Hydrogels for cell therapy

Abstract

In addition, the invention relates to hydrogels comprising the cross-linked dextran polymer of the invention, and / or hydrogels further comprising biological cells. It also relates to the therapeutic use of the hydrogel according to the invention for the treatment of a condition or disease in a mammal, said condition or disease being due to a lack or dysfunction of the endocrine function of the pancreatic organ, and to an implant comprising the hydrogel according to the invention. It also relates to an implant comprising the hydrogel of the invention and also comprising a ring and a mesh.

Description

[0001] The field of this invention is therapeutics, particularly cell therapy. More specifically, this invention relates to implants comprising a hydrogel, which can be incorporated into:

[0002] - Active ingredients, such as peptides, hormones, or proteins, or

[0003] - Secretory cells, which can be cells that secrete peptides or hormones.

[0004] The aim is to prevent, treat, or cure diseases. In particular, this can prevent and / or treat chronic diseases by completely or partially replacing the function of naturally occurring cells that are lacking in the patient's body. The invention also relates to crosslinked polymers, their precursors, methods for obtaining the crosslinked polymers, and methods for obtaining hydrogels, particularly cellular hydrogels.

[0005] Cells can be isolated or aggregated, and can be of one type or different types.

[0006] Hydrogels can be used in a variety of systems, such as:

[0007] - As a scaffold for controlled drug or active pharmaceutical ingredient release systems, or

[0008] - Used as a scaffold for implantable devices containing cells.

[0009] Hydrogels contain or consist of polymers cross-linked in a 3D network. Hydrogels can be natural or synthetic homopolymers or copolymers. They possess the ability to absorb and retain large amounts of water. This is known as hydrogel swelling.

[0010] In order to have a system that can serve as an implant capable of delivering active ingredients over a long period of time, many features must be acquired.

[0011] Among these characteristics are:

[0012] - Low degradability, especially low biodegradability, or no biodegradability, or good in vivo stability, so that the incorporated cells do not escape from the patient's body and the host cells do not infiltrate the implant.

[0013] - Good selective permeability is defined as the selective permeability of biological elements based on their size or molecular weight, by separating all or part of the incorporated cells from the host's immune system while allowing the passage of active ingredients (such as hormones, peptides, or proteins), thereby allowing a low immune response or even better, no immune response.

[0014] - Good mitigation of foreign substance response, or good biocompatibility, especially low cytotoxicity and good local tolerance.

[0015] - To enable cells to have a high survival rate, for example, with good angiogenesis near the cells and sufficient nutrient flow to the cells.

[0016] - Enables cells to function well within the hydrogel.

[0017] In order to be used as a controlled release system or scaffold for cells, hydrogels must have specific characteristics that enable them to exhibit all or some of the desired properties (such as those disclosed above) as well as good mechanical and rheological properties.

[0018] Among the highly interesting rheological and mechanical properties of hydrogels are:

[0019] - Good uniformity, which can be associated with good transparency or translucency.

[0020] - Appropriate resistance and flexibility in response to stress and strain mechanics, especially during handling and implantation, such as via laparoscopy.

[0021] - A defined mesh size, designed to maximize oxygen and nutrient exchange, controlled transport characteristics, and selective permeability.

[0022] - Good in vivo stability, meaning it is resistant to hydrolytic degradation, enzymatic degradation, or oxidative degradation.

[0023] Among the parameters that can indicate the desired rheological and mechanical properties are:

[0024] -tanδ (called the loss tangent) indicates the mechanical properties.

[0025] -G' indicates the elastic modulus (stiffness) and mesh size.

[0026] - Fracture compression and / or traction deformation, which indicate the elasticity and resistance of the hydrogel.

[0027] - Swelling property, which provides an indication of water content, size and mechanical properties.

[0028] One of the problems to be solved is obtaining hydrogels with the following properties:

[0029] - Procedures involving the implantation of hydrogels without damaging them, such as procedures performed via laparoscopy, and / or

[0030] - The gel is kept in place after implantation, for example, so that the hydrogel does not fold after implantation and / or is fixed relative to the tissue in which it is implanted.

[0031] One of the most difficult problems to be solved is obtaining implants with small thickness (to allow cells to approach the tissue), large surface area (to have a relatively large volume), good mechanical properties (to allow for minimally invasive surgery), and good biocompatibility!

[0032] Another problem to be addressed involves the sedimentation of cells or islets during cross-linking that leads to gelation.

[0033] The following are existing technologies regarding hydrogels:

[0034] -Nestor Loper Mora et al, "Evaluation of dextran(ethyleneglycol)hydrogel films for giant unilamellar lipid vesicle production and their application for the encapsulation of polymersome, Soft Matters, January 2017, Vol.13, n°33, pp 5580-5585,

[0035] -Hanwei Zhang et al., "In situ gelable interpenetrating double networkhydrogel formulated from binary components: thiolated chitosan and oxidizeddextran", Biomacromolecules, 2011, Vol.12, n°5, pp 1428-1437,

[0036] -Rongsheng Zhang et al., "A novel pH and ionic strength sensitivecarboxymethyl dextran hydrogsel, Biomaterials, 2005, Vol. 26, n°22, pp 4677-4683, and

[0037] -Taichi Ito et al., "Dextran-based in situ cross-linked injectablehydrogels to prevent peritoneal adhesion", Biomaterials, 2007, Vol.28, n°23, pp3418-3426,

[0038] The disclosed hydrogels cannot solve the technical problems in the same way as the hydrogel of this invention.

[0039] In the prior art, hydrogels containing cross-linked cells are typically designed to allow the growth of cellular objects, such as 3D cell culture. This application requires the hydrogel to simultaneously encapsulate the starting cells and make room for new cells obtained through outgrowth and / or proliferation. To meet these two opposing characteristics, the solution is to have a degradable hydrogel (e.g., degraded via cleavable bonds) that is strong enough to encapsulate cells and, after degradation, makes sufficient space for new cells. A preferred approach to achieving this degradation is to have peptide structures within the cross-linked hydrogel, particularly at the level of the cross-linking agent between the polymer backbone.

[0040] This type of behavior is completely incompatible with the purpose of the present invention, which is to obtain durable cross-linked hydrogels that encapsulate / embed cells. In this case, the hydrogel needs to have very low degradability or even better, non-degradability, because this key feature will allow the cells to remain invisible to the immune system.

[0041] The potential problems are addressed by providing a gel with physicochemical properties that allow for the fabrication of implantable devices and biocompatibility properties that allow for cell survival.

[0042] Furthermore, compared to many existing technologies, this invention allows for the preparation of hydrogels with tunable characteristics, taking into account the precursors used and the manner in which crosslinking is performed. This can result in the hydrogel enabling the controlled incorporation and release of specific objects from the hydrogel.

[0043] The suitability of a hydrogel depends on its main structure. Therefore, important parameters for characterizing the network structure of the hydrogel according to the present invention are the polymer volume fraction in the swollen state, the molecular weight of the polymer chain between two adjacent crosslinking points, and the corresponding pore size.

[0044] The problem is addressed by providing novel crosslinked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are linked by at least one central linking group L(-). iCovalent crosslinking, wherein at least one group is at least a divalent straight-chain, branched, or cyclic alkyl group comprising at least a polyethylene glycol chain, or wherein at least one group comprises at least a polyethylene glycol chain. A straight-chain, branched, or cyclic alkyl group with at least a divalent (Pox) chain.

[0045] The problem is addressed by providing novel crosslinked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are linked by at least one central linking group L(-). i Covalent crosslinking, wherein the at least one group is at least a divalent straight-chain, branched, or cyclic alkyl group comprising at least a polyethylene glycol chain.

[0046] The problem is addressed by providing novel crosslinked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are linked by at least one central linking group L(-). i Covalent crosslinking, wherein the at least one group comprises at least poly( A straight-chain, branched, or cyclic alkyl group with at least a divalent (Pox) chain.

[0047] In one embodiment, the crosslinked dextran polymer Dx has anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein

[0048] -L(-) i It is a straight-chain or branched polyether.

[0049] -i represents the valence of L and the number of W groups bound to the dextran polymer, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[0050] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[0051] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with a carboxylate group as disclosed and described in application PCT / EP2022 / 050466 filed on January 11, 2022.

[0052] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0053] -L(-) i It is a straight-chain or branched polyether with heteroatoms such as oxygen, nitrogen, or sulfur at its ends.

[0054] -i is the valence of L and -(R1) m The number of G1- groups, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[0055] -m is an integer equal to 0 or 1.

[0056] -W is -(R1) m G1-group, in which

[0057] •-R1- is a straight-chain or branched alkyl divalent group containing 1 to 6 carbon atoms and optional heteroatoms such as oxygen, nitrogen, or sulfur.

[0058] •-G1- is a straight-chain, branched, or cyclic alkyl divalent group containing 1 to 6 carbon atoms, and may contain heteroatoms such as oxygen, nitrogen, or sulfur.

[0059] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0060] -W is -(R1) m G1-group, in which

[0061] •-G1- is a sulfone derivative as described in the following formula:

[0062]

[0063] in:

[0064] n1 is an integer from 0 to 7 (0 ≤ n1 ≤ 7).

[0065] X is a sulfur atom.

[0066] * indicates the dextran backbone and the divalent group L(-). i The connection site,

[0067] ·

[0068] or

[0069] •-G1- is a succinimide derivative as described in the following formula:

[0070]

[0071] in:

[0072] ·X is a straight chain *-(CH2)n1-*, where n1 is an integer from 1 to 7 (1≤n1≤7),

[0073] * indicates the dextran backbone and the divalent group L(-). i The connection site.

[0074] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0075] -W is -(R1) m G1-group, in which

[0076] ·-(R1) m G1- is a straight-chain or branched alkyl divalent group containing fewer than 13 carbon atoms and optional heteroatoms such as oxygen, nitrogen, or sulfur.

[0077] In one embodiment, the dextran polymer is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i The result is a group generated from straight-chain or branched thiol polyethylene glycol containing at least two sulfur atoms and up to eight arms.

[0078] -Mn is 1000 to 25000 g / mol (1000 ≤ Mn ≤ 25000 g / mol), or

[0079] - The degree of polymerization (DP) is 15 to 600 (15 ≤ DP ≤ 600).

[0080] In one embodiment, the dextran polymer is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i It is not a group generated from straight-chain or branched thiol polyethylene glycol containing at least 2 sulfur atoms and at most 8 arms.

[0081] - The number-average molecular weight (Mn) is 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol), or

[0082] - The degree of polymerization (DP) is 8 to 1000 (8 ≤ DP ≤ 1000).

[0083] The properties of these hydrogels can be adjusted and customized for applications by selecting and adjusting the crosslinking reaction conditions, the degree of substitution of dextran and the molecular weight of the crosslinking agent.

[0084] The problem is solved by providing a new hydrogel, which comprises:

[0085] -Biological cells,

[0086] -Non-crosslinked hyaluronic acid salts in solution form, and

[0087] - A cross-linked dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0088] -L(-) i It is a straight-chain or branched polyether.

[0089] -i represents the valence of L and the number of W groups bound to the dextran polymer, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[0090] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[0091] In one embodiment, the crosslinked dextran polymer contained in the hydrogel according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran, which has one W group, wherein,

[0092] -L(-) i It is a straight-chain or branched polyether with heteroatoms such as oxygen, nitrogen, or sulfur at its ends.

[0093] -i is the valence of L and -(R1) m The number of G1- groups, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[0094] -m is an integer equal to 0 or 1.

[0095] -W is -(R1) m G1-group, in which

[0096] •-R1- is a straight-chain or branched alkyl divalent group containing 1 to 6 carbon atoms and optional heteroatoms such as oxygen, nitrogen, or sulfur.

[0097] •-G1- is a straight-chain, branched, or cyclic alkyl divalent group containing 1 to 6 carbon atoms, and may contain heteroatoms such as oxygen, nitrogen, or sulfur.

[0098] In one embodiment, the dextran polymer contained in the hydrogel is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i The result is a group generated from straight-chain or branched thiol polyethylene glycol containing at least two sulfur atoms and up to eight arms.

[0099] -Mn is 1000 to 25000 g / mol (1000 ≤ Mn ≤ 25000 g / mol), or

[0100] - Degree of polymerization (DP) is 15 to 600 (15 ≤ DP ≤ 600).

[0101] In one embodiment, the dextran polymer contained in the hydrogel is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i It is not a group generated from straight-chain or branched thiol polyethylene glycol containing at least 2 sulfur atoms and at most 8 arms.

[0102] - Number-average molecular weight (Mn) of 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol), or

[0103] - The degree of polymerization (DP) is 8 to 1000 (8 ≤ DP ≤ 1000).

[0104] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[0105] -L(-) i It is a linear or branched polyether, or L(-) i Is it a straight-chain or branched polymer? (zoline),

[0106] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[0107] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyethers or poly( (Azoline) derivatives, and do not contain two or more α-amino acid residues, especially α-amino acid residues linked by peptide bonds.

[0108] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[0109] -L(-) i It is a straight-chain or branched polyether.

[0110] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[0111] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[0112] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0113] -L indicates straight-chain or branched polymer ( (zoline),

[0114] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[0115] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[0116] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0117] -L(-) i It is a straight-chain or branched polyether with heteroatoms such as oxygen, nitrogen, or sulfur at its ends.

[0118] -i is the valence of L and -(R1) mThe number of G1- groups, and is an integer from 2 to 8 (2 ≤ i ≤ 8), where -m is an integer equal to 0 or 1.

[0119] -W is -(R1) m G1-group, in which

[0120] •-R1- is a straight-chain or branched alkyl divalent group containing 1 to 6 carbon atoms and optional heteroatoms such as oxygen, nitrogen, or sulfur.

[0121] •-G1- is a straight-chain, branched, or cyclic alkyl divalent group containing 1 to 6 carbon atoms, and may contain heteroatoms such as oxygen, nitrogen, or sulfur.

[0122] In one embodiment, the dextran polymer is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i The result is a group generated from straight-chain or branched thiol polyethylene glycol containing at least two sulfur atoms and up to eight arms, wherein

[0123] - The number-average molecular weight Mn is 1000 to 25000 g / mol (1000 ≤ Mn ≤ 25000 g / mol), or

[0124] - Degree of polymerization (DP) is 15 to 600 (15 ≤ DP ≤ 600).

[0125] In one embodiment, the dextran polymer is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i Not a group derived from straight-chain or branched thiol polyethylene glycol containing at least 2 sulfur atoms and at most 8 arms, wherein

[0126] - Number-average molecular weight (Mn) of 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol), or

[0127] - The degree of polymerization (DP) is 8 to 1000 (8 ≤ DP ≤ 1000).

[0128] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0129] -W is -(R1) m G1-group, in which

[0130] •-G1- is a sulfone derivative as described in the following formula:

[0131]

[0132] in:

[0133] n1 is an integer from 0 to 7 (0 ≤ n1 ≤ 7).

[0134] X is a sulfur atom.

[0135] * indicates the dextran backbone and the divalent group L(-). i The connection site,

[0136] ·

[0137] or

[0138] •-G1- is a succinimide derivative as described in the following formula:

[0139]

[0140] in:

[0141] ·X is a straight chain *-(CH2)n1-*, where n1 is an integer from 1 to 7 (1≤n1≤7),

[0142] * indicates the dextran backbone and the divalent group L(-). i The connection site.

[0143] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0144] -W is -(R1) m G1-group, in which

[0145] ·-(R1) m G1- is a straight-chain or branched alkyl divalent group containing fewer than 13 carbon atoms and optional heteroatoms such as oxygen, nitrogen, or sulfur.

[0146] It also relates to implants incorporating the hydrogel of the present invention.

[0147] The applicant unexpectedly discovered that the presence of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the crosslinking mixture helps improve the uniform redistribution of cells or islets within the hydrogel. In other words, this reduces the impact of cell or islet sedimentation.

[0148] In one embodiment, the hydrogel comprises hyaluronic acid or sodium or potassium hyaluronic acid.

[0149] In one embodiment, the weight average molecular weight (Mw) of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is from 100 to 2,500 kg / mol.

[0150] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 250 to 2500 kg / mol.

[0151] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 500 to 2250 kg / mol.

[0152] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 750 to 2000 kg / mol.

[0153] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 1,000 to 1,500 kg / mol.

[0154] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 250 to 4000 kg / mol.

[0155] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 500 to 3750 kg / mol.

[0156] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 750 to 3500 kg / mol.

[0157] In one embodiment, the Mw of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 1,000 to 3,250 kg / mol.

[0158] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is 0.5 to 30 mg / ml.

[0159] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is 0.5 to 20 mg / ml.

[0160] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is 0.5 to 10 mg / ml.

[0161] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is 0.5 to 5 mg / ml.

[0162] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is from 0.75 to 2.5 mg / ml.

[0163] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is 1.0 to 1.5 mg / ml.

[0164] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate in the hydrogel is 0.8 to 1.2 mg / ml.

[0165] In one embodiment of hyaluronic acid having a Mw of 2,000 to 4,000 kg / mol, particularly about 3,000 kg / mol, the concentration is 0.5 to 1.5 mg / ml.

[0166] In one embodiment of hyaluronic acid having a Mw of 2,000 to 4,000 kg / mol, particularly about 3,000 kg / mol, the concentration is 0.7 to 1.2 mg / ml.

[0167] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 0.5 to 2 mg / ml, with a Mw of 1,000 to 2,000 kg / mol, particularly about 1,500 kg / mol.

[0168] In one embodiment, the concentration of hyaluronic acid, sodium hyaluronate, or potassium hyaluronate is 1.0 to 1.5 mg / ml, with a concentration of 1,000 to 2,000 kg / mol, particularly about 1,500 kg / mol.

[0169] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[0170] -L(-) i It is a linear or branched polyether, or L(-) i Is it a straight-chain or branched polymer? (zoline),

[0171] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[0172] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyethers or poly( (Azoline) derivatives, and do not contain two or more α-amino acid residues, especially α-amino acid residues linked by peptide bonds.

[0173] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[0174] -L(-) i It is a straight-chain or branched polyether.

[0175] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[0176] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[0177] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[0178] -L indicates straight-chain or branched polymer ( (zoline),

[0179] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[0180] --W- is a group containing at least one straight-chain or branched alkyl group, and optionally contains heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[0181] In one implementation, -W- contains up to 60 carbon atoms.

[0182] In one implementation, -W- contains up to 60 carbon atoms without counting any -CH2-CH2O- groups.

[0183] In one implementation, -W- contains up to 50 carbon atoms.

[0184] In one embodiment, -W- contains up to 50 carbon atoms without counting any -CH2-CH2O- groups.

[0185] In one implementation, -W- contains up to 40 carbon atoms.

[0186] In one embodiment, without counting any -CH2-CH2O- groups, -W- contains up to 40 carbon atoms.

[0187] In one implementation, -W- contains up to 30 carbon atoms.

[0188] In one embodiment, -W- contains up to 30 carbon atoms without counting any -CH2-CH2O- groups.

[0189] In one implementation, -W- contains up to 20 carbon atoms.

[0190] In one embodiment, without counting any -CH2-CH2O- groups, -W- contains up to 20 carbon atoms.

[0191] In one implementation, -W- contains up to 10 carbon atoms.

[0192] In one embodiment, without counting any -CH2-CH2O- groups, -W- contains up to 10 carbon atoms.

[0193] In one implementation, -W- contains up to 10 oxygen atoms.

[0194] In one embodiment, without counting any -CH2-CH2O- groups, -W- contains up to 10 oxygen atoms.

[0195] In one implementation, -W- contains up to 5 oxygen atoms.

[0196] In one embodiment, without counting any -CH2-CH2O- groups, -W- contains up to 5 oxygen atoms.

[0197] The cross-linked dextran hydrogel according to the present invention is a dextran polymer, wherein the central linker L(-) i It is a straight-chain or branched polyethylene glycol (PEG) group.

[0198] Branched PEG refers to multiple PEG arms consisting of straight, branched, or cyclic alkyl groups or aromatic groups, containing 2 to 20 carbon atoms, and may contain heteroatoms such as nitrogen, oxygen, or sulfur.

[0199] In one embodiment, the crosslinked dextran hydrogel according to the invention is a dextran polymer, wherein the central linker L(-) i It is a branched PEG group with up to 8 arms.

[0200] In one implementation, the center connector L(-) i It is a PEG selected from formula I:

[0201]

[0202] in:

[0203] • i is an integer from 2 to 8 (2 ≤ i ≤ 8)

[0204] p is an integer equal to 0 or 1, and if i = 2, then p = 0.

[0205] ·q is an integer between 8 and 1000 (8 ≤ q ≤ 1000)

[0206] ·r is an integer equal to 0 or 1

[0207] • Q is a carbon atom, or a straight-chain, branched, or cyclic alkyl chain, or aromatic chain containing 2 to 10 carbon atoms and may contain heteroatoms such as nitrogen, oxygen, or sulfur.

[0208] * indicates the site of f4, which is an amine functional group, or an ether, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond, if the crosslinking process is carried out by native chemical linkage (NCL).

[0209] In one implementation, q is an integer from 80 to 500 (80 ≤ q ≤ 500).

[0210] In one implementation, q is an integer from 100 to 300 (100 ≤ q ≤ 300).

[0211] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group derived from straight-chain or branched thiol polyethylene glycol containing at least 2 sulfur atoms and at most 8 arms, wherein:

[0212] - Number-average molecular weight (Mn) of 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol), or

[0213] - The degree of polymerization (DP) is 8 to 1000 (8 ≤ DP ≤ 1000).

[0214] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-)i is a group generated from a straight-chain or branched thiol containing at least 2 sulfur atoms and at most 8 arms, wherein:

[0215] -Mn is 1000 to 25000 g / mol (1000 ≤ Mn ≤ 25000 g / mol), or

[0216] - Degree of polymerization (DP) is 15 to 600 (15 ≤ DP ≤ 600).

[0217] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-)i is a group according to Formula I generated from thiol polyethylene glycol or mercaptopoly(oxyethylene) listed in the table below:

[0218] Chemical name i Mn (kg / mol) Poly(ethylene glycol) dithiol 2 10 Poly(ethylene glycol) dithiol 2 3.4 Poly(ethylene glycol) dithiol 2 1 Pentaerythritol tetra(mercaptoethyl)polyoxyethylene 4 5.2 Pentaerythritol tetra(mercaptoethyl)polyoxyethylene 4 20 Pentaerythritol tetra(mercaptoethyl)polyoxyethylene 4 40 Tripentaerythritol octa(mercaptoethyl) polyoxyethylene 8 20

[0219] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to formula I generated from pentaerythritol tetra(mercaptoethyl)polyoxyethylene (CAS#188492-68-4).

[0220] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to formula I generated from linear (mercaptoethyl) polyoxyethylene (CAS#68865-60-1).

[0221] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to Formula I generated from the pentaerythritol poly(ethylene oxide) azides listed in the table below:

[0222]

[0223] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to formula I generated from pentaerythritol 4-arm PEG azide (CAS#225531-50-0).

[0224] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to Formula I generated from pentaerythritol poly(dibenzocyclooctylene)polyoxyethylene listed in the table below:

[0225]

[0226] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-)i It is a group according to formula I produced by pentaerythritol 4-arm PEG DBCO.

[0227] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i These are groups derived from maleimide polyethylene glycol according to Formula I, as listed in the table below:

[0228]

[0229] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to formula I produced by 4-arm poly(ethylene glycol)maleimide.

[0230] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group derived from norbornene polyethylene glycol according to Formula I, as listed in the table below:

[0231]

[0232] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer wherein L(-) i It is a group according to formula I produced by 4-arm poly(ethylene glycol)norbornene.

[0233] In one embodiment, the crosslinked dextran hydrogel according to the invention is a dextran polymer, wherein the central linker L is a straight-chain or branched POx group.

[0234] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which L is a straight-chain or branched POx group comprising up to 8 arms, having a number-average molecular weight (Mn) of 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol).

[0235] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which L is a straight-chain or branched POx group comprising up to 8 arms, having a number-average molecular weight (Mn) of 1,000 to 25,000 g / mol (1,000 ≤ Mn ≤ 25,000 g / mol).

[0236] In one implementation, the POx center connector is a 2-arm POx, selected from the connector of formula XII.

[0237]

[0238] in:

[0239] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[0240] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0241] In one implementation, the POx center connector is a 2-arm POx, selected from the connector of type XIIbis.

[0242]

[0243] in:

[0244] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[0245] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0246] In one implementation, the POx center connector is a 4-arm POx, selected from the connector of formula XIII.

[0247]

[0248] in:

[0249] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[0250] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0251] In one implementation, the POx center connector is a 4-arm POx, selected from the connectors of type XIV:

[0252]

[0253] in:

[0254] • The group -R1 is a straight-chain -(CH2) group.n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[0255] The divalent group -R2- is a straight-chain -(CH2) group. n2 - where n2 is an integer from 2 to 6 (2≤n2≤6).

[0256] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0257] In one implementation, the POx center connector is a 4-arm POx, selected from the connectors of type XV:

[0258]

[0259] in:

[0260] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[0261] • In one implementation, R1 = -CH2-CH2-, and R2 is a straight-chain -(CH2) n2 - where n2 is an integer from 2 to 6 (2 ≤ n2 ≤ 6),

[0262] • In another implementation, R2 = -CH2-CH2-, and R1 is a straight-chain *-(CH2) n2 -*, where n2 is an integer from 2 to 6 (2 ≤ n2 ≤ 6),

[0263] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0264] The hydroxyl functional group of the dextran polymer Dx- can be functionalized by at least one specific anionic group, such as an alkyl carboxylate, sulfate anion, sulfonate anion, phosphate anion, or phosphonate anion.

[0265] In one embodiment, the hydroxyl functional group of the dextran polymer Dx- can be functionalized with sulfate anions in salt form, and optionally with alkyl carboxylic acid ester derivatives in salt form.

[0266] In another embodiment, the hydroxyl functional group of the dextran polymer Dx- can be functionalized with a sulfonate anion in salt form, and optionally with an alkyl carboxylic acid ester derivative in salt form.

[0267] In another embodiment, the hydroxyl functional groups of the dextran polymer backbone Dx- can be functionalized with phosphate anions in salt form, and optionally with alkyl carboxylic acid ester derivatives in salt form.

[0268] In another embodiment, the hydroxyl functional group of the dextran polymer Dx- can be functionalized with a phosphonate anion in salt form, and optionally with an alkyl carboxylic acid ester derivative in salt form.

[0269] In another embodiment, the hydroxyl functional group of the dextran polymer Dx- can be functionalized with an alkyl carboxylic acid ester derivative in salt form.

[0270] In one embodiment, the hydroxyl functional group of the dextran polymer Dx- is functionalized with a specific anionic group: an alkyl carboxylate anionic functionalization.

[0271] In one embodiment, the hydroxyl functional group of the dextran polymer Dx- is functionalized by only one specific anionic group: alkyl carboxylate anionic functionalization.

[0272] The previously defined specific anionic group is selected from groups of formula II:

[0273]

[0274] in:

[0275] * indicates that it is attached to the O atom of dextran to form an ether functional group.

[0276] ·y = 2 or 3.

[0277] When y = 2, for alkyl carboxylic acid ester derivatives, then:

[0278] οY=C, and a=1.

[0279] οk = 1, l = 0, and m = 0.

[0280] οR2 = alkyl.

[0281] When y = 3, and it represents an anionic group, then:

[0282] οY=S and a=1, or Y=P and a=2.

[0283] οk = 0 or 1.

[0284] οl = 0 or 1.

[0285] οm = 0 or 1.

[0286] on = 1 or 2. Especially when n = 1,

[0287] οo = 0 or 1.

[0288] If l = 1, then m = 1.

[0289] οR3 = straight-chain, branched, or cyclic alkyl group, which may contain a heteroatom such as nitrogen, or an aromatic group, or PEG.

[0290] οR2 = alkyl.

[0291] • And Z is a counter ion, which can be an alkali metal and z = 1, or it can be an alkaline earth metal and z = 2.

[0292] In a preferred embodiment, the dextran backbone Dx- can be functionalized with sulfate anions in salt form, and optionally with alkyl carboxylic acid ester derivatives in salt form.

[0293] In another preferred embodiment, the dextran backbone Dx- can be functionalized with alkyl sulfonate anions in salt form, and optionally with alkyl carboxylic acid ester derivatives in salt form.

[0294] In another preferred embodiment, the dextran backbone Dx- can be functionalized with a sulfonate anion in salt form (which is supported by an alkyl chain containing a dimethyl-ammonium cation) and optionally with an alkyl carboxylic acid ester derivative in salt form.

[0295] In another preferred embodiment, the dextran backbone Dx- can be functionalized with an alkyl carboxylic acid ester derivative in salt form.

[0296] In one embodiment, the crosslinked dextran polymer with anionic groups according to the invention is a dextran polymer wherein the dextran polymer backbone is according to Formula III.

[0297] Where R is selected from

[0298] --H, an anionic group of formula II, or containing L(-) i The -W- group of the crosslinker has an -i value of 20 to 5000 (20 ≤ i ≤ 5000).

[0299] --W- and L(-) i The group has the meaning previously defined.

[0300] In one embodiment, the crosslinked dextran polymer with anionic groups according to the invention is a dextran polymer wherein the dextran polymer backbone is according to formula XI.

[0301]

[0302] Where R is selected from

[0303] --H, an anionic group of formula II, or containing L(-) i -(A-f2) of the crosslinking group a -G1- group,

[0304] -I is between 20 and 5000 (20 ≤ I ≤ 5000),

[0305] --(A-f2) a -G1- and L(-) I The group has the meaning previously defined.

[0306] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 1000 kDa before crosslinking and substitution.

[0307] In other words, the crosslinked dextran polymer according to the present invention is obtained by substitution and crosslinking of a natural dextran polymer with a weight-average molecular weight (Mw) of 5 to 1000 kDa.

[0308] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution.

[0309] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 100 kDa before crosslinking and substitution.

[0310] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 50 kDa before crosslinking and substitution.

[0311] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 25 kDa before crosslinking and substitution.

[0312] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution.

[0313] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is a dextran with a weight-average molecular weight (Mw) of 10 to 500 kDa before crosslinking and substitution.

[0314] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 500 kDa before crosslinking and substitution.

[0315] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution.

[0316] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 50 kDa before crosslinking and substitution.

[0317] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 40 to 250 kDa before crosslinking and substitution.

[0318] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 40 to 100 kDa before crosslinking and substitution.

[0319] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0320] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings.i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0321] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0322] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0323] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0324] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0325] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0326] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. iThe degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0327] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0328] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0329] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0330] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0331] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0332] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0333] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0334] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.2 to 0.4 (0.2≤DS1≤0.4).

[0335] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.2 to 0.4 (0.2≤DS1≤0.4).

[0336] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.2 to 0.4 (0.2≤DS1≤0.4).

[0337] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.2 to 0.3 (0.2≤DS1≤0.3).

[0338] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.2 to 0.3 (0.2≤DS1≤0.3).

[0339] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.2 to 0.3 (0.2≤DS1≤0.3).

[0340] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0341] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0342] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0343] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0344] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0345] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0346] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group aThe degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0347] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0348] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0349] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0350] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i The degree of substitution (DS1) of the dextran backbone of the crosslinking group (-W- group) is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0351] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and has an L(-) group. i -(A-f2) of the crosslinking group aThe degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0352] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 0.3 to 2.5 (0.3 ≤ DS2 ≤ 2.5).

[0353] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 0.5 to 2.3 (0.5 ≤ DS2 ≤ 2.3).

[0354] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 1.5 to 2.5 (1.5 ≤ DS2 ≤ 2.5).

[0355] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 1.7 to 2.3 (1.7 ≤ DS2 ≤ 2.3).

[0356] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 1.8 to 2.2 (1.8 ≤ DS2 ≤ 2.5).

[0357] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 0.3 to 1.5 (0.3 ≤ DS2 ≤ 1.5).

[0358] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone having total carboxylic acid methyl ester / salt grafted onto dextran is in the range of 0.3 to 0.8 (0.3 ≤ DS2 ≤ 0.8).

[0359] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone having a group of formula I or a group of formula XII, XIIbis, XIII or XIV is in the range of 0.5 to 3 (0.5 ≤ DS4 ≤ 3).

[0360] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having the groups of Formula I is in the range of 0.5 to 3 (0.5 ≤ DS4 ≤ 3).

[0361] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone having groups of formula XII, XIIbis, XIII or XIV is in the range of 0.5 to 3 (0.5 ≤ DS4 ≤ 3).

[0362] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having a group of formula I or a group of formula XII, XIIbis, XIII or XIV is in the range of 1 to 2.75 (1 ≤ DS4 ≤ 2.75).

[0363] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having the groups of Formula I is in the range of 1 to 2.75 (1 ≤ DS4 ≤ 2.75).

[0364] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone having groups of formula XII, XIIbis, XIII or XIV is in the range of 1 to 2.75 (1 ≤ DS4 ≤ 2.75).

[0365] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having a group of formula I or a group of formula XII, XIIbis, XIII or XIV is in the range of 1.5 to 2.5 (1.5 ≤ DS4 ≤ 2.5).

[0366] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having the groups of Formula I is in the range of 1.5 to 2.5 (1.5 ≤ DS4 ≤ 2.5).

[0367] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone having groups of formula XII, XIIbis, XIII or XIV is in the range of 1.5 to 2.5 (1.5 ≤ DS4 ≤ 2.5).

[0368] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having a group of formula I or a group of formula XII, XIIbis, XIII or XIV is in the range of 1.75 to 2.25 (1.75 ≤ DS4 ≤ 2.25).

[0369] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone having the groups of Formula I is in the range of 1.75 to 2.25 (1.75 ≤ DS4 ≤ 2.25).

[0370] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone having groups of formula XII, XIIbis, XIII or XIV is in the range of 1.75 to 2.25 (1.75 ≤ DS4 ≤ 2.25).

[0371] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS) of the carboxylic acid ester / salt of the dextran backbone is... C Within the range of 0.2 to 3 (0.2 ≤ DS) C ≤3).

[0372] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS) of the carboxylic acid ester / salt of the dextran backbone is... C Within the range of 0.3 to 2.5 (0.3 ≤ DS) C ≤2.5).

[0373] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS3) of the sulfate / salt, sulfonate / salt, phosphate / salt, and phosphonate / salt of the dextran backbone is in the range of 0.2 to 2.5 (0.2 ≤ DS3 ≤ 2.5).

[0374] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the degree of substitution (DS3) of the sulfate / salt, sulfonate / salt, phosphate / salt, and phosphonate / salt of the dextran backbone is in the range of 0.3 to 2.0 (0.3 ≤ DS3 ≤ 2.0).

[0375] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having a -W- group or -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio (DC) between the molar concentrations of the reactive functional groups is in the range of 0.5 to 1.5 (0.5 ≤ DC ≤ 1.5).

[0376] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the molar concentration of the -W- groups is related to the molar concentration of the crosslinking group L(-). i The molar ratio (DC) between the molar concentrations of the reactive functional groups is in the range of 0.5 to 1.5 (0.5 ≤ DC ≤ 1.5).

[0377] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio (DC) between the molar concentrations of the reactive functional groups is in the range of 0.5 to 1.5 (0.5 ≤ DC ≤ 1.5).

[0378] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having a -W- group or -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.8 to 1.2 (0.8 ≤ DC ≤ 1.2).

[0379] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the molar concentration of the -W- groups is related to the molar concentration of the crosslinking group L(-). i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.8 to 1.2 (0.8 ≤ DC ≤ 1.2).

[0380] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-)i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.8 to 1.2 (0.8 ≤ DC ≤ 1.2).

[0381] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having a -W- group or -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.9 to 1.1 (0.9 ≤ DC ≤ 1.1).

[0382] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the molar concentration of the -W- groups is related to the molar concentration of the crosslinking group L(-). i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.9 to 1.1 (0.9 ≤ DC ≤ 1.1).

[0383] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.9 to 1.1 (0.9 ≤ DC ≤ 1.1).

[0384] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having a -W- group or -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.95 to 1.05 (0.95 ≤ DC ≤ 1.05).

[0385] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the molar concentration of the -W- groups is related to the molar concentration of the crosslinking group L(-). i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.95 to 1.05 (0.95 ≤ DC ≤ 1.05).

[0386] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is in the range of 0.95 to 1.05 (0.95 ≤ DC ≤ 1.05).

[0387] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having a -W- group or -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is 1 (DC = 1).

[0388] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer in which the molar concentration of the -W- groups is related to the molar concentration of the crosslinking group L(-). i The molar ratio between the molar concentrations of the reactive functional groups is 1 (DC = 1).

[0389] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having -(A-f2) a The molar concentration of the -G1- group and the crosslinking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is 1 (DC = 1).

[0390] In one embodiment, the crosslinked dextran polymer according to the invention is composed of a reactive functional group of precursor -W- and precursor L(-). i The cross-linked dextran polymer is obtained by the reaction between reactive functional groups, wherein the reactive functional groups are present at the same concentration (DC = 1) and in the range of 5 to 25 mM.

[0391] In one implementation, it is in the range of 5 to 10 mM.

[0392] In one implementation, it is in the range of 10 to 15 mM.

[0393] In one implementation, it is in the range of 15 to 20 mM.

[0394] In one implementation, it is in the range of 20 to 25 mM.

[0395] In one embodiment, -W- is selected from groups of formula IV.

[0396]

[0397] in

[0398] * indicates the site of f1, and Indicates the site of connection with L.

[0399] ·a is an integer equal to 0 or 1.

[0400] b is an integer equal to 0 or 1.

[0401] c is an integer equal to 0 or 1.

[0402] • In one embodiment, a = 0, and f1 is an ether functional group or a carbamate functional group.

[0403] In one implementation scheme, a = 1.

[0404] The divalent group -A- is a straight-chain -(CH2). n1 -(where n1 is an integer from 1 to 7 (1 ≤ n1 ≤ 7)), branched or cyclic alkyl derivatives. It can also be branched by at least one hydroxyl group to form -CH2-CH(OH)-(CH2) where n2 is an integer from 1 to 5 (1 ≤ n2 ≤ 5). n2 -; f1 is an ether functional group or a carbamate functional group, and f2 is an amide functional group.

[0405] or,

[0406] The divalent group -A- is a linear polyether (PEG) derivative; f1 is an ether functional group or a urethane functional group, and f2 is an amide functional group.

[0407] or,

[0408] In another embodiment, the divalent group -A- is a 4-alkyl-1,4-triazole derivative or a 4-PEG-1,4-triazole derivative; f1 is an ether functional group or a carbamate functional group, and f2 is a carbon-nitrogen covalent bond.

[0409] or,

[0410] In another embodiment, the divalent group -A- is a 1-alkyl-1,4-triazole derivative or a 1-PEG-1,4-triazole derivative; f1 is an ether functional group or a carbamate functional group, and f2 is a carbon-aromatic carbon covalent bond.

[0411] • The divalent group -R1- is a straight-chain, branched or cyclic alkyl derivative, and / or an aromatic derivative, and / or a polyether (PEG) derivative, which may contain heteroatoms such as nitrogen, oxygen or sulfur.

[0412] If b = 0, then f1 is an ether functional group or a carbamate functional group.

[0413] If b = 1, then f1 is an ether functional group or a carbamate functional group, and f3 is an amide functional group, or an amine functional group, or an ether functional group, or a thioether functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond (if the crosslinking process is carried out by natural chemical linkage (NCL)).

[0414] The divalent group -G1- is a straight-chain, branched, or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, such as up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms, or up to 1 phosphorus atom. In a preferred embodiment, -G1- is a succinimide derivative, or an alkyl sulfone derivative (which may contain a heteroatom such as oxygen or sulfur), or an ethylamide derivative, or a 1,4-triazole derivative, or a polycyclic derivative derived from the Diels-Alder reaction, or an aromatic phosphine derivative generated by Staudinger ligation, or a naturally chemically linked cysteine ​​derivative.

[0415] If c = 0, then f1 is an ether functional group or a carbamate functional group.

[0416] If c = 1, then f1 is an ether functional group or a carbamate functional group, and f4 is an amine functional group, or an amide functional group, or a carbamate functional group, or a thioether functional group, or an ether functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond (if the crosslinking process is carried out by natural chemical linkage (NCL)).

[0417] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the backbone of a dextran polymer having i groups, wherein the dextran polymer is selected from dextran of formula X.

[0418]

[0419] in:

[0420] ·a is an integer equal to 0 or 1.

[0421] • i is an integer from 2 to 8 (2≤i≤8).

[0422] • L can be the same as [Dx-f1-(A-f2)] a [-G1-f3] groups, or linked to different [Dx-f1-(A-f2] groups. a [-G1-f3] group linkage.

[0423] • Dx- is the dextran moiety, which may be substituted with a specific anionic group in salt form, and optionally with an alkyl carboxylic acid ester derivative in salt form.

[0424] •f1 is an ether functional group.

[0425] The divalent group -A- is a straight-chain -(CH2) group. n1-(where n1 is an integer from 1 to 7 (1 ≤ n1 ≤ 7)), branched or cyclic alkyl derivatives. It can also be branched by at least one hydroxyl group to form -CH2-CH(OH)-(CH2) where n2 is an integer from 1 to 5 (1 ≤ n2 ≤ 5). n2 -

[0426] ·f2 is an amide functional group.

[0427] The divalent group -G1- is a straight-chain, branched, or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, such as up to 5 nitrogen atoms, up to 10 oxygen atoms, or up to 5 sulfur atoms. In a preferred embodiment, -G1- is a succinimide derivative, or an alkyl sulfone derivative (which may contain one heteroatom such as oxygen or sulfur), or a 1,4-triazole derivative.

[0428] • Integer i is the valence of the central connector L, and whether it is the same as or different from the one connected to L [Dx-f1-(A-f2)]. a The number of -G1-f3] groups.

[0429] •f3 is an amine functional group, or a thioether functional group, or an ether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0430] • Center connector L is poly( Azoline (POx) derivative, which can be linear or branched.

[0431] In this embodiment, the crosslinked dextran polymer according to the present invention is selected from dextran polymers of formula V.

[0432]

[0433] in

[0434] • f1, f2, f3, f4, -A-, -R1-, -G1- are as defined in equation IV above, and

[0435] • Dx- is the dextran moiety, which may be substituted with a specific anionic group in salt-forming form, and optionally with an alkyl carboxylic acid ester derivative in salt-forming form as previously defined.

[0436] • Integer i is the valence of the central connector L, and whether it is the same as or different from the one connected to L [Dx-f1-(A-f2)]. a -(R1-f3) b -(G1-f4) c The number of groups.

[0437] • The central connector L is a polyether (PEG) derivative, which can be linear or branched.

[0438] • In one implementation, if b = 0 and c = 1, then the center connector L can be poly( Azoline (POx) derivative, which can be linear or branched.

[0439] In this embodiment, the crosslinked dextran polymer according to the present invention is selected from dextran polymers of formula V.

[0440]

[0441] in

[0442] • f1, f2, f3, f4, -A-, -R1-, -G1- are as defined in equation IV above, and

[0443] • Dx- is the dextran moiety, which may be substituted with a specific anionic group in salt-forming form, and optionally with an alkyl carboxylic acid ester derivative in salt-forming form as previously defined.

[0444] • Integer i is the valence of the central connector L, and whether it is the same as or different from the one connected to L [Dx-f1-(A-f2)]. a -(R1-f3) b -(G1-f4) c The number of groups.

[0445] • The central connector L is a polyether (PEG) derivative, which can be linear or branched.

[0446] In one implementation, L is the same as [Dx-f1-(A-f2)]. a -(R1-f3) b -(G1-f4) c [Group linkage]

[0447] In one implementation, L is associated with different [Dx-f1-(A-f2)] a -(R1-f3) b -(G1-f4) c [Group linkage]

[0448] If a = 0, then:

[0449] • In one implementation, f1 is an ether functional group.

[0450] • In another embodiment, f1 is a carbamate functional group.

[0451] If a = 1, then:

[0452] • In one implementation, f1 is an ether functional group.

[0453] • In another embodiment, f1 is a carbamate functional group.

[0454] If a = 0 and b = 0, then:

[0455] • In one implementation, f1 is an ether functional group.

[0456] • In another embodiment, f1 is a carbamate functional group.

[0457] If a = 0 and b = 1, then:

[0458] • In one implementation, f1 is an ether functional group.

[0459] • In another embodiment, f1 is a carbamate functional group.

[0460] If a = b = c = 0, then:

[0461] • In one implementation, f1 is an ether functional group.

[0462] • In another embodiment, f1 is a carbamate functional group.

[0463] In one embodiment, corresponding to formula V, the divalent group -A- is a linear polyether (PEG) derivative selected from the following formulas:

[0464]

[0465] in:

[0466] ·n1 is an integer equal to 0 or 1.

[0467] • n2 is an integer from 1 to 7 (1≤n1≤7).

[0468] * indicates the sites f1 and f2.

[0469] • In a preferred embodiment, * denotes sites f1 and f2, where f1 and f2 are ether functional groups and amide functional groups, respectively.

[0470] In another embodiment, corresponding to formula V, the divalent group -A- is a 1-alkyl-1,4-triazole derivative selected from triazole derivatives of the following formulas or 1-PEG-1,4-triazole derivatives:

[0471]

[0472] in:

[0473] X is a straight-chain *-(CH2) n1-* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is a PEG derivative.

[0474] * indicates the site of f1, and the dashed line indicates f2.

[0475] In another embodiment, corresponding to formula V, the divalent group -A- is a 4-alkyl-1,4-triazole derivative selected from triazole derivatives of the following formulas:

[0476]

[0477] in:

[0478] X is a straight-chain *-(CH2) n1 -* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is a PEG derivative.

[0479] * indicates the site of f1, and the dashed line indicates f2.

[0480] If a = 1, then:

[0481] • In one embodiment, f2 is an amide functional group.

[0482] • In another embodiment, f2 is a carbon-nitrogen covalent bond.

[0483] • In another embodiment, f2 is a carbon-aromatic carbon covalent bond.

[0484] In one implementation, corresponding to formula X, -A- is a straight-chain -(CH2). n1 -(where n1 is an integer from 1 to 7 (1 ≤ n1 ≤ 7)), branched or cyclic alkyl derivatives. It can also be branched by at least one hydroxyl group to form -CH2-CH(OH)-(CH2) where n2 is an integer from 1 to 5 (1 ≤ n2 ≤ 5). n2 -

[0485] In one embodiment, corresponding to formula V, the divalent group -R1- is a straight-chain alkyl derivative according to the following formula:

[0486]

[0487] in:

[0488] n1 is an integer from 1 to 7 (1≤n1≤7).

[0489] • In one implementation, if a = 1, then * represents the sites f2 and f3.

[0490] • In another implementation, if a = 0, then * represents the sites f1 and f3.

[0491] In another embodiment, corresponding to formula V, the divalent group -R1- is a polyether (PEG) derivative according to the following formula:

[0492]

[0493] in:

[0494] ·n1 is an integer equal to 0 or 1.

[0495] • n2 is an integer from 1 to 7 (1≤n2≤7).

[0496] • In one implementation, if a = 1, then * represents the sites f2 and f3.

[0497] • In another implementation, if a = 0, then * represents the sites f1 and f3.

[0498] In another embodiment, corresponding to formula V, if a = 0, the divalent group -R1- can be a branched alkyl group, wherein at least one hydroxyl group is connected to an alkyl chain at the β-position relative to f1, said f1 being an ether functional group.

[0499]

[0500] in:

[0501] • n2 is an integer from 1 to 5 (1 ≤ n2 ≤ 5).

[0502] • In another implementation, if a = 0, then * represents the sites f1 and f3.

[0503] In a preferred embodiment, corresponding to formula V, the divalent group -R1- is a straight-chain alkyl derivative according to the following formula:

[0504]

[0505] in:

[0506] * indicates sites f2 and f3, where f2 is an amide functional group and f3 is an amide functional group.

[0507] In a preferred embodiment, corresponding to formula V, the divalent group -R1- is a PEG derivative according to the following formula:

[0508]

[0509] in:

[0510] n1 is an integer from 1 to 7 (1≤n1≤7).

[0511] * indicates the sites f2 and f3, which are two amide functional groups.

[0512] If b = 1, then:

[0513] • In one implementation, f3 is an amine functional group.

[0514] • In another embodiment, f3 is an ether functional group.

[0515] • In another embodiment, f3 is a thioether functional group.

[0516] • In another embodiment, f3 is an amide functional group.

[0517] • In another embodiment, f3 is a carbamate functional group.

[0518] • In another embodiment, f3 is a carbon-nitrogen covalent bond.

[0519] • In another embodiment, f3 is a carbon-aromatic carbon covalent bond.

[0520] • In another implementation, if the crosslinking process is carried out by natural chemical linkage (NCL), then f3 is a carbon-carbon covalent bond.

[0521] The properties of the G1 group depend on the crosslinking process, and different crosslinking processes with the G1 group are described below.

[0522] In one embodiment, the crosslinking process is achieved by Michael addition with a maleimide derivative, or a vinyl sulfone derivative, or an acrylamide derivative.

[0523] In one embodiment, corresponding to formula X, the integer a = 1, the crosslinking process is achieved by Michael addition with a maleimide derivative or a vinyl sulfone derivative.

[0524] In one embodiment, corresponding to formula V, integers a = c = 1, and L is a POx derivative, and the crosslinking process is achieved by Michael addition with maleimide derivatives or vinyl sulfone derivatives.

[0525] In one embodiment, corresponding to formula V, the divalent group -G1- is a succinimide derivative according to the following formula:

[0526]

[0527] in:

[0528] X is a straight-chain *-(CH2) n1-* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is an aromatic derivative, or X is a PEG derivative.

[0529] • In one implementation, if b = 1, then * represents the sites f3 and f4.

[0530] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0531] • In another implementation, if a = 1 and b = 0, then * represents the sites f2 and f4.

[0532] • In a preferred embodiment, X is an ethyl group, and * represents sites f2 and f4, where f2 is an amide functional group and f4 is a thioether functional group.

[0533] In one embodiment, corresponding to formula X, the divalent group -G1- is a succinimide derivative according to the following formula:

[0534]

[0535] in:

[0536] • R is a straight-chain, branched, or cyclic alkyl derivative, or R is an aromatic derivative, or R is a PEG derivative.

[0537] * indicates sites f2 and f3, where f2 is an amide functional group and f3 is an amine functional group, an ether functional group, or a thioether functional group.

[0538] In one embodiment, corresponding to formula V, if b = 0, c = 1, and L is a POx derivative, then the divalent group -G1- is a succinimide derivative according to the following formula:

[0539]

[0540] in:

[0541] • R is a straight-chain, branched, or cyclic alkyl derivative, or R is an aromatic derivative, or R is a PEG derivative.

[0542] * indicates sites f2 and f3, where f2 is an amide functional group and f3 is an amine functional group, an ether functional group, or a thioether functional group.

[0543] In one embodiment, corresponding to formula X, the divalent group -G1- is a succinimide derivative according to the following formula:

[0544]

[0545] in:

[0546] X is an oxygen atom, a sulfur atom, or a nitrogen atom.

[0547] • R is a straight-chain, branched, or cyclic alkyl derivative, or R is a PEG derivative.

[0548] * indicates the site of f2, which is an amide functional group, and the dashed bond indicates f3, which is a carbon-nitrogen covalent bond.

[0549] In another embodiment, corresponding to formula V, if b = 0, c = 1, and L is a POx derivative, then the divalent group -G1- is a succinimide derivative according to the following formula:

[0550]

[0551] in:

[0552] X is an oxygen atom, a sulfur atom, or a nitrogen atom.

[0553] • R is a straight-chain, branched, or cyclic alkyl derivative, or R is a PEG derivative.

[0554] * indicates the site of f2, which is an amide functional group, and the dashed bond indicates f3, which is a carbon-nitrogen covalent bond.

[0555] In another embodiment, corresponding to formula V, the divalent group -G1- is a diethyl sulfone derivative according to the following formula:

[0556]

[0557] in:

[0558] • In one implementation, if b = 1, then * represents the sites f3 and f4.

[0559] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0560] • In another implementation, if b = 0, then * represents the sites f2 and f4.

[0561] • In a preferred embodiment, * denotes sites f3 and f4, which are thioether functional groups.

[0562] In another embodiment, corresponding to formula V, the divalent group -G1- is a sulfone derivative according to the following formula:

[0563]

[0564] in:

[0565] n1 is an integer from 0 to 7 (0≤n1≤7).

[0566] ·X is an oxygen atom, or a sulfur atom, or a CH2 group.

[0567] • In one implementation, if b = 1, then * represents the sites f3 and f4.

[0568] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0569] • In another implementation, if a = 1 and b = 0, then * represents the sites f2 and f4.

[0570] • In a preferred embodiment, a = 1, b = 0, X is a sulfur atom, n1 = 2, f2 is an amide functional group, and f4 is a thioether functional group.

[0571] In another embodiment, corresponding to formula X, the divalent group -G1- is a sulfone derivative according to the following formula:

[0572]

[0573] in:

[0574] n1 is an integer from 0 to 7 (0≤n1≤7).

[0575] ·X is an oxygen atom, or a sulfur atom, or a CH2 group.

[0576] * indicates sites f2 and f3, where f2 is an amide functional group and f3 is an amine functional group, an ether functional group, or a thioether functional group.

[0577] In another embodiment, corresponding to formula V, if b = 0, c = 1, and L is a POx derivative, then the divalent group -G1- is a sulfone derivative according to the following formula:

[0578]

[0579] in:

[0580] n1 is an integer from 0 to 7 (0≤n1≤7).

[0581] ·X is an oxygen atom, or a sulfur atom, or a CH2 group.

[0582] * indicates sites f2 and f3, where f2 is an amide functional group and f3 is an amine functional group, an ether functional group, or a thioether functional group.

[0583] In another embodiment, corresponding to formula V, the divalent group -G1- is an acrylamide derivative according to the following formula:

[0584]

[0585] in:

[0586] • In one embodiment, * denotes the site of f3, which is an amine functional group, an ether functional group, or a thioether functional group, and the dashed bond denotes f4, which is a carbon-nitrogen covalent bond.

[0587] • In another embodiment, the dashed bond represents f3, which is a carbon-nitrogen covalent bond, and * represents the site of f4, which is an amine functional group, or an ether functional group, or a thioether functional group.

[0588] In one embodiment, the crosslinking process is achieved through a 1,3-cycloaddition between an alkyne and an azide derivative, referred to as a 1,3-dipolar cycloaddition or Huisgen reaction.

[0589] In one embodiment, corresponding to formula V, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0590]

[0591] In this context, the two dashed bonds represent f3 and f4, which are covalent bonds or chemical functional groups defined before and after the bond.

[0592] In one embodiment, corresponding to formula X, where integer a = 1, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0593]

[0594] in:

[0595] R1 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or R1 is an aromatic derivative, or R1 is a PEG derivative.

[0596] * indicates the site of f2, where f2 is an amide functional group, and the dashed bond indicates f3, where f3 is a carbon-nitrogen covalent bond or a carbon-aromatic carbon covalent bond.

[0597] In one embodiment, corresponding to formula V, the integers a = c = 1, and L is a Pox derivative, and the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0598]

[0599] in:

[0600] X1 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or X1 is an aromatic derivative, or X1 is a PEG derivative.

[0601] * indicates the site of f2, where f2 is an amide functional group, and the dashed bond indicates f4, where f4 is a carbon-nitrogen covalent bond or a carbon-aromatic carbon covalent bond.

[0602] In another embodiment, corresponding to formula X, where integer a = 0, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0603]

[0604] in:

[0605] * indicates the site of f1, which is an ether functional group, and the dashed bond indicates f3, which is a carbon-nitrogen covalent bond.

[0606] In another embodiment, corresponding to formula V, integers a = 0, b = 0, c = 1, and L is a Pox derivative, and the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0607]

[0608] in:

[0609] * indicates the site of f1, which is an ether functional group, and the dashed bond indicates f4, which is a carbon-nitrogen covalent bond.

[0610] In one implementation, the crosslinking process is achieved through a 1,3-cycloaddition between the strained alkyne and the azide derivative, known as strain-promoted azide-alkyne cycloaddition, or SPAAC.

[0611] In one implementation, corresponding to formula X, where integer a = 1, the crosslinking process is achieved through a 1,3-cycloaddition between the strained alkyne and the azide derivative, referred to as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0612] In one implementation, corresponding to formula V, integers a = c = 1, b = 0, and L is a POx derivative, the crosslinking process is achieved through a 1,3-cycloaddition between the strained alkyne and the azide derivative, referred to as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0613] In one embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0614]

[0615] -in:

[0616] • The dashed circle represents a cyclooctene derivative from strained cyclooctene, which may contain a heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized as: a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen, or sulfur, or halogen, especially fluorine.

[0617] • The two dashed bonds represent f3 and f4, which are covalent bonds or chemical functional groups defined before and after the bond.

[0618] • In another implementation, if a = b = 0, then the two dashed keys represent f1 and f4.

[0619] • In another implementation, if a = 1 and b = 0, then the two dashed keys represent f2 and f4.

[0620] • In a preferred embodiment, the dashed bond represents f2 and f4, where f2 is an amide functional group and f4 is a carbon-nitrogen covalent bond.

[0621] In one embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0622]

[0623] in:

[0624] • The dashed circle represents a cyclooctene derivative from strained cyclooctene, which may contain a heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized as: a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen, or sulfur, or halogen, especially fluorine.

[0625] X is a straight-chain *-(CH2) n1 -* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is a PEG derivative.

[0626] • The two dashed bonds represent f3 and f4, which are covalent bonds or chemical functional groups defined before and after the bond.

[0627] • In another implementation, if a = b = 0, then * represents the site of f1, and the dashed key represents f4.

[0628] • In another implementation, if a = 1 and b = 0, then * represents the site of f1, and the dashed key represents f4.

[0629] • In a preferred embodiment, X is a PEG derivative, and the dashed bonds represent f2 and f4, which are amide functional groups.

[0630] In another embodiment, corresponding to formula X, the divalent group -G1- is a triazole derivative according to the following formula:

[0631]

[0632] in:

[0633] • The dashed circle represents a cyclooctene derivative from strained cyclooctene, which may contain a heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized as: a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen, or sulfur, or halogen, especially fluorine.

[0634] R1 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or R1 is an aromatic derivative, or R1 is a PEG derivative.

[0635] R2 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or R2 is an aromatic derivative, or R2 is a PEG derivative.

[0636] * indicates the site of f2, which is an amide functional group, and the dashed bond indicates f3, which is a carbon-nitrogen covalent bond, an amide functional group, or a carbamate functional group.

[0637] In another embodiment, corresponding to formula V, b = 0 and c = 1, the divalent group -G1- is a triazole derivative according to the following formula:

[0638]

[0639] in:

[0640] • The dashed circle represents a cyclooctene derivative from strained cyclooctene, which may contain a heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized as: a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen, or sulfur, or halogen, especially fluorine.

[0641] X1 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or X1 is an aromatic derivative, or X1 is a PEG derivative.

[0642] X2 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or X2 is an aromatic derivative, or X2 is a PEG derivative.

[0643] * indicates the site of f2, which is an amide functional group, and the dashed bond indicates f4, which is a carbon-nitrogen covalent bond, an amide functional group, or a carbamate functional group.

[0644] In a preferred embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0645]

[0646] Wherein: * represents the site of f2, which is an amide functional group, and the dashed bond represents f4, which is a carbon-nitrogen covalent bond.

[0647] In a preferred embodiment, corresponding to formula X, the divalent group -G1- is a triazole derivative according to the following formula:

[0648]

[0649] Wherein: * represents the site of f2, which is an amide functional group, and the dashed bond represents f3, which is a carbon-nitrogen covalent bond.

[0650] In another preferred embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0651]

[0652] in:

[0653] X is a PEG derivative.

[0654] * indicates the sites f2 and f4, where f2 and f4 are amide functional groups.

[0655] In another preferred embodiment, corresponding to formula X, the divalent group -G1- is a triazole derivative according to the following formula:

[0656]

[0657] in:

[0658] R1 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or R1 is an aromatic derivative, or R1 is a PEG derivative.

[0659] * indicates the sites f2 and f3, where f2 and f3 are amide functional groups.

[0660] In another preferred embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0661]

[0662] in:

[0663] X1 is a straight-chain, branched, or cyclic alkyl derivative that may contain heteroatoms such as oxygen, or X1 is an aromatic derivative, or X1 is a PEG derivative.

[0664] * indicates the sites f2 and f4, where f2 and f4 are amide functional groups.

[0665] In another embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0666]

[0667] Where: * represents the site of f3, and the dashed key represents f4.

[0668] In another embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0669]

[0670] Wherein: the dashed key represents f3, and * represents the site of f4.

[0671] In one embodiment, the crosslinking process is achieved via Diels-Alder cycloaddition between maleimide and furan derivative.

[0672] In one embodiment, corresponding to formula V, the divalent group -G1- is a polycyclic derivative consisting of a succinimide moiety according to the following formula:

[0673]

[0674] in:

[0675] X is a straight-chain *-(CH2) n1 -* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is a PEG derivative.

[0676] X1 is a straight-chain *-(CH2) n1 -* (where n1 is an integer from 0 to 7 (0≤n1≤7)), branched or cyclic alkyl derivatives.

[0677] X2 is either -H or -Me.

[0678] • In one implementation, if a = b = 1, then * represents the sites f3 and f4.

[0679] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0680] • In another implementation, if a = 1 and b = 0, then * represents the sites f2 and f4.

[0681] In one embodiment, the crosslinking process is achieved via an inverse electron-demand Diels-Alder reaction, or IEDDA, ​​between tetrazine and norbornene derivatives.

[0682] In one embodiment, corresponding to formula V, the divalent group -G1- is a polycyclic derivative consisting of a pyridazine moiety according to the following formula:

[0683]

[0684] in:

[0685] X is a straight-chain *-(CH2) n1 -* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is an aromatic derivative, or X is a PEG derivative.

[0686] • In one implementation, if a = b = 1, then * represents the sites f3 and f4.

[0687] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0688] • In another implementation, if a = 1 and b = 0, then * represents the sites f2 and f4.

[0689] In another embodiment, corresponding to formula V, the divalent group -G1- is a polycyclic derivative consisting of a pyridazine moiety according to the following formula:

[0690]

[0691] in:

[0692] X is a straight-chain *-(CH2) n1 -* (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives, or X is an aromatic derivative, or X is a PEG derivative.

[0693] • In one implementation, if a = b = 1, then * represents the sites f3 and f4.

[0694] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0695] • In another implementation, if a = 1 and b = 0, then * represents the sites f2 and f4.

[0696] In one implementation, the crosslinking process is achieved through Staudinger linkage between the aromatic phosphine and the azide derivative.

[0697] In one embodiment, corresponding to formula V, the divalent group -G1- is an aromatic derivative according to the following formula:

[0698]

[0699] in:

[0700] • In one implementation, if a = b = 1, then * represents the sites f3 and f4.

[0701] • In another implementation, if a = b = 0, then * represents the sites f1 and f4.

[0702] • In another implementation, if a = 1 and b = 0, then * represents the sites f2 and f4.

[0703] In one implementation, corresponding to formula V, the crosslinking process is achieved through a natural chemical link (NCL) between the thioester and the N-terminal cysteine ​​derivative.

[0704] In one implementation, the divalent group -G1- can be formalized according to the following formula:

[0705]

[0706] in:

[0707] • Dashed lines represent carbon-nitrogen covalent bonds.

[0708] • In one implementation, if a = b = 1, then * and dashed lines represent sites f3 and f4.

[0709] • In another implementation, if a = b = 0, then * and dashed lines represent sites f1 and f4.

[0710] • In another implementation, if a = 1 and b = 0, then * and dashed lines represent sites f2 and f4.

[0711] If c = 1, then:

[0712] • In one implementation, f4 is an amine functional group.

[0713] • In another embodiment, f4 is an ether functional group.

[0714] • In another embodiment, f4 is a thioether functional group.

[0715] • In another embodiment, f4 is an amide functional group.

[0716] • In another embodiment, f4 is a carbamate functional group.

[0717] • In another embodiment, f4 is a carbon-nitrogen covalent bond.

[0718] • In another implementation, f4 is a carbon-aromatic carbon covalent bond.

[0719] • In another embodiment, if the crosslinking process is carried out by natural chemical linkage (NCL), then f4 is a carbon-carbon covalent bond.

[0720] In one implementation, f3 is an amine functional group.

[0721] In another embodiment, f3 is an ether functional group.

[0722] In another embodiment, f3 is a thioether functional group.

[0723] In another embodiment, f3 is an amide functional group.

[0724] In another embodiment, f3 is a carbamate functional group.

[0725] In another implementation, f3 is a carbon-nitrogen covalent bond.

[0726] In another implementation, f3 is a carbon-aromatic carbon covalent bond.

[0727] The crosslinked dextran polymer according to the present invention is selected from dextran polymers of formula V.

[0728]

[0729] in:

[0730] ·i is an integer from 2 to 8 (2≤i≤8),

[0731] ·a=1,

[0732] b = 1,

[0733] c = 1,

[0734] • Dx is the dextran derivative described in Formula III.

[0735] • L is the PEG center connector described in Formula I.

[0736] • f1 is an ether functional group or a carbamate functional group.

[0737] The divalent group -A- is a straight-chain -(CH2) group. n1 -(where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives,

[0738] ·f2 is an amide functional group.

[0739] The divalent group -R1- is a straight-chain -(CH2) group. n1 -(where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives,

[0740] •f3 is an amide functional group.

[0741] The divalent group -G1- is a 1,4-triazole derivative.

[0742] •f4 is a carbon-nitrogen covalent bond, especially since the nitrogen atom is inside the triazole ring.

[0743] In a preferred embodiment, the integer i equals 4, i = 4.

[0744] According to the above embodiments, the triazole derivative is composed of a cyclooctene derivative derived from a strained cyclooctene, which may contain a heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized as: a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen, or sulfur, or halogen, especially fluorine.

[0745] According to the two embodiments described above, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains 4 to 6 nitrogen atoms.

[0746] According to one implementation scheme, the 1,4-triazole derivative is obtained through a copper-free reaction.

[0747] The crosslinked dextran polymer according to the present invention is selected from dextran polymers of formula V.

[0748]

[0749] in:

[0750] • I is an integer from 2 to 8 (2 ≤ I ≤ 8).

[0751] ·a=1,

[0752] b = 0,

[0753] c = 1,

[0754] • Dx is the dextran derivative described in Formula III.

[0755] • L is the PEG center connector described in Formula I.

[0756] The divalent group -A- is a straight-chain -(CH2) group. n1 -(where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives,

[0757] • f1 is an ether functional group or a carbamate functional group.

[0758] • The divalent group -G1- is a 1,4-triazole derivative, which is a cyclooctene derivative derived from strained cyclooctyne, which may contain a heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized as: a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen, or sulfur, or halogen, especially fluorine.

[0759] The 1,4-triazole derivative contains nitrogen, which is covalently linked to -CORa- (Ra is an alkyl group containing 1 to 4 carbon atoms) to form an amide functional group, and Ra is linked to f2, which is an amide functional group, and f4 is a carbon-nitrogen covalent bond, wherein the nitrogen atom is inside the triazole ring.

[0760] In a preferred embodiment, the integer i equals 4, i = 4.

[0761] According to one embodiment, 1,4-triazole is a polycyclic group containing an acyl group, the acyl group being connected to nitrogen via an amide functional group, the nitrogen being within a ring but not derived from the triazole ring.

[0762] According to one embodiment, 1,4-triazole comprises cyclooctyne with a nitrogen in the cyclooctyne ring.

[0763] According to one embodiment, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains 4 to 6 nitrogen atoms.

[0764] In one embodiment, the divalent group -G1- is a 1,4-triazole derivative as described by the following formula:

[0765]

[0766] Wherein: * represents the site of f2, which is an amide functional group, and the dashed bond represents f4, which is a carbon-nitrogen bond.

[0767] •f4 is a carbon-nitrogen covalent bond, in which the nitrogen atom is inside the triazole ring.

[0768] According to one implementation scheme, triazole 1,4 is obtained through a copper-free reaction.

[0769] According to one implementation, Dx is the dextran derivative described in Formula III:

[0770]

[0771] Where R is selected from

[0772] --H, an anionic group of formula II, or containing L(-) i The -W- group of the crosslinking group,

[0773] -i is between 20 and 5000 (20 ≤ i ≤ 5000),

[0774] --W- and L(-) i The group has the meaning previously defined.

[0775] According to one implementation, L is the PEG center connector described in Formula I:

[0776]

[0777] in:

[0778] ·i is an integer equal to 4,

[0779] · p is an integer equal to 1,

[0780] ·q is an integer between 8 and 1000 (8 ≤ q ≤ 1000),

[0781] ·r is an integer equal to 0 or 1.

[0782] • Q is a branched alkyl chain containing 2 to 10 carbon atoms.

[0783] * indicates the site of f4, which is a carbon-nitrogen covalent bond, wherein the nitrogen atom is inside the triazole ring.

[0784] Triazole 1,4 was obtained through a copper-free reaction.

[0785] The present invention also relates to dextran polymers of formula VIII prior to crosslinking reactions.

[0786]

[0787] in

[0788] If a, a', b, and b' are all not equal to 0, then f1, f2, f3, and Dx are as defined above.

[0789] ·and

[0790] x equals 0 or 1.

[0791] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[0792] • If either b' or c is not equal to 0, then -A'- is -A- as defined above.

[0793] • If b', b, and c are equal to 0, then a is equal to 0, and A' is the precursor A before the crosslinking reaction.

[0794] • If c is not equal to 0, then -R'1- is -R1- as defined above, and -G'1- is the precursor -G1-.

[0795] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[0796] In one embodiment, corresponding to formula VIII, A' is an alkyl carboxylic acid ester derivative or a poly(oxyethylene) carboxylic acid ester derivative, or an alkyl azide derivative, or a poly(oxyethylene) azide derivative, or a propargyl derivative, or a poly(oxyethylene) propargyl derivative, or a 2-hydroxyalkyl carboxylic acid ester, or a 2-hydroxyalkylamine.

[0797] The present invention also relates to dextran polymers of formula XVIII prior to crosslinking reactions.

[0798]

[0799] in

[0800] • If a is not equal to 0, then f1, f2, and Dx are as defined above.

[0801] ·and

[0802] • If a equals 0, then f1 is an ether functional group and G'1 is a propargyl derivative; if a does not equal 0, then -A' is A as defined above.

[0803] In one embodiment, corresponding to formula XVIII, if a = 1, then A' is an alkyl carboxylic acid ester derivative, or a 2-hydroxyalkyl carboxylic acid ester, or a 2-hydroxyalkylamine.

[0804] In one embodiment, corresponding to formula VIII, if a = 1, b = 0, and L is a POx derivative, then A is an alkyl carboxylic acid ester derivative, or a 2-hydroxyalkyl carboxylic acid ester, or a 2-hydroxyalkylamine.

[0805] A' as a carboxylic acid ester derivative can be formalized using the following formula:

[0806]

[0807] A', as an azide derivative, corresponds to formula VIII and can be formalized using the following formula:

[0808]

[0809] A', as a propargyl derivative, corresponds to formula VIII and can be formalized using the following formula:

[0810]

[0811] A' as a 2-hydroxyalkyl carboxylic acid ester derivative can be formalized using the following formula:

[0812]

[0813] A' as a 2-hydroxyalkylamine derivative can be formalized using the following formula:

[0814]

[0815] in:

[0816] -n is an integer from 1 to 7 (1≤n≤7).

[0817] -m is an integer between 1 and 5 (1 ≤ m ≤ 5).

[0818] -f1 is as previously specified.

[0819] In one embodiment, corresponding to formula VIII, if a = 1, then R'1 is a poly(oxyethylene) group or alkyl group with a terminal amine, or terminal hydroxyl group, or terminal thiol, or terminal carboxylic acid ester, or terminal azide or terminal alkyne.

[0820] R'1 can be formalized as follows:

[0821]

[0822] R'1, as a carboxylic acid ester derivative, can be formalized using the following formula:

[0823]

[0824] R'1 as an azide derivative can be formalized using the following formula:

[0825]

[0826] in:

[0827] -n is an integer from 1 to 7 (1 ≤ n ≤ 7)

[0828] -X = -NH2, or -OH, or -SH

[0829] -f2 is as previously specified.

[0830] Alternatively, in another embodiment, corresponding to formula VIII, if a = 0, R'1 is a branched alkyl group, wherein at least one hydroxyl group is connected to an alkyl chain at the β position relative to f1, and has a terminal hydroxyl group, or a terminal thiol, or a terminal azide or a terminal alkyne.

[0831] R'1 can be formalized as follows:

[0832]

[0833] in:

[0834] -n is an integer from 1 to 5 (1 ≤ n ≤ 5).

[0835] -X = -NH2, or -OH, or -SH, or -N3, or -C≡CH.

[0836] -f1 is as previously specified.

[0837] Alternatively, in another embodiment, R'1 is a propargyl derivative and can be formalized as follows:

[0838]

[0839] in:

[0840] -n is an integer from 1 to 7 (1 ≤ n ≤ 7)

[0841] -f1 is as previously specified.

[0842] In one embodiment, corresponding to formula VIII, -G'1 is a maleimide derivative, or a vinyl sulfone derivative, or a strained cyclooctylene derivative, or an azide derivative, or a propargyl derivative, or a furan derivative, or an acrylamide derivative, or a norbornene derivative, or a trans-cyclooctene derivative, or a tetrazine derivative, or an aromatic phosphine, or cysteine, or a thioester, or a thiol derivative, or an amine derivative, or a hydroxyl derivative.

[0843] In one embodiment, corresponding to formula XVIII, if a = 1, then G'1 is a maleimide derivative, or a vinyl sulfone derivative, or a strained cyclooctyne derivative, or an azide derivative, or a propargyl derivative, or a thiol derivative, or an amine derivative, or a hydroxyl derivative.

[0844] In one embodiment, corresponding to formula VIII, if a = 1, b = 0, and L is a POx derivative, then G'1 is a maleimide derivative, or a vinyl sulfone derivative, or a strained cyclooctyne derivative, or an azide derivative, or a propargyl derivative, or a thiol derivative, or an amine derivative, or a hydroxyl derivative.

[0845] -G'1, as a thiol, amine, or hydroxyl derivative, corresponding to formula VIII, can be formalized as follows:

[0846]

[0847] G'1, as a thiol, amine, or hydroxyl derivative, corresponding to formula XVIII, can be formalized as follows:

[0848]

[0849] G'1, as a maleimide, corresponding to formula VIII, can be formalized as follows:

[0850]

[0851] G'1, as a maleimide, corresponding to formula XVIII, can be formalized as follows:

[0852]

[0853] G'1, as a vinyl sulfone, corresponding to formula VIII, can be formalized as follows:

[0854]

[0855] G'1, as a vinyl sulfone, corresponding to formula XVIII, can be formalized as follows:

[0856]

[0857] G'1, as a strained octylene, corresponding to equation VIII, can be formalized as follows:

[0858]

[0859] G'1, as an azide derivative, corresponds to formula VIII and can be formalized using the following formula:

[0860]

[0861] G'1, as an azide derivative corresponding to formula XVIII, can be formalized using the following formula:

[0862]

[0863] G'1, as a propargyl derivative, corresponds to formula VIII and can be formalized using the following formula:

[0864]

[0865] G'1, as a propargyl derivative corresponding to formula XVIII, can be formalized using the following formula:

[0866]

[0867] in:

[0868] n is an integer from 0 to 7 (0 ≤ n ≤ 7).

[0869] • R is a straight-chain, branched, or cyclic alkyl derivative, or R is a PEG derivative.

[0870] X = -NH2, or -OH, or -SH

[0871] X1 is an oxygen atom, a sulfur atom, or a CH2 group.

[0872] •f2 as previously defined.

[0873] In one implementation, corresponding to formula XVIII, if a = 0, then G'1 is a propargyl derivative.

[0874] G'1, as a propargyl derivative, corresponding to formula XVIII, can be formalized as follows:

[0875]

[0876] in:

[0877] •f1 as previously defined.

[0878] G'1, as a furan derivative, corresponds to formula VIII and can be formalized using the following formula:

[0879]

[0880] G'1, as an acrylamide derivative, corresponds to formula VIII and can be formalized using the following formula:

[0881]

[0882] G'1, as a norbornene derivative, corresponds to formula VIII and can be formalized using the following formula:

[0883]

[0884] G'1, as a trans-cyclooctene derivative, corresponds to formula VIII and can be formalized using the following formula:

[0885]

[0886] G'1, as a tetrazine derivative, corresponds to formula VIII and can be formalized using the following formula:

[0887]

[0888] G'1, as an aromatic phosphine derivative, corresponds to formula VIII and can be formalized using the following formula:

[0889]

[0890] G'1, as a cysteine ​​derivative, corresponds to formula VIII and can be formalized using the following formula:

[0891]

[0892] G'1, as a thioester derivative, corresponds to formula VIII and can be formalized using the following formula:

[0893]

[0894] in:

[0895] X = -NH2, or -OH, or -SH

[0896] ·X1=-O- or -S-

[0897] n is an integer equal to 0 or 1.

[0898] R1 is an alkyl group.

[0899] ·X2=-CH2- or aromatics

[0900] R2 = -H or -CH3

[0901] •f3 as previously defined.

[0902] • The dashed bond represents f3, which is a carbon-nitrogen covalent bond or a carbon-carbon covalent bond.

[0903] The present invention also relates to hydrogels comprising crosslinked dextran polymers according to the present invention.

[0904] In one implementation, the hydrogel is transparent.

[0905] "Transparent" means that, under the conditions for visual inspection disclosed in Example C21 of application PCT / EP2022 / 050466, the observer considers the sample to be transparent compared to Standard 2 (6 NTU), and / or the UV absorbance of the hydrogel measured as in Example C21 of application PCT / EP2022 / 050466 is less than 0.06 (absorbance units).

[0906] In one embodiment, the hydrogel is visually transparent and has a UV absorbance of <0.06 (absorbance units).

[0907] In one embodiment, the hydrogel according to the invention is characterized in that Tanδ is less than 1.

[0908] In this specification, Tanδ is the ratio of loss modulus G” to storage modulus (also known as elastic modulus) G’ (Tanδ=G” / G).

[0909] In one embodiment, the hydrogel according to the invention is characterized in that Tanδ is less than or equal to 0.5.

[0910] In one embodiment, the hydrogel according to the invention is characterized in that Tanδ is less than or equal to 0.1.

[0911] In one embodiment, the hydrogel according to the invention is characterized in that Tanδ is less than or equal to 0.05.

[0912] In one embodiment, the hydrogel according to the invention is characterized in that Tanδ is less than or equal to 0.01.

[0913] In one embodiment, the hydrogel according to the invention is characterized in that the concentration of the crosslinked dextran polymer after swelling in water is 0.01 to 0.2 g / g.

[0914] In one embodiment, the hydrogel according to the invention is characterized in that the concentration of the crosslinked dextran polymer after swelling in water is 0.03 to 0.1 g / g.

[0915] In one embodiment, the hydrogel according to the invention is characterized in that the concentration of the crosslinked dextran polymer after swelling in water is 0.05 to 0.1 g / g.

[0916] In one embodiment, the hydrogel is translucent.

[0917] In another embodiment, the hydrogel is transparent.

[0918] In one embodiment, the hydrogel has a Young's modulus of 1 to 200 kPa.

[0919] In one embodiment, the hydrogel has a Young's modulus of 5 to 200 kPa.

[0920] In one embodiment, the hydrogel has a Young's modulus of 20 to 200 kPa.

[0921] In one embodiment, the hydrogel has a Young's modulus of 30 to 200 kPa.

[0922] In one embodiment, the hydrogel has a Young's modulus of 50 to 200 kPa.

[0923] In one embodiment, the hydrogel has a Young's modulus of 30 to 180 kPa.

[0924] In one embodiment, the hydrogel has a Young's modulus of 50 to 150 kPa.

[0925] In one embodiment, the hydrogel has a Young's modulus of 5 to 100 kPa.

[0926] In one embodiment, the hydrogel has a Young's modulus of 10 to 90 kPa.

[0927] In one embodiment, the hydrogel has a Young's modulus of 10 to 75 kPa.

[0928] In one embodiment, the hydrogel has a G' of 0.5 to 70 kPa.

[0929] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 10%.

[0930] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 15%.

[0931] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 20%.

[0932] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 25%.

[0933] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 30%.

[0934] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 35%.

[0935] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 40%.

[0936] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 45%.

[0937] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 50%.

[0938] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 55%.

[0939] In one embodiment, the hydrogel exhibits a fracture compressive deformation greater than or equal to 60%.

[0940] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 10%.

[0941] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 15%.

[0942] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 20%.

[0943] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 25%.

[0944] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 30%.

[0945] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 35%.

[0946] In one embodiment, the tensile deformation at break of the hydrogel is greater than or equal to 40%.

[0947] In one embodiment, the swelling ratio of the hydrogel is greater than 0.7.

[0948] In one embodiment, the swelling ratio of the hydrogel is greater than 0.8.

[0949] In one embodiment, the swelling ratio of the hydrogel is greater than 0.9.

[0950] In one embodiment, the swelling ratio of the hydrogel is greater than 1.

[0951] In one embodiment, the swelling ratio of the hydrogel is greater than 1.1.

[0952] In one embodiment, the swelling ratio of the hydrogel is greater than or equal to 1.2.

[0953] In one embodiment, the swelling ratio of the hydrogel is greater than or equal to 1.3.

[0954] In one embodiment, the swelling ratio of the hydrogel is greater than or equal to 1.4.

[0955] In one embodiment, the swelling ratio of the hydrogel is greater than or equal to 1.5.

[0956] In one embodiment, the swelling ratio of the hydrogel is greater than or equal to 1.6.

[0957] In one embodiment, the swelling ratio of the hydrogel is less than or equal to 5.

[0958] In one embodiment, the swelling ratio of the hydrogel is less than or equal to 4.

[0959] In one embodiment, the swelling ratio of the hydrogel is less than or equal to 3.

[0960] In one embodiment, the swelling ratio of the hydrogel is less than or equal to 2.8.

[0961] In one embodiment, the swelling ratio of the hydrogel is less than or equal to 2.5.

[0962] In one embodiment, the swelling ratio of the hydrogel is less than or equal to 2.3.

[0963] In one embodiment, the hydrogel has a water content of at least 80 wt%.

[0964] In one embodiment, the hydrogel has a water content of at least 85 wt%.

[0965] In one embodiment, the hydrogel has a water content of at least 90 wt%.

[0966] In one embodiment, the hydrogel has a water content of at least 97 wt%.

[0967] In one embodiment, the hydrogel has a water content of at least 96 wt%.

[0968] In one embodiment, the hydrogel has a water content of at least 95 wt%.

[0969] In one embodiment, the hydrogel has a water content of at least 94 wt%.

[0970] In one embodiment, the hydrogel has a water content of at least 93 wt%.

[0971] In one embodiment, the water content of the hydrogel is up to 99 wt%.

[0972] In one embodiment, the water content of the hydrogel is up to 98 wt%.

[0973] In one embodiment, the hydrogel according to the invention is characterized in that it further comprises biological cells.

[0974] In one implementation, the cells are cells derived from human or animal sources.

[0975] In one implementation, the cell is a cell line.

[0976] In one implementation, the cells are derived from stem cells.

[0977] In one implementation, the stem cells are selected from embryonic stem cells, induced pluripotent stem cells, or mesenchymal stem cells.

[0978] In one implementation, the cells are primary cells.

[0979] In one implementation, the cell is a protein, hormone, or peptide-secreting cell.

[0980] In one implementation, the cells are selected from:

[0981] Insulin-secreting cells used in diabetes treatment

[0982] - Factor VIII or Factor IX secretory cells used for the treatment of hemophilia, and - β-glucocerebrosidase secretory cells used for Gaucher disease.

[0983] In one implementation, the cells are selected from those that secrete the following:

[0984] Growth hormone

[0985] Human growth hormone (hGH)

[0986] • Recombinant human growth hormone (rhGH)

[0987] -Growth hormone-releasing hormone (GHRH)

[0988] - Thyroid-stimulating hormone (TSH)

[0989] - Thyrotropin-releasing hormone (TRH)

[0990] -Adrenocorticotropic hormone (ACTH), and

[0991] - Parathyroid hormone (PTH).

[0992] In one implementation, the cells are selected from those that secrete the following:

[0993] -glucagon

[0994] -Insulin, and

[0995] -GLP-1.

[0996] In one implementation, the cells are selected from cells that secrete insulin.

[0997] In one implementation, the cells are selected from cells that secrete insulin.

[0998] In one implementation, the cells are selected from cells that secrete the following growth factors:

[0999] - Vascular endothelial growth factor (VEGF)

[1000] - Nerve growth factor (NGF)

[1001] Platelet-derived growth factor (PDGF)

[1002] - Fibroblast growth factor (FGF)

[1003] - Epidermal growth factor (EGF)

[1004] - Transforming growth factor (TGF), and

[1005] - Insulin-like growth factor I and II (IGF-I and IGF-II).

[1006] In one embodiment, the cells are selected from cells that secrete the following clotting factors or blood coagulation factors:

[1007] Factor I (e.g., fibrinogen),

[1008] Factor II (e.g., prothrombin),

[1009] Factor III (e.g., tissue factor),

[1010] - Factor V (e.g., pre-accelerator, unstable factor),

[1011] -Factor VI,

[1012] - Factor VII (e.g., stabilizing factor, pre-inverting factor),

[1013] Factor VIII (e.g., antihemophilic factor A),

[1014] -Factor VIIIC,

[1015] Factor IX (e.g., antihemophilic factor B),

[1016] - Factor X (e.g., Stuart-Prower factor),

[1017] Factor XI (e.g., plasma prothrombin kinase precursor),

[1018] - Factor XII (e.g., the Hagerman factor),

[1019] - Factor XIII (e.g., fibroblast stabilizer),

[1020] - von Willebrand factor (vWF)

[1021] -prokallikrein,

[1022] -Heparin cofactor II,

[1023] - High molecular weight kininogens (e.g., Fitzgerald factor),

[1024] -Antithrombin III, and

[1025] -Fibronectin.

[1026] In one embodiment, the cell is selected from cells that secrete immunoglobulin chains (heavy or light chains) or fragments thereof, the immunoglobulin chains (heavy or light chains) or fragments thereof comprising at least one immunoglobulin variable domain sequence and optionally comprising an immunoglobulin Fc region.

[1027] In one embodiment, the cells are selected from cells that secrete cytokines or cytokine receptors, or chimeric proteins containing cytokines or their receptors.

[1028] In one embodiment, the cells are selected from cells that secrete erythropoietin.

[1029] In one embodiment, the cells are selected from cells that secrete interleukins (ILs) such as:

[1030] -IL-1, IL-2 to IL-10.

[1031] In one embodiment, the cells are selected from cells that secrete the following alternative enzymes:

[1032] -α-galactosidase A (GLA),

[1033] -α-L-iduroglycoside (IDVA),

[1034] -Arylsulfatase B (ARSB),

[1035] -glucocerebroside lipase, and

[1036] -N-sulfoglucosamine sulfohydrolase (SGSH).

[1037] In one embodiment, the hydrogel according to the invention is characterized in that insulin-secreting cells are selected from the pancreatic cell group.

[1038] In one embodiment, the hydrogel according to the invention is characterized in that the insulin-secreting cells are Langherans islets.

[1039] In one embodiment, the hydrogel according to the invention is characterized in that the biological cells are pseudo-islets.

[1040] The present invention also relates to the use of the cross-linked dextran copolymer according to the invention in the form of a hydrogel for the preparation of cell compositions.

[1041] In one embodiment, the cells are selected from one or more types of isolated or aggregated cells that can secrete active ingredients.

[1042] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 2, 4 or 8.

[1043] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 2, 4 or 8.

[1044] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 2.

[1045] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 2.

[1046] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 4.

[1047] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 4.

[1048] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 8.

[1049] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 8.

[1050] The crosslinking step is the gelling step that leads to the formation of the hydrogel according to the invention.

[1051] Hydrogel formation kinetics are a function of temperature and can be modulated by reactant concentration, pH, and temperature.

[1052] In one embodiment, the time to obtain the hydrogel according to the invention is from 1 minute to 6 hours.

[1053] In one implementation, the crosslinking step is performed for 1 hour.

[1054] In one embodiment, the temperature of the crosslinking step is from 4°C to room temperature (20°C to 25°C) and can vary between the mixing step and the gelling and molding steps.

[1055] In one embodiment, mixing is carried out at 4°C, and gelation is carried out at room temperature (20°C to 25°C) for 1 hour.

[1056] In one embodiment, mixing is carried out at room temperature (20°C to 25°C).

[1057] In one embodiment, mixing is carried out at 4°C or room temperature (20°C to 25°C), and gelation is carried out at room temperature (20°C to 25°C) for 1 hour.

[1058] In one implementation, gelation is carried out at 37°C.

[1059] In one embodiment, after crosslinking or gelation, the hydrogel swells in a buffer solution with a pH of 5 to 8, preferably 6 to 8, and more preferably 6.8 to 7.5.

[1060] In one implementation, the buffer solution is a PBS solution with pH 7.4.

[1061] In one implementation, the buffer solution is a Tris solution with pH 7.4.

[1062] In one implementation, the buffer solution is a Tris solution with pH 8.

[1063] In one embodiment, swelling increases the mass of the hydrogel by 1, 2, 3, or 4 times compared to its initial mass.

[1064] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention into a hydrogel form, comprising the following steps:

[1065] a) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -W- of dextran.

[1066] b) Preparation of precursor L(-) i sterile solution,

[1067] c) Add the sterile solution obtained in step b) to the solution obtained in step a).

[1068] d) Add directly to the mold, or introduce the solution into the mold after mixing.

[1069] e) Perform crosslinking and gelation, for example, at room temperature (20°C to 25°C) or at 37°C.

[1070] f) Demolding and swelling are performed to obtain a hydrogel.

[1071] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention into a hydrogel form, comprising the following steps:

[1072] a) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -(A-f2). a -G1-、-(A'-f2) a -G'1-dextran,

[1073] b) Preparation of precursor L(-) i sterile solution,

[1074] c) Add the sterile solution obtained in step b) to the solution obtained in step a).

[1075] d) Add directly to the mold, or introduce the solution into the mold after mixing.

[1076] e) Perform crosslinking and gelation, for example, at room temperature (20°C to 25°C) or at 37°C.

[1077] f) Demolding and swelling are performed to obtain a hydrogel.

[1078] In one implementation, steps c) and d) are performed simultaneously.

[1079] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[1080] Dextran containing an anionic group of Formula II is prepared by grafting or substituting a hydroxyl group onto the dextran. In one embodiment, dextran containing an anionic group of Formula II is prepared by grafting or substituting a carboxymethyl group onto a methyl dextran carboxylate / salt.

[1081] In one embodiment of the method according to the invention, the active pharmaceutical ingredient (API) is embedded in a hydrogel.

[1082] The present invention also relates to the therapeutic use of the hydrogel according to the invention as a therapeutic implant for administering APIs to mammals.

[1083] The present invention also relates to a method for preparing hydrogels containing biological cells, comprising the following steps:

[1084] a) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -W- of dextran.

[1085] b) Preparation of precursor L(-) i sterile solution,

[1086] c) Prepare suspensions of biological cells.

[1087] d) Mix the biological cell suspension obtained from step c) with the solution obtained from step b) or a).

[1088] e) Add the sterile solution obtained from step a) or b) that was not used in step d) to the solution obtained from step d).

[1089] f) The addition in step e) can be done directly in the mold, or the solution can be introduced into the mold after mixing.

[1090] g) The crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1091] h) Demolding and swelling are performed to obtain a hydrogel containing biological cells.

[1092] The present invention also relates to a method for preparing hydrogels containing biological cells, comprising the following steps:

[1093] a) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -(A-f2). a -G1-、-(A'-F2) a -G'1-dextran,

[1094] b) Preparation of precursor L(-) i sterile solution,

[1095] c) Prepare suspensions of biological cells.

[1096] d) Mix the biological cell suspension obtained from step c) with the solution obtained from step b) or a).

[1097] e) Add the sterile solution obtained from step a) or b) that was not used in step d) to the solution obtained from step d).

[1098] f) The addition in step e) can be done directly in the mold, or the solution can be introduced into the mold after mixing.

[1099] g) The crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1100] h) Demolding and swelling are performed to obtain a hydrogel containing biological cells.

[1101] The present invention also relates to a method for preparing hydrogels containing biological cells, comprising the following steps:

[1102] a) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -W- of dextran.

[1103] b) Preparation of precursor L(-) selected from the following i Sterile solutions: thiol polyethylene glycol, mercapto-poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide, or pentaerythritol poly(dibenzocyclooctylene) polyoxyethylene.

[1104] c) Prepare a sterile solution of sodium hyaluronate.

[1105] d) Prepare a sterile suspension of biological cells.

[1106] e) Mix the sodium hyaluronate solution obtained in step c) with the precursor solution obtained in step b).

[1107] f) Mix the biological cell suspension obtained from step d) with the solution obtained from step e) or step a).

[1108] g) Mix the solution obtained in step f) with the solution obtained in step e).

[1109] h) Add the unused sterile solution obtained from step a) or e) in step g) to the solution obtained from step f).

[1110] i) The addition in step g) can be done directly in the mold, or the solution can be introduced into the mold after mixing.

[1111] j) The crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1112] k) Demolding and swelling are performed to obtain a hydrogel containing biological cells.

[1113] In one implementation, the mold is a ring net.

[1114] The ring is an outer ring and consists of an upper and a lower part that clamps the mesh in the middle. The two parts of the ring are glued together with the mesh.

[1115] In one embodiment, the crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1116] In one embodiment, the crosslinking and gelation reactions are carried out at a controlled temperature of 15°C to 37°C.

[1117] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[1118] In one embodiment, the hydrogel according to the invention is characterized in that it further comprises biological cells.

[1119] In one implementation, the cells are cells derived from human or animal sources.

[1120] In one implementation, the cell is a cell line.

[1121] In one implementation, the cells are derived from stem cells.

[1122] In one implementation, the stem cells are selected from embryonic stem cells, induced pluripotent stem cells, or mesenchymal stem cells.

[1123] In one implementation, the cells are primary cells.

[1124] In one implementation, the cell is a protein, hormone, or peptide-secreting cell.

[1125] In one implementation, the cells are selected from:

[1126] Insulin-secreting cells used in diabetes treatment

[1127] - Factor VIII or Factor IX secretory cells used for the treatment of hemophilia, and

[1128] - β-glucocerebroside secretory cells used for Gaucher disease.

[1129] In one embodiment, the hydrogel according to the invention is characterized in that insulin-secreting cells are selected from the pancreatic cell group.

[1130] In one embodiment, the hydrogel according to the invention is characterized in that the insulin-secreting cells are Langerhans islands.

[1131] In one embodiment, the hydrogel according to the invention is characterized in that the biological cells are pseudo-islets.

[1132] The present invention also relates to the therapeutic use of the hydrogel according to the invention for treating a condition or disease in mammals, wherein the condition or disease is caused by a lack of endocrine function or dysfunction of the pancreatic organ.

[1133] The present invention also relates to hydrogels used as pharmaceuticals.

[1134] The present invention also relates to hydrogels for treating diseases such as diabetes.

[1135] The present invention also relates to an implantable device comprising at least one hydrogel according to the invention and obtained by the method according to the invention.

[1136] The present invention also relates to implants composed of hydrogels according to the invention.

[1137] The present invention also relates to implants comprising the hydrogel according to the invention.

[1138] The present invention also relates to implants comprising a hydrogel according to the invention and cells or islets of Langerhans.

[1139] The present invention also relates to a reagent kit comprising:

[1140] - Solution of the dextran polymer of formula VIII prior to the crosslinking reaction:

[1141]

[1142] in

[1143] If a, a', b, and b' are all equal to 0, then f1, f2, f3, f4, and Dx are defined according to equation IV.

[1144] ·and

[1145] x equals 0 or 1.

[1146] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1147] • If either b' or c is not equal to 0, then -A' is A according to the definition above.

[1148] • If b', b, and c equal 0, then a equals 0, and A' is the precursor A prior to the crosslinking reaction.

[1149] • If c is not equal to 0, then R'1 is R1 as defined above, and G'1 is the precursor G1.

[1150] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1151] - A solution of thiol polyethylene glycol, mercapto polyethylene (oxyethylene), pentaerythritol polyethylene (oxyethylene) azide, or pentaerythritol polyethylene (dibenzocyclooctylene) polyethylene (oxyethylene).

[1152] - Biological cells.

[1153] The present invention also relates to a reagent kit comprising:

[1154] - Solution of the dextran polymer of formula VIII prior to the crosslinking reaction:

[1155]

[1156] in

[1157] If a, a', b, and b' are all equal to 0, then f1, f2, f3, f4, and Dx are defined according to equation IV.

[1158] ·and

[1159] x equals 0 or 1.

[1160] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1161] • If either b' or c is not equal to 0, then -A' is A according to the definition above.

[1162] • If b', b, and c equal 0, then a equals 0, and A' is the precursor A prior to the crosslinking reaction.

[1163] • If c is not equal to 0, then R'1 is R1 as defined above, and G'1 is the precursor G1.

[1164] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1165] - A solution of thiol polyethylene glycol, mercapto polyethylene (oxyethylene), pentaerythritol polyethylene (oxyethylene) azide, or pentaerythritol polyethylene (dibenzocyclooctylene) polyethylene (oxyethylene).

[1166] -Biological cells,

[1167] - Non-crosslinked sodium hyaluronate solution.

[1168] The present invention also relates to a reagent kit comprising:

[1169] - Solution of the dextran polymer of formula VIII prior to the crosslinking reaction:

[1170]

[1171] in

[1172] If a, a', b, and b' are all equal to 0, then f1, f2, f3, f4, and Dx are defined according to equation IV.

[1173] ·and

[1174] x equals 0 or 1.

[1175] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1176] • If either b' or c is not equal to 0, then -A' is A according to the definition above.

[1177] • If b', b, and c equal 0, then a equals 0, and A' is the precursor A prior to the crosslinking reaction.

[1178] • If c is not equal to 0, then R'1 is R1 as defined above, and G'1 is the precursor G1.

[1179] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1180] - A solution of thiol polyethylene glycol, mercapto polyethylene (oxyethylene), pentaerythritol polyethylene (oxyethylene) azide, or pentaerythritol polyethylene (dibenzocyclooctylene) polyethylene (oxyethylene).

[1181] - Biological cells.

[1182] In one implementation, at least 50% of the hydrogel surface is in direct contact with the medium in which the hydrogel is embedded.

[1183] In one embodiment, at least 75% of the hydrogel surface is in direct contact with the medium in which the hydrogel is embedded.

[1184] In one implementation, at least 90% of the hydrogel surface is in direct contact with the medium in which the hydrogel is embedded.

[1185] In one embodiment, at least 95% of the hydrogel surface is in direct contact with the medium in which the hydrogel is embedded.

[1186] In one implementation, 99% of the hydrogel surface is in direct contact with the medium in which the hydrogel is embedded.

[1187] In one implementation, at least 50% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1188] "Direct contact with the outside" means that there is no separation between the hydrogel and the outside, for example, there is no wall made of non-hydrogel material between the hydrogel and the outside of the device or implant.

[1189] In one implementation, at least 75% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1190] In one implementation, at least 90% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1191] In one implementation, at least 95% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1192] In one implementation, at least 99% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1193] In one implementation, 100% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1194] Cells or APIs are embedded in a maze of cross-linked dextran hydrogels.

[1195] In this specification, the term “embed” is equivalent to “encapsulation” or “encapsulation”.

[1196] Hydrogel matrices allow small molecules (such as nutrients and APIs, which are either embedded in the hydrogel or secreted by the embedded cells) to pass through.

[1197] Typically, APIs are selected from the following hormones and peptides: PTH protein, insulin, and clotting factors.

[1198] In one embodiment, the mesh size of the matrix is ​​immune-isolated and blocks T lymphocytes in order to preserve cells.

[1199] In one implementation, the mesh size is less than 1 μm.

[1200] In another embodiment, it is less than 100 nanometers, preferably less than 10 nanometers, and more preferably about 5 nanometers.

[1201] In one embodiment, the present invention relates to implants comprising rings, nets, hydrogels according to the invention, and cells.

[1202] In one implementation, the thickness of the implant is less than 3,000 μm.

[1203] In one implementation, the thickness of the implant is less than 2,000 μm.

[1204] In one implementation, the thickness of the implant is less than 1,000 μm.

[1205] In one implementation, the thickness of the implant is less than 900 μm.

[1206] In one implementation, the thickness of the implant is greater than 300 μm.

[1207] In one implementation, the thickness of the implant is greater than 400 μm.

[1208] In one implementation, the thickness of the implant is greater than 500 μm.

[1209] In one implementation, the total surface area of ​​the implant is 10 cm². 2 Up to 200cm 2 .

[1210] In one implementation, the total surface area of ​​the implant is 15 cm². 2 Up to 100cm 2 .

[1211] In one implementation, the implant contains 0.5 to 20 ml of hydrogel.

[1212] In one embodiment, the implant contains 0.75 to 10 ml of hydrogel.

[1213] In one implementation, the implant contains 0.8 to 5 ml of hydrogel.

[1214] In one embodiment, the ring and implant are parallelepiped rectangles, particularly parallelepiped rectangles with rounded corners.

[1215] The ring and network structure makes the hydrogel easy to manipulate and resistant to manipulations, including those for implantation. This is even true for hydrogels with larger pore sizes (e.g., those with lower DS- and lower concentrations of reactive groups during crosslinking).

[1216] The ring and network structure makes the hydrogel easy to manipulate and provides good resistance to procedures, including those for implantation. This is even true for hydrogels with larger pore sizes (e.g., -(A-f2) with lower DS during crosslinking). a The same applies to -G1- and lower concentrations of reactive groups.

[1217] In one embodiment, the inner diameter of the ring is 10 to 100 mm.

[1218] In one implementation, the inner diameter of the ring is 15 to 50 mm.

[1219] In one embodiment, the diameter of the ring is 0.5 to 5 mm.

[1220] In one embodiment, the diameter of the ring is 0.5 to 10 mm.

[1221] In one embodiment, the diameter of the ring is 0.5 to 5 mm.

[1222] In one embodiment, the total thickness of the ring (lower part plus upper part and adhesive) is 100 to 3000 μm.

[1223] In one embodiment, the total thickness of the ring (lower part plus upper part and adhesive) is 150 to 2000 μm.

[1224] In one embodiment, the total thickness of the ring is 200 to 5000 μm.

[1225] In one embodiment, the total thickness of the ring is 500 to 3000 μm.

[1226] In one implementation, the ring has a rectangular, square, or circular cross-section.

[1227] In one implementation, the ring is made of a bio-inert material.

[1228] In one implementation, the ring is made of a biocompatible elastomer.

[1229] In one embodiment, the material of the ring is selected from silicone, particularly PDMS, polyurethane, polyether, polyether polyester copolymer and polypropylene oxide.

[1230] In one embodiment, the ring is made of silicone.

[1231] In one implementation, the ring is made of PDMS.

[1232] In one implementation, the net is non-biodegradable.

[1233] In one implementation, the net is biocompatible.

[1234] In one implementation, the net is non-absorbable.

[1235] In one implementation, the mesh is a surgical mesh.

[1236] In one embodiment, the filament material in the mesh is selected from polypropylene, polyethylene, polyester, especially PET, PTFE, PVDF (polyvinylidene fluoride) and ePVDF (stretched PVDF).

[1237] In one specific implementation, the filament material in the mesh is selected from PTFE, PVDF, and ePVDF.

[1238] In one embodiment, the filament material in the mesh is selected from polypropylene, polyester, particularly PET, PTFE, and PVDF (polyvinylidene fluoride).

[1239] In one embodiment, the filament material in the mesh is selected from polypropylene and polyester, particularly PET.

[1240] In one embodiment, the filament material in the mesh is selected from polypropylene.

[1241] In one embodiment, the filament material in the mesh is selected from polyester, particularly PET.

[1242] In one implementation, the thickness of the mesh ranges from 50 to 500 μm.

[1243] In one implementation, the thickness of the m-mesh ranges from 100 to 300 μm.

[1244] In one embodiment, the diameter of the filament ranges from 0.08 to 0.2 mm.

[1245] In one implementation, the mesh aperture size ranges from 0.4 to 4 mm.

[1246] In one implementation, the mesh aperture size ranges from 0.6 to 2 mm.

[1247] In one embodiment, the mesh has holes with side dimensions ranging from 0.4 to 4 mm.

[1248] In one embodiment, the mesh has holes with side dimensions ranging from 0.6 to 3 mm.

[1249] In one implementation, the fabric of the net is selected from knitted fabrics, warp-knitted fabrics, woven fabrics, and nonwoven fabrics.

[1250] In one embodiment, the fabric of the net is selected from warp-knitted fabrics, particularly multifilament fabrics.

[1251] In one implementation, the net is treated to improve its hydrophilicity.

[1252] In one implementation, the mesh is treated with alkali, particularly for polyester, and even more so for PET.

[1253] In one embodiment, the treatment involves functionalizing the surface with reactive functional groups such as -OH, -COOH, and reactive molecules or polymers.

[1254] The grafted polymer can thus expose reactive functional groups, such as thiol functional groups, for further reaction with the hydrogel or hydrogel precursor.

[1255] In one embodiment, the treatment is performed by adsorbing a synthetic polymer (e.g., poloxamer or polyvinyl pyrrolidone, PVP) or a natural polymer (e.g., collagen) or a surfactant after chemical or physical treatment.

[1256] In one implementation, the net is held below the outer end of the ring.

[1257] In one implementation, the mesh does not come into contact with the exterior of the implant, which comprises the ring, mesh, and hydrogel.

[1258] In one implementation, the adhesive is biocompatible.

[1259] In one embodiment, the adhesive is a biocompatible silicone glue, such as Silbione MED ADH 4200 supplied by Elkem.

[1260] In one embodiment, the adhesive remains below the outer end of the ring.

[1261] For example, it will be generated by SurgicalNet TM The provided warp-knitted polyester surgical mesh fabric, type PETKM3002 (1×0.9mm aperture), was treated in 1M NaOH at 70°C for 5 hours and then rinsed with deionized water and 96% ethanol. This treatment resulted in increased hydrophilicity of the mesh fabric, thereby leading to improved wettability with aqueous solutions.

[1262] For example, a ring network structure can be obtained using the following method:

[1263] - Use a stainless steel punch to cut biocompatible PDMS sheets supplied by Grace Biolabs or Interstate Speciality Products into squares incorporating a circular empty disk.

[1264] - Introduce a portion of the treated polyester surgical mesh between two square PDMS sheets: Glue the two PDMS sheets and the surgical mesh together using biocompatible silicone adhesive (Silbione MED ADH 4200, supplied by Elkem). Align the circular empty discs and maintain tension on the surgical mesh during gluing.

[1265] -Then, a square construct is cut using a stainless steel punch to obtain a final object consisting of two PDMS rings that sandwich the surgical mesh in the middle and are glued together.

[1266] Wash the tablets with a 1% poloxamer F127 solution and rinse with water before steam sterilization.

[1267] In one implementation scheme, the implant can be obtained through the following methods:

[1268] - The hydrogel composition was incorporated into the ring network construct. The concentrated polymer solution was mixed using a pipette, and a controlled volume of the mixture was introduced into the ring network construct adhered to a glass slide.

[1269] - Perform cross-linking that leads to gelation. The ring-and-hydrogel composition is then introduced into a Tris 150mM / NaCl 30mM / cysteine ​​10mM solution at pH 8 or into PBS at pH 7.4.

[1270] - Rinse the hydrogel with cysteine-free PBS solution and then immerse it in the PBS solution overnight at 37°C. The hydrogel block is then stored in PBS solution at 4°C until use.

[1271] Hydrogel / ring implants can therefore be easily manipulated with forceps and can be folded for surgical implantation as needed. Furthermore, the ring can be secured with sutures.

[1272] The hydrogel volume can be adjusted by the inner diameter and thickness of the ring network structure. For the same ring network structure, the hydrogel volume can be adjusted to control the convexity / concavity of the hydrogel above the ring level.

[1273] In one embodiment, the hydrogel comprises a first layer of hydrogel without cells or islets and a second layer of hydrogel containing cells or islets.

[1274] Such a structure can be obtained by the method disclosed in this application, but with two steps of adding a hydrogel precursor: the first step is to add a cellless or islet-free hydrogel precursor as a first layer, and the second step is to add a cell- or islet-containing hydrogel precursor as a second layer while the gelation of the first step is not yet complete, particularly at a time corresponding to 5% to 25% of the gelation time of the first hydrogel.

[1275] Figure 1 An implant (1) is shown, comprising a hydrogel (11) containing cells or islets of Langerhans (not shown), a ring (12), and a mesh (13). The upper portion of the ring, the lower portion of the ring, and the mesh may be glued together (not shown).

[1276] Figure 2 An implant (1) is shown, comprising a hydrogel (11) containing cells or islets (not shown), a ring (12) and a mesh (13), wherein the hydrogel is concave.

[1277] Figure 3 The image shows a top view of an implant (1) comprising a hydrogel (11) containing cells or islets of Langerhans (not shown), a ring (12), and a mesh (13).

[1278] Figure 4 An implant (1) is shown, comprising a cell-free hydrogel (20) sandwiching a cell-containing hydrogel (21) in between. The upper portion of 20 is optional.

[1279] Figure 5 The non-fasting blood glucose levels (in g / L) measured in control rats (rats 1, 2, and 3) and rats implanted with C16B-18 (rats 4) or rats implanted with C16B-3A / C16B-3B (rats 338) are shown. Rats 4 received 6200 IEQ, and rats 338 received 4000 IEQ. The vertical dashed lines represent implantation, removal in rat 4, and removal in rat 338, respectively. The gray areas with dashed lines represent blood glucose values ​​(minimum, maximum, average) measured in the animals before streptozotocin-induced diabetes. Blood glucose values ​​>6 g / L were unmeasurable on the glucometer (measured as high by the device) and were arbitrarily set to 6.5 g / L.

[1280] This invention relates to all embodiments listed below.

[1281] A cross-linked dextran polymer with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are connected by at least one central linker group L(-). iCovalent crosslinking, wherein at least one group is at least a divalent straight-chain, branched, or cyclic alkyl group comprising at least a polyethylene glycol chain, or wherein at least one group comprises at least a polyethylene glycol chain. A straight-chain, branched, or cyclic alkyl group with at least a divalent alkyl group of azoline (POx) chain.

[1282] The problem is solved by providing novel crosslinked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are connected by at least one central linker group L(-). i Covalent crosslinking, wherein the at least one group is at least a divalent straight-chain, branched, or cyclic alkyl group comprising at least a polyethylene glycol chain.

[1283] The problem is solved by providing novel crosslinked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are connected by at least one central linker group L(-). i Covalent crosslinking, wherein the at least one group comprises at least poly( A straight-chain, branched, or cyclic alkyl group with at least a divalent alkyl group of azoline (POx) chain.

[1284] In one embodiment, the crosslinked dextran polymer according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1285] -L(-) i It is a straight-chain or branched polyether with heteroatoms (such as oxygen, nitrogen, or sulfur) at its ends.

[1286] -i represents the valence of L and -(R1) m The number of G1- groups, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[1287] -m is an integer equal to 0 or 1.

[1288] -W is -(R1) m G1-group, in which

[1289] •-R1- is a straight-chain or branched alkyl divalent group containing 1 to 6 carbon atoms and optionally heteroatoms (e.g., oxygen, nitrogen, or sulfur).

[1290] •-G1- is a straight-chain or branched or cyclic alkyl divalent group containing 1 to 6 carbon atoms and may contain heteroatoms (e.g., oxygen, nitrogen or sulfur).

[1291] In one embodiment, the dextran polymer is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups.i The product is a group generated from straight-chain or branched mercapto polyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, wherein:

[1292] -Mn is 1000 to 25000 g / mol (1000 ≤ Mn ≤ 25000 g / mol) or

[1293] - Degree of polymerization (DP) is 15 to 600 (15 ≤ DP ≤ 600).

[1294] In one embodiment, the dextran polymer is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i Not a group derived from straight-chain or branched mercapto polyethylene glycol containing at least 2 sulfur atoms and at most 8 arms, wherein:

[1295] - Number-average molecular weight (Mn) of 500 to 40000 g / mol (500 ≤ Mn ≤ 40000 g / mol), or

[1296] - The degree of polymerization (DP) is 8 to 1000 (8 ≤ DP ≤ 1000).

[1297] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1298] -L(-) i It is a linear or branched polyether, or L(-) i Is it a straight-chain or branched polymer? (zoline)

[1299] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1300] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyethers, or poly( (Azoline) derivatives, and do not contain two or more α-amino acid residues, especially α-amino acid residues linked by peptide bonds.

[1301] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1302] -L(-) i It is a straight-chain or branched polyether.

[1303] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1304] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1305] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1306] -L indicates straight-chain or branched polymer ( (zoline),

[1307] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1308] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1309] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1310] -L(-) i It is a linear or branched polyether, or L(-) i Is it a straight-chain or branched polymer? (zoline)

[1311] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1312] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyethers, or poly( (Azoline) derivatives, and do not contain two or more α-amino acid residues, especially α-amino acid residues linked by peptide bonds.

[1313] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1314] -L(-) i It is a straight-chain or branched polyether.

[1315] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1316] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1317] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1318] -L indicates straight-chain or branched polymer ( (zoline)

[1319] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1320] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1321] In one embodiment, the crosslinked dextran hydrogel according to the invention is a dextran polymer, wherein the central linker L(-) i It is a straight-chain or branched polyethylene glycol (PEG) group.

[1322] In one implementation, the center connector L(-) i It is a PEG selected from formula I:

[1323]

[1324] in:

[1325] • i is an integer from 2 to 8 (2 ≤ i ≤ 8)

[1326] p is an integer equal to 0 or 1, and if i = 2, then p = 0.

[1327] ·q is an integer between 8 and 1000 (8 ≤ q ≤ 1000)

[1328] ·r is an integer equal to 0 or 1

[1329] Q is a carbon atom, or a straight-chain, branched, or cyclic alkyl chain, or an aromatic compound containing 2 to 10 carbon atoms and may contain heteroatoms (e.g., nitrogen, oxygen, or sulfur).

[1330] * indicates the site of f4, which is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond (if the crosslinking process is carried out by natural chemical linkage (NCL)).

[1331] In one embodiment, the crosslinked dextran hydrogel according to the invention is a dextran polymer, wherein the central linker L is a straight-chain or branched POx group.

[1332] In one implementation, the POx center connector is a 2-arm POx, selected from type XII connectors.

[1333]

[1334] in:

[1335] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[1336] * indicates the site of f3, which is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1337] In one implementation, the POx center connector is a 2-arm POx, selected from the XIIbis connector.

[1338]

[1339] in:

[1340] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[1341] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1342] In one implementation, the POx center connector is a 4-arm POx, selected from type XIII connectors.

[1343]

[1344] in:

[1345] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[1346] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1347] In another embodiment, the POx center connector is a 4-arm POx, selected from the XIV connector:

[1348]

[1349] in:

[1350] • The group -R1 is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0≤n1≤4)), branched or cyclic alkyl derivatives.

[1351] The divalent group -R2- is a straight-chain -(CH2) group. n2 - where n2 is an integer from 2 to 6 (2≤n2≤6).

[1352] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1353] In another embodiment, the POx center connector is a 4-arm POx, selected from the XV connector:

[1354]

[1355] in:

[1356] • The group -R is a straight-chain -(CH2) group. n1 -CH3 (where n1 is an integer from 0 to 4 (0 ≤ n1 ≤ 4)), branched or cyclic alkyl derivatives,

[1357] • In one implementation, R1 = -CH2-CH2-, and R2 is a straight-chain -(CH2) n2- where n2 is an integer from 2 to 6 (2 ≤ n2 ≤ 6),

[1358] • In another implementation, R2 = -CH2-CH2-, and R1 is a straight-chain *-(CH2) n2 -*, where n2 is an integer from 2 to 6 (2 ≤ n2 ≤ 6),

[1359] * indicates the site of f3, where f3 is an amine functional group, or an ether functional group, or a thioether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1360] In one embodiment, -W- is selected from group IV.

[1361]

[1362] in

[1363] * indicates the site of f1, and This indicates the connection point with L.

[1364] ·a is an integer equal to 0 or 1.

[1365] b is an integer equal to 0 or 1.

[1366] c is an integer equal to 0 or 1.

[1367] • In one embodiment, a = 0, and f1 is an ether functional group or a carbamate functional group.

[1368] In one implementation scheme, a = 1.

[1369] The divalent group -A- is a straight-chain -(CH2). n1 -(where n1 is an integer from 1 to 7 (1 ≤ n1 ≤ 7)), branched or cyclic alkyl derivatives. It can also be branched by at least one hydroxyl group, to form -CH2-CH(OH)-(CH2) where n2 is an integer from 1 to 5 (1 ≤ n2 ≤ 5). n2 -; f1 is an ether functional group or a carbamate functional group, and f2 is an amide functional group.

[1370] or,

[1371] The divalent group -A- is a linear polyether (PEG) derivative; f1 is an ether functional group or a urethane functional group, and f2 is an amide functional group.

[1372] or,

[1373] The divalent group -A- is a 4-alkyl-1,4-triazole derivative or a 4-PEG-1,4-triazole derivative; f1 is an ether functional group or a carbamate functional group, and f2 is a carbon-nitrogen covalent bond.

[1374] or,

[1375] The divalent group -A- is an l-alkyl-1,4-triazole derivative or an l-PEG-1,4-triazole derivative; f1 is an ether functional group or a carbamate functional group, and f2 is a carbon-aromatic carbon covalent bond.

[1376] • The divalent group -R1- is a straight-chain, branched or cyclic alkyl derivative, and / or an aromatic derivative, and / or a polyether (PEG) derivative, which may contain heteroatoms such as nitrogen, oxygen or sulfur.

[1377] If b = 0, then f1 is an ether functional group or a carbamate functional group.

[1378] If b = l, then f1 is an ether functional group or a carbamate functional group, and f3 is an amide functional group, or an amine functional group, or an ether functional group, or a thioether functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond (if the crosslinking process is carried out by natural chemical linkage (NCL)).

[1379] The divalent group -G1- is a straight-chain, branched, or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, such as up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms, or up to 1 phosphorus atom. In a preferred embodiment, -G1- is a succinimide derivative, or an alkyl sulfone derivative that may contain one heteroatom (e.g., oxygen or sulfur), or an ethylamide derivative, or a 1,4-triazole derivative, or a polycyclic derivative derived from a Diels-Alder reaction, or an aromatic phosphine derivative generated by Staudinger ligation, or a naturally chemically linked cysteine ​​derivative.

[1380] If c = 0, then f1 is an ether functional group or a carbamate functional group.

[1381] If c = 1, then f1 is an ether functional group or a carbamate functional group, and f4 is an amine functional group, or an amide functional group, or a carbamate functional group, or a thioether functional group, or an ether functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond (if the crosslinking process is carried out by natural chemical linkage (NCL)).

[1382] The crosslinked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least one divalent group L(-) is present.i It is covalently bonded to the backbone of a dextran polymer having i groups, wherein the dextran polymer is selected from dextran of formula X.

[1383]

[1384] in:

[1385] ·a is an integer equal to 0 or 1.

[1386] • i is an integer from 2 to 8 (2≤i≤8).

[1387] L can be the same as [Dx-f1-(A-f2)]. a It can be linked to [-G1-f3] groups, or to different [Dx-f1-(A-f2] groups. a [-G1-f3] group linkage.

[1388] • Dx- is the dextran moiety, which can be substituted by a specific anionic group in salt form, and optionally by an alkyl carboxylic acid ester derivative in salt form.

[1389] •f1 is an ether functional group.

[1390] The divalent group -A- is a straight-chain -(CH2) group. n1 -(where n1 is an integer from 1 to 7 (1 ≤ n1 ≤ 7)), branched or cyclic alkyl derivatives. It can also be branched by at least one hydroxyl group, to form -CH2-CH(OH)-(CH2) where n2 is an integer from 1 to 5 (1 ≤ n2 ≤ 5). n2 -

[1391] ·f2 is an amide functional group.

[1392] The divalent group -G1- is a straight-chain, branched, or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, such as up to 5 nitrogen atoms, up to 10 oxygen atoms, or up to 5 sulfur atoms. In a preferred embodiment, -G1- is a succinimide derivative, or an alkyl sulfone derivative that may contain one heteroatom (e.g., oxygen or sulfur), or a 1,4-triazole derivative.

[1393] • Integer i represents the valence of the central connector L and whether it is the same as or different from that connected to L [Dx-f1-(A-f2)]. a The number of -G1-f3] groups.

[1394] •f3 is an amine functional group, or a thioether functional group, or an ether functional group, or an amide functional group, or a carbamate functional group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1395] • Center connector L is poly( Azoline (POx) derivative, which can be linear or branched.

[1396] In this embodiment, the crosslinked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1397]

[1398] in

[1399] • f1, f2, f3, f4, -A-, -R1-, -G1- are defined according to equation IV above, and

[1400] • Dx- is the dextran moiety, which may be substituted by a specific anionic group in salt form, and optionally by an alkyl carboxylic acid ester derivative in salt form as defined above.

[1401] • Integer i represents the valence of the central connector L and whether it is the same as or different from that connected to L [Dx-f1-(A-f2)]. a -(R1-f3) b -(G1-f4) c The number of groups.

[1402] • The central connector L is a polyether (PEG) derivative, which can be linear or branched.

[1403] • In one implementation, if b = 0 and c = 1, then the center connector L can be poly( Azoline (POx) derivative, which can be linear or branched.

[1404] In this embodiment, the crosslinked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1405]

[1406] in

[1407] • f1, f2, f3, f4, -A-, -R1-, -G1- are defined according to equation IV above, and

[1408] • Dx- is the dextran moiety, which may be substituted with a specific anionic group in salt-forming form, and optionally with an alkyl carboxylic acid ester derivative in salt-forming form as defined above.

[1409] • Integer i represents the valence of the central connector L and whether it is the same as or different from that connected to L [Dx-f1-(A-f2)a-(R1-f3)] b -(G1-f4) c The number of groups.

[1410] • The central connector L is a polyether (PEG) derivative, which can be linear or branched.

[1411] The hydroxyl functional group of the dextran polymer Dx- can be functionalized by at least one specific anionic group, such as: alkyl carboxylate, sulfate anion, sulfonate anion, phosphate anion, or phosphonate anion.

[1412] In one embodiment, the hydroxyl functional group of the dextran polymer Dx- is functionalized with a specific anionic group, said anionic group being an alkylcarboxylate anion.

[1413] In one embodiment, the crosslinked dextran polymer with anionic groups according to the invention is a dextran polymer wherein the dextran polymer backbone is according to Formula III.

[1414]

[1415] Where R is selected from

[1416] --H, an anionic group of formula II, or containing L(-) i The -W- group of the crosslinking group,

[1417] -i is between 20 and 5000 (20 ≤ i ≤ 5000),

[1418] --W- and L(-) i The group has the meaning as previously defined.

[1419] In one embodiment, the crosslinked dextran polymer with anionic groups according to the invention is a dextran polymer wherein the dextran polymer backbone is according to formula XI.

[1420]

[1421] Where R is selected from

[1422] --H, an anionic group of formula II, or containing L(-) i -(A-f2) of the crosslinking group a -G1- group,

[1423] -I is between 20 and 5000 (20 ≤ I ≤ 5000),

[1424] --(A-f2) a -G1- and L(-) i The group has the meaning previously defined.

[1425] In one embodiment, the crosslinked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1426]

[1427] in:

[1428] • i is an integer from 2 to 8 (2 ≤ i ≤ 8)

[1429] ·a=1,

[1430] b = 1,

[1431] c = 1,

[1432] • Dx is the dextran derivative described in Formula III.

[1433] • L is the PEG center connector described in Formula I.

[1434] • f1 is an ether functional group or a carbamate functional group.

[1435] The divalent group -A- is a straight-chain -(CH2) group. n1 -(where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives,

[1436] ·f2 is an amide functional group.

[1437] • The divalent group -R1- is a straight-chain -(CH2)n1- (where n1 is an integer from 1 to 7 (1≤n1≤7)), branched, or cyclic alkyl derivative.

[1438] •f3 is an amide functional group.

[1439] The divalent group -G1- is a 1,4-triazole derivative.

[1440] •f4 is a carbon-nitrogen covalent bond, specifically in which the nitrogen atom is inside the triazole ring.

[1441] ·

[1442] In a preferred embodiment, the integer i equals 4, i = 4.

[1443] According to the above embodiments, the triazole derivative comprises a cyclooctene derivative derived from strained cyclooctene, the cyclooctene derivative containing a heteroatom, such as nitrogen, oxygen or sulfur, and optionally functionalized by a straight-chain, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms, or by an aromatic derivative, or by a heteroatom (e.g., nitrogen, oxygen or sulfur, or halogen (especially fluorine)).

[1444] According to the two embodiments described above, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains 4 to 6 nitrogen atoms.

[1445] According to one implementation scheme, the 1,4-triazole derivative is obtained through a copper-free reaction.

[1446] In one embodiment, the crosslinked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1447]

[1448] in:

[1449] • i is an integer from 2 to 8 (2 ≤ i ≤ 8)

[1450] ·a=1,

[1451] b = 0,

[1452] c = 1,

[1453] • Dx is the dextran derivative described in Formula III.

[1454] • L is the PEG center connector described in Formula I.

[1455] The divalent group -A- is a straight-chain -(CH2) group. n1 -(where n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives,

[1456] • f1 is an ether functional group or a carbamate functional group.

[1457] The divalent group -G1- is a 1,4-triazole derivative, which is a cyclooctene derivative derived from strained cyclooctene. This cyclooctene derivative may contain a heteroatom, such as nitrogen, oxygen, or sulfur, and is optionally functionalized with a straight-chain, branched, or cyclic alkyl derivative containing 2 to 20 carbon atoms, or with an aromatic derivative, or with a heteroatom (e.g., nitrogen, oxygen, or sulfur, or a halogen (especially fluorine)).

[1458] The 1,4-triazole derivative contains nitrogen, which is covalently linked to -CORa-, where Ra is an alkyl group containing 1 to 4 carbon atoms, thereby forming an amide functional group. Ra is also linked to f2, which is an amide functional group, and f4 is a carbon-nitrogen covalent bond, wherein the nitrogen atom is inside the triazole ring.

[1459] In a preferred embodiment, the integer i equals 4, i = 4.

[1460] According to one embodiment, 1,4-triazole is a polycyclic group containing an acyl group, the acyl group being connected to nitrogen via an amide functional group, the nitrogen being within a ring but not derived from the triazole ring.

[1461] According to one embodiment, 1,4-triazole comprises cyclooctyne with a nitrogen in the cyclooctyne ring.

[1462] According to one embodiment, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally, it contains 4 to 6 nitrogen atoms.

[1463] In one embodiment, the divalent group -G1- is a 1,4-triazole derivative described by the following formula:

[1464]

[1465] Wherein: * represents the site of f2, which is an amide functional group, and the dashed bond represents f4, which is a carbon-nitrogen bond.

[1466] •f4 is a carbon-nitrogen covalent bond, in which the nitrogen atom is inside the triazole ring.

[1467] According to one implementation scheme, triazole 1,4 is obtained through a copper-free reaction.

[1468] According to one implementation, Dx is the dextran derivative described in Formula III:

[1469]

[1470] Where R is selected from

[1471] --H, an anionic group of formula II, or containing L(-) i The -W- group of the crosslinking group,

[1472] -i is between 20 and 5000 (20 ≤ i ≤ 5000),

[1473] --W- and L(-) i The group has the meaning previously defined.

[1474] According to one implementation, L is the PEG center connector described in Formula I:

[1475]

[1476] in:

[1477] ·i is an integer equal to 4,

[1478] · p is an integer equal to 1,

[1479] ·q is an integer between 8 and 1000 (8 ≤ q ≤ 1000)

[1480] ·r is an integer equal to 0 or 1

[1481] • Q is a branched alkyl chain containing 2 to 10 carbon atoms.

[1482] * indicates the site of f4, which is a carbon-nitrogen covalent bond, wherein the nitrogen atom is inside the triazole ring.

[1483] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 2, 4 or 8.

[1484] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 2, 4 or 8.

[1485] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 2.

[1486] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 2.

[1487] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 4.

[1488] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 4.

[1489] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, where i is 8.

[1490] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer with an anionic group of formula II, wherein at least the divalent group L(-) is present. i It is covalently bonded to the polymer backbone of dextran having i groups, where i is 8.

[1491] In one embodiment, the crosslinked dextran polymer according to the invention is a dextran polymer in which the dextran polymer backbone is dextran with a weight-average molecular weight (Mw) of 5 to 1000 kDa before crosslinking and substitution.

[1492] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer having L(-) markings. i The -W- group or -(A-f2) of the crosslinking group a The degree of substitution (DS1) of the dextran backbone of the -G1- group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[1493] In one embodiment, the crosslinked dextran polymer according to the invention is a crosslinked dextran polymer wherein the degree of substitution (DS3) of the sulfate / salt, sulfonate / salt, phosphate / salt, and phosphonate / salt of the dextran backbone is in the range of 0.2 to 2.5 (0.2 ≤ DS3 ≤ 2.5).

[1494] The present invention also relates to a dextran polymer of formula VIII prior to the crosslinking reaction.

[1495]

[1496] in

[1497] If none of a, a', b, and b' are equal to 0, then f1, f2, f3, and Dx are defined as above.

[1498] ·and

[1499] x equals 0 or 1.

[1500] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1501] • If either b' or c is not equal to 0, then -A'- is -A- as defined above.

[1502] • If b', b, and c equal 0, then a equals 0, and A' is the precursor A prior to the crosslinking reaction.

[1503] • If c is not equal to 0, then -R'1- is -R1- as defined above, and -G'1- is the precursor -G1-.

[1504] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1505] This invention relates to hydrogels, which comprise:

[1506] - A cross-linked dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1507] -L(-) i It is a linear or branched polyether

[1508] -i represents the valence of L and the number of W groups bound to the dextran polymer, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[1509] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1510] In one embodiment, the hydrogel comprises a non-crosslinked hyaluronic acid salt in solution form.

[1511] In one embodiment, the present invention relates to a hydrogel comprising:

[1512] -Biological cells,

[1513] -Non-crosslinked hyaluronic acid salts in solution form, and

[1514] - A cross-linked dextran polymer Dx with anionic groups, wherein at least one divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1515] -L(-) i It is a linear or branched polyether

[1516] -i represents the valence of L and the number of W groups bound to the dextran polymer, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[1517] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1518] In one embodiment, the crosslinked dextran polymer contained in the hydrogel according to the invention is not a dextran polymer with carboxylic acid groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1519] -L(-) i It is a straight-chain or branched polyether with heteroatoms (such as oxygen, nitrogen, or sulfur) at its ends.

[1520] -i is the valence of L and -(R1) m The number of G1- groups, and is an integer from 2 to 8 (2 ≤ i ≤ 8).

[1521] -m is an integer equal to 0 or 1.

[1522] -W is -(R1) m G1-group, in which

[1523] •-R1- is a straight-chain or branched divalent alkyl group containing 1 to 6 carbon atoms and optionally heteroatoms (e.g., oxygen, nitrogen, or sulfur).

[1524] •-G1- is a straight-chain, branched, or cyclic alkyl divalent group containing 1 to 6 carbon atoms, and may contain heteroatoms such as oxygen, nitrogen, or sulfur.

[1525] In one embodiment, the dextran polymer contained in the hydrogel is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i The product is a group derived from straight-chain or branched thiol polyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, wherein:

[1526] -Mn is 1000 to 25000 g / mol (1000 ≤ Mn ≤ 25000 g / mol) or

[1527] - Degree of polymerization (DP) is 15 to 600 (15 ≤ DP ≤ 600).

[1528] In one embodiment, the dextran polymer contained in the hydrogel is not such a dextran polymer, wherein at least a divalent group L(-) is covalently bonded to the dextran polymer backbone having i W groups. i Not a group derived from straight-chain or branched mercapto polyethylene glycol containing at least 2 sulfur atoms and at most 8 arms, wherein:

[1529] - Number-average molecular weight (Mn) of 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol), or

[1530] - The degree of polymerization (DP) is 8 to 1000 (8 ≤ DP ≤ 1000).

[1531] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1532] -L(-) i It is a linear or branched polyether, or L(-) i Is it a straight-chain or branched polymer? (zoline),

[1533] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1534] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyethers, or poly( (Azoline) derivatives, and do not contain two or more α-amino acid residues, especially α-amino acid residues linked by peptide bonds.

[1535] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1536] -L(-) i It is a straight-chain or branched polyether.

[1537] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1538] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1539] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1540] -L indicates straight-chain or branched polymer ( (zoline),

[1541] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1542] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1543] In one embodiment, the hydrogel comprises hyaluronic acid or sodium or potassium hyaluronic acid.

[1544] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1545] -L(-) i It is a linear or branched polyether, or L(-) i Is it a straight-chain or branched polymer? (zoline)

[1546] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1547] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyethers, or poly( (Azoline) derivatives, and do not contain two or more α-amino acid residues, especially α-amino acid residues linked by peptide bonds.

[1548] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) is present. i It is covalently bonded to the dextran polymer backbone having i -W- groups, wherein,

[1549] -L(-) i It is a linear or branched polyether

[1550] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1551] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1552] The crosslinked dextran polymer contained in the hydrogel according to the invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bonded to the dextran polymer backbone having i W groups, wherein,

[1553] -L indicates a straight-chain or branched polymer ( (zoline)

[1554] -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8).

[1555] --W- is a group containing at least a straight-chain or branched alkyl group, and optionally contains heteroatoms (e.g., oxygen, nitrogen, or sulfur), aromatic rings, polyether derivatives, and does not contain two or more α-amino acid residues, particularly α-amino acid residues linked by peptide bonds.

[1556] In one embodiment, the hydrogel according to the invention is characterized in that Tanδ is less than 1.

[1557] In one implementation, the hydrogel is transparent.

[1558] In one implementation, the hydrogel is translucent.

[1559] In one embodiment, the hydrogel according to the invention is characterized in that, after swelling in water, the concentration of the crosslinked dextran polymer is 0.01 to 0.2 g / g.

[1560] In one embodiment, the Young's modulus of the hydrogel is 1 to 200 kPa.

[1561] In one embodiment, the G' of the hydrogel is 0.5 to 70 kPa.

[1562] In one embodiment, the fracture compressive deformation of the hydrogel is greater than or equal to 10%.

[1563] In one embodiment, the swelling ratio of the hydrogel is greater than 0.7.

[1564] In one embodiment, the hydrogel has a water content of at least 80 wt%. In another embodiment, the hydrogel according to the invention is characterized in that it further contains biological cells.

[1565] In one implementation, the cell is a protein, hormone, or peptide-secreting cell.

[1566] In one implementation, the cells are selected from:

[1567] Insulin-secreting cells used in diabetes treatment

[1568] - Factor VIII or Factor IX secretory cells used for the treatment of hemophilia and β-glucocerebrosidase secretory cells used for Gaucher disease.

[1569] In one embodiment, the hydrogel according to the invention is characterized in that the biological cells are pseudo-islets.

[1570] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention into a hydrogel form, comprising the following steps:

[1571] g) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -W- of dextran.

[1572] h) Preparation of precursor L(-) i sterile solution,

[1573] i) Add the sterile solution obtained in step b) to the solution obtained in step a).

[1574] j) Add the solution directly into the mold, or introduce the solution into the mold after mixing.

[1575] k) Perform crosslinking and gelation, for example, at room temperature (20°C to 25°C) or at 37°C.

[1576] l) Demolding and swelling are performed to obtain a hydrogel.

[1577] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention into a hydrogel form, comprising the following steps:

[1578] g) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -(A-f2). a -G1-、-(A'-f2) a -G'1-dextran,

[1579] h) Preparation of precursor L(-) i sterile solution,

[1580] i) Add the sterile solution obtained in step b) to the solution obtained in step a).

[1581] j) Add the solution directly into the mold, or introduce the solution into the mold after mixing.

[1582] k) Perform crosslinking and gelation, for example, at room temperature (20°C to 25°C) or at 37°C.

[1583] l) Demolding and swelling are performed to obtain a hydrogel.

[1584] In one implementation, steps c) and d) are performed simultaneously.

[1585] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[1586] Dextran bearing an anionic group of Formula II is prepared by grafting or substituting a hydroxyl group on the dextran. In one embodiment, dextran bearing an anionic group of Formula II is prepared by grafting or substituting a carboxymethyl group on a methyl dextran carboxylate / salt.

[1587] The crosslinking step is the gelling step that leads to the formation of the hydrogel according to the invention.

[1588] The present invention also relates to a method for preparing hydrogels containing biological cells, comprising the following steps:

[1589] i) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -W- of dextran.

[1590] j) Preparation of precursor L(-) i sterile solution,

[1591] k) Prepare biological cell suspensions,

[1592] l) Mix the biological cell suspension obtained in step c) with the solution obtained in step b) or a).

[1593] m) Add the sterile solution obtained from step a) or b) that was not used in step d) to the solution obtained from step d).

[1594] n) The addition in step e) can be done directly in the mold, or the solution can be introduced into the mold after mixing.

[1595] o) Crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1596] p) Demolding and swelling are performed to obtain a hydrogel containing biological cells.

[1597] The present invention also relates to a method for preparing hydrogels containing biological cells, comprising the following steps:

[1598] i) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -(A-f2). a-G1-、-(A'-f2) a -G'1-dextran,

[1599] j) Preparation of precursor L(-) i sterile solution,

[1600] k) Prepare biological cell suspensions,

[1601] l) Mix the biological cell suspension obtained in step c) with the solution obtained in step b) or a).

[1602] m) Add the sterile solution obtained from step a) or b) that was not used in step d) to the solution obtained from step d).

[1603] n) The addition in step e) can be done directly in the mold, or the solution can be introduced into the mold after mixing.

[1604] o) Crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1605] p) Demolding and swelling are performed to obtain a hydrogel containing biological cells.

[1606] The present invention also relates to a method for preparing hydrogels containing biological cells, comprising the following steps:

[1607] l) Prepare a sterile solution comprising an anionic group of formula II and at least two precursors -W- of dextran.

[1608] m) Prepare precursor L(-) selected from the following i Sterile solutions: thiol polyethylene glycol, mercapto-poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide, or pentaerythritol poly(dibenzocyclooctylene) polyoxyethylene.

[1609] n) Prepare a sterile solution of sodium hyaluronate.

[1610] o) Prepare a sterile suspension of biological cells.

[1611] p) The sodium hyaluronate solution obtained in step c) is mixed with the precursor solution from step b).

[1612] q) Mix the biological cell suspension obtained from step d) with the solution obtained from step e) or from step a).

[1613] r) Mix the solution obtained in step f) with the solution obtained in step e).

[1614] s) Add the unused sterile solution obtained from step a) or e) in step g) to the solution obtained from step f).

[1615] t) The addition in step g) can be done directly in the mold, or the solution can be introduced into the mold after mixing.

[1616] u) Crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1617] v) Demolding and swelling are performed to obtain a hydrogel containing biological cells.

[1618] According to one embodiment, in the molding step, the solution contains a penetrant, which is a nonionic penetrant, such as trehalose.

[1619] According to one implementation, in the molding step, the solution contains a nonionic penetrant with a weight ratio of NaCl greater than 2, particularly greater than 5, and even more particularly greater than 10.

[1620] According to one embodiment, during the molding step, the solution contains 5 to 50 mg / ml of a nonionic penetrant, particularly trehalose.

[1621] In one implementation, the mold is a ring network.

[1622] In one embodiment, the crosslinking and gelation reactions are carried out at room temperature (20°C to 25°C).

[1623] The present invention also relates to a reagent kit comprising:

[1624] - Solution of the dextran polymer of formula VIII prior to the crosslinking reaction:

[1625]

[1626] in

[1627] If a, a', b, and b' are all equal to 0, then f1, f2, f3, f4, and Dx are defined according to equation IV.

[1628] ·and

[1629] x equals 0 or 1.

[1630] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1631] • If either b' or c is not equal to 0, then -A' is A as defined above.

[1632] • If b', b, and c are equal to 0, then a is equal to 0, and A' is the precursor A before the crosslinking reaction.

[1633] • If c is not equal to 0, then R'1 is R1 as defined above, and G'1 is the precursor G1.

[1634] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1635] - A solution of thiol polyethylene glycol, mercapto polyethylene (oxyethylene), pentaerythritol polyethylene (oxyethylene) azide, or pentaerythritol polyethylene (dibenzocyclooctylene) polyethylene (oxyethylene).

[1636] - Biological cells.

[1637] The present invention also relates to a reagent kit comprising:

[1638] - Solution of the dextran polymer of formula VIII prior to the crosslinking reaction:

[1639]

[1640] in

[1641] If a, a', b, and b' are all equal to 0, then f1, f2, f3, f4, and Dx are defined according to equation IV.

[1642] ·and

[1643] x equals 0 or 1.

[1644] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1645] • If either b' or c is not equal to 0, then -A' is A according to the definition above.

[1646] • If b', b, and c are equal to 0, then a is equal to 0, and A' is the precursor A before the crosslinking reaction.

[1647] • If c is not equal to 0, then R'1 is R1 as defined above, and G'1 is the precursor G1.

[1648] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1649] - A solution of thiol polyethylene glycol, mercapto polyethylene (oxyethylene), pentaerythritol polyethylene (oxyethylene) azide, or pentaerythritol polyethylene (dibenzocyclooctylene) polyethylene (oxyethylene).

[1650] - Biological cells.

[1651] - Non-crosslinked sodium hyaluronate solution.

[1652] The present invention also relates to a reagent kit comprising:

[1653] - Solution of the dextran polymer of formula VIII prior to the crosslinking reaction:

[1654]

[1655] in

[1656] If a, a', b, and b' are all equal to 0, then f1, f2, f3, f4, and Dx are defined according to equation IV.

[1657] ·and

[1658] x equals 0 or 1.

[1659] • If a, a', b, and b' are equal to 0, then x is equal to 0, and Dx is the dextran polymer backbone according to Formula III, wherein R is selected from -H or anionic groups of Formula II.

[1660] • If either b' or c is not equal to 0, then -A' is A according to the definition above.

[1661] • If b', b, and c are equal to 0, then a is equal to 0, and A' is the precursor A before the crosslinking reaction.

[1662] • If c is not equal to 0, then R'1 is R1 as defined above, and G'1 is the precursor G1.

[1663] • If c equals 0, then b equals 0, and R'1 is the precursor R1 prior to the crosslinking reaction.

[1664] - A solution of thiol polyethylene glycol, mercapto polyethylene (oxyethylene), pentaerythritol polyethylene (oxyethylene) azide, or pentaerythritol polyethylene (dibenzocyclooctylene) polyethylene (oxyethylene).

[1665] - Biological cells.

[1666] The present invention also relates to the use of cross-linked dextran copolymers according to the invention in the form of hydrogels for the preparation of cell compositions.

[1667] The present invention also relates to the therapeutic use of the hydrogel according to the invention as a therapeutic implant for administering at least an API to mammals.

[1668] The present invention also relates to the therapeutic use of the hydrogel according to the invention for treating a symptom or disease in mammals, wherein the symptom or disease is caused by a lack of endocrine function or dysfunction of the pancreatic organ.

[1669] The present invention also relates to hydrogels used as pharmaceuticals.

[1670] The present invention also relates to hydrogels for treating diseases such as diabetes.

[1671] The present invention also relates to implants comprising the hydrogel of the present invention.

[1672] In one embodiment, the present invention relates to an implant comprising a mesh, a ring, a hydrogel according to the invention, and cells.

[1673] The ring and network structures make hydrogels easy to manipulate and resistant to manipulations, including implantation. This is true even for hydrogels with larger pore sizes (e.g., those with lower DS- and lower concentrations of reactive groups during crosslinking).

[1674] The ring and network structure makes the hydrogel easy to manipulate and resistant to manipulations, including implantation. This is even true for hydrogels with larger pore sizes (e.g., -(A-f2) with lower DS during crosslinking). a The same applies to hydrogels containing -G1- and lower concentrations of reactive groups.

[1675] The ring and network structure makes the hydrogel easy to manipulate and provides good resistance to manipulations, including implantation, even when the hydrogel is very thin but has a fairly large planar surface.

[1676] In one embodiment, the implant is a parallelepiped rectangle with rounded corners.

[1677] In one implementation, the thickness of the implant is less than 3,000 μm.

[1678] In one implementation, the total surface area of ​​the implant is 10 cm². 2 Up to 200cm 2 .

[1679] In one implementation, the implant contains 0.5 to 20 ml of hydrogel.

[1680] In one embodiment, the inner diameter of the ring is 10 to 100 mm.

[1681] In one implementation, the ring is a parallelepiped rectangle with rounded corners.

[1682] In one embodiment, the ring is made of a bio-inert material.

[1683] In one embodiment, the ring is made of a biocompatible elastomer.

[1684] In one embodiment, the material of the ring is selected from silicone, particularly PDMS, polyurethane, polyether, polyether polyester copolymer and polypropylene oxide.

[1685] In one implementation, the net is non-biodegradable.

[1686] In one implementation, the net is biocompatible.

[1687] In one implementation, the net is non-absorbable.

[1688] In one implementation, the mesh is a surgical mesh.

[1689] In one embodiment, the filament material in the mesh is selected from polypropylene, polyethylene, polyester, especially PET, PTFE, PVDF (polyvinylidene fluoride) and ePVDF (stretched PVDF).

[1690] In one implementation, the thickness of the mesh ranges from 50 to 500 μm.

[1691] In one implementation, the mesh aperture size ranges from 0.4 to 4 mm.

[1692] In one embodiment, the mesh has holes with side dimensions ranging from 0.4 to 4 mm.

[1693] In one implementation, the fabric of the net is selected from knitted fabrics, warp-knitted fabrics, woven fabrics, and nonwoven fabrics.

[1694] In one implementation, the implant can be obtained through the following process:

[1695] - The hydrogel composition is incorporated into the ring network construct. A concentrated polymer solution is mixed using a pipette, and a controlled volume of the mixture is introduced into the ring network construct adhered to a glass slide.

[1696] - Perform cross-linking to induce gelation, then introduce the ring-and-hydrogel composition into a Tris 150mM / NaCl 30mM / cysteine ​​10mM solution at pH 8 or into PBS at pH 7.4.

[1697] - Rinse the hydrogel with cysteine-free PBS solution and then immerse it in the PBS solution overnight at 37°C. The hydrogel block is then stored in PBS solution at 4°C until use. Example

[1698] Partial A-Chemistry

[1699] Example A1: Synthesis of substituted dextran

[1700] Table 1: List of synthesized polysaccharides

[1701]

[1702]

[1703]

[1704]

[1705]

[1706]

[1707]

[1708]

[1709] Polysaccharide 1-dextran carboxylic acid methyl ester / salt sulfate / salt and maleimide

[1710] Polysaccharide 1,1-dextran carboxylic acid methyl ester / salt

[1711] 50 g (0.31 mol glucoside units, 0.93 mol hydroxyl functional groups) of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol was dissolved in water (225 g / L) at 30 °C. NaBH4 (2 × 58 mg, 2 × 1.54 mmol) was added every 30 minutes, and the mixture was stirred at 30 °C for 1 hour. Sodium chloroacetate (72 g, 0.62 mol) was added to the solution, and the mixture was heated at 65 °C for 1 hour. Then, 10N NaOH (103 mL, 1.03 mol) was slowly added over 1.5 hours, and the mixture was stirred at 65 °C for 1 hour. The mixture was diluted with water (85 mL), cooled to room temperature, neutralized with acetic acid, and subsequently purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7 followed by water. The concentration of polysaccharide 1.1 in the final solution was determined by dry extract, followed by acid / base determination to determine the degree of substitution of methyl carboxylate / salt.

[1712] Based on the dry extract: [polysaccharide 1.1] = 46.3 mg / g.

[1713] According to the acid / base determination, the degree of substitution (DS2) of methyl carboxylate / salt is 0.8.

[1714] Polysaccharide 1,2-methylcarboxylic acid dextran

[1715] A 550 g solution of the polysaccharide 1.1 obtained above (46.3 mg / g, DS2 = 0.8, 25.5 g, 112.6 mmol glucoside unit) was protonated with acidified sulfonic acid resin (Purolite C100H, 2.0 equivalents / L, 200 mL, 400 mmol) for 2 hours. The resulting solution was filtered and then freeze-dried. Polysaccharide 1,3-dextran carboxylic acid methyl ester / salt sulfate / salt

[1716] 10.0 g (48.0 mmol glucoside unit) of freeze-dried polysaccharide 1.2 was dissolved in a mixture of DMF (320 mL) and formamide (80 mL). After complete dissolution, 2-methyl-2-butene (80 mL, 755.2 mmol) was slowly added. SO3·DMF complex (58.8 g, 383.9 mmol) was added rapidly, and the reaction mixture was stirred at 30 °C for 3 hours. The mixture was neutralized by slow addition of 5% NaHCO3 aqueous solution (800 mL) and purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with EtOH in NaCl (9 g / L in water), NaCl (9 g / L in water), and then water. The concentration of polysaccharide 1.3 in the final solution was determined by the dry extract, and the degree of sulfate / salt substitution was determined by liquid chromatography following complete sulfate / salt hydrolysis of a representative sample.

[1717] Based on the dry extract: [polysaccharide 1.3] = 39.6 mg / g.

[1718] According to LC analysis, the degree of substitution (DS3) of sulfate ester / salt is 1.5.

[1719] Polysaccharide 1-dextran carboxylic acid methyl ester / salt sulfate / salt and maleimide

[1720] Add 1.76 g (15.82 mmol) of 2-hydroxypyridine 1-oxide (HOPO) to a 303 g solution of the polysaccharide 1.3 obtained above (39.6 mg / g, DS3 = 1.5, DS2 = 0.8, 12.0 g, 31.64 mmol glucoside unit), and cool the mixture to 4 °C. Add N-(2-aminoethyl)maleimide hydrochloride (Mal) (1.68 g, 9.49 mmol) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (3.03 g, 15.82 mmol) to the solution, and stir the reaction mixture at 4 °C for 2 hours. Two additional additions of EDC (3.03 g, 15.82 mmol) were made every 2 hours. The mixture was diluted with phosphate buffer at pH 7 and then purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 1 in the final solution was determined by the dry extract, and the degree of substitution of maleimide was determined by the concentration of D2O. 1 Identified by ¹H NMR. The final solution was stored at -20°C.

[1721] Based on the dry extract: [Polysaccharide 1] = 22.2 mg / g.

[1722] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.25.

[1723] Polysaccharide 2-dextran carboxylic acid methyl ester / salt sulfate / salt and maleimide

[1724] Polysaccharide 2,1-dextran carboxylic acid methyl ester / salt

[1725] Using a method similar to that used to prepare polysaccharide 1.1, polysaccharide 2.1 was obtained from dextran (0.25 mol glucoside units, 0.75 mol hydroxyl functional groups, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol, and with sodium chloroacetate (35.9 g, 0.31 mol) and 10N NaOH (82 mL, 0.82 mol) at 60 °C.

[1726] Based on the dry extract: [polysaccharide 2.1] = 44.2 mg / g.

[1727] According to the acid / base determination, the degree of substitution (DS2) of methyl carboxylate / salt is 0.5.

[1728] Polysaccharide 2,2-methylcarboxylic acid dextran

[1729] Polysaccharide 2.2 was obtained from polysaccharide 2.1 (44.2 mg / g, DS2 = 0.5, 12.0 g, 59.4 mmol glucoside unit) using a method similar to that used to prepare polysaccharide 1.2.

[1730] Polysaccharide 2,3-dextran carboxylic acid methyl ester / salt sulfate / salt

[1731] Polysaccharide 2.3 was obtained from polysaccharide 2.2 (4.0 g, 20.9 mmol glucoside unit) using a method similar to that used to prepare polysaccharide 1.3.

[1732] Based on the dry extract: [polysaccharide 2.3] = 22.0 mg / g.

[1733] According to LC analysis, the degree of substitution (DS3) of sulfate ester / salt is 1.9.

[1734] Polysaccharide 2-dextran carboxylic acid methyl ester / salt sulfate / salt and maleimide

[1735] Polysaccharide 2 was obtained from polysaccharide 2.3 (22.0 mg / g, DS2 = 0.5, DS3 = 1.9, 4.6 g, 11.6 mmol glucoside unit) using a method similar to that used to prepare polysaccharide 1, and was obtained with N-(2-aminoethyl)maleimide hydrochloride (615 mg, 3.48 mmol).

[1736] Based on the dry extract: [Polysaccharide 2] = 12.3 mg / g.

[1737] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.23.

[1738] Polysaccharide 3-dextran carboxylic acid methyl ester / salt sulfate / salt and vinyl sulfone

[1739] HOPO (1.46 g, 13.18 mmol) was added to 280 g of a solution of polysaccharide 1.3 (35.7 mg / g, DS3 = 1.5, DS2 = 0.8, 10.0 g, 26.37 mmol glucoside unit), and the mixture was cooled to 4 °C. 2-[[2-(vinylsulfonyl)ethyl]thio]ethylamine hydrochloride (VS) (1.83 g, 7.91 mmol) (according to the following synthesis: SASTéwart et al., Soft Matter, 2018, 14, 8317), Et3N (1.10 mL, 7.91 mmol), and EDC (2.53 g, 13.18 mmol) were added to the solution, and the reaction mixture was stirred at 4 °C to 25 °C for 2 hours. Two additional additions of EDC (2.53 g, 13.18 mmol) were made every 2 hours. The mixture was diluted with NaCl (9 g / L in water) and then purified by ultrafiltration over a PES membrane (MWCO 5 kDa) using NaCl (9 g / L in water), carbonate buffer at pH 10, NaCl (9 g / L in water), phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 3 in the final solution was determined by the dry extract, and the degree of substitution of vinyl sulfone was determined by the concentration of polysaccharide 3 in D2O. 1 Identified by ¹H NMR. The final solution was stored at -20°C.

[1740] Based on the dry extract: [Polysaccharide 3] = 21.8 mg / g.

[1741] according to 1 H NMR (D2O), degree of substitution of vinyl sulfone (DS1) = 0.28.

[1742] Polysaccharide 4-dextran carboxylic acid methyl ester / salt sulfonate / salt and maleimide

[1743] Polysaccharide 4,1-dextran carboxylic acid methyl ester / salt

[1744] 65 g (0.4 mol glucoside units, 1.2 mol hydroxyl functional groups) of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol was dissolved in water (285 g / L) at 30 °C. Then, NaBH4 (74 mg, 1.95 mmol) was added, and the mixture was stirred at 30 °C for 2 hours. Sodium chloroacetate (140 g, 1.2 mol) was added to the solution, and the mixture was heated at 65 °C for 1 hour. Then, 10 N NaOH (200 mL, 2 mol) was slowly added over 1.5 hours, and the mixture was stirred at 65 °C for 1 hour. The mixture was diluted with water (120 mL), cooled to room temperature, neutralized with acetic acid, and subsequently purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, followed by water. The intermediate polysaccharide (polysaccharide 4.1.1) was then freeze-dried.

[1745] At 65°C, 90 g (0.35 mol glycoside units) of freeze-dried polysaccharide 4.1.1 was dissolved in water (260 g / L), followed by the addition of sodium chloroacetate (204 g, 1.75 mol), and the mixture was maintained at 65°C for 1 hour. Then, 10N NaOH (175 mL, 1.75 mol) was slowly added over 1 hour, and the mixture was stirred at 65°C for another hour. Next, another portion of sodium chloroacetate (122 g, 1.05 mol) was added, and the mixture was maintained at 65°C for 0.5 hours. Then, 10N NaOH (105 mL, 1.05 mol) was slowly added over 1 hour, and the mixture was stirred at 65°C for another hour. The mixture was diluted with water, cooled to room temperature, neutralized with acetic acid, and subsequently purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with pH 7 phosphate buffer, followed by water. The concentration of polysaccharide 4.1 in the final solution was determined by dry extract, followed by acid / base determination to determine the degree of substitution of methyl carboxylate / salt.

[1746] Based on the dry extract: [Polysaccharide 4.1] = 48.4 mg / g.

[1747] According to the acid / base determination, the degree of substitution (DS2) of methyl carboxylate / salt is 2.1.

[1748] Polysaccharide 4,2-dextran carboxylic acid methyl ester / salt sulfonate / salt

[1749] To a 400 g solution of the polysaccharide 4.1 obtained above (48.4 mg / g, DS2 = 2.1, 19.4 g, 58.63 mmol glucoside units), HOPO (9.77 g, 87.95 mmol), 3-amino-1-propanesulfonic acid (high taurine) (9.79 g, 70.36 mmol), Et3N (9.81 mL, 70.36 mmol), and EDC (16.86 g, 87.95 mmol) were added, and the reaction mixture was stirred at 25 °C for 2 hours. Two additional EDC additions (16.86 g, 87.95 mmol) were performed every 2 hours. The mixture was diluted with phosphate buffer at pH 7 and then purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The final polysaccharide concentration of 4.2 in the solution was determined by the dry extract, and the degree of substitution of high taurine was determined by the amount of taurine in D2O. 1 Identified by 1H NMR. The final solution was stored at 4°C.

[1750] Based on the dry extract: [polysaccharide 4.2] = 40.9 mg / g.

[1751] according to 1 H NMR (D2O), degree of substitution of high taurine (DS3) = 1.15.

[1752] Polysaccharide 4-dextran carboxylic acid methyl ester / salt sulfonate / salt and maleimide

[1753] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 4.2 (40.9 mg / g, DS2 = 2.1, DS3 = 1.15, 9.4 g, 20.0 mmol glucoside unit) was started, and polysaccharide 4 was obtained with N-(2-aminoethyl)maleimide hydrochloride (1.06 g, 6.0 mmol).

[1754] Based on the dry extract: [Polysaccharide 4] = 19.9 mg / g.

[1755] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.25.

[1756] Polysaccharide methyl 5-dextran carboxylate / ammonium sulfonate and maleimide

[1757] Polysaccharide 5,1-dextran carboxylic acid methyl ester / ammonium hydrochloride

[1758] Polysaccharide 5.1 was prepared from polysaccharide 4.1 (48.4 mg / g, DS2 = 2.1, 7.3 g, 22.11 mmol glucoside unit) using a method similar to that used to prepare polysaccharide 4.2. The polysaccharide 5.1 was obtained using HOPO (2.95 g, 26.53 mmol), 3-((2-aminoethyl)-dimethylammonium)propane-l-sulfonate / salt (SB) (7.51 g, 26.53 mmol) (according to the following synthesis: L. Yang et al., J. Mater. Chem. B, 2013, 1, 1421), Et3N (7.4 mL, 53.06 mmol), and EDC (3 × 5.09 g, 3 × 26.53 mmol).

[1759] Based on the dry extract: [Polysaccharide 5.1] = 34.6 mg / g.

[1760] according to 1 H NMR (D2O), degree of substitution of SB (DS3) = 0.7.

[1761] Polysaccharide methyl 5-dextran carboxylate / ammonium sulfonate and maleimide

[1762] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 5 was obtained from polysaccharide 5.1 (34.6 mg / g, DS2 = 2.1, DS3 = 0.7, 7.5 g, 16.7 mmol glucoside unit) and N-(2-aminoethyl)maleimide hydrochloride (885 mg, 5.01 mmol).

[1763] Based on the dry extract: [Polysaccharide 5] = 15.0 mg / g.

[1764] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.22.

[1765] Polysaccharide methyl 6-dextran carboxylate / salt and cyclooctyne (DBCO)

[1766] HOPO (244 mg, 2.20 mmol) and DMF (25 mL) were added to a 30 g solution of polysaccharide 4.1 (48.4 mg / g, DS2 = 2.1, 1.45 g, 4.39 mmol glucoside unit), and the mixture was cooled to 4 °C. 3-amino-1-(11,12-disodehydrodibenzo[b,f]azo-5(6H)-yl)-1-propanone (DBCO-NH2) (365 mg, 1.32 mmol) and EDC (422 mg, 2.20 mmol) were added to this solution in DMF (5 mL), and the reaction mixture was stirred at 4 °C to 25 °C for 2 hours. Two additional additions of EDC (422 mg, 2.20 mmol) were made every 2 hours. The mixture was diluted with phosphate buffer at pH 7 and then purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 6 in the final solution was determined by the dry extract, and the degree of substitution of DBCO-NH2 was determined by the amount of D2O. 1 Identified by ¹H NMR. The final solution was stored at -20°C.

[1767] Based on the dry extract: [Polysaccharide 6] = 6.1 mg / g.

[1768] according to 1 H NMR (D2O), degree of substitution (DS1) of DBCO-NH2 = 0.28.

[1769] Polysaccharide methyl 7-dextran carboxylate / salt and azide

[1770] Using a method similar to that used in the final step for preparing polysaccharide 1 (the step from polysaccharide 1.3 to polysaccharide 1), polysaccharide 7 was obtained from polysaccharide 4.1 (48.4 mg / g, DS2 = 2.1, 6.2 g, 18.8 mmol glucoside unit) and 11-azido-3,6,9-trioxaundecan-1-amine (N3-PEG3-NH2) (1.23 g, 5.64 mmol).

[1771] Based on the dry extract: [Polysaccharide 7] = 24.6 mg / g.

[1772] according to 1 H NMR (D2O), the degree of substitution (DS1) of N3-PEG3-NH2 is 0.28.

[1773] Polysaccharide 8-dextran carboxylic acid methyl ester / phosphonate / salt and maleimide

[1774] Polysaccharide 8,1-dextran carboxylic acid methyl ester / salt phosphonate / salt

[1775] Using a method similar to that used to prepare polysaccharide 4.2, polysaccharide 4.1 (41.3 mg / g, DS2 = 2.1, 4.13 g, 12.51 mmol glucoside unit) was started, and polysaccharide 8.1 was obtained with HOPO (695 mg, 6.25 mmol), 3-aminopropylphosphonic acid (261 mg, 1.88 mmol), Et3N (262 μL, 1.88 mmol) and EDC (3 × 1.20 g, 3 × 6.25 mmol).

[1776] Based on the dry extract: [Polysaccharide 8.1] = 16.0 mg / g.

[1777] according to 1 ¹H NMR (D₂O), degree of substitution of 3-aminopropylphosphonic acid (DS₃) = 0.05. Polysaccharide methyl 8-dextran carboxylate / phosphonate / salt and maleimide.

[1778] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 8 was obtained from polysaccharide 8.1 (16.0 mg / g, DS2 = 2.1, DS3 = 0.05, 2.64 g, 7.83 mmol glucoside unit) and N-(2-aminoethyl)maleimide hydrochloride (415 mg, 2.35 mmol).

[1779] Based on the dry extract: [Polysaccharide 8] = 8.0 mg / g.

[1780] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.25.

[1781] Polysaccharide 9-dextran carboxylic acid methyl ester / phosphonate / salt and maleimide

[1782] Polysaccharide 9,1-dextran carboxylic acid methyl ester / salt phosphonate / salt

[1783] Using a method similar to that used to prepare polysaccharide 4.2, polysaccharide 4.1 (41.3 mg / g, DS2 = 2.1, 4.13 g, 12.51 mmol glucoside unit) was started, and a solution was obtained with 3-aminopropylphosphonic acid (2.09 g, 15.01 mmol). The pH of this solution was adjusted to pH 2 by adding 1 M HCl. After 16 hours, the solution was neutralized with 1 M NaOH and then purified by ultrafiltration with NaCl (9 g / L in water) followed by water on a PES membrane (MWCO 5 kDa).

[1784] Based on the dry extract: [Polysaccharide 9.1] = 13.3 mg / g.

[1785] according to 1¹H NMR (D₂O), degree of substitution of 3-aminopropylphosphonic acid (DS₃) = 0.75. Polysaccharide methyl 9-dextran carboxylate / phosphonate and maleimide.

[1786] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 9.1 (13.3 mg / g, DS2 = 2.1, DS3 = 0.75, 3.32 g, 7.59 mmol glucoside unit) was started, and polysaccharide 9 was obtained with N-(2-aminoethyl)maleimide hydrochloride (402 mg, 2.28 mmol).

[1787] Based on the dry extract: [Polysaccharide 9] = 11.5 mg / g.

[1788] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.21.

[1789] Polysaccharide 10-dextran carboxylic acid methyl ester / salt sulfate / salt and cyclooctyne (DBCO)

[1790] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 10 was obtained from polysaccharide 1.3 (33.3 mg / g, DS2 = 0.8, DS3 = 1.5, 10.0 g, 26.37 mmol glucoside unit) and with DBCO-NH2 (2.19 g, 7.91 mmol).

[1791] Based on the dry extract: [Polysaccharide 10] = 23.3 mg / g.

[1792] according to 1 H NMR (D2O), degree of substitution (DS1) of DBCO-NH2 = 0.23.

[1793] Polysaccharide methyl 11-dextran carboxylate / salt and cyclooctyne (DBCO)

[1794] Polysaccharide 11,1-dextran carboxylic acid methyl ester / salt

[1795] Using a method similar to that used to prepare polysaccharide 4.1, polysaccharide 11.1 was obtained starting from dextran with a weight-average molar mass of 250 kg / mol.

[1796] Based on the dry extract: [Polysaccharide 11.1] = 44.6 mg / g.

[1797] According to the acid / base determination, the degree of substitution (DS2) of methyl carboxylate / salt is 2.0.

[1798] Polysaccharide methyl 11-dextran carboxylate / salt and cyclooctyne (DBCO)

[1799] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 11 was obtained from polysaccharide 11.1 (44.6 mg / g, DS2 = 2.0, 15.6 g, 48.42 mmol glucoside unit) and with DBCO-NH2 (803 mg, 2.91 mmol).

[1800] Based on the dry extract: [Polysaccharide 11] = 20.4 mg / g.

[1801] According to 1H NMR (D2O), the degree of substitution (DS1) of DBCO-NH2 is 0.06.

[1802] Polysaccharide methyl 12-dextran carboxylate / salt and cyclooctyne (DBCO)

[1803] Polysaccharide 12,1-dextran carboxylic acid methyl ester / salt

[1804] Using a method similar to that used to prepare polysaccharide 4.1, polysaccharide 12.1 was obtained starting from dextran with a weight-average molar mass of 500 kg / mol.

[1805] Based on the dry extract: [Polysaccharide 12.1] = 45.3 mg / g.

[1806] According to the acid / base determination, the degree of substitution (DS2) of methyl carboxylate / salt is 2.0.

[1807] Polysaccharide methyl 12-dextran carboxylate / salt and cyclooctyne (DBCO)

[1808] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 12.1 (45.3 mg / g, DS2 = 2.0, 9.06 g, 28.12 mmol glucoside unit) was started, and polysaccharide 12 was obtained with DBCO-NH2 (466 mg, 1.69 mmol).

[1809] Based on the dry extract: [Polysaccharide 12] = 21.7 mg / g.

[1810] According to 1H NMR (D2O), the degree of substitution (DS1) of DBCO-NH2 is 0.06.

[1811] Polysaccharide methyl 14-dextran carboxylate / salt and vinyl sulfone

[1812] Divinyl sulfone (38.8 mL, 387.3 mmol) was rapidly added to a 100 mM NaOH solution of polysaccharide 4.1.1 (20.0 mg / mL, DS2 = 1.2, 5.0 g, 19.37 mmol glucoside unit). The reaction was stopped by adjusting the pH to 6 with 1 M HCl after 4 minutes at room temperature. The mixture was diluted with phosphate buffer at pH 7 and then purified by ultrafiltration over a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 14 in the final solution was determined by the dry extract, and the degree of substitution of vinyl sulfone was determined by the concentration of divinyl sulfone in D2O. 1 Identified by ¹H NMR. The final solution was stored at -20°C.

[1813] Based on the dry extract: [Polysaccharide 14] = 20.7 mg / g.

[1814] according to 1 H NMR (D2O), degree of substitution of vinyl sulfone (DS1) = 0.17.

[1815] Polysaccharide 15-dextran carboxylic acid methyl ester / salt and triazole-PEG-azide

[1816] Polysaccharide 15,1-dextran carboxylic acid methyl ester / salt and propargyl

[1817] 20 g (123.4 mmol glucoside units, 370.1 mmol hydroxyl functional groups) of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol was dissolved in water (300 g / L) at 30 °C. Then, NaBH4 (2 × 23 mg, 2 × 0.6 mmol) was added every 30 minutes, and the mixture was stirred at 30 °C for 1 hour. The mixture was cooled at 10 °C, and then KOH (5.54 g, 98.7 mmol) and benzyltriethylammonium chloride (1.69 g, 7.4 mmol) were added all at once. Then, propargyl bromide (5.87 g, 49.3 mmol) in toluene (43% w:w) was slowly added over 30 minutes, and the mixture was stirred at 10 °C for 20 minutes and then at 25 °C for 18 hours. The mixture was heated at 60 °C, and sodium chloroacetate (71.8 g, 616.8 mmol) was added all at once. One hour later, 10N NaOH (56.7 mL, 567 mmol) was slowly added over 1.5 hours, and the mixture was stirred at 60 °C for 1 hour. Another portion of sodium chloroacetate (43.1 g, 370.1 mmol) was added at once. One hour later, 10N NaOH (37.0 mL, 370 mmol) was slowly added over 1.5 hours, and the mixture was stirred at 60 °C for 1 hour. The mixture was diluted with water (54 mL), cooled to room temperature, and neutralized with acetic acid. Phosphate buffer (750 mL) and EtOH (575 mL) at pH 7 were added, the mixture was filtered, and subsequently purified by ultrafiltration over a PES membrane (MWCO 5 kDa) using phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The polysaccharide concentration of the final solution was determined by the dry extract, and the degree of substitution of propargyl groups was determined by the concentration of polysaccharides in D2O. 1 H NMR was used to determine the degree of substitution of the methyl carboxylate / salt, followed by acid / base determination.

[1818] Based on the dry extract: [Polysaccharide 15.1] = 40.9 mg / g.

[1819] according to 1 H NMR (D2O), degree of substitution of propargyl group = 0.24.

[1820] According to the acid / base determination, the degree of substitution (DS2) of methyl carboxylate / salt is 1.8.

[1821] Polysaccharide 15-dextran carboxylic acid methyl ester / salt and triazole-PEG-azide

[1822] To a 39 g solution of polysaccharide 15.1 (40.9 mg / g, DS1 = 0.24, DS2 = 1.8, 1.6 g, 5.06 mmol glucoside unit), sodium ascorbate (48.1 mg, 0.24 mmol), copper sulfate pentahydrate (30.3 mg, 0.12 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (105.5 mg, 0.24 mmol), and 1,17-diazido-3,6,9,12,15-pentadecane (4.03 g, 12.1 mmol) were sequentially added. The reaction mixture was stirred at room temperature for 18 hours, diluted with phosphate buffer at pH 7, and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 15 in the final solution was determined by the dry extract, and the degree of substitution of the azide was determined by the concentration of polysaccharide in D2O. 1 Identified by ¹H NMR. The final solution was stored at -20°C.

[1823] Based on the dry extract: [Polysaccharide 15] = 7.0 mg / g.

[1824] according to 1 H NMR (D2O), degree of substitution (DS1) of azide = 0.24.

[1825] Polysaccharide methyl 17-dextran carboxylate / salt and methyl furan

[1826] Using a method similar to that used in the final step (the step from polysaccharide 1.3 to polysaccharide 1) for preparing polysaccharide 1, polysaccharide 17 was obtained from polysaccharide 4.1 (50.8 mg / g, DS2 = 2.1, 17.8 g, 53.9 mmol glucoside unit) and 5-methylfurfurylamine (1.80 g, 16.17 mmol).

[1827] Based on the dry extract: [Polysaccharide 17] = 30.2 mg / g.

[1828] according to 1 H NMR (D2O), degree of substitution of furan (DS1) = 0.30.

[1829] Polysaccharide methyl 18-dextran carboxylate / salt and tetrazine

[1830] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 4.1 (50.8 mg / g, DS2 = 2.1, 5.08 g, 15.38 mmol glucoside unit) was started and polysaccharide 18 was obtained by using (4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)methylamine hydrochloride (1.1 g, 4.62 mmol).

[1831] Based on the dry extract: [Polysaccharide 18] = 11.7 mg / g.

[1832] according to 1 ¹H NMR (D₂O), degree of substitution of tetrazine (DS₁) = 0.25.

[1833] Polysaccharide 24-dextran glycine carbamate and cyclooctyne (DBCO)

[1834] Polysaccharide 24,1-dextran glycine carbamate

[1835] 100 g (0.62 mol glucoside units, 1.85 mol hydroxyl functional groups) of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol was dissolved in water (300 g / L) at 30 °C. Then, NaBH4 (2 × 116 mg, 2 × 3.08 mmol) was added every 30 minutes, and the mixture was stirred at 30 °C for 1 hour. The mixture was diluted with phosphate buffer at pH 7, cooled to room temperature, and subsequently purified by ultrafiltration through a PES membrane (MWCO 5 kDa) with phosphate buffer at pH 7, followed by water. The resulting solution was filtered and then lyophilized to give intermediate polysaccharide 24.1.1.

[1836] 25 g (154.2 mmol glucoside units, 462.6 mmol hydroxyl functional groups) of polysaccharide 24.1.1 was dissolved in DMF / DMSO 50:50 (185 g / L) at 80 °C, followed by the addition of toluene (25 mL) and 1,4-diazabicyclo[2.2.2]octane (6.92 g, 61.7 mmol). 32 g of the reaction mixture was distilled, followed by the slow addition of ethyl isocyanate (51.9 mL, 462.6 mmol) over 30 minutes. The mixture was diluted with DMF (150 mL), stirred at 80 °C for 18 hours, and then cooled to room temperature. Water (900 mL) was added, and the resulting precipitate was filtered off. The precipitate was suspended in H₂O / EtOH (70:30) (2.5 L), and the pH of the solution was adjusted to pH 13 by adding 10 N NaOH. After 2 hours, the mixture was neutralized with 6 M HCl, filtered, and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) with NaCl (9 g / L in water) followed by water. The polysaccharide concentration of 24.1 in the final solution was determined by the dry extract, and an acid / base assay was subsequently performed to determine the degree of substitution of glycine carbamate.

[1837] Based on the dry extract: [polysaccharide 24.1] = 47.8 mg / g.

[1838] According to the acid / base test, the degree of substitution (DS2) of glycine carbamate is 2.4.

[1839] Polysaccharide 24-dextran glycine carbamate and cyclooctyne (DBCO)

[1840] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 24 was obtained from polysaccharide 24.1 (47.8 mg / g, DS2 = 2.4, 5.98 g, 13.07 mmol glucoside unit) and with DBCO-NH2 (1.08 g, 3.92 mmol).

[1841] Based on the dry extract: [Polysaccharide 24] = 20.8 mg / g.

[1842] according to 1 H NMR (D2O), degree of substitution (DS1) of DBCO-NH2 = 0.28.

[1843] Polysaccharide 25-dextran glycine carbamate and maleimide

[1844] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 24.1 (47.8 mg / g, DS2 = 2.4, 5.98 g, 13.07 mmol glucoside unit) was started, and polysaccharide 25 was obtained with N-(2-aminoethyl)maleimide hydrochloride (693 mg, 2.92 mmol).

[1845] Based on the dry extract: [Polysaccharide 25] = 22.8 mg / g.

[1846] according to 1 ¹H NMR (D₂O), degree of substitution of maleimide (DS₁) = 0.26.

[1847] Example A2: Polyethylene glycol derivatives containing at least two reactive functional groups

[1848] Commercial polyethylene glycol (PEG) derivatives functionalized with reactive functional groups were purchased. The reactive functional groups included thiols (“PEG-SH”), azides (PEG-N3), and alkynes (PEG-DBCO). Linear isofunctionalized bifunctional and multi-arm isofunctionalized derivatives with different molecular weights and functional groups were used, and are shown in Table 2 below.

[1849] Table 2: List of commercial PEG derivatives used

[1850]

[1851]

[1852] These PEG derivatives correspond to the precursor of Formula I -L

[1853] Partial B-biology

[1854] Example B1A: Preparation of pseudo-islets

[1855] The Min-6 cell line (Caltag Medsystems) was cultured in the medium shown in Table 3 in an incubator at 37°C and 5% CO2. Cells were isolated three times a week using 0.05% trypsin / EDTA and passaged in the medium at a 5-fold dilution.

[1856] Table 3: Culture medium composition

[1857] Culture medium for Min-6 pseudo-islets Dulbecco Modified Eagle Medium (DMEM) 10% FBS 1% penicillin / streptomycin 1mM pyruvate 0.05mM β-mercaptoethanol

[1858] Using the Min-6 cell line, 500 cells were seeded per well in a 400 μm microwell Eplasia plate (Corning) and incubated at 37°C and 5% CO2 for 3 days to form pseudoislets with an average diameter of 150 μm. The pseudoislets were then collected, concentrated by centrifugation, and finally suspended in 0.9% NaCl.

[1859] Example B2A: Isolation of primary human pancreatic islets

[1860] Pancreas was obtained from a human brain-dead donor. Islets were produced according to the method described in the Pancreas Retrieval Technique for Islet Isolation (Pattou et al., Anchir 2005). In short, the pancreas was isolated from the tissue and perfused into Wirsung tubes to be treated with collagenases I and II (… The mixture from Roche (France) was digested to ensure the release of islets. The islets were then purified using density gradient centrifugation (EuroFicoll, Sigma-Aldrich). Finally, the purified islets were cultured in culture flasks in CMRL medium supplemented with 0.625% BSA and 1% penicillin / streptomycin at 37°C and 5% CO2. The medium was changed every 2 to 3 days.

[1861] Example B2B: Isolation of primary rat pancreatic islets

[1862] Pancreatic islets were isolated from male Wistar or Lewis rats (approximately 300 g) using a method similar to that described in A Pratical Guide to Rodent Islet Isolation and Assessment, Carter et al. Biological Procedures Online 2009.

[1863] In short, the pancreas is perfused via injection of collagenase into the common bile duct. Following perfusion, the pancreas is removed and digested at 37°C for 10 minutes. The islets are then purified by density gradient centrifugation. The purified islets are cultured in non-adherent culture flasks in DMEM (Gibco) medium supplemented with 10% fetal bovine serum, 2 g / L glucose, and 1% penicillin / streptomycin at 37°C and 5% CO2. The medium is changed every 2 to 3 days.

[1864] Example 3A: Islet Equivalent Count

[1865] To normalize the amount of islets or pseudo-islets used in each experiment, islets or pseudo-islets are counted to determine the islet equivalent count (IEQ). One IEQ corresponds to the volume of a perfectly spherical islet / pseudo-islet with a diameter of 150 μm. During counting, a multiplication factor is applied to each islet based on its size. This mathematical compensation for different islet diameters allows for normalization between formulations (see NIH CIT Consortium Chemistry Manufacturing Controls Monitoring Committee; Purified Human Islets: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ): NIH Clinical Islet Transplantation Consortium Standard Operating Procedure. Cell R.4 Repair Replace RegenReprogram).

[1866] Two 50 μL samples from each batch of islets or pseudo-islets were counted on a glass slide with a 50 μm grid. Islets or pseudo-islets were classified by size according to Table 4A.

[1867] Table 4A: Multiplication factor for islet size used in islet equivalent determination

[1868] Pancreatic islet size Multiplication factor 50 to 100 μm 1 / 6 100 to 150 μm 1 / 1.5 150 to 200 μm 1.7 200 to 250 μm 3.5 250 to 300 μm 6.3

[1869] The islet equivalent count is determined by averaging the counts from two independent samples.

[1870] Example B3B: Islet Equivalent Count

[1871] To normalize the amount of islets used in each experiment, islets were counted to determine the islet equivalent count (IEQ). One IEQ corresponds to the volume of a perfectly spherical islet with a diameter of 150 μm. During counting, a multiplication factor was applied to each islet based on its size. This mathematical compensation for the different diameters of the islets enabled normalization between islet formulations (see NIH CIT Consortium Chemical Manufacturing Control Monitoring Committee; Purified Human Islets: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ): NIH Clinical Islet Transplantation Consortium Standard Operating Procedure. Cell R.4 Repair Replace Regen Reprogram).

[1872] Two 50 μL samples from each batch of islets were counted on a glass slide with a 50 μm grid. The islets were classified by size according to Table 4B.

[1873] Table 4B: Multiplication factor for islet size used in islet equivalent determination

[1874] Pancreatic islet size Multiplication factor 50 to 100 μm 1 / 6 100 to 150 μm 1 / 1.5 150 to 200 μm 1.7 200 to 250 μm 3.5 250 to 300 μm 6.3

[1875] The islet equivalent count is determined by averaging the counts from two independent samples.

[1876] Partial C-physical chemistry

[1877] Example C1A: Preparation of a solution of concentrated polysaccharide functionalized with maleimide (Mal) groups

[1878] A concentrated polysaccharide solution was prepared by weighing an appropriate weight of sterile freeze-dried polysaccharide obtained according to part A1 and adding an appropriate weight of sterile deionized water. The solution was placed on an orbital shaker at 70 rpm overnight to ensure complete dissolution. The pH of the solution was adjusted to pH 4 by adding concentrated hydrochloric acid, followed by sterile filtration (0.22 μm). The mass concentration (mg / g) of the polysaccharide solution was determined by the dry extract. The volume concentration (mg / mL) of the polysaccharide solution was determined by density measurement, with 100 μL of solution weighed three times. The solution was frozen at -20°C until use.

[1879] Example C1B: Preparation of concentrated polysaccharide solutions functionalized with vinyl sulfone (VS), DBCO, or azide groups

[1880] A concentrated polysaccharide solution was prepared by weighing an appropriate weight of sterile freeze-dried polysaccharide obtained according to part A1 and adding an appropriate weight of sterile deionized water. The solution was placed on an orbital shaker at 70 rpm overnight to ensure complete dissolution. The pH of the solution was adjusted to pH 7.4 by adding NaOH before sterile filtration (0.22 μm). The mass concentration (mg / g) of the polysaccharide solution was determined by the dry extract. The volume concentration (mg / mL) of the polysaccharide solution was determined by density measurement, with 100 μL of solution weighed three times. The solution was frozen at -20°C until use.

[1881] Example C2: Preparation of concentrated PEG derivative solution

[1882] A concentrated solution of PEG (from the list in Table 2) was prepared by weighing an appropriate weight of PEG powder and adding an appropriate weight of sterile deionized water. The solution was placed on a roller shaker at 15 rpm for 2 hours to ensure complete dissolution before sterile filtration (0.22 μm). The mass concentration (mg / g) of the PEG solution was determined by dry extraction. The volume concentration (mg / mL) of the PEG solution was determined by density measurement, with 100 μL of solution weighed three times. The sterile solution was frozen at -20°C until use.

[1883] Example C3: Preparation of concentrated sodium hyaluronate solution

[1884] A concentrated solution of sodium hyaluronate (Pharma grade 150 from Novamatrix) is prepared by weighing an appropriate amount of sodium hyaluronate powder and adding an appropriate amount of sterile deionized water. Alternatively, another hyaluronate salt (Pharma grade 300 from Echelon Biosens, HTL Biotechnologies), specifically for C4B-16 gels, can be used. The solution is placed on a roller shaker at 10 rpm overnight until completely dissolved before sterile filtration (0.22 μm).

[1885] Example C3bis: Preparation of concentrated sodium hyaluronate solution

[1886] A concentrated solution of trehalose dihydrate D(+) (Sigma-Aldrich) was prepared by weighing an appropriate amount of trehalose dihydrate D(+) powder and adding an appropriate amount of sterile deionized water. The solution was heated to 45°C until completely dissolved under mixing and cooled to ambient temperature before sterile filtration (0.22 μm).

[1887] Example C3A: Hydrogel Preparation

[1888] The hydrogel was prepared under sterile conditions.

[1889] Concentrated sterile solutions of polysaccharides and PEG derivatives prepared according to Examples C1A or C1B and Example C2 were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg) and equilibrated at room temperature (20°C to 25°C) or 4°C. Optionally, concentrated polysaccharide solutions with VS or DBCO groups were supplemented with Tris buffer at pH 7.4 or pH 8.

[1890] A concentrated PEG solution was added to a concentrated polysaccharide solution in 2 mL Eppendorf solution. The volume ratio of PEG solution to polysaccharide solution was 70:30 (%:%) or 80:20 (%:%). The solutions were mixed using a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator adhered to a glass slide. Different molded hydrogel geometries were prepared.

[1891] Table 5A: Conditions for molding hydrogel geometry.

[1892]

[1893] The cross-linking process leading to gelation was carried out at room temperature (20°C to 25°C) or at 37°C for 1 hour. The hydrogel was demolded and introduced into a Tris 150mM / NaCl 30mM / cysteine ​​10mM solution (2 mL) or PBS at pH 7.4 at 37°C for 1 hour.

[1894] The hydrogel was rinsed with 20 mL of cysteine-free PBS solution and then immersed in 10 mL of the same PBS solution overnight at 37°C. The hydrogel block was then stored in 10 mL of PBS solution at 4°C until use.

[1895] Example C3B: Hydrogel Preparation

[1896] The hydrogel was prepared under sterile conditions.

[1897] Concentrated sterile solutions of polysaccharides and PEG derivatives prepared according to Examples C1A or C1B and Example C2 were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg) and equilibrated at room temperature (20°C to 25°C) or 4°C. Alternatively, osmotic pressure can be adjusted using a combination of NaCl and nonionic reagents (e.g., trehalose), particularly in Example C4B24. Optionally, concentrated polysaccharide solutions with VS or DBCO groups were supplemented with Tris buffer at pH 7.4 or pH 8.

[1898] A concentrated solution of PEG / hyaluronic acid salt was prepared by mixing a concentrated PEG solution prepared according to Example C2 with a concentrated sodium hyaluronate solution prepared according to Example C3.

[1899] A polysaccharide concentrate supplemented with pluronic F127 (Sigma-Aldrich) was prepared by mixing the polysaccharide concentrate prepared according to Example C1B.

[1900] In 2 mL Eppendorf, concentrated solutions of PEG or PEG / hyaluronic acid were added to concentrated solutions of polysaccharides or polysaccharides / poloxam (especially polysaccharides / poloxam F127). When hyaluronic acid was added to the PEG solution, the volume ratio of PEG solution to polysaccharide solution was 70:30 (%:%) or 80:20 (%:%). The solutions were mixed using a pipette, and controlled volumes of the mixture were introduced into circular silicone spacers adhered to a glass slide. Different molded hydrogel geometries were prepared.

[1901] Adding poloxamer-type nonionic surfactants can help improve the wettability of the composition.

[1902] Table 5B: Conditions for molding hydrogel geometry.

[1903]

[1904] Crosslinking leading to gelation was performed at room temperature (20°C to 25°C) or at 37°C for 1 hour. The hydrogel was demolded and introduced into a solution of Tris 150mM / NaCl 30mM / cysteine ​​10mM at pH 8 or PBS at pH 7.4 for 1 hour at 37°C.

[1905] The hydrogel was rinsed with cysteine-free PBS solution and then immersed in the PBS solution overnight at 37°C. The hydrogel block was then stored in PBS solution at 4°C until use.

[1906] Example C4A: Hydrogel Composition

[1907] Different hydrogel compositions were prepared according to the scheme described in Example C3A, which are shown in Table 6A. The concentrations of the two reactive groups (maleimide (Mal) or vinyl sulfone (VS) reacting with thiols (SH) or alkyne (DBCO) reacting with azides (N3)) and the polymer (polysaccharide and PEG derivative) correspond to the final concentrations when the polymer solution is mixed.

[1908] Table 6A: Compositions of various hydrogels made from polysaccharides and PEG derivatives.

[1909]

[1910]

[1911]

[1912] Obtain a solid, disc-shaped hydrogel block. The hydrogel block is easily demolded and handled with tweezers for characterization.

[1913] Table 7A: Structure of hydrogels

[1914]

[1915]

[1916]

[1917]

[1918]

[1919]

[1920]

[1921]

[1922]

[1923]

[1924]

[1925]

[1926]

[1927]

[1928] Example C4B: Hydrogel Composition

[1929] Different hydrogel compositions were prepared according to the scheme described in Example C3B (Table 6B). The concentrations of the two reactive groups (maleimide (Mal) or ethyl sulfone (VS) reacting with thiols (SH), or alkynes (DBCO) reacting with azides (N3)) and the polymers (polysaccharides and PEG derivatives) correspond to the final concentrations after mixing the polymer solutions. Table 6B: Compositions of various hydrogels made from polysaccharides and PEG derivatives and optionally incorporating sodium hyaluronate.

[1930]

[1931]

[1932] *HA 3,000 kDa, all other HAs are 1,500 kg / mol.

[1933] #Add Prolactin F127 (10mg / ml)

[1934] Obtain a solid, disc-shaped hydrogel block. The hydrogel block is easily demolded and handled with tweezers for characterization.

[1935] Table 7B: Structure of Hydrogels

[1936]

[1937]

[1938]

[1939]

[1940]

[1941] Example C5A: Rheological Characterization of Hydrogels

[1942] Oscillatory shear tests were performed using a rotational rheometer (AR2000, TA instrument) equipped with a cone-plate geometry. The crosslinking process leading to gelation was completed "in situ," meaning that before initiating oscillatory measurements, a concentrated solution of polysaccharide and PEG was dropwise introduced between the cone and plate and mixed by rotation of the geometry. Oscillatory time-scan tests were performed at 25°C or 37°C, with a constant strain of 0.1% and a constant oscillation frequency of 1 Hz. Storage modulus G' (i.e., elasticity) and Tanδ (ratio G" / G') values ​​were reported as a function of time in the plateau region of the measured (G', G") at 1600 seconds.

[1943] Table 8A: Results of hydrogel rheological characterization.

[1944]

[1945]

[1946]

[1947] The hydrogel exhibits a low Tanδ value, meaning that G' is much higher than G”, which is a typical characteristic of chemically cross-linked hydrogels exhibiting solid elasticity (see Polysaccharide Hydrogels: Characterization and Biomedical Applications, 2016 Pan Stanford Publishing Pte. Ltd.; Chapter 3, p. 97). Increasing the concentration of Mal:SH leads to an increase in the elastic modulus G'.

[1948] Increasing temperature and pH are two ways to accelerate the cross-linking process that leads to the gelation of a hydrogel prepared from polysaccharides with VS groups and PEG-SH. For example, proceeding from a neutral pH to pH 8 and / or increasing the temperature from 25°C to 37°C results in faster gelation.

[1949] This allows for fine-tuning of the gelation rate, which can be convenient because rapid gelation can help avoid cell sedimentation, while slow gelation can facilitate casting of the polymer mixture prior to gelation.

[1950] Example C5B: Rheological Characterization of Hydrogels

[1951] Oscillatory shear tests were performed using a rotational rheometer (AR2000, TA instrument) equipped with a cone-plate geometry. The crosslinking leading to gelation was completed "in situ," meaning that before initiating oscillatory measurements, a concentrated solution of polysaccharide and PEG was dropwise introduced between the cone and plate and mixed by rotation of the geometry. Oscillatory time-scan tests were performed at 25°C or 37°C, with a constant strain of 0.1% and a constant oscillation frequency of 1 Hz. Storage modulus G' (i.e., elastic modulus) and Tanδ (ratio G" / G') values ​​were reported as a function of time in the plateau region of the measured (G"G') at 1600 seconds.

[1952] Table 8B: Results of hydrogel rheological characterization.

[1953]

[1954] The hydrogel exhibits a low Tanδ value, meaning that G' is much higher than G”, which is a typical characteristic of chemically cross-linked hydrogels that behave as solid elastic materials (see Polysaccharide Hydrogels: Characterization and Biomedical Applications, 2016 Pan Stanford Publishing Pte. Ltd.; Chapter 3, p. 97).

[1955] An increase in the concentration of reactive groups leads to an increase in the elastic modulus G'.

[1956] This allows for fine-tuning of the crosslinking rate that leads to gelation, which can be convenient because rapid gelation can help avoid cell sedimentation, while slow gelation can facilitate casting of the polymer mixture prior to gelation.

[1957] The gelation properties are maintained in the presence of non-reactive sodium hyaluronate.

[1958] This allows for further tuning of the hydrogel composition to avoid cell sedimentation, even for slow-gel compositions.

[1959] Example C6A: Hydrogel swelling and water content

[1960] The hydrogel blocks were weighed immediately after demolding (w0) and immediately after swelling overnight in PBS solution (wovernight). The swelling ratio was defined as the mass ratio wovernight / w0. The water content of the hydrogel was determined by measuring the mass of the hydrogel in the swollen state and by controlling the concentration of the polymer precursor used to synthesize the hydrogel.

[1961] Table 9A: Hydrogel swelling and water content.

[1962]

[1963]

[1964] Hydrogels contain a high water content. The water content varies depending on the structure and concentration of the polymer precursor.

[1965] Example C6B: Hydrogel swelling and water content

[1966] The hydrogel blocks were weighed immediately after demolding (w0) and immediately after swelling in PBS solution overnight (wovernight). The swelling ratio was defined as mass ratioovernight / w0. The water content of the hydrogel was determined by measuring the mass of the hydrogel in the swollen state and by controlling the concentration of the polymer precursor used to synthesize the hydrogel.

[1967] Table 9B: Hydrogel swelling and water content.

[1968]

[1969] *HA 3,000 kg / mol, all other examples HA 1,500 kg / mol

[1970] Hydrogels contain a high water content.

[1971] Example C7A: Stability of hydrogel in physiological media at 37°C

[1972] Hydrogel blocks were stored at 37°C in PBS (pH 7.4) or serum (FBS fetal bovine serum) and weighed at different time points. Disc-shaped hydrogels with a diameter of 9 mm and a thickness of 1.6 mm were tested.

[1973] Table 10A: Stability of hydrogels in physiological media

[1974]

[1975] The hydrogel was completely recovered and stored in a physiological medium at 37°C, with no significant change in its mass. For example, in the case of hydrolysis side reactions or changes in network structure, swelling (increase in mass) or dissolution (decrease in mass) of the hydrogel is expected. This indicates that the hydrogel is stable under physiological conditions.

[1976] Example C7B: Stability of hydrogel in physiological media at 37°C

[1977] Hydrogel blocks were stored at 37°C in PBS (pH 7.4), serum (FBS fetal bovine serum), or 100 mM acetate buffer (pH 4). The blocks were weighed at different time points. Disc-shaped hydrogels with a diameter of 9 mm and a thickness of 1.6 mm were tested.

[1978] Table 10B: Stability of hydrogels in physiological media

[1979]

[1980]

[1981] The hydrogel was completely recovered and stored in a physiological medium at 37°C, with no significant change in its mass. For example, in the case of hydrolysis side reactions or changes in network structure, swelling (increase in mass) or dissolution (decrease in mass) of the hydrogel is expected. This indicates that the hydrogel is stable under physiological conditions.

[1982] At acidic pH, fairly good gel stability is obtained, as no significant mass reduction has been reported.

[1983] Example C8A: Encapsulation of macromolecular probes within hydrogels

[1984] Commercially available fluorescent dextran-FITC 3kDa and dextran-FITC 70kDa were each dissolved in water to obtain concentrated stock solutions.

[1985] The concentrated sterile solutions of polysaccharide DMCmal and PEG-SH prepared according to Examples C1A and C2, respectively, were equilibrated at 4°C. For DMCVS-based hydrogels, the concentrated solutions of polysaccharide DMCmal and PEG-SH prepared according to Examples C1B and C2, respectively, were equilibrated at 20°C to 25°C.

[1986] A solution of polysaccharide DMCmal or DMCVS was mixed with fluorescent dextran. 100 μL of concentrated PEG-SH solution was added to 100 μL of the concentrated solution of polysaccharide DMCmal or DMCVS and fluorescent dextran. The solutions were mixed using a pipette, and 200 μL of the latter mixture was introduced into a rectangular silicone mold (IBIDI 12 × 7.75 mm). For DMCmal-based hydrogels, the crosslinking process leading to gelation was carried out at room temperature (20°C to 25°C) for 1 hour, or for DMCVS-based hydrogels, the crosslinking process leading to gelation was carried out at 37°C for 1 hour.

[1987] The hydrogel block was demolded and introduced into the wells (12-well multi-well plate), and soaked in 1.3 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution containing the same concentration of encapsulated fluorescent dextran. The hydrogel was allowed to swell overnight at 37 °C, and the supernatant was weighed to estimate the degree of swelling and the amount (mg) in the hydrogel volume.

[1988] Prior to the release experiment, the hydrogel was rapidly washed twice with 1 mL of pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution.

[1989] Table 11A: Compositions of hydrogels with encapsulated fluorescent macromolecular probes

[1990]

[1991] Example C8B: Encapsulation of macromolecular probes within hydrogels

[1992] Commercially available fluorescent dextran-FITC 3kDa and dextran-FITC 70kDa were each dissolved in water to obtain concentrated stock solutions.

[1993] Concentrated sterile solutions of polysaccharides and PEG, or polysaccharides DMCmal and PEG-SH, prepared according to Examples C1A, C1B, and C2, were equilibrated at 4°C. For DMCVS-based hydrogels, concentrated solutions of polysaccharides DMCmal and PEG-SH, prepared according to Examples C1B and C2, were equilibrated at 20°C to 25°C.

[1994] A solution of polysaccharide DMCmal, DMCVS, or DMCDBCO was mixed with fluorescent dextran. 100 μL of a concentrated solution of PEG-SH or PEG-N3 was added to 100 μL of a concentrated solution of polysaccharide, DMCmal, or DMCVS with fluorescent dextran. The solutions were mixed using a pipette, and 200 μL of the latter mixture was introduced into a rectangular silicone mold (IBIDI 12 × 7.75 mm). For DMCmal and DMC-DBCO-based hydrogels, crosslinking leading to gelation was performed at room temperature (20°C to 25°C) for 1 hour, or for DMCVS-based hydrogels, crosslinking leading to gelation was performed at 37°C for 1 hour.

[1995] The hydrogel block was demolded and introduced into the wells (12-well multi-well plate), and soaked in 1.3 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution containing the same concentration of encapsulated fluorescent dextran. The hydrogel was allowed to swell overnight at 37 °C, and the supernatant was weighed to estimate the degree of swelling and the amount (mg) in the hydrogel volume.

[1996] Prior to the release experiment, the hydrogel was rapidly washed twice with 1 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution (shown in Example C7B).

[1997] Table 11B: Compositions of hydrogels with encapsulated fluorescent macromolecular probes

[1998]

[1999] Example C9A: Release of macromolecular probes within hydrogels

[2000] Hydrogels containing encapsulated fluorescent probes, produced according to Example C8A, were introduced into each well (12-well multi-well plate) and soaked in 2 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution. The plate was covered with a membrane and placed in an oven at 37°C. 200 μL of buffer was sampled at different time points and replaced with fresh buffer. The concentration of fluorescent probes in the samples was determined by fluorescence (using a fluorescent plate reader, SAFAS) using a calibration curve. The cumulative fraction of fluorescent probes released at each time point corresponds to the ratio of the cumulative amount of fluorescent probes released in the swollen hydrogel to the initial amount of fluorescent probes.

[2001] Table 12A: Cumulative fractions of macromolecular probes released from the hydrogel at different time points.

[2002]

[2003] Increasing the size of the macromolecular probe leads to slower release kinetics. This demonstrates the selective permeation properties of the hydrogel network structure.

[2004] Example C9B: Release of macromolecular probes from hydrogels

[2005] Hydrogels containing encapsulated fluorescent probes, produced according to Example C8B, were introduced into each well (12-well multi-well plate) and soaked in 2 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution. The plate was covered with a membrane and placed in an oven at 37°C. 200 μL of buffer was sampled at different time points and replaced with fresh buffer. The concentration of fluorescent probes in the samples was determined by fluorescence (using a fluorescent plate reader, SAFAS) using a calibration curve. The cumulative fraction of fluorescent probes released at each time point corresponds to the ratio of the cumulative amount of fluorescent probes released in the swollen hydrogel to the initial amount of fluorescent probes.

[2006] Table 12B: Cumulative fractions of macromolecular probes released from the hydrogel at different time points.

[2007]

[2008] Increasing the size of the macromolecular probe leads to slower release kinetics. This demonstrates the selective permeation properties of the hydrogel network structure.

[2009] Example C10: Molding of a thin hydrogel disc

[2010] According to the procedure described in Example C3A, the concentrated polymer solution was equilibrated at 4°C. The solution was mixed using a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator (10 mm in diameter and 0.5 mm, 432 μm or 356 μm thick) adhered to a glass slide. A second glass slide was placed on the droplet to promote its diffusion throughout the entire diameter of the mold.

[2011] After a cross-linking process leading to gelation was carried out at 20°C to 25°C for 1 hour, the hydrogel disc was demolded. The thickness was inferred from the diameter and volume of the gel.

[2012] Table 13: Thickness of Hydrogel Compositions

[2013]

[2014] This method allows for the acquisition of hydrogel discs with controlled diameter and thickness by adjusting the diameter of the mold and the volume of the hydrogel.

[2015] Example C11A: Determination of the mechanical resistance of hydrogels.

[2016] For compression, a swollen rectangular hydrogel block as described in Example C3A was introduced into a flat glass crystallizer and immersed in PBS. Uniaxial compression was performed at 0.2 mm / min in 0.9% NaCl at 20°C to 25°C using a general mechanical testing apparatus (Zwickroell) equipped with a flat compression plate. The initial thickness of the sample was determined from the contact between the plate and the hydrogel as the force began to increase. Deformation was defined as the ratio of compressive displacement (mm) to initial thickness (mm). The deformation at fracture was determined by the force / displacement curve. Fracture was defined as when a decrease in force relative to displacement was observed.

[2017] For traction, dog-bone shaped hydrogel blocks were prepared by molding the hydrogel in a dog-bone shaped silicone mold. Uniaxial traction was performed at 3 mm / min in 0.9% NaCl at 20°C to 25°C using a general mechanical testing apparatus (Zwickroell) equipped with screw clamps. The initial length of the sample was measured between the clamps using a ruler. Deformation was defined as the ratio of traction displacement (mm) to initial length (mm). Deformation at fracture was determined by the force / displacement curve. Fracture was defined as the observation of a decrease in force relative to displacement. Young's modulus was determined by the slope of the true strain / deformation curve. True strain (kPa) corresponds to the ratio of pressure (N) to the instantaneous surface area (mm²) of the sample under compression.

[2018] Table 14A: Mechanical resistance of hydrogels

[2019]

[2020]

[2021] NM means not measured.

[2022] The hydrogel according to the invention exhibits a very good combination of deformability and stiffness suitable for surgical implantation, providing excellent mechanical resistance characteristics.

[2023] Example C11B: Determination of the mechanical resistance of hydrogels.

[2024] For compression, a swollen rectangular hydrogel block as described in Example C3B was introduced into a flat glass crystallizer and immersed in PBS. Uniaxial compression was performed at 0.2 mm / min in 0.9% NaCl at 20°C to 25°C using a general mechanical testing apparatus (Zwickroell) equipped with a flat compression plate. The initial thickness of the sample was determined from the contact between the plate and the hydrogel as the force began to increase. Deformation was defined as the ratio of compressive displacement (mm) to initial thickness (mm). The deformation at fracture was determined by the force / displacement curve. Fracture was defined as when a decrease in force relative to displacement was observed.

[2025] Young's modulus is determined by the slope of the true strain / deformation curve. True strain (kPa) corresponds to the ratio of pressure (N) to the instantaneous surface area (mm2) of the sample under compression.

[2026] Table 14B: Mechanical resistance of hydrogels

[2027]

[2028] The hydrogel according to the invention exhibits a very good combination of deformability and stiffness suitable for surgical implantation, providing excellent mechanical resistance characteristics.

[2029] Example C12: Surgical net

[2030] Example C12A: Alkali Treatment of Polyester Surgical Mesh

[2031] A PETKM3002 warp-knitted polyester multifilament surgical mesh (1×0.9 mm aperture) supplied by SurgicalMeshTM was treated in 1M NaOH at 70°C for 5 hours, followed by washing with deionized water and 96% ethanol. This treatment resulted in increased hydrophilicity of the mesh, thereby improving the wettability of the aqueous solutions of the polymers constituting the hydrogel.

[2032] Example C12B: PVDF surgical mesh fabric

[2033] The warp-knitted PVDF surgical mesh fabric (1×1mm aperture) supplied by Dynamesh is formed from knitted monofilaments with a diameter of 100μm.

[2034] Example C12C: PTFE perforated surgical membrane

[2035] A 150μm thick PTFE surgical membrane supplied by Aran Biomedical was perforated in a star pattern (1×2mm pore size).

[2036] Example C13: Construction of the construct

[2037] Example C13A: Manufacturing of Ring Network Structures

[2038] Use a stainless steel punch to cut biocompatible PDMS sheets supplied by Grace Biolabs, Interstate Speciality Products, or Limitless Shielding into a square shape that incorporates a circular empty disc.

[2039] A portion of the treated polyester surgical mesh, or a portion of the surgical mesh described in Example C12, is introduced between two square PDMS sheets: the two PDMS sheets are glued together with the surgical mesh using biocompatible silicone adhesive (Silbione MED ADH 4200 supplied by Elkem). The circular empty discs are aligned, and the surgical mesh is kept taut during gluing.

[2040] Finally, the square structure is cut with a stainless steel punch to obtain the final object consisting of two PDMS rings that clamp the surgical mesh in the middle and glue them together.

[2041] Before steam or ETO (ethylene oxide) sterilization, wash the tablets with 1% poloxamer F127 solution and rinse with water.

[2042] Example C13B: Fabrication of a proportional rectangular structure

[2043] For human surgery, to allow for easier surgical implantation through a 15mm inner diameter cannula, the implant can have an elongated form, such as a rectangle with a width of less than 100mm.

[2044] Biocompatible PDMS sheets supplied by Grace Biolabs, Interstate Specialty Products, or Limitless Shielding are cut into various forms, such as elongated rectangular empty frames.

[2045] A portion of the surgical mesh described in Example C12 is introduced between the frame PDMS sheets: the two PDMS sheets are glued together with the surgical mesh using biocompatible silicone adhesive (Silbione MED ADH 4200 supplied by Elkem). The empty frame is aligned, and the surgical mesh is kept taut during gluing.

[2046] Finally, the excess mesh around the frame is cut to obtain the final object consisting of two PDMS frames that sandwich the surgical mesh in the middle and glue them together.

[2047] Excess mesh around the frame may be deliberately left uncut to make it easier to secure to the tissue using absorbable or non-absorbable tacks.

[2048] Before steam or ETO sterilization, wash the tablets with 1% poloxamer F127 solution and rinse with water.

[2049] Example C14: Constructs of different sizes

[2050] Example C14A: Ring network structures of different sizes

[2051] According to Example C13A, different ring network structures can be produced by changing parameters (e.g., the inner / outer diameter and thickness of the PDMS rings and the thickness of the adhesive).

[2052] Table 15A: Dimensions of ring network structures.

[2053]

[2054] Example C14B: Scaled-up structures of different sizes

[2055] According to Example C13B, different constructs are produced by changing parameters (e.g., the inner / outer diameter and thickness of the PDMS ring and the thickness of the adhesive).

[2056] Table 15B: Dimensions of scaled-up structures.

[2057]

[2058] Example C15: Hydrogel / Construction Composite Composition

[2059] Example C15A: Hydrogel / Ring Network Composite Composition

[2060] The hydrogel composition prepared according to Example C3B and described in Example C4B was incorporated into the ring network structure described in Example C14. The concentrated polymer solution was mixed using a pipette, and a controlled volume of the mixture was introduced into the ring network structure adhered to a glass slide.

[2061] Crosslinking leading to gelation was performed at room temperature (20°C to 25°C) or at 37°C for 1 hour. The ring-and-hydrogel composition was then introduced into a Tris 150mM / NaCl 30mM / cysteine ​​10mM solution at pH 8 or into PBS at pH 7.4 for 1 hour at 37°C.

[2062] The hydrogel was washed with cysteine-free PBS solution and then soaked in PBS solution at 37°C overnight. The hydrogel block was then stored in PBS solution at 4°C until use.

[2063] Preparation of different hydrogel / ring network composite compositions (Table 16A):

[2064] Table 16A: Hydrogel / ring network or proportional composite material compositions.

[2065]

[2066] Therefore, the hydrogel / ring composite material is easy to manipulate with forceps and can be folded for surgical implantation, especially for minimally invasive procedures. Furthermore, the ring can be fixed to the tissue using sutures.

[2067] The volume of the hydrogel can be adjusted by the inner diameter and thickness, or by the ring network structure. For the same ring network structure, the volume of the hydrogel can be adjusted to control the convexity of the hydrogel above the ring level.

[2068] Example C15B: Hydrogel / proportional composite material composition

[2069] To facilitate implantation, ensure the absence of filling defects, and limit islet subsidence, proportional implants for human testing are manufactured using devices that allow horizontal or vertical filling with hydrogel.

[2070] The device consists of glass plates, with silicone sheets ranging from 50 to 2000 μm in thickness fixed to one side of each plate. Two holes, 1 to 6 mm in diameter, are drilled in one of the glass plates and the corresponding sheet. A female luerlock is glued to the center of one of the holes. The empty implant is placed between the two sheets of the laminated glass. To ensure the desired thickness and seal during hydrogel casting, the assembly is held tightly against both sides of the silicone frame of the structure.

[2071] Two methods were used to inject hydrogel compositions of appropriate volumes into proportionally proportioned constructs:

[2072] a. Reconstruction and Injection: After mixing the gel precursor solution, place the gel solution into a syringe with a Luer lock. Screw the syringe onto the Luer lock on the glass plate and then inject the gel into the implant.

[2073] b. Dual-injector injection with mixing chamber (from) The dual-syringe biomaterial delivery system (M-System) delivers the hydrogel precursor solution into two separate syringes. The flow coupling between the two syringes is extended via a mixing chamber with a male Luer lock screwed onto the filling device. This is achieved by using... The clamp simultaneously pushes two syringes, reconstructing the hydrogel and immediately injecting it into the device.

[2074] Table 16B: Hydrogel / ring network or proportional composite material compositions.

[2075]

[2076] Example C16A: Encapsulation of insulin-producing islets in a hydrogel

[2077] Islet encapsulation was performed in a sterile environment.

[2078] The concentrated sterile solutions of polysaccharides and PEG prepared according to Examples C1A or C1B and C2 were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg). The solutions were equilibrated at room temperature (20°C to 25°C or at 4°C).

[2079] A concentrated mixture of PEG and islets was prepared by mixing an equal volume of isotonic PEG solution (prepared according to Example C2) with islet or pseudo-islet suspension (prepared according to Examples B1A or B2A). The mixture containing PEG and islets was then gently mixed with an isotonic concentrated solution of polysaccharide at a volume ratio of 70:30 (islet suspension / PEG:polysaccharide).

[2080] The solution was gently mixed using a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator from Grace Biolab attached to a glass slide. Different hydrogel sizes were prepared based on the volume and silicone mold diameter.

[2081] Table 17A: Volume and Dimensions of Molded Hydrogels

[2082]

[2083] The cross-linking process leading to gelation was carried out for 1 hour at room temperature (RT, 20°C to 25°C) or in an oven at a controlled temperature, such as 20°C, 25°C, or 37°C. The hydrogel containing the islets was demolded and introduced into a pH 8 solution of Tris 150 mM / NaCl 30 mM / cysteine ​​10 mM at room temperature or in culture medium for 15 minutes. The hydrogel was then immersed in a culture medium containing 10 mM cysteine ​​at 37°C for 1 hour. After 1 hour, the cysteine-containing culture medium was removed and replaced with fresh culture medium. The sterile hydrogel containing cells was stored at 37°C and 5% CO2 prior to further in vitro testing or in vivo implantation.

[2084] According to this method, different hydrogel compositions were prepared using pseudo-islets or primary islets.

[2085] Table 18A: Hydrogel compositions incorporating islets of different densities and properties.

[2086]

[2087] RT refers to room temperature, between 20°C and 25°C.

[2088] Example C16B: Encapsulation of insulin-producing islets of Langerhans cells in a hydrogel

[2089] The encapsulation of pancreatic islets was performed in a sterile environment.

[2090] The concentrated sterile solutions of polysaccharides and PEG prepared according to Examples C1A or C1B and C2 were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg). The solutions were equilibrated at room temperature (20°C to 25°C or at 4°C).

[2091] A concentrated mixture of PEG and islets was prepared by mixing equal volumes of isotonic PEG solution or PEG / sodium hyaluronate solution (prepared according to Examples C2, C3, and C3B) with islet suspension (prepared according to Example B1B). The mixture containing PEG and islets was then gently mixed with an isotonic concentrated solution of polysaccharide at a volume ratio of 70:30 (islet suspension / PEG:polysaccharide) or 80:20 (islet suspension / PEG / hyaluronate:polysaccharide).

[2092] The solution was gently mixed with a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator (from Grace Biolab) or a ring-like construct adhered to a glass slide. Different hydrogel sizes were prepared by using different silicone molds (Table 5B) or ring-like constructs (Table 16).

[2093] Crosslinking leading to gelation was performed for 1 hour at room temperature (RT, 20°C to 25°C) or in an oven at a controlled temperature, such as 20°C, 25°C, or 37°C. Hydrogel discs or hydrogel / rings incorporating islets were introduced into a pH 8 solution of Tris 150 mM / NaCl 30 mM / cysteine ​​10 mM (Mal or VS:SH crosslinking) or in culture medium for 15 minutes (DBCO:N3 crosslinking) at room temperature. The hydrogel was then immersed in a medium containing 10 mM cysteine ​​for 1 hour at 37°C or in a medium without cysteine. After 1 hour, the cysteine-containing medium was removed and replaced with fresh medium. The sterile hydrogel containing cells was stored at 37°C and 5% CO2 prior to further in vitro testing or in vivo implantation.

[2094] Different hydrogel / islet compositions were prepared according to this method.

[2095] Table 18B: Hydrogel compositions incorporating islets of different densities and properties.

[2096]

[2097]

[2098]

[2099]

[2100] *HA 3,000 kg / mol, all other examples HA 1,500 kg / mol

[2101] #Add 10 mg / ml Pranic F127 during the hydrogel preparation process

[2102] Example C17: Spatial uniformity of cells encapsulated within the thickness of the hydrogel

[2103] Hydrogel discs incorporating the islets were prepared as described in Example C16B. A side-view photograph of the hydrogel was taken to evaluate the uniformity of the islets by the thickness of the hydrogel. Uniformity was achieved when the islets were located throughout the entire thickness of the hydrogel and not substantially on one side.

[2104] Table 17B: Homogeneity of islets in hydrogels

[2105]

[2106] The addition of sodium hyaluronate increases the viscosity of the mixture and allows for uniform spatial positioning of islets within the thickness of the hydrogel.

[2107] Using longer hyaluronic acid salt chains allows for a higher level of uniformity.

[2108] Example C18: Evaluation and control of hydrogel transparency

[2109] The hydrogel compositions described in Examples C4A and C4B, and some other hydrogels disclosed in Example C, are visually transparent.

[2110] To quantify the transparency properties of the hydrogel, UV absorbance was measured by casting 80 μL of the precursor solution into a 96-well plate. SAFAS). 100 μL of PBS was added to the top of the gel to prevent drying. Absorbance measurements at 400 nm were used to quantify the turbidity level of the sample. Five measurements were averaged at different locations. Measurements were taken 3 hours after casting in a fully swollen equilibrium state. The samples in the table below were also measured under standard conditions on a black panel with light levels ranging from 2,000 to 3,750 Lux. Perform a visual inspection before the Apollo II liquid detection unit.

[2111] Hydrogels of composition C4B-13 were prepared similarly to those in Example C3B. Concentrated sterile solutions of polysaccharide / Prönnicke DMCDBCO and PEG-N3 prepared according to Examples C1 and C2, respectively, were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg) and equilibrated at 4°C. Concentrated sterile solutions of PEG-N3 prepared according to Example C2 were adjusted with a combination of trehalose and concentrated NaCl to obtain isotonic stock solutions (300 mOsm / kg) and equilibrated at 4°C.

[2112] Table 17C: Hydrogel Compositions

[2113]

[2114] Table 17B: Visual appearance and UV absorbance of hydrogel compositions

[2115]

[2116] All gels are transparent under "normal" conditions (in real life).

[2117] However, because the change in absorbance is used to assess the variable homogeneity state of the macroscopic network, and considering the effect of variations in the permeate on transparency, we had to use the stringent conditions described above. These results indicate that, in the presence of HA, the turbidity of the hydrogel decreases when the permeate NaCl is replaced with trehalose.

[2118] Other nonionic penetrants such as mannitol or glycerin can also be used to improve transparency in the presence of HA.

[2119] This characteristic does not imply a change in gel properties or impermeability. Therefore, the diffusion of small molecules, such as insulin, is maintained regardless of the degree of turbidity.

[2120] Example C19: Laparoscopic implantation of hydrogel / proportional composite material

[2121] The implantability of the hydrogel / proportional composite material was evaluated in domestic pigs via laparoscopic surgery.

[2122] First, both implants (C15B-1 and C15B-4) are inserted into surgical bags, which are then rolled up for insertion into the peritoneal cavity via a 15mm cannula. The implants are then removed from the bags and subsequently secured to the peritoneum (C15B-1) or at the level of the peritoneal tear (C15B-4) in contact with muscle using sutures or non-absorbable surgical staples fixed with a silicone frame.

[2123] The implant remained intact / undamaged throughout the multiple surgical procedures and at the end of the implantation process.

[2124] Part D - Biological Evaluation of Hydrogels

[2125] Example D1A: In vitro evaluation of hydrogel cytotoxicity profile by extract assay

[2126] In accordance with ISO 10993-5: Recommendations for the biological evaluation of medical devices, extraction methods are used to evaluate the cytotoxicity profile of hydrogels.

[2127] The hydrogel was placed 3 cm deep in a 24-well plate. 2 3T3 cells were incubated in DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2 with steady-state stirring (70 rpm) for 24 hours to obtain the hydrogel extraction medium. In parallel, 3T3 cells were seeded at 5000 cells / well in 96-well plates and incubated overnight at 37°C and 5% CO2. The next day, the cell culture medium was removed and replaced with the hydrogel incubation medium. After incubating the 3T3 cells with the extraction medium at 37°C and 5% CO2 for 24 hours, viability was measured by quantifying intracellular ATP concentration using the ATPLite kit (Perkin Elmer) according to the manufacturer's instructions.

[2128] Calculate the survival percentage using the following formula:

[2129]

[2130] The cytotoxicity of the hydrogel composition C4A-21 was evaluated using this extract test. The results are shown in Table 19 below. The percentage of viability was compared with the untreated control. The standard deviation (SD) of the mean viability of triplicate wells was calculated (n = 2 hydrogels, each extract stored in triplicate wells).

[2131] Table 19A: Viability of hydrogels by extract test (ISO 10993-5).

[2132]

[2133] N = 2 independent experiments.

[2134] Compared with untreated cells, the selected composition did not show any significant cytotoxicity (t-test, p = 0.48) (see Table 19).

[2135] Example D1B: In vitro evaluation of the cytotoxicity profiles of C16B-18, C16B-19, and C16B-B20 hydrogels by extract assays in the presence or absence of human islets.

[2136] Following ISO 10993-5: Recommendations for the biological evaluation of medical devices, extraction methods were used to evaluate the cytotoxicity profiles of C16B-18, C16B-19, and C16B-20. The test items for C16B-18, C16B-19, and C16B-20 were performed with or without primary human islets. Primary human islets were isolated according to Example B3A and encapsulated as described in Examples C16B-18, C16B-19, and C16B-20.

[2137] With or without islets of Langerhans, C16B-18, C16B-19, and C16B-20 were placed in a 6-well plate at a depth of 6 cm. 2 3T3 cells were incubated in DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2 with steady-state stirring (70 rpm) for 24 hours to obtain the hydrogel extraction medium. In parallel, 3T3 cells were seeded at 5000 cells / well in 96-well plates and incubated overnight at 37°C and 5% CO2. The next day, the cell culture medium was removed and replaced with the hydrogel incubation medium. After 24 hours of incubation with the extraction medium at 37°C and 5% CO2, viability was measured by quantifying intracellular ATP concentration using the ATPLite kit (Perkin Elmer) according to the manufacturer's instructions.

[2138] Calculate the survival percentage using the following formula:

[2139]

[2140] The control group consisted of untreated 3T3 cells.

[2141] Cytotoxicity of C16B-18, C16B-19, and C16B-20 was evaluated by extract assays. Results are shown in Table 19B. Values ​​were normalized relative to untreated 3T3.

[2142] Table 19B: Viability of 3T3 cells as measured by extract test (ISO 10993-5) after treatment with C16B-18, C16B-19 and C16B-20, with or without islet cells.

[2143]

[2144] The negative control showed a viability of 75.2 ± 24.8% (mean ± SD). SD refers to the standard deviation. N = 2 independent experiments, n = 3 independent hydrogels / experiments.

[2145] With or without encapsulated human islets, the selected compositions showed no effect on 3T3 cell viability (one-way ANOVA, p = 0.7868; p = 0.7293; p = 0.5853; p = 0.9105; p = 0.8582; p = 0.6743 for C16B-18, C16B-19, C16B-19, C16B-20 and C16B-20 with islets, respectively).

[2146] In summary, C16B-18, C16B-19, and C16B-20 are not associated with any cytotoxicity, whether or not they are present in human islets...

Claims

1. A hydrogel comprising: - a cross-linked dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bonded to a dextran polymer backbone having i W groups, wherein -L(-) i It is a linear or branched polyether. -i is the valence of L and the number of W groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8), --W- is a group comprising at least one linear or branched alkyl group, and optionally comprising heteroatoms such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether derivative, and does not comprise two or more alpha amino acid residues, especially alpha amino acid residues linked by a peptide bond.

2. The hydrogel according to claim 1, further comprising non-cross-linked hyaluronate in the form of a solution.

3. The hydrogel according to any one of the preceding claims, wherein it further comprises biological cells.

4. The hydrogel according to any one of the preceding claims, wherein it is selected from the dextran polymer Dx carrying anionic groups, wherein the at least divalent group L(-) i covalently bonded to the dextran polymer backbone having i -W- groups, wherein -L(-) i It is a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly( Oxazoline), -i is the valence of L and the number of -W- groups bound to the dextran polymer, and is an integer from 2 to 8 (2≤i≤8), --W- is a group containing at least one linear or branched alkyl group, and optionally containing heteroatoms such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether or a poly( The invention does not contain any α-amino acid residues, particularly α-amino acid residues linked by a peptide bond.

5. The hydrogel according to any one of the preceding claims, characterized in that Tanδ is less than 1.

6. The hydrogel according to any one of the preceding claims, wherein it is a transparent hydrogel.

7. The hydrogel according to any one of the preceding claims, wherein it is a translucent hydrogel.

8. The hydrogel of any one of the preceding claims, wherein the cross-linked dextran polymer concentration after swelling in water is 0.01 to 0.2 g / g.

9. The hydrogel according to any one of the preceding claims, wherein its Young's modulus is from 1 to 200 kPa.

10. The hydrogel according to any one of the preceding claims, wherein its G' is from 0.5 to 70 kPa.

11. The hydrogel according to any one of the preceding claims, wherein its compression set at break is greater than or equal to 10%.

12. The hydrogel according to any one of the preceding claims, wherein its swelling ratio is greater than 0.

7.

13. A hydrogel according to any preceding claim, wherein the water content is at least 80 wt%.

14. The hydrogel according to any one of claims 1 to 13, wherein the biological cells are protein, hormone or peptide secreting cells.

15. The hydrogel according to any one of claims 1 to 14, wherein the biological cells are selected from: - Insulin-secreting cells for the treatment of diabetes, - Factor VIII-secreting cells or Factor IX-secreting cells for the treatment of hemophilia and β-glucocerebrosidase-secreting cells for Gaucher disease.

16. The hydrogel according to any one of claims 1 to 15, wherein the biological cells are pseudo-islets.

17. A method for synthesizing the cross-linked dextran polymer according to any one of claims 1 to 16 in the form of a hydrogel, comprising the following steps: a) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-, b) Preparation of precursor L(-) i of sterile solution, c) adding the sterile solution obtained from step b) to the solution obtained from step a), d) the addition is carried out directly in the mold, or the solution is introduced into the mold after mixing, e) performing crosslinking and gelation, for example at room temperature (20 to 25° C.) or 37° C., f) demoulding and swelling to obtain a hydrogel.

18. A method for synthesizing the cross-linked dextran polymer according to any one of claims 1 to 16 in the form of a hydrogel, comprising the following steps: a) preparing a sterile solution comprising an anionic group having formula II and at least two precursors -(A-f2) a -G1-、-(A'-f2) a -G'1- dextran, b) Preparation of precursor L(-) i of sterile solution, c) adding the sterile solution obtained from step b) to the solution obtained from step a), d) the addition is carried out directly in the mold, or the solution is introduced into the mold after mixing, e) performing crosslinking and gelation, for example at room temperature (20 to 25° C.) or 37° C., f) demoulding and swelling to obtain a hydrogel.

19. The method according to claims 17 to 18, wherein the cross-linking step is a gelling step resulting in the formation of a hydrogel.

20. A method for preparing a hydrogel containing biological cells according to claims 3 to 16, comprising the following steps: a) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-, b) Preparation of precursor L(-) i of sterile solution, c) preparing a suspension of biological cells, d) mixing the biological cell suspension obtained from step c) with the solution obtained from step b) or a), e) adding the sterile solution obtained from step a) or b) not used in step d) to the solution obtained from step d), f) This addition of step e) can be carried out directly in the mold, or the solution can be introduced into the mold after mixing, g) performing crosslinking and gelation reactions at room temperature (20 to 25° C.), h) performing demoulding and swelling to obtain a hydrogel containing biological cells.

21. A method for preparing a hydrogel containing biological cells according to claims 3 to 16, comprising the following steps: a) preparing a sterile solution comprising an anionic group having formula II and at least two precursors -(A-f2) a -G1-、-(A'-f2) a -G'1- dextran dextran, b) Preparation of precursor L(-) i of sterile solution, c) preparing a suspension of biological cells, d) mixing the biological cell suspension obtained from step c) with the solution obtained from step b) or a), e) adding the sterile solution obtained from step a) or b) not used in step d) to the solution obtained from step d), f) This addition of step e) can be carried out directly in the mold, or the solution can be introduced into the mold after mixing, g) performing crosslinking and gelation reactions at room temperature (20 to 25° C.), h) performing demoulding and swelling to obtain a hydrogel containing biological cells.

22. A method for preparing a hydrogel containing biological cells according to claims 3 to 16, comprising the following steps: a) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-, b) preparing a precursor L(-) selected from the following i Sterile solution of: thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene, c) preparing a sterile solution of sodium hyaluronate, d) preparing a sterile suspension of biological cells, e) mixing the sodium hyaluronate solution obtained from step c) with the precursor solution from step b), f) mixing the biological cell suspension obtained from step d) with the solution obtained from step e) or step a), g) mixing the solution obtained from step f) with the solution obtained from step e), h) adding the sterile solution obtained from step a) or e) not used in step g) to the solution obtained from step f), i) the addition of step g) is carried out directly in the mold, or the solution is introduced into the mold after mixing, j) performing crosslinking and gelation reactions at room temperature (20 to 25° C.), k) performing demoulding and swelling to obtain a hydrogel containing biological cells.

23. Method for preparing a hydrogel according to claims 1 to 16, wherein in the step of molding, the solution comprises an osmotic agent, which is a non-ionic osmotic agent, such as trehalose.

24. Method for preparing a hydrogel according to claims 1 to 16, wherein in the molding step, the solution comprises a weight ratio of non-ionic osmotic agent to NaCl greater than 2, in particular greater than 5, more in particular greater than 10.

25. A method of preparing a hydrogel according to claims 1 to 16, wherein the mould is a ring net.

26. A method for preparing a hydrogel according to claims 1 to 16, wherein the cross-linking and gelation reactions are carried out at room temperature (20 to 25°C).

27. A kit comprising: - Solution of dextran polymer of formula VIII before cross-linking reaction: in If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to Formula IV, ·and x is equal to 0 or 1, If a, a', b and b' are equal to 0, then x is equal to 0, and Dx is a dextran polymer backbone according to formula III, wherein R is selected from -H or an anionic group of formula II, If one of b' and c is not equal to 0, then -A' is A as defined above, If b', b and c are equal to 0, then a is equal to 0, and A' is the precursor A before the cross-linking reaction, If c is not equal to 0, R'1 is R1 as defined above, and G'1 is the precursor G1, If c is equal to 0, then b is equal to 0, and R'1 is the precursor R1 before the cross-linking reaction; - a solution of thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; -Biological cells.

28. A kit comprising: - Solution of dextran polymer of formula VIII before cross-linking reaction: in If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to Formula IV, ·and x is equal to 0 or 1, If a, a', b and b' are equal to 0, then x is equal to 0, and Dx is a dextran polymer backbone according to formula III, wherein R is selected from -H or an anionic group of formula II, If one of b' and c is not equal to 0, then -A' is A as defined above, If b', b and c are equal to 0, then a is equal to 0, and A' is the precursor A before the cross-linking reaction, If c is not equal to 0, R'1 is R1 as defined above, and G'1 is the precursor G1, If c is equal to 0, then b is equal to 0, and R'1 is the precursor R1 before the cross-linking reaction; - a solution of thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; - Biological cells; -Solution of non-cross-linked sodium hyaluronate.

29. A kit comprising: - Solution of dextran polymer of formula VIII before cross-linking reaction: in If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to Formula IV, ·and x is equal to 0 or 1, If a, a', b and b' are equal to 0, then x is equal to 0, and Dx is a dextran polymer backbone according to formula III, wherein R is selected from -H or an anionic group of formula II, If one of b' and c is not equal to 0, then -A' is A as defined above, If b', b and c are equal to 0, then a is equal to 0, and A' is the precursor A before the cross-linking reaction, If c is not equal to 0, R'1 is R1 as defined above, and G'1 is the precursor G1, If c is equal to 0, then b is equal to 0, and R'1 is the precursor R1 before the cross-linking reaction; - a solution of thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; -Biological cells.

30. Use of the cross-linked dextran copolymer according to any one of claims 1 to 16 in the form of a hydrogel for preparing a cell composition.

31. Therapeutic use of a hydrogel according to any one of claims 1 to 16 as a therapeutic implant for administering at least one API to a mammal.

32. Therapeutic use of a hydrogel according to any one of claims 1 to 16 for treating a condition or disease in a mammal, wherein the condition or disease is due to a deficiency or dysfunction of the endocrine function of the pancreatic organ.

33. The hydrogel according to any one of claims 1 to 16 for use as a medicament.

34. A hydrogel according to any one of claims 1 to 16 for use in treating a disease such as diabetes.

35. An implant comprising the hydrogel according to any one of claims 1 to 16.

36. An implant comprising a ring, a mesh, a hydrogel according to any one of claims 1 to 16 and cells.

37. An implant according to any one of claims 35 to 36, wherein it is a parallelepiped rectangle with rounded corners.

38. An implant according to any one of claims 35 to 37, wherein its thickness is less than 3 000 μm.

39. An implant according to any one of claims 35 to 37, wherein the total surface area is 10 cm 2 Up to 200cm 2 .

40. An implant according to any one of claims 35 to 38, wherein it comprises 0.5 to 20 ml of hydrogel.

41. An implant according to any one of claims 35 to 40, wherein the inner diameter of the ring is 10 to 100 mm.

42. An implant according to any one of claims 35 to 41, wherein the ring is a parallelepiped rectangle with rounded corners.

43. An implant according to any one of claims 35 to 42, wherein the material of the ring is a bioinert material.

44. An implant according to any one of claims 35 to 43, wherein the material of the ring is a biocompatible elastomer.

45. Implant according to any one of claims 35 to 44, wherein the material of the ring is selected from silicone, in particular PDMS, polyurethane, polyether, polyether polyester copolymer and polypropylene oxide.

46. ​​An implant according to any one of claims 35 to 45, wherein the mesh is non-biodegradable.

47. An implant according to any one of claims 35 to 45, wherein the mesh is biocompatible.

48. An implant according to any one of claims 35 to 45, wherein the mesh is non-absorbable.

49. An implant according to any one of claims 35 to 48, wherein the mesh is a surgical mesh.

50. An implant according to any one of claims 35 to 49, wherein the silk material in the material of the mesh is selected from polypropylene, polyethylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride) and ePVDF (stretched PVDF).

51. An implant according to any one of claims 35 to 37, wherein the mesh has a thickness of 50 to 500 μm.

52. An implant according to any one of claims 35 to 51, wherein the mesh has a pore size of 0.4 to 4 mm.

53. An implant according to any one of claims 35 to 37, wherein the fabric of the mesh is selected from the group consisting of knitted fabric, warp knitted fabric, woven fabric, non-woven fabric.

54. An implant according to any one of claims 35 to 53, wherein it is obtained by the following method: - incorporating the hydrogel composition into the ring network construct, mixing the concentrated polymer solution with a pipette and introducing a controlled volume of the mixture into the ring network construct adhered to a glass slide, - cross-linking leading to gelation is performed, and then the ring network + hydrogel composition is introduced into a solution of Tris 150mM / NaCl 30mM / cysteine ​​10mM at pH 8 or PBS at pH 7.4, - The hydrogel was washed with cysteine-free PBS solution and further immersed in the PBS solution at 37°C overnight, and then the hydrogel block was stored in the PBS solution at 4°C until use.

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