA molecules.

2. 10. The method of claim 1, further comprising the step of cross-linking activated carboxyl groups of HA molecules using the cross-linking agent to obtain cross-linked HA molecules, wherein the cross-linking is performed prior to the alkaline hydrolysis.

3. 3. The method of claim 2, further comprising activating the carboxyl groups of the HA molecules with a coupling agent to form activated carboxyl groups, wherein the activation occurs prior to or simultaneously with the cross-linking.

4. 4. The method of claim 3, wherein the coupling agent is a triazine-based coupling reagent.

5. 5. The method of claim 4, wherein the coupling agent is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).

6. The method of claim 3, wherein the activation step and the crosslinking step are performed simultaneously.

7. The method of claim 1, wherein the cross-linking agent is a trehalose residue.

8. 8. The method of claim 7, wherein the cross-linking agent is diaminotrehalose (DATH).

9. 2. The method of claim 1, wherein after the hydrolysis, at least 90% of the bonds between the HA molecules and the cross-linking agent are amide bonds.

10. 10. The method of claim 1, wherein after the hydrolysis, less than 5% of the bonds between the HA molecules and the crosslinker are ester bonds.

11. 10. The method of claim 1, wherein said hydrolysis comprises swelling the cross-linked HA molecules in an alkaline solution for at least 1 hour.

12. 10. The method of claim 1, wherein less than 20% by weight of the hydrogel product comprises non-crosslinked HA molecules.

13. 3. The method of claim 2, wherein the HA molecule has a molecular weight of about 10 kDa to 10 MDa before cross-linking.

14. 10. The method of claim 1, wherein the hydrogel product has an effective crosslinking ratio (CrR) of at least 0.

43.

15. 2. The method of claim 1, wherein the cross-linked HA molecule comprises GAG in its native state together with a cross-linker consisting of or consisting essentially of a carbohydrate-type structure or a derivative thereof.

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