Hydrogels for cell therapy

Abstract

The invention relates to a cross-linked dextran polymer Dx bearing anionic groups in which the at least divalent group L (-) i is covalently bound to the main chain of a dextran polymer having i-W-groups, where-L (-) i is a linear or branched polyether or a linear or branched poly (# imgabs0 # oxazoline), -i is the valence of L and the number of-W-groups bound to the dextran polymer and is an integer of 2 to 8 (2 < = i < = 8)--W-is a group comprising at least one linear or branched alkyl group and optionally comprising a heteroatom such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether derivative or a poly (# imgabs 1 # oxazoline) derivative, and which does not comprise two or more alpha amino acid residues, in particular alpha amino acid residues linked by peptide linkages. The invention also relates to a dextran polymer prior to the cross-linking reaction and to a method for synthesizing the cross-linked dextran polymer according to the invention into a hydrogel form. The invention also relates to hydrogels comprising the cross-linked dextran polymer of the invention and / or to hydrogels further comprising biological cells. The invention also relates to the therapeutic use of the hydrogel according to the invention for the treatment of a condition or disease in a mammal, wherein the condition or disease is caused by an endocrine deficiency or dysfunction of the pancreatic organ, and to an implant comprising the hydrogel according to the invention.

Description

[0001] The field of the invention is therapy, in particular cell therapy. More specifically, the invention relates to implants comprising a hydrogel that can incorporate:

[0002] - an active principle, such as a peptide, hormone or protein, or

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

[0004] The goal is to prevent, treat, or cure disease. In particular, this can be used to prevent and / or treat chronic diseases by fully or partially replacing the function of naturally occurring cells that are deficient in the patient's body. The present invention also relates to crosslinked polymers, precursors thereof, methods for obtaining crosslinked polymers, and methods for obtaining hydrogels, particularly hydrogels containing cells.

[0005] The cells may be isolated or aggregated and may be of one type or of different types.

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

[0007] - stents as controlled drug or active pharmaceutical ingredient delivery systems, or

[0008] - Use as scaffolds 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 have the ability to absorb and retain large amounts of water. This is known as the swelling of the hydrogel.

[0010] In order to have a system that can serve as an implant capable of long-term delivery of active ingredients, a number of characteristics must be achieved.

[0011] Among these characteristics may be mentioned:

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

[0013] - good permselectivity, defined as the ability to selectively permeate biological elements according to their size or molecular weight, allowing a low or even better no immune response by isolating the incorporated cells in whole or in part from the host's immune system while allowing the passage of active ingredients (e.g. hormones, peptides or proteins),

[0014] - well-reduced foreign body response, or good biocompatibility, in particular low cytotoxicity and good local tolerance,

[0015] - to enable cells to have high survival rates, such as good vascularization near the cells and sufficient nutrient flow to the cells,

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

[0017] In order to be used as a controlled release system or scaffold for cells, the hydrogel must have specific characteristics such that it displays 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 one can mention:

[0019] - good homogeneity, which can be associated with good transparency or translucency,

[0020] - adequate resistance and flexibility to stress and strain mechanics, particularly during handling and implantation, e.g. by laparoscopy,

[0021] - defined mesh size for maximizing oxygen and nutrient exchange, controlled transport properties and selective permeability,

[0022] -Good in vivo stability, i.e. resistance to hydrolytic, enzymatic or oxidative degradation.

[0023] Among the parameters that may give an indication of the desired rheological and mechanical properties, one may cite:

[0024] - tan δ (called loss tangent), which gives an indication of the mechanical properties,

[0025] - G', which gives an indication of the elastic modulus (stiffness) and mesh size,

[0026] - compression and / or traction deformation at break, which gives an indication of the elasticity and resistance of the hydrogel,

[0027] - Swelling properties, which give an indication on the water content, dimensional and mechanical properties.

[0028] One of the problems to be solved is to obtain hydrogels with properties that allow:

[0029] - procedures to implant the hydrogel without destroying it, such as by laparoscopic procedures, and / or

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

[0031] A very difficult problem to be solved is to obtain an implant with a small thickness (in order to allow cells to be close to the tissue), a large surface (in order to have a relatively large volume) and with good mechanical properties (in order to allow minimally invasive surgery) as well as with good biocompatibility!

[0032] Another problem to be solved relates to the sedimentation of cells or islets during cross-linking leading to gelation.

[0033] The following are the existing technologies related to 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 doublenetwork hydrogel formulated from binary components: thiolated chitosan and oxidized dextran'', 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 are not able to solve the technical problem like the hydrogels of the present invention.

[0039] In the prior art, hydrogels containing cross-linked cells are very often intended to allow the growth of cell objects, such as 3D cell culture. Such applications require hydrogels that can simultaneously embed the starting cells and make room for new cells obtained by outgrowth and / or proliferation. In order to meet these two opposing characteristics, the solution is to have a degradable hydrogel (e.g., degradable by cleavable bonds) that is strong enough to embed the cells and make enough room for new cells after degradation. The method of choice to achieve this degradation is to have peptide structures in the cross-linked hydrogel, in particular at the level of the cross-linker between the polymer backbones.

[0040] This type of behavior is completely incompatible with the purpose of the present invention, which is to obtain durable cross-linked hydrogel encapsulation / embedded cells. In this case, the hydrogel needs to have very little degradability or even better, no degradability, because this key feature will allow the cells to remain invisible to the immune system.

[0041] Potential problems are addressed by providing a gel that has physicochemical properties that allow for the preparation of implantable devices and biocompatible properties that allow for cell survival.

[0042] Furthermore, compared to many prior art techniques, the present invention allows for the preparation of hydrogels with tunable characteristics, given the precursors used and the manner in which cross-linking is performed. This can result in hydrogels that allow for the controlled incorporation and release of specific targets from the hydrogel.

[0043] The applicability of the hydrogel depends on its main structure, so the 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 cross-linking points and the corresponding mesh size.

[0044] The problem is solved by providing novel cross-linked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are connected via at least one central linking group L(-) iCovalent cross-linking, the at least one group is at least a divalent linear, branched or cyclic alkyl group containing at least a polyethylene glycol chain, or the at least one group is at least a poly ( An at least divalent linear, branched or cyclic alkyl group containing an oxazoline (Pox) chain.

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

[0046] The problem is solved by providing novel cross-linked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are connected via at least one central linking group L(-) i Covalent cross-linking, the at least group is at least composed of poly ( An at least divalent linear, branched or cyclic alkyl group containing an oxazoline (Pox) chain.

[0047] In one embodiment, the cross-linked dextran polymer Dx carries anionic groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

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

[0049] -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),

[0050] --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, in particular alpha amino acid residues linked by a peptide bond.

[0051] In one embodiment, the cross-linked dextran polymer according to the present 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 cross-linked dextran polymer according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[0053] -L(-) i is a linear or branched polyether having heteroatoms such as oxygen, nitrogen or sulfur at its terminal ends,

[0054] -i is the valence of L and -(R1) m G1- the number of 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, wherein

[0057] -R1- is a linear or branched alkyl divalent radical containing 1 to 6 carbon atoms and optionally heteroatoms such as oxygen, nitrogen or sulfur,

[0058] -G1- is a linear or 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 cross-linked dextran polymer according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

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

[0061] -G1- is a sulfone derivative as described below:

[0062]

[0063] in:

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

[0065] X is a sulfur atom,

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

[0067] ·

[0068] or

[0069] -G1- is a succinimide derivative as described below:

[0070]

[0071] in:

[0072] X is a linear *-(CH2)n1-*, wherein n1 is an integer from 1 to 7 (1≤n1≤7),

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

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

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

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

[0077] In one embodiment, the dextran polymer is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i The result is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, which

[0078] - Mn is from 1000 to 25 000 g / mol (1000 ≤ Mn ≤ 25 000 g / mol), or

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

[0080] In one embodiment, the dextran polymer is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i A radical other than a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms,

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

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

[0083] By selecting and adjusting the cross-linking reaction conditions, the degree of substitution and molecular weight of dextran and cross-linker, the properties of such hydrogels can be tuned and customized for specific applications.

[0084] The problem is solved by providing a new hydrogel comprising:

[0085] - Biological cells,

[0086] - non-cross-linked hyaluronate in solution, and

[0087] - a cross-linked dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

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

[0089] -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),

[0090] --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, in particular alpha amino acid residues linked by a peptide bond.

[0091] In one embodiment, the cross-linked dextran polymer contained in the hydrogel according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having 1 W group, wherein

[0092] -L(-) i is a linear or branched polyether having heteroatoms such as oxygen, nitrogen or sulfur at its terminal ends,

[0093] -i is the valence of L and -(R1) m G1- the number of 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, wherein

[0096] -R1- is a linear or branched alkyl divalent radical containing 1 to 6 carbon atoms and optionally heteroatoms such as oxygen, nitrogen or sulfur,

[0097] -G1- is a linear or 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 a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i The result is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, which

[0099] - Mn is from 1000 to 25 000 g / mol (1000 ≤ Mn ≤ 25 000 g / mol), or

[0100] - The degree of polymerization (DP) is from 15 to 600 (15≤DP≤600).

[0101] In one embodiment, the dextran polymer contained in the hydrogel is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i A radical other than a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms,

[0102] - a 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 from 8 to 1000 (8≤DP≤1000).

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

[0105] -L(-) i Is a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly ( oxazoline),

[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 comprising at least one linear or branched alkyl group, and optionally comprising heteroatoms such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether or a poly( and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

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

[0109] -L(-) i It is a linear 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 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, in particular alpha amino acid residues linked by a peptide bond.

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

[0113] -L is a straight chain or branched poly ( oxazoline),

[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 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, in particular alpha amino acid residues linked by a peptide bond.

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

[0117] -L(-) i is a linear or branched polyether having heteroatoms such as oxygen, nitrogen or sulfur at its terminal ends,

[0118] -i is the valence of L and -(R1) mG1- the number of groups, and is an integer from 2 to 8 (2≤i≤8),

[0119] -m is an integer equal to 0 or 1,

[0120] -W is -(R1) m G1-group, wherein

[0121] -R1- is a linear or branched alkyl divalent radical containing 1 to 6 carbon atoms and optionally heteroatoms such as oxygen, nitrogen or sulfur,

[0122] -G1- is a linear or branched or cyclic alkyl divalent group containing 1 to 6 carbon atoms, and may contain heteroatoms such as oxygen, nitrogen or sulfur.

[0123] In one embodiment, the dextran polymer is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i The result is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, wherein

[0124] - a number average molecular weight Mn of 1000 to 25 000 g / mol (1000≤Mn≤25 000 g / mol), or

[0125] - The degree of polymerization (DP) is from 15 to 600 (15≤DP≤600).

[0126] In one embodiment, the dextran polymer is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i a radical not derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms, wherein

[0127] - a number average molecular weight (Mn) of 500 to 40 000 g / mol (500≤Mn≤40 000 g / mol), or

[0128] - The degree of polymerization (DP) is from 8 to 1000 (8≤DP≤1000).

[0129] In one embodiment, the cross-linked dextran polymer according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[0130] -W is -(R1) m G1-group, wherein

[0131] -G1- is a sulfone derivative as described below:

[0132]

[0133] in:

[0134] n1 is an integer from 0 to 7 (0≤n1≤7),

[0135] X is a sulfur atom,

[0136] * represents the dextran backbone and the divalent group L(-) i The connection site,

[0137] ·

[0138] or

[0139] -G1- is a succinimide derivative as described below:

[0140]

[0141] in:

[0142] X is a linear *-(CH2)n1-*, wherein n1 is an integer from 1 to 7 (1≤n1≤7),

[0143] * represents the dextran backbone and the divalent group L(-) i connection site.

[0144] In one embodiment, the cross-linked dextran polymer according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[0145] -W is -(R1) m G1-group, wherein

[0146] -(R1) m G1- is a straight or branched chain alkyl divalent radical containing less than 13 carbon atoms and optionally heteroatoms such as oxygen, nitrogen or sulfur.

[0147] Also contemplated are implants comprising the hydrogels of the invention.

[0148] The Applicant has surprisingly found that the presence of hyaluronic acid or sodium or potassium hyaluronic acid in the cross-linked mixture contributes to an improved uniform redistribution of cells or islets in the hydrogel. In other words, this reduces the effects of cell or islet sedimentation.

[0149] In one embodiment, the hydrogel comprises hyaluronic acid or the sodium salt or potassium salt of hyaluronic acid.

[0150] In one embodiment, the weight average molecular weight (Mw) of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt is 100 to 2500 kg / mol.

[0151] In one embodiment, the Mw of hyaluronic acid or sodium or potassium hyaluronic acid salt is from 250 to 2500 kg / mol.

[0152] In one embodiment, the Mw of hyaluronic acid or sodium or potassium hyaluronic acid salt is from 500 to 2250 kg / mol.

[0153] In one embodiment, the Mw of hyaluronic acid or sodium or potassium hyaluronic acid salt is from 750 to 2000 kg / mol.

[0154] In one embodiment, the Mw of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt is from 1 000 to 1500 kg / mol.

[0155] In one embodiment, the Mw of hyaluronic acid or sodium or potassium hyaluronic acid salt is from 250 to 4000 kg / mol.

[0156] In one embodiment, the Mw of hyaluronic acid or sodium or potassium hyaluronic acid salt is from 500 to 3750 kg / mol.

[0157] In one embodiment, the Mw of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt is from 750 to 3500 kg / mol.

[0158] In one embodiment, the Mw of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt is from 1 000 to 3250 kg / mol.

[0159] In one embodiment, the concentration of hyaluronic acid or sodium salt of hyaluronic acid or potassium salt of hyaluronic acid in the hydrogel is 0.5 to 30 mg / ml.

[0160] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt in the hydrogel is 0.5 to 20 mg / ml.

[0161] In one embodiment, the concentration of hyaluronic acid or sodium or potassium hyaluronic acid in the hydrogel is 0.5 to 10 mg / ml.

[0162] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt in the hydrogel is 0.5 to 5 mg / ml.

[0163] In one embodiment, the concentration of hyaluronic acid or sodium or potassium hyaluronic acid in the hydrogel is 0.75 to 2.5 mg / ml.

[0164] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt in the hydrogel is 1.0 to 1.5 mg / ml.

[0165] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronic acid salt or potassium hyaluronic acid salt in the hydrogel is 0.8 to 1.2 mg / ml.

[0166] In one embodiment of hyaluronic acid having a Mw of 2 000 to 4 000 kg / mol, in particular about 3 000 kg / mol, its concentration is 0.5 to 1.5 mg / ml.

[0167] In one embodiment of hyaluronic acid having a Mw of 2 000 to 4 000 kg / mol, in particular about 3 000 kg / mol, its concentration is 0.7 to 1.2 mg / ml.

[0168] In one embodiment of hyaluronic acid or sodium or potassium hyaluronic acid having an Mw of 1 000 to 2 000 kg / mol, in particular about 1 500 kg / mol, the concentration is 0.5 to 2 mg / ml.

[0169] In one embodiment with 1 000 to 2 000 kg / mol, in particular about 1 500 kg / mol, of hyaluronic acid or sodium or potassium hyaluronic acid, the concentration is 1.0 to 1.5 mg / ml.

[0170] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[0171] -L(-) i Is a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly ( oxazoline),

[0172] -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),

[0173] --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 or a poly( and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

[0174] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[0175] -L(-) i It is a linear or branched polyether.

[0176] -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),

[0177] --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, in particular alpha amino acid residues linked by a peptide bond.

[0178] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bound to a dextran polymer backbone having i W groups, wherein:

[0179] -L is a straight chain or branched poly ( oxazoline),

[0180] -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),

[0181] --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, in particular alpha amino acid residues linked by a peptide bond.

[0182] In one embodiment, -W- comprises up to 60 carbon atoms.

[0183] In one embodiment, -W- contains up to 60 carbon atoms, not counting any -CH2-CH2O- groups.

[0184] In one embodiment, -W- comprises up to 50 carbon atoms.

[0185] In one embodiment, -W- contains up to 50 carbon atoms, not counting any -CH2-CH2O- groups.

[0186] In one embodiment, -W- comprises up to 40 carbon atoms.

[0187] In one embodiment, -W- contains up to 40 carbon atoms, not counting any -CH2-CH2O- groups.

[0188] In one embodiment, -W- comprises up to 30 carbon atoms.

[0189] In one embodiment, -W- contains up to 30 carbon atoms, not counting any -CH2-CH2O- groups.

[0190] In one embodiment, -W- comprises up to 20 carbon atoms.

[0191] In one embodiment, -W- contains up to 20 carbon atoms, not counting any -CH2-CH2O- groups.

[0192] In one embodiment, -W- comprises up to 10 carbon atoms.

[0193] In one embodiment, -W- contains up to 10 carbon atoms, not counting any -CH2-CH2O- groups.

[0194] In one embodiment, -W- comprises up to 10 oxygen atoms.

[0195] In one embodiment, -W- contains up to 10 oxygen atoms, not counting any -CH2-CH2O- groups.

[0196] In one embodiment, -W- comprises up to 5 oxygen atoms.

[0197] In one embodiment, -W- contains up to 5 oxygen atoms, not counting any -CH2-CH2O- groups.

[0198] The cross-linked dextran hydrogel according to the present invention is a dextran polymer in which the central linker L(-) i is a linear or branched polyethylene glycol (PEG) group.

[0199] Branched PEG means multiple PEG arms containing from 2 to 20 carbon atoms linked by linear, branched or cyclic alkyl groups or by aromatic groups, and may contain heteroatoms such as nitrogen, oxygen or sulfur.

[0200] In one embodiment, the cross-linked dextran hydrogel according to the present invention is a dextran polymer wherein the central linker L(-) i It is a branched PEG group with up to 8 arms.

[0201] In one embodiment, the central linker L(-) i is a PEG selected from the group consisting of PEGs of Formula I:

[0202]

[0203] in:

[0204] i is an integer from 2 to 8 (2≤i≤8)

[0205] p is an integer equal to 0 or 1, and if i=2, then p=0

[0206] q is an integer from 8 to 1000 (8≤q≤1000)

[0207] r is an integer equal to 0 or 1

[0208] Q is a carbon atom, or a linear, branched or cyclic alkyl chain containing 2 to 10 carbon atoms and which may contain heteroatoms such as nitrogen, oxygen or sulfur, or an aromatic

[0209] * 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 cross-linking process is performed by native chemical ligation (NCL).

[0210] In one embodiment, q is an integer from 80 to 500 (80≤q≤500).

[0211] In one embodiment, q is an integer from 100 to 300 (100≤q≤300).

[0212] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms, which:

[0213] - a number average molecular weight (Mn) of 500 to 40 000 g / mol (500≤Mn≤40 000 g / mol), or

[0214] - The degree of polymerization (DP) is from 8 to 1000 (8≤DP≤1000).

[0215] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms, which:

[0216] - Mn is from 1000 to 25 000 g / mol (1000 ≤ Mn ≤ 25 000 g / mol), or

[0217] - The degree of polymerization (DP) is from 15 to 600 (15≤DP≤600).

[0218] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical according to formula I derived from a thiol polyethylene glycol or a thiol poly(ethylene oxide) as listed in the table below:

[0219] Chemical name 1 Mn (kg / mol) Poly(ethylene glycol) dithiol 2 10 Poly(ethylene glycol) dithiol 2 3.4 Poly(ethylene glycol) dithiol 2 1 Pentaerythritol tetrakis(mercaptoethyl)polyoxyethylene 4 5.2 Pentaerythritol tetrakis(mercaptoethyl)polyoxyethylene 4 20 Pentaerythritol tetrakis(mercaptoethyl)polyoxyethylene 4 40 Tripentaerythritol Octa(mercaptoethyl)polyoxyethylene 8 20

[0220] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical according to formula I derived from pentaerythritol tetrakis(mercaptoethyl)polyoxyethylene (CAS# 188492-68-4).

[0221] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a group according to formula I derived from linear (mercaptoethyl)polyoxyethylene (CAS# 68865-60-1).

[0222] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical according to formula I derived from the pentaerythritol poly(oxyethylene) azide listed in the table below:

[0223]

[0224] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a group according to formula I generated from pentaerythritol 4-arm PEG azide (CAS# 225531-50-0).

[0225] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) iis a radical according to formula I derived from the pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene listed in the table below:

[0226]

[0227] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical according to formula I derived from pentaerythritol 4-arm PEG DBCO.

[0228] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a group according to formula I derived from the maleimide polyethylene glycols listed in the table below:

[0229]

[0230] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a group according to formula I derived from 4-arm poly(ethylene glycol)maleimide.

[0231] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a radical according to formula I derived from the norbornene polyethylene glycols listed in the table below:

[0232]

[0233] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L(-) i is a group according to formula I derived from 4-arm poly(ethylene glycol) norbornene.

[0234] In one embodiment, the cross-linked dextran hydrogel according to the present invention is a dextran polymer wherein the central linker L is a linear or branched POx group.

[0235] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L is a linear or branched POx group comprising up to 8 arms and having a number average molecular weight (Mn) of 500 to 40 000 g / mol (500≤Mn≤40 000 g / mol).

[0236] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein L is a linear or branched POx group comprising up to 8 arms and having a number average molecular weight (Mn) of 1000 to 25 000 g / mol (1 000≤Mn≤25 000 g / mol).

[0237] In one embodiment, the POx central linker is a 2-arm POx selected from the linker of Formula XII.

[0238]

[0239] in:

[0240] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[0241] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0242] In one embodiment, the POx central linker is a 2-arm POx selected from linkers of formula XIIbis.

[0243]

[0244] in:

[0245] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[0246] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0247] In one embodiment, the POx central linker is a 4-arm POx selected from the linkers of Formula XIII.

[0248]

[0249] in:

[0250] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[0251] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0252] In one embodiment, the POx central linker is a 4-arm POx selected from the linker of Formula XIV:

[0253]

[0254] in:

[0255] The group -R1 is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[0256] The divalent group -R2- is a straight chain -(CH2) n2 -, wherein n2 is an integer from 2 to 6 (2≤n2≤6).

[0257] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0258] In one embodiment, the POx central linker is a 4-arm POx selected from the linker of formula XV:

[0259]

[0260] in:

[0261] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[0262] In one embodiment, R1 = -CH2-CH2-, and R2 is a linear -(CH2) n2 -, wherein n2 is an integer from 2 to 6 (2≤n2≤6),

[0263] In another embodiment, R2 = -CH2-CH2-, and R1 is a linear *-(CH2) n2 -*, wherein n2 is an integer from 2 to 6 (2≤n2≤6),

[0264] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0265] The hydroxyl groups of the dextran polymer Dx- can be functionalized with at least one specific anionic group, such as an alkyl carboxylate, a sulfate anion, a sulfonate anion, a phosphate anion, or a phosphonate anion.

[0266] In one embodiment, the hydroxyl functional groups of the dextran polymer Dx- can be functionalized with sulfate anions in salt form and optionally with alkyl carboxylate derivatives in salt form.

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

[0268] 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 carboxylate derivatives in salt form.

[0269] In another embodiment, the hydroxyl functional groups of the dextran polymer Dx- can be functionalized with phosphonate anions in salt form and optionally with alkyl carboxylate derivatives in salt form.

[0270] In another embodiment, the hydroxyl functional groups of the dextran polymer Dx- can be functionalized with alkyl carboxylate derivatives in salt form.

[0271] In one embodiment, the hydroxyl functional groups of the dextran polymer Dx- are functionalized with a specific anionic group: an alkyl carboxylate anion.

[0272] In one embodiment, the hydroxyl functional groups of the dextran polymer Dx- are functionalized with only one specific anionic group: an alkyl carboxylate anion.

[0273] The specific anionic groups previously defined are selected from groups of formula II:

[0274]

[0275] in:

[0276] * indicates that it is connected to the O atom of dextran to form an ether functional group.

[0277] y = 2 or 3.

[0278] When y=2, alkyl carboxylate derivative, then:

[0279] oY=C, and a=1.

[0280] o k=1, l=0, and m=0.

[0281] oR2 = alkyl.

[0282] When y=3, anionic group, then:

[0283] oY=S and a=1, or Y=P and a=2.

[0284] ok=0 or 1.

[0285] οl=0 or 1.

[0286] οm=0 or 1.

[0287] On = 1 or 2. In particular, n = 1,

[0288] οo=0 or 1.

[0289] oIf l=1, then m=1.

[0290] oR3 = linear, branched or cyclic alkyl, which may contain one heteroatom such as nitrogen, or aromatic, or PEG.

[0291] oR2 = alkyl.

[0292] • And, Z is a counterion, which may be an alkali metal, and z=1, or it may be an alkaline earth metal, and z=2.

[0293] In a preferred embodiment, the dextran backbone Dx- can be functionalized with sulfate anions in salt form and optionally with alkyl carboxylate derivatives in salt form.

[0294] In another preferred embodiment, the dextran backbone Dx- can be functionalized with an alkylsulfonate anion in salt form and optionally with an alkylcarboxylate derivative in salt form.

[0295] In another preferred embodiment, the dextran backbone Dx- can be functionalized with a sulfonate anion in salt form supported by an alkyl chain comprising a dimethyl-ammonium cation, and optionally with an alkyl carboxylate derivative in salt form.

[0296] In another preferred embodiment, the dextran backbone Dx- can be functionalized with an alkyl carboxylate derivative in salt form.

[0297] In one embodiment, the cross-linked dextran polymer carrying anionic groups according to the present invention is a dextran polymer wherein the dextran polymer backbone is according to formula III,

[0298] Where R is selected from

[0299] --H, anionic group of formula II, or with L(-) i -W- group of a crosslinker,

[0300] -i is 20 to 5000 (20≤i≤5000),

[0301] --W- and L(-) i The groups have the meanings defined previously.

[0302] In one embodiment, the cross-linked dextran polymer carrying anionic groups according to the present invention is a dextran polymer wherein the dextran polymer backbone is according to formula XI,

[0303]

[0304] Where R is selected from

[0305] --H, anionic group of formula II, or with L(-) i Cross-linked -(A-f2) a -G1- group,

[0306] -l is 20 to 5000 (20≤l≤5000),

[0307] --(A-f2) a -G1- and L(-) I The groups have the meanings defined previously.

[0308] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 1000 kDa before cross-linking and substitution.

[0309] In other words, the cross-linked dextran polymer according to the present invention is obtained after substitution and cross-linking of a natural dextran polymer having a weight average molecular weight (Mw) of 5 to 1000 kDa.

[0310] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 250 kDa before cross-linking and substitution.

[0311] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 100 kDa before cross-linking and substitution.

[0312] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 50 kDa before cross-linking and substitution.

[0313] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 25 kDa before cross-linking and substitution.

[0314] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution.

[0315] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 10 to 500 kDa before cross-linking and substitution.

[0316] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 500 kDa before cross-linking and substitution.

[0317] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution.

[0318] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 50 kDa before cross-linking and substitution.

[0319] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 40 to 250 kDa before cross-linking and substitution.

[0320] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 40 to 100 kDa before cross-linking and substitution.

[0321] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0322] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0323] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0324] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0325] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking group is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0326] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0327] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0328] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking group is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0329] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0330] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0331] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0332] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0333] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 250 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0334] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 250 kDa before cross-linking and substitution, and having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0335] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 250 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0336] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.2 to 0.4 (0.2≤DS1≤0.4).

[0337] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution, and having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking groups is in the range of 0.2 to 0.4 (0.2≤DS1≤0.4).

[0338] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.2 to 0.4 (0.2≤DS1≤0.4).

[0339] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.2 to 0.3 (0.2≤DS1≤0.3).

[0340] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution, and having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking groups is in the range of 0.2 to 0.3 (0.2≤DS1≤0.3).

[0341] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 20 to 100 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.2 to 0.3 (0.2≤DS1≤0.3).

[0342] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0343] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking group is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0344] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[0345] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0346] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking group is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0347] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) aThe degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.01 to 0.4 (0.01≤DS1≤0.4).

[0348] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0349] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking group is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0350] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.05 to 0.4 (0.05≤DS1≤0.4).

[0351] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0352] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-)i The degree of substitution (DS1) of the dextran backbone with -W- groups of the cross-linking groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0353] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 250 to 1000 kDa before cross-linking and substitution, and having L(-) i Cross-linked -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.1 to 0.4 (0.1≤DS1≤0.4).

[0354] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 0.3 to 2.5 (0.3≤DS2≤2.5).

[0355] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 0.5 to 2.3 (0.5≤DS2≤2.3).

[0356] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 1.5 to 2.5 (1.5≤DS2≤2.5).

[0357] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 1.7 to 2.3 (1.7≤DS2≤2.3).

[0358] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 1.8 to 2.2 (1.8≤DS2≤2.5).

[0359] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 0.3 to 1.5 (0.3≤DS2≤1.5).

[0360] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS2) of the dextran backbone with total carboxylic acid methyl esters / salts grafted onto the dextran is in the range of 0.3 to 0.8 (0.3≤DS2≤0.8).

[0361] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with 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).

[0362] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula I is in the range of 0.5 to 3 (0.5≤DS4≤3).

[0363] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula XII, XIIbis, XIII or XIV is in the range of 0.5 to 3 (0.5≤DS4≤3).

[0364] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with 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).

[0365] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula I is in the range of 1 to 2.75 (1≤DS4≤2.75).

[0366] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula XII, XIIbis, XIII or XIV is in the range of 1 to 2.75 (1≤DS4≤2.75).

[0367] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with 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).

[0368] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula I is in the range of 1.5 to 2.5 (1.5≤DS4≤2.5).

[0369] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula XII, XIIbis, XIII or XIV is in the range of 1.5 to 2.5 (1.5≤DS4≤2.5).

[0370] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with 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).

[0371] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula I is in the range of 1.75 to 2.25 (1.75≤DS4≤2.25).

[0372] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS4) of the dextran backbone with groups of formula XII, XIIbis, XIII or XIV is in the range of 1.75 to 2.25 (1.75≤DS4≤2.25).

[0373] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS) of the carboxylate / salt of the dextran backbone is C ) in the range of 0.2 to 3 (0.2≤DS C ≤3).

[0374] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS) of the carboxylate / salt of the dextran backbone isC ) in the range of 0.3 to 2.5 (0.3≤DS C ≤2.5).

[0375] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS3) of sulfate, sulfonate, phosphate or phosphonate of the dextran backbone is in the range of 0.2 to 2.5 (0.2≤DS3≤2.5).

[0376] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS3) of sulfate, sulfonate, phosphate or phosphonate of the dextran backbone is in the range of 0.3 to 2.0 (0.3≤DS3≤2.0).

[0377] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer whose -W- group or -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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 cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having a molar concentration of -W- groups that is equal to the molar concentration of the cross-linking groups 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).

[0379] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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).

[0380] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer whose -W- group or -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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 cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having a molar concentration of -W- groups that is equal to the molar concentration of the cross-linking groups 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).

[0382] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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).

[0383] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer whose -W- group or -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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 cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having a molar concentration of -W- groups that is equal to the molar concentration of the cross-linking groups 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).

[0385] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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).

[0386] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer whose -W- group or -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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 cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having a molar concentration of -W- groups that is equal to the molar concentration of the cross-linking groups 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).

[0388] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein -(A-f2) a The molar concentration of -G1- groups and the cross-linking 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).

[0389] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer whose -W- group or -(A-f2) a The molar concentration of -G1- groups and the cross-linking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is 1 (DC=1).

[0390] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer having a molar concentration of -W- groups that is equal to the molar concentration of the cross-linking groups L(-) i The molar ratio between the molar concentrations of the reactive functional groups is 1 (DC=1).

[0391] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein -(A-f2) a The molar concentration of -G1- groups and the cross-linking group L(-) i The molar ratio between the molar concentrations of the reactive functional groups is 1 (DC=1).

[0392] In one embodiment, the cross-linked dextran polymer according to the present invention is composed of a reactive functional group of a precursor -W- and a precursor L(-) i The cross-linked dextran polymer is obtained by the reaction between the reactive functional groups of , wherein the reactive functional groups are present at the same concentration (DC=1) and are in the range of 5 to 25 mM.

[0393] In one embodiment it is in the range of 5 to 10 mM.

[0394] In one embodiment it is in the range of 10 to 15 mM.

[0395] In one embodiment it is in the range of 15 to 20 mM.

[0396] In one embodiment it is in the range of 20 to 25 mM.

[0397] In one embodiment, -W- is selected from a group of formula IV.

[0398]

[0399] in

[0400] * indicates the site of f1, and Indicates the site of attachment to L.

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

[0402] b is an integer equal to 0 or 1.

[0403] c is an integer equal to 0 or 1.

[0404] In one embodiment, a=0 and f1 is an ether function or a carbamate function.

[0405] In one embodiment, a=1,

[0406] o The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives. It may also be branched by at least one hydroxyl group, such as -CH2-CH(OH)-(CH2) wherein 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.

[0407] or,

[0408] o The divalent group -A- is a linear polyether (PEG) derivative; f1 is an ether functional group or a carbamate functional group, and f2 is an amide functional group.

[0409] or,

[0410] o 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 function or a carbamate function, and f2 is a carbon-nitrogen covalent bond.

[0411] or,

[0412] o 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.

[0413] The divalent group -R1- is a linear, 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.

[0414] o If b=0, f1 is an ether function or a carbamate function.

[0415] o If b=1, f1 is an ether function or a carbamate function, and f3 is an amide function, or an amine function, or an ether function, or a thioether function, or a carbamate function, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond (if the cross-linking process is carried out by native chemical ligation (NCL)).

[0416] The divalent group -G1- is a linear, branched or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, for example, 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 alkylsulfone derivative (which may contain one heteroatom such as oxygen or sulfur), or an ethylamide derivative, or a 1,4-triazole derivative, or a polycyclic derivative from a Diels-Alder reaction, or an aromatic phosphine derivative produced by Staudinger ligation, or a cysteine ​​derivative from natural chemical ligation.

[0417] o If c=0, f1 is an ether function or a carbamate function.

[0418] o If c=1, 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 cross-linking process is carried out by native chemical ligation (NCL)).

[0419] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least the divalent group L(-) i covalently bound to a dextran polymer backbone having i groups, the dextran polymer being selected from dextran of formula X,

[0420] in:

[0421] a is an integer equal to 0 or 1.

[0422] ·i is an integer from 2 to 8 (2≤i≤8).

[0423] ·L can be used with the same [Dx-f1-(A-f2)a -G1-f3] group, or with different [Dx-f1-(A-f2) a -G1-f3] group is connected.

[0424] • Dx- is a dextran moiety which may be substituted with specific anionic groups in salt form and optionally with alkyl carboxylate derivatives in salt form.

[0425] f1 is an ether functional group.

[0426] The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives. It may also be branched by at least one hydroxyl group, such as -CH2-CH(OH)-(CH2) wherein n2 is an integer from 1 to 5 (1≤n2≤5) n2 -.

[0427] f2 is an amide functional group.

[0428] The divalent group -G1- is a linear, branched or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, for example, up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms. In a preferred embodiment, -G1- is a succinimide derivative, or an alkylsulfone derivative (which may contain one heteroatom such as oxygen or sulfur), or a 1,4-triazole derivative.

[0429] The integer i is the valence of the central linker L, and the same or different [Dx-f1-(A-f2) a -G1-f3] groups.

[0430] f3 is an amine functional group, a thioether functional group, an ether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0431] ·The central joint L is poly ( Oxazoline) (POx) derivatives, which may be linear or branched.

[0432] In this embodiment, the cross-linked dextran polymer according to the present invention is selected from the dextran polymers of formula V.

[0433]

[0434] in

[0435] f1, f2, f3, f4, -A-, -R1-, -G1- are as defined in Formula IV above, and

[0436] • Dx- is a dextran moiety which may be substituted with specific anionic groups in salt form and optionally with alkyl carboxylate derivatives as defined previously in salt form.

[0437] The integer i is the valence of the central linker L, and the same or different [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ]The number of groups.

[0438] • The central linker L is a polyether (PEG) derivative, which can be linear or branched.

[0439] In one embodiment, if b=0 and c=1, the central linker L can be poly( Oxazoline) (POx) derivatives, which may be linear or branched.

[0440] In this embodiment, the cross-linked dextran polymer according to the present invention is selected from the dextran polymers of formula V.

[0441]

[0442] in

[0443] f1, f2, f3, f4, -A-, -R1-, -G1- are as defined in Formula IV above, and

[0444] • Dx- is a dextran moiety which may be substituted with specific anionic groups in salt form and optionally with alkyl carboxylate derivatives as defined previously in salt form.

[0445] The integer i is the valence of the central linker L, and the same or different [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ]The number of groups.

[0446] • The central linker L is a polyether (PEG) derivative, which can be linear or branched.

[0447] In one embodiment, L is the same as [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ] group connection.

[0448] In one embodiment, L is different from [Dx-f1-(A-f2) a -(R1-f3) b-(G1-f4) c ] group connection.

[0449] If a=0, then:

[0450] • In one embodiment, f1 is an ether functional group.

[0451] • In another embodiment, f1 is a carbamate functional group.

[0452] If a=1, then:

[0453] • In one embodiment, f1 is an ether functional group.

[0454] • In another embodiment, f1 is a carbamate functional group.

[0455] If a=0 and b=0, then:

[0456] • In one embodiment, f1 is an ether functional group.

[0457] • In another embodiment, f1 is a carbamate functional group.

[0458] If a=0 and b=1, then:

[0459] • In one embodiment, f1 is an ether functional group.

[0460] • In another embodiment, f1 is a carbamate functional group.

[0461] If a=b=c=0, then:

[0462] • In one embodiment, f1 is an ether functional group.

[0463] • In another embodiment, f1 is a carbamate functional group.

[0464] In one embodiment, corresponding to Formula V, the divalent group -A- is a linear polyether (PEG) derivative of PEG selected from the following formulas:

[0465]

[0466] in:

[0467] n1 is an integer equal to 0 or 1.

[0468] n2 is an integer from 1 to 7 (1≤n1≤7).

[0469] * indicates the sites of f1 and f2.

[0470] In a preferred embodiment, * represents the sites of f1 and f2, said f1 and f2 being an ether functional group and an amide functional group, respectively.

[0471] In another embodiment, corresponding to Formula V, the divalent group -A- is a 1-alkyl-1,4-triazole derivative or a 1-PEG-1,4-triazole derivative selected from triazole derivatives of the formula:

[0472]

[0473] in:

[0474] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is a PEG derivative.

[0475] · * indicates the site of f1, and the dotted bond indicates f2.

[0476] In another embodiment, corresponding to Formula V, the divalent group -A- is a 4-alkyl-1,4-triazole derivative or a 4-PEG-1,4-triazole derivative selected from triazole derivatives of the formula:

[0477]

[0478] in:

[0479] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is a PEG derivative.

[0480] · * indicates the site of f1, and the dotted bond indicates f2.

[0481] If a=1, then:

[0482] • In one embodiment, f2 is an amide functional group.

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

[0484] • In another embodiment, f2 is a carbon-aromatic carbon covalent bond.

[0485] In one embodiment, corresponding to formula X, -A- is a linear -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivatives. It may also be branched by at least one hydroxyl group, such as -CH2-CH(OH)-(CH2) wherein n2 is an integer from 1 to 5 (1≤n2≤5) n2 -.

[0486] In one embodiment, corresponding to Formula V, the divalent group -R1- is a linear alkyl derivative according to the formula:

[0487]

[0488] in:

[0489] n1 is an integer from 1 to 7 (1≤n1≤7).

[0490] In one embodiment, if a=1, * represents the positions of f2 and f3.

[0491] In another embodiment, if a=0, * represents the position of f1 and f3.

[0492] In another embodiment, corresponding to Formula V, the divalent group -R1- is a polyether (PEG) derivative according to the following formula:

[0493]

[0494] in:

[0495] n1 is an integer equal to 0 or 1.

[0496] n2 is an integer from 1 to 7 (1≤n2≤7).

[0497] In one embodiment, if a=1, * represents the positions of f2 and f3.

[0498] In another embodiment, if a=0, * represents the position of f1 and f3.

[0499] 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 attached to the alkyl chain in the beta position relative to f1, said f1 being an ether function.

[0500]

[0501] in:

[0502] n2 is an integer from 1 to 5 (1≤n2≤5).

[0503] In another embodiment, if a=0, * represents the position of f1 and f3.

[0504] In a preferred embodiment, corresponding to formula V, the divalent group -R1- is a linear alkyl derivative according to the following formula:

[0505]

[0506] in:

[0507] * indicates the sites of f2 and f3, wherein f2 is an amide functional group and f3 is an amide functional group.

[0508] In a preferred embodiment, corresponding to formula V, the divalent group -R1- is a PEG derivative according to the following formula:

[0509]

[0510] in:

[0511] n1 is an integer from 1 to 7 (1≤n1≤7).

[0512] * indicates the sites of f2 and f3, which are two amide functional groups.

[0513] If b=1, then:

[0514] • In one embodiment, f3 is an amine functional group.

[0515] • In another embodiment, f3 is an ether function.

[0516] • In another embodiment, f3 is a thioether functional group.

[0517] • In another embodiment, f3 is an amide functional group.

[0518] • In another embodiment, f3 is a carbamate functional group.

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

[0520] • In another embodiment, f3 is a carbon-aromatic carbon covalent bond.

[0521] In another embodiment, if the cross-linking process is performed by native chemical ligation (NCL), then f3 is a carbon-carbon covalent bond.

[0522] The nature of the group G1 depends on the crosslinking process, and different crosslinking processes are described below in conjunction with the G1 group.

[0523] In one embodiment, the cross-linking process is achieved by Michael addition with a maleimide derivative, or a vinyl sulfone derivative, or an acrylamide derivative.

[0524] In one embodiment, corresponding to formula X, integer a=1, the crosslinking process is achieved by Michael addition with a maleimide derivative or a vinyl sulfone derivative.

[0525] In one embodiment, corresponding to formula V, integer a=c=1, and L is a POx derivative, the crosslinking process is achieved by Michael addition with a maleimide derivative or a vinyl sulfone derivative.

[0526] In one embodiment, corresponding to formula V, the divalent group -G1- is a succinimide derivative according to the following formula:

[0527]

[0528] in:

[0529] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is an aromatic derivative, or X is a PEG derivative.

[0530] In one embodiment, if b=1, * indicates the positions of f3 and f4.

[0531] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0532] In another embodiment, if a=1 and b=0, then * represents the positions of f2 and f4.

[0533] In a preferred embodiment, X is an ethyl group, and * represents the position of f2, which is an amide functional group, and f4, which is a thioether functional group.

[0534] In one embodiment, corresponding to formula X, the divalent group -G1- is a succinimide derivative according to the following formula:

[0535]

[0536] in:

[0537] • R is a linear, branched or cyclic alkyl derivative, or R is an aromatic derivative, or R is a PEG derivative.

[0538] * represents the sites of f2 and f3, wherein f2 is an amide functional group and f3 is an amine functional group, an ether functional group, or a thioether functional group.

[0539] In one embodiment, corresponding to formula V, if b=0, c=1, and L is a POx derivative, the divalent group -G1- is a succinimide derivative according to the following formula:

[0540]

[0541] in:

[0542] • R is a linear, branched or cyclic alkyl derivative, or R is an aromatic derivative, or R is a PEG derivative.

[0543] * represents the sites of f2 and f3, wherein f2 is an amide functional group and f3 is an amine functional group, an ether functional group, or a thioether functional group.

[0544] In one embodiment, corresponding to formula X, the divalent group -G1- is a succinimide derivative according to the following formula:

[0545]

[0546] in:

[0547] X is an oxygen atom, a sulfur atom or a nitrogen atom.

[0548] • R is a linear, branched or cyclic alkyl derivative, or R is a PEG derivative.

[0549] * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f3, which is a carbon-nitrogen covalent bond.

[0550] 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 formula:

[0551]

[0552] in:

[0553] X is an oxygen atom, a sulfur atom or a nitrogen atom.

[0554] • R is a linear, branched or cyclic alkyl derivative, or R is a PEG derivative.

[0555] * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f3, which is a carbon-nitrogen covalent bond.

[0556] In another embodiment, corresponding to Formula V, the divalent group -G1- is a diethylsulfone derivative according to the formula:

[0557]

[0558] in:

[0559] In one embodiment, if b=1, * indicates the positions of f3 and f4.

[0560] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0561] In another embodiment, if b=0, * represents the position of f2 and f4.

[0562] In a preferred embodiment, * represents the position of f3 and f4, said f3 and f4 being thioether functional groups.

[0563] In another embodiment, corresponding to Formula V, the divalent group -G1- is a sulfone derivative according to the formula:

[0564]

[0565] in:

[0566] n1 is an integer from 0 to 7 (0≤n1≤7).

[0567] X is an oxygen atom, a sulfur atom or a CH2 group.

[0568] In one embodiment, if b=1, * indicates the positions of f3 and f4.

[0569] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0570] In another embodiment, if a=1 and b=0, then * represents the positions of f2 and f4.

[0571] In a preferred embodiment, a=1, b=0, X is a sulfur atom, n1=2, f2 is an amide function, and f4 is a thioether function.

[0572] In another embodiment, corresponding to formula X, the divalent group -G1- is a sulfone derivative according to the formula:

[0573]

[0574] in:

[0575] n1 is an integer from 0 to 7 (0≤n1≤7).

[0576] X is an oxygen atom, a sulfur atom or a CH2 group.

[0577] * represents the sites of f2 and f3, wherein f2 is an amide functional group and f3 is an amine functional group, an ether functional group or a thioether functional group.

[0578] In another embodiment, corresponding to formula V, if b=0, c=1, and L is a POx derivative, the divalent group -G1- is a sulfone derivative according to the following formula:

[0579]

[0580] in:

[0581] n1 is an integer from 0 to 7 (0≤n1≤7).

[0582] X is an oxygen atom, a sulfur atom or a CH2 group.

[0583] * represents the sites of f2 and f3, wherein f2 is an amide functional group and f3 is an amine functional group, an ether functional group or a thioether functional group.

[0584] In another embodiment, corresponding to Formula V, the divalent group -G1- is an acrylamide derivative according to the formula:

[0585]

[0586] in:

[0587] • In one embodiment, * indicates the site of f3, which is an amine function, or an ether function, or a thioether function, and the dotted bond indicates f4, which is a carbon-nitrogen covalent bond.

[0588] • In another embodiment, the dotted bond represents f3, which is a carbon-nitrogen covalent bond, and * represents the site of f4, which is an amine function, or an ether function, or a thioether function.

[0589] In one embodiment, the cross-linking process is achieved via a 1,3-cycloaddition between an alkyne and an azide derivative, known as a 1,3-dipolar cycloaddition or Huisgen reaction.

[0590] In one embodiment, corresponding to formula V, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0591]

[0592] Therein, the two dashed bonds represent f3 and f4, which are covalent bonds or chemical functional groups defined previously and later.

[0593] In one embodiment, corresponding to formula X, integer a=1, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0594]

[0595] in:

[0596] • R1 is a linear, branched or cyclic alkyl derivative which may contain heteroatoms such as oxygen, or R1 is an aromatic derivative, or R1 is a PEG derivative.

[0597] * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f3, which is a carbon-nitrogen covalent bond or a carbon-aromatic carbon covalent bond.

[0598] In one embodiment, corresponding to formula V, the integer a=c=1, and L is a Pox derivative, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0599]

[0600] in:

[0601] X1 is a linear, branched or cyclic alkyl derivative, which may contain heteroatoms such as oxygen, or X1 is an aromatic derivative, or X1 is a PEG derivative.

[0602] * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f4, which is a carbon-nitrogen covalent bond or a carbon-aromatic carbon covalent bond.

[0603] In another embodiment, corresponding to formula X, integer a=0, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0604]

[0605] in:

[0606] * indicates the site of f1, which is an ether functional group, and the dotted bond indicates f3, which is a carbon-nitrogen covalent bond.

[0607] In another embodiment, corresponding to Formula V, integers a=0, b=0, c=1, and L is a Pox derivative, the divalent group -G1- is a 1,4-triazole derivative according to the following formula:

[0608]

[0609] in:

[0610] * indicates the site of f1, which is an ether functional group, and the dotted bond indicates f4, which is a carbon-nitrogen covalent bond.

[0611] In one embodiment, the cross-linking process is achieved via a 1,3-cycloaddition between a strained alkyne and an azide derivative, termed strain-promoted azide-alkynecycloaddition, or SPAAC.

[0612] In one embodiment, corresponding to formula X, integer a=1, the cross-linking process is achieved by a 1,3-cycloaddition between a strained alkyne and an azide derivative, referred to as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0613] In one embodiment, corresponding to formula V, integers a=c=1, b=0, and L is a POx derivative, the crosslinking process is achieved by a 1,3-cycloaddition between a strained alkyne and an azide derivative, referred to as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0614] In one embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0615]

[0616] -in:

[0617] The dotted circle represents a cyclooctene derivative from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur and is optionally functionalized with: a linear, branched or cyclic alkyl derivative containing from 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, especially fluorine.

[0618] • The two dashed bonds represent f3 and f4, which are covalent bonds or chemical functional groups defined previously and later.

[0619] • In another embodiment, if a=b=0, the two dashed bonds represent f1 and f4.

[0620] • In another embodiment, if a=1 and b=0, the two dashed bonds represent f2 and f4.

[0621] In a preferred embodiment, the dotted bond represents f2 and f4, wherein f2 is an amide functional group and f4 is a carbon-nitrogen covalent bond.

[0622] In one embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0623]

[0624] in:

[0625] The dotted circle represents a cyclooctene derivative from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur and is optionally functionalized with: a linear, branched or cyclic alkyl derivative containing from 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, especially fluorine.

[0626] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is a PEG derivative.

[0627] • The two dashed bonds represent f3 and f4, which are covalent bonds or chemical functional groups defined previously and later.

[0628] • In another embodiment, if a=b=0, then * represents the position of f1 and the dotted bond represents f4.

[0629] • In another embodiment, if a=1 and b=0, then * represents the position of f1 and the dotted bond represents f4.

[0630] In a preferred embodiment, X is a PEG derivative and the dotted bond represents f2 and f4, said f2 and f4 being amide functional groups.

[0631] In another embodiment, corresponding to formula X, the divalent group -G1- is a triazole derivative according to the following formula:

[0632]

[0633] in:

[0634] The dotted circle represents a cyclooctene derivative from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur and is optionally functionalized with: a linear, branched or cyclic alkyl derivative containing from 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, especially fluorine.

[0635] • R1 is a linear, branched or cyclic alkyl derivative which may contain heteroatoms such as oxygen, or R1 is an aromatic derivative, or R1 is a PEG derivative.

[0636] • R2 is a linear, branched or cyclic alkyl derivative which may contain heteroatoms such as oxygen, or R2 is an aromatic derivative, or R2 is a PEG derivative.

[0637] * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f3, which is a carbon-nitrogen covalent bond, or an amide functional group, or a carbamate functional group.

[0638] 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:

[0639]

[0640] in:

[0641] The dotted circle represents a cyclooctene derivative from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur and is optionally functionalized with: a linear, branched or cyclic alkyl derivative containing from 2 to 20 carbon atoms; or an aromatic derivative; or a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, especially fluorine.

[0642] X1 is a linear, branched or cyclic alkyl derivative, which may contain heteroatoms such as oxygen, or X1 is an aromatic derivative, or X1 is a PEG derivative.

[0643] • X2 is a linear, branched or cyclic alkyl derivative which may contain heteroatoms such as oxygen, or X2 is an aromatic derivative, or X2 is a PEG derivative.

[0644] * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f4, which is a carbon-nitrogen covalent bond, or an amide functional group, or a carbamate functional group.

[0645] In a preferred embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0646]

[0647] Wherein: * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f4, which is a carbon-nitrogen covalent bond.

[0648] In a preferred embodiment, corresponding to formula X, the divalent group -G1- is a triazole derivative according to the following formula:

[0649]

[0650] Wherein: * indicates the site of f2, which is an amide functional group, and the dotted bond indicates f3, which is a carbon-nitrogen covalent bond.

[0651] In another preferred embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0652]

[0653] in:

[0654] • X is a PEG derivative.

[0655] * indicates the sites of f2 and f4, which are amide functional groups.

[0656] In another preferred embodiment, corresponding to formula X, the divalent group -G1- is a triazole derivative according to the following formula:

[0657]

[0658] in:

[0659] • R1 is a linear, branched or cyclic alkyl derivative which may contain heteroatoms such as oxygen, or R1 is an aromatic derivative, or R1 is a PEG derivative.

[0660] * indicates the sites of f2 and f3, which are amide functional groups.

[0661] In another preferred embodiment, corresponding to formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0662]

[0663] in:

[0664] X1 is a linear, branched or cyclic alkyl derivative, which may contain heteroatoms such as oxygen, or X1 is an aromatic derivative, or X1 is a PEG derivative.

[0665] * indicates the sites of f2 and f4, which are amide functional groups.

[0666] In another embodiment, corresponding to Formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0667]

[0668] Wherein: * indicates the site of f3, and the dotted bond indicates f4.

[0669] In another embodiment, corresponding to Formula V, the divalent group -G1- is a triazole derivative according to the following formula:

[0670]

[0671] Wherein: the dotted bond represents f3, and * represents the site of f4.

[0672] In one embodiment, the cross-linking process is achieved via a Diels-Alder cycloaddition between maleimide and furan derivatives.

[0673] In one embodiment, corresponding to formula V, the divalent group -G1- is a polycyclic derivative consisting of one succinimide moiety according to the following formula:

[0674]

[0675] in:

[0676] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is a PEG derivative.

[0677] ·X1 is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 0 to 7 (0≤n1≤7)), a branched or cyclic alkyl derivative.

[0678] X2 is -H or -Me.

[0679] In one embodiment, if a=b=1, * indicates the positions of f3 and f4.

[0680] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0681] In another embodiment, if a=1 and b=0, then * represents the positions of f2 and f4.

[0682] In one embodiment, the cross-linking process is achieved through an inverse electron-demand Diels-Alder reaction, or IEDDA, ​​between tetrazine and norbornene derivatives.

[0683] In one embodiment, corresponding to formula V, the divalent group -G1- is a polycyclic derivative consisting of one pyridazine moiety according to the following formula:

[0684]

[0685] in:

[0686] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is an aromatic derivative, or X is a PEG derivative.

[0687] In one embodiment, if a=b=1, * indicates the positions of f3 and f4.

[0688] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0689] In another embodiment, if a=1 and b=0, then * represents the positions of f2 and f4.

[0690] In another embodiment, corresponding to formula V, the divalent group -G1- is a polycyclic derivative consisting of one pyridazine moiety according to the formula:

[0691]

[0692] in:

[0693] ·X is a straight chain*-(CH2) n1 -* (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative, or X is an aromatic derivative, or X is a PEG derivative.

[0694] In one embodiment, if a=b=1, * indicates the positions of f3 and f4.

[0695] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0696] In another embodiment, if a=1 and b=0, then * represents the positions of f2 and f4.

[0697] In one embodiment, the cross-linking process is achieved via a Staudinger linkage between an aromatic phosphine and an azide derivative.

[0698] In one embodiment, corresponding to formula V, the divalent group -G1- is an aromatic derivative according to the following formula:

[0699]

[0700] in:

[0701] In one embodiment, if a=b=1, * indicates the positions of f3 and f4.

[0702] In another embodiment, if a=b=0, then * represents the positions of f1 and f4.

[0703] In another embodiment, if a=1 and b=0, then * represents the positions of f2 and f4.

[0704] In one embodiment, corresponding to Formula V, the cross-linking process is achieved by native chemical ligation (NCL) between a thioester and an N-terminal cysteine ​​derivative.

[0705] In one embodiment, the divalent group -G1- can be formalized according to the following formula:

[0706]

[0707] in:

[0708] • Dashed lines represent carbon-nitrogen covalent bonds.

[0709] • In one embodiment, if a=b=1, the * and dotted lines indicate the positions of f3 and f4.

[0710] In another embodiment, if a=b=0, the * and the dotted line indicate the positions of f1 and f4.

[0711] • In another embodiment, if a=1 and b=0, the * and dotted lines represent the sites of f2 and f4.

[0712] If c=1, then:

[0713] • In one embodiment, f4 is an amine functional group.

[0714] • In another embodiment, f4 is an ether functional group.

[0715] • In another embodiment, f4 is a thioether functional group.

[0716] • In another embodiment, f4 is an amide functional group.

[0717] • In another embodiment, f4 is a carbamate functional group.

[0718] • In another embodiment, f4 is a carbon-nitrogen covalent bond.

[0719] • In another embodiment, f4 is a carbon-aromatic carbon covalent bond.

[0720] In another embodiment, if the cross-linking process is performed by native chemical ligation (NCL), f4 is a carbon-carbon covalent bond.

[0721] In one embodiment, f3 is an amine functional group.

[0722] In another embodiment, f3 is an ether functional group.

[0723] In another embodiment, f3 is a thioether functional group.

[0724] In another embodiment, f3 is an amide functional group.

[0725] In another embodiment, f3 is a carbamate functional group.

[0726] In another embodiment, f3 is a carbon-nitrogen covalent bond.

[0727] In another embodiment, f3 is a carbon-aromatic carbon covalent bond.

[0728] The cross-linked dextran polymer according to the present invention is selected from the dextran polymers of formula V.

[0729]

[0730] in:

[0731] i is an integer from 2 to 8 (2≤i≤8),

[0732] a=1,

[0733] b = 1,

[0734] c = 1,

[0735] Dx is a dextran derivative described in formula III,

[0736] L is the PEG central linker described in Formula I,

[0737] f1 is an ether functional group or a carbamate functional group,

[0738] The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative,

[0739] f2 is an amide functional group,

[0740] The divalent group -R1- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative,

[0741] f3 is an amide functional group,

[0742] The divalent group -G1- is a 1,4-triazole derivative,

[0743] • f4 is a carbon-nitrogen covalent bond, particularly wherein the nitrogen atom is within the triazole ring.

[0744] In a preferred embodiment, the integer i is equal to 4, i=4.

[0745] According to the above embodiment, the triazole derivative is composed of a cyclooctene derivative from a strained cyclooctyne, which may contain a heteroatom such as nitrogen, oxygen or sulfur and is optionally functionalized with: a linear, 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 a halogen, in particular fluorine.

[0746] According to the two above-mentioned embodiments, the triazole derivative comprises more than 10 and less than 30 carbon atoms, optionally it comprises 4 to 6 nitrogen atoms.

[0747] According to one embodiment, the 1,4-triazole derivative is obtained by a copperless reaction.

[0748] The cross-linked dextran polymer according to the present invention is selected from the dextran polymers of formula V.

[0749]

[0750] in:

[0751] I is an integer from 2 to 8 (2≤I≤8),

[0752] a=1,

[0753] b = 0,

[0754] c = 1,

[0755] Dx is a dextran derivative described in formula III,

[0756] L is the PEG central linker described in Formula I,

[0757] The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative,

[0758] f1 is an ether functional group or a carbamate functional group,

[0759] The divalent group -G1- is a 1,4-triazole derivative, which is a cyclooctene derivative from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur, and is optionally functionalized with: a linear, 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 a halogen, especially fluorine.

[0760] The 1,4-triazole derivative carries 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, wherein f2 is an amide functional group, and f4 is a carbon-nitrogen covalent bond, wherein the nitrogen atom is within the triazole ring.

[0761] In a preferred embodiment, the integer i is equal to 4, i=4.

[0762] According to one embodiment, the 1,4-triazole is a polycyclic radical comprising an acyl group which is linked via an amide function to a nitrogen which is within a ring but not from a triazole ring.

[0763] According to one embodiment, the 1,4-triazole comprises a cyclooctyne with nitrogen in the cyclooctyne ring.

[0764] According to one embodiment, the triazole derivative comprises more than 10 and less than 30 carbon atoms, optionally it comprises from 4 to 6 nitrogen atoms.

[0765] In one embodiment, the divalent group -G1- is a 1,4-triazole derivative as described by the following formula:

[0766]

[0767] Wherein: * indicates the position of f2, which is an amide functional group, and the dotted bond indicates f4, which is a carbon-nitrogen bond.

[0768] f4 is a carbon-nitrogen covalent bond, where the nitrogen atom is within the triazole ring.

[0769] According to one embodiment, triazole 1,4 is obtained by a copper-free reaction.

[0770] According to one embodiment, Dx is a dextran derivative described in formula III:

[0771]

[0772] Where R is selected from

[0773] --H, anionic group of formula II, or with L(-) i -W- group of the cross-linking group,

[0774] -i is 20 to 5000 (20≤i≤5000),

[0775] --W- and L(-) i The groups have the meanings defined previously.

[0776] According to one embodiment, L is a PEG central linker described in Formula I:

[0777]

[0778] in:

[0779] i is an integer equal to 4,

[0780] p is an integer equal to 1,

[0781] q is an integer from 8 to 1000 (8≤q≤1000),

[0782] r is an integer equal to 0 or 1,

[0783] Q is a branched alkyl chain containing 2 to 10 carbon atoms,

[0784] * indicates the position of f4, which is a carbon-nitrogen covalent bond, wherein the nitrogen atom is within the triazole ring.

[0785] Triazoles 1,4 were obtained via a copper-free reaction.

[0786] The present invention also relates to the dextran polymer of formula VIII prior to the cross-linking reaction.

[0787]

[0788] in

[0789] If a, a', b and b' are not equal to 0, then f1, f2, f3, Dx are as defined above,

[0790] ·and

[0791] x is equal to 0 or 1.

[0792] 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,

[0793] If one of b' and c is not equal to 0, then -A'- is -A- as defined above,

[0794] 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.

[0795] If c is not equal to 0, -R'1- is -R1- as defined above, and -G'1- is the precursor -G1-.

[0796] If c is equal to 0, then b is equal to 0 and R'1 is the precursor R1 before the cross-linking reaction.

[0797] In one embodiment, corresponding to Formula VIII, A' is an alkyl carboxylate derivative or a poly(oxyethylene) carboxylate 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 carboxylate, or a 2-hydroxyalkylamine.

[0798] The present invention also relates to the dextran polymer of formula XVIII prior to the cross-linking reaction.

[0799]

[0800] in

[0801] If a is not equal to 0, then f1, f2, and Dx are as defined above,

[0802] ·and

[0803] If a is equal to 0, f1 is an ether function and G'1 is a propargyl derivative, if a is not equal to 0, -A' is A as defined above.

[0804] In one embodiment, corresponding to Formula XVIII, if a=1, then A' is an alkyl carboxylate derivative, or a 2-hydroxyalkyl carboxylate, or a 2-hydroxyalkylamine.

[0805] In one embodiment, corresponding to Formula VIII, if a=1, b=0, and L is a POx derivative, then A is an alkyl carboxylate derivative, or a 2-hydroxyalkyl carboxylate, or a 2-hydroxyalkylamine.

[0806] A' as a carboxylic acid ester derivative can be formalized as follows:

[0807]

[0808] A' as an azide derivative, corresponding to Formula VIII, can be formalized as follows:

[0809]

[0810] A' as a propargyl derivative, corresponding to formula VIII, can be formalized as follows:

[0811]

[0812] A' as a 2-hydroxyalkyl carboxylate derivative can be formalized by the following formula:

[0813]

[0814] A' as a 2-hydroxyalkylamine derivative can be formalized as follows:

[0815]

[0816] in:

[0817] -n is an integer from 1 to 7 (1≤n≤7),

[0818] -m is an integer between 1 and 5 (1≤m≤5),

[0819] -f1 is as defined previously.

[0820] In one embodiment, corresponding to formula VIII, if a=1, then R'1 is a poly(ethylene oxide) group or an alkyl group with a terminal amine, or a terminal hydroxyl, or a terminal thiol, or a terminal carboxylate, or a terminal azide, or a terminal alkyne.

[0821] R'1 can be formalized as follows:

[0822]

[0823] R'1 as a carboxylate derivative can be formalized as follows:

[0824]

[0825] R'1 as an azide derivative can be formalized as follows:

[0826]

[0827] in:

[0828] -n is an integer from 1 to 7 (1≤n≤7)

[0829] -X=-NH2, or -OH, or -SH

[0830] -f2 is as defined previously.

[0831] Alternatively, in another embodiment, corresponding to Formula VIII, if a=0, then R'1 is a branched alkyl group wherein at least one hydroxyl group is attached to the alkyl chain at a beta position relative to f1 and having a terminal hydroxyl group, or a terminal thiol, or a terminal azide or a terminal alkyne.

[0832] R'1 can be formalized as follows:

[0833]

[0834] in:

[0835] -n is an integer from 1 to 5 (1≤n≤5),

[0836] -X=-NH2, or -OH, or -SH, or -N3, or -C≡CH,

[0837] -f1 is as defined previously.

[0838] Alternatively, in another embodiment, R'1 is a propargyl derivative and can be formalized as follows:

[0839]

[0840] in:

[0841] -n is an integer from 1 to 7 (1≤n≤7)

[0842] -f1 is as defined previously.

[0843] In one embodiment, corresponding to Formula VIII, -G' is a maleimide derivative, or a vinyl sulfone derivative, or a strained cyclooctyne 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 a cysteine, or a thioester, or a thiol derivative, or an amine derivative, or a hydroxyl derivative.

[0844] 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 hydroxy derivative.

[0845] 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 hydroxy derivative.

[0846] -G'1 as a thiol, amine or hydroxy derivative, corresponding to Formula VIII, can be formalized as follows:

[0847]

[0848] G'1 as a thiol, amine or hydroxy derivative, corresponding to Formula XVIII, can be formalized as follows:

[0849]

[0850] G'1 as a maleimide, corresponding to Formula VIII, can be formalized as follows:

[0851]

[0852] G'1 as a maleimide, corresponding to Formula XVIII, can be formalized as follows:

[0853]

[0854] G'1 as vinyl sulfone, corresponding to formula VIII, can be formalized as follows:

[0855]

[0856] G'1 as vinyl sulfone, corresponding to Formula XVIII, can be formalized as follows:

[0857]

[0858] G'1 as a strained cyclooctyne, corresponding to formula VIII, can be formalized as follows:

[0859]

[0860] G'1 as an azide derivative, corresponding to Formula VIII, can be formalized as follows:

[0861]

[0862] G'1 as an azide derivative, corresponding to Formula XVIII, can be formalized as follows:

[0863]

[0864] G'1 as a propargyl derivative, corresponding to formula VIII, can be formalized as follows:

[0865]

[0866] G'1 as a propargyl derivative, corresponding to formula XVIII, can be formalized as follows:

[0867]

[0868] in:

[0869] n is an integer from 0 to 7 (0≤n1≤7),

[0870] R is a linear, branched or cyclic alkyl derivative, or R is a PEG derivative,

[0871] X=-NH2, or -OH, or -SH,

[0872] X1 is an oxygen atom, a sulfur atom, or a CH2 group,

[0873] • f2 is as defined previously.

[0874] In one embodiment, corresponding to Formula XVIII, if a=0, then G'1 is a propargyl derivative.

[0875] G'1 as a propargyl derivative, corresponding to formula XVIII, can be formalized as follows:

[0876]

[0877] in:

[0878] • f1 is as defined previously.

[0879] G'1 as a furan derivative, corresponding to formula VIII, can be formalized as follows:

[0880]

[0881] G'1, as an acrylamide derivative, corresponds to Formula VIII and can be formalized as follows:

[0882]

[0883] G'1, as a norbornene derivative, corresponds to Formula VIII and can be formalized as follows:

[0884]

[0885] G'1, as a trans-cyclooctene derivative, corresponds to formula VIII and can be formalized as follows:

[0886]

[0887] G'1 as a tetrazine derivative, corresponding to formula VIII, can be formalized as follows:

[0888]

[0889] G'1 as an aromatic phosphine derivative, corresponding to formula VIII, can be formalized as follows:

[0890]

[0891] G'1 as a cysteine ​​derivative, corresponding to Formula VIII, can be formalized as follows:

[0892]

[0893] G'1 as a thioester derivative, corresponding to Formula VIII, can be formalized as follows:

[0894]

[0895] in:

[0896] X=-NH2, or -OH, or -SH

[0897] X1=-O- or -S-

[0898] n is an integer equal to 0 or 1

[0899] R1 is an alkyl group

[0900] ·X2=-CH2- or aromatic

[0901] R2=-H or -CH3

[0902] • f3 is as defined previously.

[0903] • The dotted bond represents f3, which is a carbon-nitrogen covalent bond or a carbon-carbon covalent bond.

[0904] The present invention also relates to a hydrogel comprising the cross-linked dextran polymer according to the invention.

[0905] In one embodiment, the hydrogel is transparent.

[0906] "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 a standard of 2 (6 NTU), and / or the UV absorbance of the hydrogel as measured in Example C21 of application PCT / EP2022 / 050466 is lower than 0.06 (absorbance units).

[0907] In one embodiment, the hydrogel is visually clear and has a UV absorbance of <0.06 (absorbance units).

[0908] In one embodiment, the hydrogel according to the invention is characterized by a Tan δ of less than 1.

[0909] In this specification, Tan δ is the ratio of loss modulus G″ to storage modulus (also referred to as elastic modulus) G′ (Tan δ=G″ / G).

[0910] In one embodiment, the hydrogel according to the invention is characterized by a Tan δ less than or equal to 0.5.

[0911] In one embodiment, the hydrogel according to the invention is characterized by a Tan δ less than or equal to 0.1.

[0912] In one embodiment, the hydrogel according to the invention is characterized by a Tan δ less than or equal to 0.05.

[0913] In one embodiment, the hydrogel according to the invention is characterized by a Tan δ less than or equal to 0.01.

[0914] In one embodiment, the hydrogel according to the invention is characterized by a cross-linked dextran polymer concentration of 0.01 to 0.2 g / g after swelling in water.

[0915] In one embodiment, the hydrogel according to the invention is characterized by a cross-linked dextran polymer concentration of 0.03 to 0.1 g / g after swelling in water.

[0916] In one embodiment, the hydrogel according to the invention is characterized by a cross-linked dextran polymer concentration of 0.05 to 0.1 g / g after swelling in water.

[0917] In one embodiment, the hydrogel is translucent.

[0918] In another embodiment, the hydrogel is transparent.

[0919] In one embodiment, the hydrogel has a Young's modulus of 1 to 200 kPa.

[0920] In one embodiment, the hydrogel has a Young's modulus of 5 to 200 kPa.

[0921] In one embodiment, the hydrogel has a Young's modulus of 20 to 200 kPa.

[0922] In one embodiment, the hydrogel has a Young's modulus of 30 to 200 kPa.

[0923] In one embodiment, the hydrogel has a Young's modulus of 50 to 200 kPa.

[0924] In one embodiment, the hydrogel has a Young's modulus of 30 to 180 kPa.

[0925] In one embodiment, the hydrogel has a Young's modulus of 50 to 150 kPa.

[0926] In one embodiment, the hydrogel has a Young's modulus of 5 to 100 kPa.

[0927] In one embodiment, the hydrogel has a Young's modulus of 10 to 90 kPa.

[0928] In one embodiment, the hydrogel has a Young's modulus of 10 to 75 kPa.

[0929] In one embodiment, the hydrogel has a G' of 0.5 to 70 kPa.

[0930] In one embodiment, the hydrogel has a compression set at break greater than or equal to 10%.

[0931] In one embodiment, the hydrogel has a compression set at break greater than or equal to 15%.

[0932] In one embodiment, the hydrogel has a compression set at break greater than or equal to 20%.

[0933] In one embodiment, the hydrogel has a compression set at break greater than or equal to 25%.

[0934] In one embodiment, the hydrogel has a compression set at break greater than or equal to 30%.

[0935] In one embodiment, the hydrogel has a compression set at break greater than or equal to 35%.

[0936] In one embodiment, the hydrogel has a compression set at break greater than or equal to 40%.

[0937] In one embodiment, the hydrogel has a compression set at break greater than or equal to 45%.

[0938] In one embodiment, the hydrogel has a compression set at break greater than or equal to 50%.

[0939] In one embodiment, the hydrogel has a compression set at break greater than or equal to 55%.

[0940] In one embodiment, the hydrogel has a compression set at break greater than or equal to 60%.

[0941] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 10%.

[0942] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 15%.

[0943] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 20%.

[0944] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 25%.

[0945] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 30%.

[0946] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 35%.

[0947] In one embodiment, the hydrogel has a tensile strain at break greater than or equal to 40%.

[0948] In one embodiment, the hydrogel has a swelling ratio greater than 0.7.

[0949] In one embodiment, the hydrogel has a swelling ratio greater than 0.8.

[0950] In one embodiment, the hydrogel has a swelling ratio greater than 0.9.

[0951] In one embodiment, the hydrogel has a swelling ratio greater than 1.

[0952] In one embodiment, the hydrogel has a swelling ratio greater than 1.1.

[0953] In one embodiment, the hydrogel has a swelling ratio greater than or equal to 1.2.

[0954] In one embodiment, the hydrogel has a swelling ratio greater than or equal to 1.3.

[0955] In one embodiment, the hydrogel has a swelling ratio greater than or equal to 1.4.

[0956] In one embodiment, the hydrogel has a swelling ratio greater than or equal to 1.5.

[0957] In one embodiment, the hydrogel has a swelling ratio greater than or equal to 1.6.

[0958] In one embodiment, the hydrogel has a swelling ratio of less than or equal to 5.

[0959] In one embodiment, the hydrogel has a swelling ratio of 4 or less.

[0960] In one embodiment, the hydrogel has a swelling ratio of less than or equal to 3.

[0961] In one embodiment, the hydrogel has a swelling ratio less than or equal to 2.8.

[0962] In one embodiment, the hydrogel has a swelling ratio less than or equal to 2.5.

[0963] In one embodiment, the hydrogel has a swelling ratio less than or equal to 2.3.

[0964] In one embodiment, the hydrogel has a water content of at least 80 wt%.

[0965] In one embodiment, the hydrogel has a water content of at least 85 wt%.

[0966] In one embodiment, the hydrogel has a water content of at least 90 wt%.

[0967] In one embodiment, the hydrogel has a water content of at least 97 wt%.

[0968] In one embodiment, the hydrogel has a water content of at least 96 wt%.

[0969] In one embodiment, the hydrogel has a water content of at least 95 wt%.

[0970] In one embodiment, the hydrogel has a water content of at least 94 wt%.

[0971] In one embodiment, the hydrogel has a water content of at least 93 wt%.

[0972] In one embodiment, the hydrogel has a water content of up to 99 wt%.

[0973] In one embodiment, the hydrogel has a water content of up to 98 wt%.

[0974] In one embodiment, the hydrogel according to the invention is characterized in that it also comprises biological cells.

[0975] In one embodiment, the cell is a cell from human or animal origin.

[0976] In one embodiment, the cell is a cell line.

[0977] In one embodiment, the cells are of stem cell origin.

[0978] In one embodiment, the stem cells are selected from embryonic stem cells, induced pluripotent stem cells, or mesenchymal stem cells.

[0979] In one embodiment, the cell is a primary cell.

[0980] In one embodiment, the cell is a protein, hormone or peptide secreting cell.

[0981] In one embodiment, the cell is selected from the group consisting of:

[0982] -Insulin-secreting cells for diabetes treatment

[0983] - Factor VIII or Factor IX secreting cells for the treatment of hemophilia, and

[0984] - For β-glucocerebrosidase-secreting cells in Gaucher disease.

[0985] In one embodiment, the cell is selected from cells that secrete:

[0986] - Growth hormone

[0987] Human growth hormone (hGH),

[0988] Recombinant human growth hormone (rhGH)

[0989] -Growth hormone-releasing hormone (GHRH),

[0990] -thyroid stimulating hormone (TSH),

[0991] -thyrotropin-releasing hormone (TRH),

[0992] - adrenocorticotropic hormone (ACTH), and

[0993] -Parathyroid hormone (PTH).

[0994] In one embodiment, the cell is selected from cells that secrete:

[0995] -Glucagon

[0996] -Insulin, and

[0997] -GLP-1.

[0998] In one embodiment, the cells are selected from cells that secrete insulin.

[0999] In one embodiment, the cells are selected from cells that secrete insulin.

[1000] In one embodiment, the cell is selected from cells that secrete a growth factor selected from:

[1001] -vascular endothelial growth factor (VEGF),

[1002] -Nerve growth factor (NGF),

[1003] - Platelet-derived growth factor (PDGF),

[1004] - fibroblast growth factor (FGF),

[1005] - epidermal growth factor (EGF),

[1006] - Transforming growth factor (TGF), and

[1007] -Insulin-like growth factors I and II (IGF-I and IGF-II).

[1008] In one embodiment, the cell is selected from a cell that secretes a coagulation factor or blood clotting factor selected from:

[1009] - Factor I (e.g., fibrinogen),

[1010] - Factor II (e.g., prothrombin),

[1011] - Factor III (e.g., tissue factor),

[1012] - Factor V (e.g., proaccelerin, labile factor),

[1013] - Factor VI,

[1014] - Factor VII (e.g., stabilizing factor, proconvertin),

[1015] - Factor VIII (e.g., antihemophilic factor A),

[1016] - Factor VIIIC,

[1017] - Factor IX (eg, antihemophilic factor B),

[1018] - Factor X (e.g., Stuart-Prower factor),

[1019] - Factor XI (e.g., plasma thromboplastin precursor),

[1020] - Factor XII (eg, Hagerman factor),

[1021] - Factor XIII (eg, fibrin stabilizing factor),

[1022] -von Willebrand factor (vWF),

[1023] - Prekallikrein,

[1024] - Heparin cofactor II,

[1025] - high molecular weight kininogens (e.g., Fitzgerald factor),

[1026] - Antithrombin III, and

[1027] -Fibronectin.

[1028] In one embodiment, the cell is selected from cells that secrete immunoglobulin chains (heavy or light) or fragments thereof, comprising at least one immunoglobulin variable domain sequence and optionally comprising an immunoglobulin Fc region.

[1029] In one embodiment, the cell is selected from a cell that secretes a cytokine or a cytokine receptor, or a chimeric protein comprising a cytokine or its receptor.

[1030] In one embodiment, the cells are selected from cells that secrete erythropoietin.

[1031] In one embodiment, the cell is selected from a cell that secretes an interleukin (IL) selected from:

[1032] -IL-1, IL-2 to IL-10.

[1033] In one embodiment, the cell is selected from cells that secrete a replacement enzyme selected from:

[1034] -α-galactosidase A (GLA),

[1035] -α-L-iduronidase (IDVA),

[1036] - arylsulfatase B (ARSB),

[1037] -glucocerebrosidase, and

[1038] -N-sulfoglucosamine sulfohydrolase (SGSH).

[1039] In one embodiment, the hydrogel according to the invention is characterized in that the insulin-secreting cells are selected from the group of pancreatic cells.

[1040] In one embodiment, the hydrogel according to the invention is characterized in that the insulin-secreting cells are islets of Langerhans.

[1041] In one embodiment, the hydrogel according to the invention is characterized in that the biological cells are pseudo-islets.

[1042] The present invention also relates to the use of the cross-linked dextran copolymer according to the present invention in the form of a hydrogel to prepare a cell composition.

[1043] In one embodiment, the cells are selected from one or more types of isolated or aggregated cells that secrete an active ingredient.

[1044] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 2, 4, or 8.

[1045] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-)i Covalently bound to a dextran polymer backbone having i groups, where i is 2, 4, or 8.

[1046] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 2.

[1047] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 2.

[1048] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 4.

[1049] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 4.

[1050] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 8.

[1051] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 8.

[1052] The cross-linking step is the gelling step leading to the formation of the hydrogel according to the invention.

[1053] The hydrogel formation kinetics is a function of temperature and can be modulated by the concentration of reactants, pH, and temperature.

[1054] In one embodiment, the time to obtain the hydrogel according to the invention is from 1 minute to 6 hours.

[1055] In one embodiment, the cross-linking step is performed for 1 hour.

[1056] In one embodiment, the temperature of the cross-linking step is from 4°C to room temperature (20°C to 25°C) and may vary between the mixing step and the gelling and molding steps.

[1057] In one embodiment, mixing is performed at 4°C and gelling is performed at room temperature (20 to 25°C) for 1 hour.

[1058] In one embodiment, mixing is performed at room temperature (20°C to 25°C).

[1059] In one embodiment, mixing is performed at 4°C or room temperature (20°C to 25°C), and gelling is performed at room temperature (20°C to 25°C) for 1 hour.

[1060] In one embodiment, gelation is performed at 37°C.

[1061] In one embodiment, after cross-linking or gelling, the hydrogel is swollen in a buffer solution having a pH of 5 to 8, preferably 6 to 8, and more preferably 6.8 to 7.5.

[1062] In one embodiment, the buffer solution is a PBS solution at pH 7.4.

[1063] In one embodiment, the buffer solution is a Tris solution at pH 7.4.

[1064] In one embodiment, the buffer solution is a Tris solution at pH 8.

[1065] In one embodiment, swelling increases the mass of the hydrogel by 1, 2, 3, or 4 times compared to its initial mass.

[1066] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention in the form of a hydrogel, which comprises the following steps:

[1067] a) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-,

[1068] b) Preparation of precursor L(-) i of sterile solution,

[1069] c) adding the sterile solution obtained from step b) to the solution obtained from step a),

[1070] d) Addition is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1071] e) performing crosslinking and gelling, for example at room temperature (20° C. to 25° C.) or at 37° C.,

[1072] f) demoulding and swelling to obtain a hydrogel.

[1073] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention in the form of a hydrogel, which comprises the following steps:

[1074] 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,

[1075] b) Preparation of precursor L(-) i of sterile solution,

[1076] c) adding the sterile solution obtained from step b) to the solution obtained from step a),

[1077] d) Addition is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1078] e) performing crosslinking and gelling, for example at room temperature (20° C. to 25° C.) or at 37° C.,

[1079] f) demoulding and swelling to obtain a hydrogel.

[1080] In one embodiment, steps c) and d) are performed simultaneously.

[1081] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[1082] Dextran with anionic groups of formula II is prepared by grafting or substituting the hydroxyl groups on dextran. In one embodiment, dextran with anionic groups of formula II is prepared by grafting or substituting the carboxymethyl groups on dextran carboxylate methyl ester.

[1083] In one embodiment of the method according to the invention, the active pharmaceutical ingredient (API) is embedded in the hydrogel.

[1084] The present invention also relates to the therapeutic use of the hydrogel according to the invention as a therapeutic implant for administering an API to a mammal.

[1085] The present invention also relates to a method for preparing a hydrogel containing biological cells, comprising the following steps:

[1086] a) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-,

[1087] b) Preparation of precursor L(-) i of sterile solution,

[1088] c) preparing a suspension of biological cells,

[1089] d) mixing the biological cell suspension obtained from step c) with the solution obtained from step b) or a),

[1090] e) adding the sterile solution obtained from step a) or b) not used in step d) to the solution obtained from step d),

[1091] f) the addition of step e) is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1092] g) performing cross-linking and gelation reactions at room temperature (20° C. to 25° C.),

[1093] h) performing demoulding and swelling to obtain a hydrogel containing biological cells.

[1094] The present invention also relates to a method for preparing a hydrogel containing biological cells, comprising the following steps:

[1095] 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,

[1096] b) Preparation of precursor L(-) i of sterile solution,

[1097] c) preparing a suspension of biological cells,

[1098] d) mixing the biological cell suspension obtained from step c) with the solution obtained from step b) or a),

[1099] e) adding the sterile solution obtained from step a) or b) not used in step d) to the solution obtained from step d),

[1100] f) the addition of step e) is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1101] g) performing cross-linking and gelation reactions at room temperature (20° C. to 25° C.),

[1102] h) performing demoulding and swelling to obtain a hydrogel containing biological cells.

[1103] The present invention also relates to a method for preparing a hydrogel containing biological cells, comprising the following steps:

[1104] a) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-,

[1105] b) preparing a precursor L(-) selected from the group consisting of i Sterile solution of: mercaptan polyethylene glycol, mercapto poly (ethylene oxide), pentaerythritol poly (ethylene oxide) azide or pentaerythritol poly (dibenzocyclooctyne) polyoxyethylene,

[1106] c) preparing a sterile solution of sodium hyaluronate,

[1107] d) preparing a sterile suspension of biological cells,

[1108] e) mixing the sodium hyaluronate solution obtained from step c) with the precursor solution of step b),

[1109] f) mixing the biological cell suspension obtained from step d) with the solution obtained from step e) or from step a),

[1110] g) mixing the solution obtained from step f) with the solution obtained from step e),

[1111] h) adding the sterile solution obtained from step a) or e) not used in step g) to the solution obtained from step f),

[1112] i) the addition of step g) is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1113] j) performing cross-linking and gelation reactions at room temperature (20° C. to 25° C.),

[1114] k) performing demoulding and swelling to obtain a hydrogel containing biological cells.

[1115] In one embodiment, the mold is a ring net.

[1116] The ring is an outer ring and is composed of an upper part and a lower part that sandwich the net in the middle. The two parts of the ring and the net are glued together.

[1117] In one embodiment, the cross-linking and gelling reactions are carried out at room temperature (20°C to 25°C).

[1118] In one embodiment, the cross-linking and gelling reactions are carried out at a controlled temperature of 15°C to 37°C.

[1119] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[1120] In one embodiment, the hydrogel according to the invention is characterized in that it also comprises biological cells.

[1121] In one embodiment, the cell is a cell from human or animal origin.

[1122] In one embodiment, the cell is a cell line.

[1123] In one embodiment, the cells are of stem cell origin.

[1124] In one embodiment, the stem cells are selected from embryonic stem cells, induced pluripotent stem cells, or mesenchymal stem cells.

[1125] In one embodiment, the cell is a primary cell.

[1126] In one embodiment, the cell is a protein, hormone or peptide secreting cell.

[1127] In one embodiment, the cell is selected from the group consisting of:

[1128] -Insulin-secreting cells for diabetes treatment

[1129] - Factor VIII or Factor IX secreting cells for the treatment of hemophilia, and

[1130] - For β-glucocerebrosidase-secreting cells in Gaucher disease.

[1131] In one embodiment, the hydrogel according to the invention is characterized in that the insulin-secreting cells are selected from the group of pancreatic cells.

[1132] In one embodiment, the hydrogel according to the invention is characterized in that the insulin-secreting cells are islets of Langerhans.

[1133] In one embodiment, the hydrogel according to the invention is characterized in that the biological cells are pseudo-islets.

[1134] The present invention also relates to the therapeutic use of a hydrogel according to the invention for treating a disorder or disease in a mammal, wherein the disorder or disease is due to an endocrine deficiency or dysfunction of the pancreatic organ.

[1135] The present invention also relates to a hydrogel for use as a medicament.

[1136] The present invention also relates to hydrogels for use in treating diseases such as diabetes.

[1137] The invention also relates to an implantable device comprising at least one hydrogel according to the invention and obtained by a method according to the invention.

[1138] The invention also relates to an implant consisting of the hydrogel according to the invention.

[1139] The invention also relates to an implant comprising the hydrogel according to the invention.

[1140] The invention also relates to an implant comprising a hydrogel according to the invention and cells or pancreatic islets.

[1141] The present invention also relates to a kit comprising:

[1142] -Solution of the dextran polymer of formula VIII before the cross-linking reaction:

[1143]

[1144] in

[1145] If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to formula IV,

[1146] ·and

[1147] x is equal to 0 or 1,

[1148] 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,

[1149] If one of b' and c is not equal to 0, then -A' is A as defined above,

[1150] 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,

[1151] If c is not equal to 0, R'1 is R1 as defined above and G'1 is the precursor G1,

[1152] If c is equal to 0, then b is equal to 0, and R'1 is the precursor R1 before the cross-linking reaction,

[1153] - a solution of thiol polyethylene glycol, mercapto poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene,

[1154] -Biological cells.

[1155] The present invention also relates to a kit comprising:

[1156] -Solution of the dextran polymer of formula VIII before the cross-linking reaction:

[1157]

[1158] in

[1159] If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to formula IV,

[1160] ·and

[1161] x is equal to 0 or 1,

[1162] 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,

[1163] If one of b' and c is not equal to 0, then -A' is A as defined above,

[1164] 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,

[1165] If c is not equal to 0, R'1 is R1 as defined above and G'1 is the precursor G1,

[1166] If c is equal to 0, then b is equal to 0, and R'1 is the precursor R1 before the cross-linking reaction,

[1167] - a solution of thiol polyethylene glycol, mercapto poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene,

[1168] - Biological cells,

[1169] -Non-cross-linked sodium hyaluronate solution.

[1170] The present invention also relates to a kit comprising:

[1171] -Solution of the dextran polymer of formula VIII before the cross-linking reaction:

[1172]

[1173] in

[1174] If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to formula IV,

[1175] ·and

[1176] x is equal to 0 or 1,

[1177] 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,

[1178] If one of b' and c is not equal to 0, then -A' is A as defined above,

[1179] 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,

[1180] If c is not equal to 0, R'1 is R1 as defined above and G'1 is the precursor G1,

[1181] If c is equal to 0, then b is equal to 0, and R'1 is the precursor R1 before the cross-linking reaction,

[1182] - a solution of thiol polyethylene glycol, mercapto poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene,

[1183] -Biological cells.

[1184] In one embodiment, at least 50% of the surface of the hydrogel is in direct contact with the medium in which the hydrogel is embedded.

[1185] In one embodiment, at least 75% of the surface of the hydrogel is in direct contact with the medium in which the hydrogel is embedded.

[1186] In one embodiment, at least 90% of the surface of the hydrogel is in direct contact with the medium in which the hydrogel is embedded.

[1187] In one embodiment, at least 95% of the surface of the hydrogel is in direct contact with the medium in which the hydrogel is embedded.

[1188] In one embodiment, 99% of the hydrogel surface is in direct contact with the medium in which the hydrogel is embedded.

[1189] In one embodiment, at least 50% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1190] By "in direct contact with the exterior" is meant that there is no separation between the hydrogel and the exterior, eg there is no wall made of non-hydrogel material between the hydrogel and the exterior of the device or implant.

[1191] In one embodiment, at least 75% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1192] In one embodiment, at least 90% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1193] In one embodiment, at least 95% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1194] In one embodiment, at least 99% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1195] In one embodiment, 100% of the hydrogel surface is in direct contact with the exterior of the device or implant.

[1196] Cells or API are embedded in the maze of cross-linked dextran hydrogel.

[1197] In this specification, the word "embedding" is equivalent to "encapsulating" or "encapsulating".

[1198] The hydrogel matrix allows the passage of small molecules such as nutrients and APIs, which are either entrapped in the hydrogel or secreted by the entrapped cells.

[1199] Typically, the API is a hormone and peptide drug selected from the group consisting of: PTH protein, insulin, and coagulation factors.

[1200] In one embodiment, the pore size of the matrix is ​​immunoisolating and blocks T lymphocytes so as to preserve the cells.

[1201] In one embodiment, the mesh size is less than 1 μm.

[1202] In another embodiment, it is less than 100 nanometers, preferably less than 10 nanometers, and more preferably about 5 nanometers.

[1203] In one embodiment, the present invention relates to an implant comprising a ring, a mesh, a hydrogel according to the present invention and cells.

[1204] In one embodiment, the thickness of the implant is less than 3 000 μm.

[1205] In one embodiment, the thickness of the implant is less than 2000 μm.

[1206] In one embodiment, the thickness of the implant is less than 1000 μm.

[1207] In one embodiment, the thickness of the implant is less than 900 μm.

[1208] In one embodiment, the thickness of the implant is greater than 300 μm.

[1209] In one embodiment, the thickness of the implant is greater than 400 μm.

[1210] In one embodiment, the thickness of the implant is greater than 500 μm.

[1211] In one embodiment, the total surface area of ​​the implant is 10 cm 2 Up to 200cm 2 .

[1212] In one embodiment, the total surface area of ​​the implant is 15 cm 2 Up to 100cm 2 .

[1213] In one embodiment, the implant comprises 0.5 to 20 ml of hydrogel.

[1214] In one embodiment, the implant comprises 0.75 to 10 ml of hydrogel.

[1215] In one embodiment, the implant comprises 0.8 to 5 ml of hydrogel.

[1216] In one embodiment, the ring and the implant are parallelepiped rectangles, in particular parallelepiped rectangles with rounded corners.

[1217] The ring and mesh structure makes the hydrogel easy to handle and has good resistance to manipulation, including for implantation. This is true even for hydrogels with larger mesh sizes (e.g., -W- with lower DS and lower concentration of reactive groups during cross-linking).

[1218] The ring and mesh structure makes the hydrogel easy to handle and has good resistance to manipulation, including for implantation. This is even for hydrogels with larger mesh sizes (such as -(A-f2) with lower DS during cross-linking). a -G1- and lower concentrations of reactive groups).

[1219] In one embodiment, the inner diameter of the ring is from 10 to 100 mm.

[1220] In one embodiment, the inner diameter of the ring is from 15 to 50 mm.

[1221] In one embodiment, the ring has a diameter of 0.5 to 5 mm.

[1222] In one embodiment, the ring has a diameter of 0.5 to 10 mm.

[1223] In one embodiment, the ring has a diameter of 0.5 to 5 mm.

[1224] In one embodiment, the total (lower part plus upper part and glue) thickness of the ring is 100 to 3000 μm.

[1225] In one embodiment, the total (lower part plus upper part and glue) thickness of the ring is 150 to 2000 μm.

[1226] In one embodiment, the total thickness of the ring is from 200 to 5000 μm.

[1227] In one embodiment, the total thickness of the ring is from 500 to 3000 μm.

[1228] In one embodiment, the ring has a rectangular, square or circular cross-section.

[1229] In one embodiment, the material of the ring is a bioinert material.

[1230] In one embodiment, the material of the ring is a biocompatible elastomer.

[1231] In one embodiment, the material of the ring is selected from silicone, in particular PDMS, polyurethane, polyether, polyether polyester copolymer and polypropylene oxide.

[1232] In one embodiment, the material of the ring is silicone.

[1233] In one embodiment, the material of the ring is PDMS.

[1234] In one embodiment, the net is non-biodegradable.

[1235] In one embodiment, the mesh is biocompatible.

[1236] In one embodiment, the mesh is non-absorbable.

[1237] In one embodiment, the mesh is a surgical mesh.

[1238] In one embodiment, the filament material in the material of the mesh is selected from polypropylene, polyethylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride) and ePVDF (elongated PVDF).

[1239] In a specific embodiment, the silk material in the material of the mesh is selected from PTFE, PVDF and ePVDF.

[1240] In one embodiment, the filament material in the mesh is selected from polypropylene, polyester, in particular PET, PTFE and PVDF (polyvinylidene fluoride).

[1241] In one embodiment, the filament material in the mesh is selected from polypropylene and polyester, in particular PET.

[1242] In one embodiment, the filament material in the mesh is selected from polypropylene.

[1243] In one embodiment, the filament material in the mesh is selected from polyester, in particular PET.

[1244] In one embodiment, the mesh has a thickness in the range of 50 to 500 μm.

[1245] In one embodiment, the thickness of the m-mesh ranges from 100 to 300 μm.

[1246] In one embodiment, the diameter of the filaments ranges from 0.08 to 0.2 mm.

[1247] In one embodiment, the mesh has a pore size ranging from 0.4 to 4 mm.

[1248] In one embodiment, the mesh has a pore size ranging from 0.6 to 2 mm.

[1249] In one embodiment, the mesh has pores with side dimensions ranging from 0.4 to 4 mm.

[1250] In one embodiment, the mesh has pores with side dimensions ranging from 0.6 to 3 mm.

[1251] In one embodiment, the fabric of the web is selected from the group consisting of knitted fabric, warp knitted fabric, woven fabric, nonwoven fabric.

[1252] In one embodiment, the fabric of the mesh is selected from warp knitted fabrics, in particular multifilament fabrics.

[1253] In one embodiment, the web is treated to increase hydrophilicity.

[1254] In one embodiment, the web is treated with a base, particularly for polyester, more particularly for PET.

[1255] In one embodiment, the treatment is the functionalization of the surface with reactive functional groups such as -OH, -COOH and reactive molecules or polymers.

[1256] The grafted polymer can thereby expose reactive functional groups, such as thiol functional groups, for further reaction with the hydrogel or hydrogel precursor.

[1257] In one embodiment, the treatment is performed by adsorption of a synthetic polymer (such as poloxamer or polyvinyl pyrrolidone (PVP)) or a natural polymer (such as collagen) or a surfactant after chemical or physical treatment.

[1258] In one embodiment, the mesh is retained beneath the outer ends of the loops.

[1259] In one embodiment, the mesh is not in contact with the exterior of the implant comprising the ring, mesh, and hydrogel.

[1260] In one embodiment, the glue is biocompatible.

[1261] In one embodiment, the glue is a biocompatible silicone glue, such as Silbione MED ADH 4200 supplied by Elkem.

[1262] In one embodiment, the glue remains under the outer end of the ring.

[1263] For example, SurgicalNet TM The provided warp knitted polyester surgical mesh type PETKM3002 (1×0.9 mm pore size) was treated in NaOH 1M at 70° C. for 5 hours and rinsed with deionized water and 96% ethanol. This treatment resulted in an increase in the hydrophilicity of the mesh, leading to improved wettability with aqueous solutions.

[1264] For example, the ring network structure can be obtained as follows:

[1265] - Using a stainless steel punch, cut a biocompatible PDMS sheet supplied by Grace Biolabs or Interstate Speciality Product into a square with a circular empty disk incorporated.

[1266] - Introduce the treated polyester surgical mesh part between two square PDMS sheets: glue the two PDMS sheets and the surgical mesh together with biocompatible silicone glue (Silbione MED ADH 4200, supplied by Elkem). Align the circular empty discs and keep the surgical mesh taut during gluing.

[1267] - The square construct was then cut with a stainless steel punch to obtain a final object consisting of two PDMS rings sandwiching the surgical mesh and glued together, and

[1268] - The sheets were washed with a 1% solution of Poloxamer F127 and rinsed with water before steam sterilization.

[1269] In one embodiment, the implant may be obtained by:

[1270] - Incorporating the hydrogel composition into the ring network construct. The concentrated polymer solution was mixed with a pipette and a controlled volume of the mixture was introduced into the ring network construct adhered to a glass slide,

[1271] - Cross-linking leading to gelation was performed. The ring network + hydrogel composition was then introduced into a solution of Tris 150 mM / NaCl 30 mM / Cysteine ​​10 mM at pH 8 or PBS at pH 7.4.

[1272] The hydrogels were rinsed with cysteine-free PBS solution and further immersed in the PBS solution overnight at 37° C. The hydrogel pieces were then stored in PBS solution at 4° C. until use.

[1273] The hydrogel / ring mesh implant can therefore be easily manipulated with forceps and can be folded for surgical implantation. Additionally, the ring can be secured with sutures.

[1274] The volume of the hydrogel can be adjusted by the inner diameter and thickness of the ring network structure. For the same ring network structure, the volume of the hydrogel can be adjusted to control the convexity / concavity of the hydrogel above the ring level.

[1275] In one embodiment, the hydrogel comprises a first layer of hydrogel that does not contain cells or islets and a second layer of hydrogel that contains cells or islets.

[1276] Such a structure can be obtained by the method disclosed in the present application, but with two steps of adding hydrogel precursors: the first step is to add a hydrogel precursor without cells or islets as a first layer, and the second step is to add a hydrogel precursor containing cells or islets as a second layer while the gelation of the first step is not completed, in particular at a time corresponding to 5% to 25% of the gelation time of the first hydrogel.

[1277] Figure 1 An implant (1) is shown comprising a hydrogel (11) containing cells or islets (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).

[1278] 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.

[1279] Figure 3 It is a top view of an implant (1) comprising a hydrogel (11) containing cells or islets (not shown), a ring (12) and a mesh (13).

[1280] Figure 4 An implant (1) is shown comprising a hydrogel (20) without cells sandwiching a hydrogel (21) containing cells. The upper portion of 20 is optional.

[1281] Figure 5Shown are the non-fasting blood glucose (in g / L) measured in control rats (rat 1, 2 and 3) and the rat (rat 4) that is implanted with C16B-18 or the rat (rat 338) that is implanted with C16B-3A / C16B-3B. Rat 4 has received 6200IEQ, and rat 338 has received 4 000IEQ.Vertical dotted lines represent the removal of implantation, rat 4 and rat 338 respectively.The grey area with dotted lines represents the blood glucose value (minimum, maximum, average) measured in animal before inducing diabetes with streptozotocin.The blood glucose value of>6g / L is less than (being measured as high by device) on glucometer, and is arbitrarily set to 6.5g / L.

[1282] The present invention relates to all embodiments listed below.

[1283] Cross-linked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are connected via at least one central linker group L(-) i Covalent cross-linking, the at least one group is a linear, branched or cyclic alkyl group of at least two valences containing at least a polyethylene glycol chain, or the at least one group is a linear, branched or cyclic alkyl group containing at least a polyethylene glycol chain. An at least divalent linear, branched or cyclic alkyl group of the oxazoline (POx) chain.

[1284] The problem is solved by providing novel cross-linked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are linked via at least one central linker group L(-) i Covalent cross-linking, the at least one group is an at least divalent linear, branched or cyclic alkyl group containing at least a polyethylene glycol chain.

[1285] The problem is solved by providing novel cross-linked dextran polymers with anionic groups, wherein at least two sugar units of dextran belonging to two different polymer chains are linked via at least one central linker group L(-) i Covalent cross-linking, the at least group is composed of at least poly ( An at least divalent linear, branched or cyclic alkyl group of the oxazoline (POx) chain.

[1286] In one embodiment, the cross-linked dextran polymer according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[1287] -L(-) i is a linear or branched polyether having heteroatoms (such as oxygen, nitrogen or sulfur) at its terminal ends,

[1288] -i is the valence of L and -(R1) m G1- the number of groups, and is an integer from 2 to 8 (2≤i≤8),

[1289] -m is an integer equal to 0 or 1,

[1290] -W is -(R1) m G1-group, wherein

[1291] -R1- is a linear or branched alkyl divalent radical containing 1 to 6 carbon atoms and optionally heteroatoms (such as oxygen, nitrogen or sulfur),

[1292] -G1- is a linear or branched or cyclic alkyl divalent radical containing 1 to 6 carbon atoms and which may contain heteroatoms such as oxygen, nitrogen or sulfur.

[1293] In one embodiment, the dextran polymer is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i The result is a radical derived from a linear or branched thiol polyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, wherein:

[1294] - Mn is 1000 to 25 000 g / mol (1000 ≤ Mn ≤ 25 000 g / mol) or

[1295] - The degree of polymerization (DP) is from 15 to 600 (15≤DP≤600).

[1296] In one embodiment, the dextran polymer is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i A radical not derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms, wherein:

[1297] - a number average molecular weight (Mn) of 500 to 40,000 g / mol (500 ≤ Mn ≤ 40,000 g / mol), or

[1298] - The degree of polymerization (DP) is from 8 to 1000 (8≤DP≤1000).

[1299] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1300] -L(-) iIs a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly ( oxazoline)

[1301] -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),

[1302] --W- is a group comprising at least a linear or branched alkyl group, and optionally comprising heteroatoms (such as oxygen, nitrogen or sulfur), an aromatic ring, a polyether or a poly( and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

[1303] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1304] -L(-) i It is a linear or branched polyether.

[1305] -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),

[1306] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1307] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bound to a dextran polymer backbone having i W groups, wherein:

[1308] -L is a straight chain or branched poly ( oxazoline),

[1309] -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),

[1310] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1311] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1312] -L(-) i Is a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly ( oxazoline)

[1313] -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),

[1314] --W- is a group comprising at least a linear or branched alkyl group, and optionally comprising heteroatoms (such as oxygen, nitrogen or sulfur), an aromatic ring, a polyether or a poly( and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

[1315] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1316] -L(-) i It is a linear or branched polyether.

[1317] -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),

[1318] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1319] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bound to a dextran polymer backbone having i W groups, wherein:

[1320] -L is a straight chain or branched poly ( oxazoline)

[1321] -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),

[1322] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1323] In one embodiment, the cross-linked dextran hydrogel according to the present invention is a dextran polymer wherein the central linker L(-) i is a linear or branched polyethylene glycol (PEG) group.

[1324] In one embodiment, the central linker L(-) i is a PEG selected from the group consisting of PEGs of Formula I:

[1325]

[1326] in:

[1327] i is an integer from 2 to 8 (2≤i≤8)

[1328] p is an integer equal to 0 or 1, and if i=2, then p=0

[1329] q is an integer from 8 to 1000 (8≤q≤1000)

[1330] r is an integer equal to 0 or 1

[1331] Q is a carbon atom, or a linear, branched or cyclic alkyl chain, or an aromatic group containing from 2 to 10 carbon atoms and which may contain heteroatoms such as nitrogen, oxygen or sulfur,

[1332] * 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 cross-linking process is performed by native chemical ligation (NCL)).

[1333] In one embodiment, the cross-linked dextran hydrogel according to the present invention is a dextran polymer wherein the central linker L is a linear or branched POx group.

[1334] In one embodiment, the POx central linker is a 2-arm POx selected from the linker of Formula XII.

[1335]

[1336] in:

[1337] The group -R is a straight chain -(CH2) n1-CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[1338] * indicates the site of f3, which is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1339] In one embodiment, the POx central linker is a 2-arm POx selected from the linker of formula XIIbis.

[1340]

[1341] in:

[1342] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[1343] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1344] In one embodiment, the POx central linker is a 4-arm POx selected from the linker of Formula XIII.

[1345]

[1346] in:

[1347] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[1348] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1349] In another embodiment, the POx central linker is a 4-arm POx selected from the linker of Formula XIV:

[1350]

[1351] in:

[1352] The group -R1 is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative.

[1353] The divalent group -R2- is a straight chain -(CH2) n2 -, wherein n2 is an integer from 2 to 6 (2≤n2≤6).

[1354] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1355] In another embodiment, the POx central linker is a 4-arm POx selected from a linker of formula XV:

[1356]

[1357] in:

[1358] The group -R is a straight chain -(CH2) n1 -CH3 (wherein n1 is an integer from 0 to 4 (0≤n1≤4)), a branched or cyclic alkyl derivative,

[1359] In one embodiment, R1 = -CH2-CH2-, and R2 is a linear -(CH2) n2 -, wherein n2 is an integer from 2 to 6 (2≤n2≤6),

[1360] In another embodiment, R2 = -CH2-CH2-, and R1 is a linear *-(CH2) n2 -*, wherein n2 is an integer from 2 to 6 (2≤n2≤6),

[1361] * indicates the site of f3, wherein f3 is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1362] In one embodiment, -W- is selected from a group of formula IV.

[1363]

[1364] in

[1365] * indicates the position of f1, and Indicates the attachment site to L.

[1366] a is an integer equal to 0 or 1.

[1367] b is an integer equal to 0 or 1.

[1368] c is an integer equal to 0 or 1.

[1369] In one embodiment, a=0 and f1 is an ether function or a carbamate function.

[1370] In one embodiment, a=1,

[1371] o The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative. It may also be branched with at least one hydroxyl group, such as -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.

[1372] or,

[1373] o The divalent group -A- is a linear polyether (PEG) derivative; f1 is an ether functional group or a carbamate functional group, and f2 is an amide functional group.

[1374] or,

[1375] o 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.

[1376] or,

[1377] o 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.

[1378] The divalent group -R1- is a linear, 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.

[1379] o If b=0, then f1 is an ether function, or a carbamate function.

[1380] o If b=1, 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 cross-linking process is carried out by native chemical ligation (NCL)).

[1381] The divalent group -G1- is a linear, branched or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, for example, 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 alkylsulfone derivative which may contain one heteroatom (e.g., oxygen or sulfur), or an ethylamide derivative, or a 1,4-triazole derivative, or a polycyclic derivative from a Diels-Alder reaction, or an aromatic phosphine derivative produced by Staudinger ligation, or a cysteine ​​derivative from natural chemical ligation.

[1382] o If c=0, f1 is an ether function or a carbamate function.

[1383] o If c=1, 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 cross-linking process is carried out by native chemical ligation (NCL)).

[1384] The cross-linked dextran polymer according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least the divalent group L(-) i covalently bound to a dextran polymer backbone having i groups, the dextran polymer being selected from dextran of formula X,

[1385]

[1386] in:

[1387] a is an integer equal to 0 or 1.

[1388] ·i is an integer from 2 to 8 (2≤i≤8).

[1389] ·L can be combined with the same [Dx-f1-(A-f2) a -G1-f3] group, or with different [Dx-f1-(A-f2) a -G1-f3] group is connected.

[1390] • Dx- is a dextran moiety which may be substituted with specific anionic groups in salt form and optionally with alkyl carboxylate derivatives in salt form.

[1391] f1 is an ether functional group.

[1392] The divalent group -A- is a straight chain -(CH2) n1-(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative. It may also be branched with at least one hydroxyl group, such as -CH2-CH(OH)-(CH2) where n2 is an integer from 1 to 5 (1≤n2≤5) n2 -.

[1393] f2 is an amide functional group.

[1394] The divalent group -G1- is a linear, branched or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, for example, up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms. In a preferred embodiment, -G1- is a succinimide derivative, or an alkylsulfone derivative which may contain one heteroatom (e.g., oxygen or sulfur), or a 1,4-triazole derivative.

[1395] The integer i is the valence of the central linker L and the same or different valence of the linker L [Dx-f1-(A-f2) a -G1-f3] groups.

[1396] f3 is an amine functional group, a thioether functional group, an ether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[1397] ·The central joint L is poly ( Oxazoline) (POx) derivatives, which may be linear or branched.

[1398] In this embodiment, the cross-linked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1399]

[1400] in

[1401] f1, f2, f3, f4, -A-, -R1-, -G1- are as defined in Formula IV above, and

[1402] • Dx- is a dextran moiety which may be substituted with specific anionic groups in salt form and optionally with alkyl carboxylate derivatives as defined above in salt form.

[1403] The integer i is the valence of the central linker L and the same or different valence of the linker L [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ]The number of groups.

[1404] • The central linker L is a polyether (PEG) derivative, which can be linear or branched.

[1405] In one embodiment, if b=0 and c=1, the central linker L can be poly( Oxazoline) (POx) derivatives, which may be linear or branched.

[1406] In this embodiment, the cross-linked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1407]

[1408] in

[1409] f1, f2, f3, f4, -A-, -R1-, -G1- are as defined in Formula IV above, and

[1410] • Dx- is a dextran moiety which may be substituted with specific anionic groups in salt form and optionally with alkyl carboxylate derivatives as defined above in salt form.

[1411] The integer i is the valence of the central linker L and the same or different valence of the linker L [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ]The number of groups.

[1412] • The central linker L is a polyether (PEG) derivative, which can be linear or branched.

[1413] The hydroxyl groups of the dextran polymer Dx- can be functionalized with at least one specific anionic group, such as an alkyl carboxylate, a sulfate anion, a sulfonate anion, a phosphate anion, or a phosphonate anion.

[1414] In one embodiment, the hydroxyl functional groups of the dextran polymer Dx- are functionalized with a specific anionic group, which is an alkyl carboxylate anion.

[1415] In one embodiment, the cross-linked dextran polymer carrying anionic groups according to the present invention is a dextran polymer wherein the dextran polymer backbone is according to formula III,

[1416] Where R is selected from

[1417] --H, anionic group of formula II, or with L(-) i -W- group of the cross-linking group,

[1418] -i is 20 to 5000 (20≤i≤5000),

[1419] --W- and L(-) i The groups have the meanings defined previously.

[1420] In one embodiment, the cross-linked dextran polymer carrying anionic groups according to the present invention is a dextran polymer wherein the dextran polymer backbone is according to formula XI,

[1421]

[1422] Where R is selected from

[1423] --H, anionic group of formula II, or with L(-) i Cross-linked -(A-f2) a -G1- group,

[1424] -I is 20 to 5000 (20≤I≤5000),

[1425] --(A-f2) a -G1- and L(-) i The groups have the meanings defined previously.

[1426] In one embodiment, the cross-linked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1427]

[1428] in:

[1429] i is an integer from 2 to 8 (2≤i≤8)

[1430] a=1,

[1431] b = 1,

[1432] c = 1,

[1433] Dx is a dextran derivative described in formula III,

[1434] L is the PEG central linker described in Formula I,

[1435] f1 is an ether functional group or a carbamate functional group,

[1436] The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative,

[1437] f2 is an amide functional group,

[1438] The divalent group -R1- is a linear -(CH2)n1- (wherein n1 is an integer from 1 to 7 (1≤n1≤7)), branched or cyclic alkyl derivative,

[1439] f3 is an amide functional group,

[1440] The divalent group -G1- is a 1,4-triazole derivative

[1441] • f4 is a carbon-nitrogen covalent bond, particularly wherein the nitrogen atom is within the triazole ring.

[1442] ·

[1443] In a preferred embodiment, the integer i is equal to 4, i=4.

[1444] According to the above embodiment, the triazole derivative comprises a cyclooctene derivative from a strained cyclooctyne, which may contain a heteroatom such as nitrogen, oxygen or sulfur and is optionally functionalized with a linear, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms, or with an aromatic derivative, or with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, especially fluorine.

[1445] According to the two above-mentioned embodiments, the triazole derivative comprises more than 10 and less than 30 carbon atoms, optionally it comprises 4 to 6 nitrogen atoms.

[1446] According to one embodiment, the 1,4-triazole derivative is obtained by a copper-free reaction.

[1447] In one embodiment, the cross-linked dextran polymer according to the present invention is selected from the dextran polymer of formula V.

[1448]

[1449] in:

[1450] i is an integer from 2 to 8 (2≤i≤8)

[1451] a=1,

[1452] b = 0,

[1453] c = 1,

[1454] Dx is a dextran derivative described in formula III,

[1455] L is the PEG central linker described in Formula I,

[1456] The divalent group -A- is a straight chain -(CH2) n1 -(wherein n1 is an integer from 1 to 7 (1≤n1≤7)), a branched or cyclic alkyl derivative,

[1457] f1 is an ether functional group or a carbamate functional group,

[1458] the divalent group -G1- is a 1,4-triazole derivative which is a cyclooctene derivative from a strained cyclooctyne, which cyclooctene derivative may contain one heteroatom, such as nitrogen, oxygen or sulfur, and is optionally functionalized with a linear, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms, or with an aromatic derivative, or with a heteroatom, such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine,

[1459] The 1,4-triazole derivative carries 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, 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 within the triazole ring.

[1460] In a preferred embodiment, the integer i is equal to 4, i=4.

[1461] According to one embodiment, the 1,4-triazole is a polycyclic radical comprising an acyl group which is linked via an amide function to a nitrogen which is within a ring but not from a triazole ring.

[1462] According to one embodiment, the 1,4-triazole comprises a cyclooctyne with nitrogen in the cyclooctyne ring.

[1463] According to one embodiment, the triazole derivative comprises more than 10 and less than 30 carbon atoms, optionally it comprises from 4 to 6 nitrogen atoms.

[1464] In one embodiment, the divalent group -G1- is a 1,4-triazole derivative described by the formula:

[1465]

[1466] Wherein: * indicates the position of f2, which is an amide functional group, and the dotted bond indicates f4, which is a carbon-nitrogen bond.

[1467] f4 is a carbon-nitrogen covalent bond, where the nitrogen atom is within the triazole ring.

[1468] According to one embodiment, triazole 1,4 is obtained by a copper-free reaction.

[1469] According to one embodiment, Dx is a dextran derivative described in formula III:

[1470]

[1471] Where R is selected from

[1472] --H, anionic group of formula II, or with L(-) i -W- group of the cross-linking group,

[1473] -i is 20 to 5000 (20≤i≤5000),

[1474] --W- and L(-) i The groups have the meanings defined previously.

[1475] According to one embodiment, L is a PEG central linker described in Formula I:

[1476]

[1477] in:

[1478] i is an integer equal to 4,

[1479] p is an integer equal to 1,

[1480] q is an integer from 8 to 1000 (8≤q≤1000)

[1481] r is an integer equal to 0 or 1

[1482] Q is a branched alkyl chain containing 2 to 10 carbon atoms,

[1483] * indicates the position of f4, which is a carbon-nitrogen covalent bond, wherein the nitrogen atom is within the triazole ring.

[1484] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 2, 4, or 8.

[1485] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 2, 4, or 8.

[1486] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 2.

[1487] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 2.

[1488] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 4.

[1489] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 4.

[1490] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i -W- groups, where i is 8.

[1491] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer with anionic groups of formula II, wherein the at least divalent group L(-) i Covalently bound to a dextran polymer backbone having i groups, where i is 8.

[1492] In one embodiment, the cross-linked dextran polymer according to the present invention is a dextran polymer wherein the dextran polymer backbone is dextran having a weight average molecular weight (Mw) of 5 to 1000 kDa before cross-linking and substitution.

[1493] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the cross-linked dextran polymer has L(-) i -W- group of cross-linking group or -(A-f2) a The degree of substitution (DS1) of the dextran backbone of -G1- groups is in the range of 0.001 to 0.4 (0.001≤DS1≤0.4).

[1494] In one embodiment, the cross-linked dextran polymer according to the present invention is a cross-linked dextran polymer wherein the degree of substitution (DS3) of sulfate, sulfonate, phosphate or phosphonate of the dextran backbone is in the range of 0.2 to 2.5 (0.2≤DS3≤2.5).

[1495] The present invention also relates to the dextran polymer of formula VIII prior to the cross-linking reaction.

[1496]

[1497] in

[1498] If none of a, a', b and b' is equal to 0, then f1, f2, f3, Dx are defined as above,

[1499] ·and

[1500] x is equal to 0 or 1,

[1501] 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,

[1502] If one of b' and c is not equal to 0, then -A'- is -A- as defined above,

[1503] 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,

[1504] If c is not equal to 0, -R'1- is -R1- as defined above, and -G'1- is the precursor -G1-,

[1505] If c is equal to 0, then b is equal to 0 and R'1 is the precursor R1 before the cross-linking reaction.

[1506] The present invention relates to a hydrogel comprising:

[1507] - a cross-linked dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[1508] -L(-) i Is a linear or branched polyether

[1509] -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),

[1510] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1511] In one embodiment, the hydrogel comprises non-cross-linked hyaluronate in solution.

[1512] In one embodiment, the present invention relates to a hydrogel comprising:

[1513] - Biological cells,

[1514] - non-cross-linked hyaluronate in solution, and

[1515] - a cross-linked dextran polymer Dx with anionic groups, wherein at least a divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[1516] -L(-) i Is a linear or branched polyether

[1517] -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),

[1518] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1519] In one embodiment, the cross-linked dextran polymer contained in the hydrogel according to the present invention is not a dextran polymer with carboxylic acid groups, wherein the at least divalent group L(-) i covalently bound to a dextran polymer backbone having i W groups, wherein

[1520] -L(-) i is a linear or branched polyether having heteroatoms (such as oxygen, nitrogen or sulfur) at its terminal ends,

[1521] -i is the valence of L and -(R1) m G1- the number of groups, and is an integer from 2 to 8 (2≤i≤8),

[1522] -m is an integer equal to 0 or 1,

[1523] -W is -(R1) m G1-group, wherein

[1524] -R1- is a linear or branched divalent alkyl group containing 1 to 6 carbon atoms and optionally heteroatoms (such as oxygen, nitrogen or sulfur),

[1525] -G1- is a linear or branched or cyclic alkyl divalent group containing 1 to 6 carbon atoms, and may contain heteroatoms such as oxygen, nitrogen or sulfur.

[1526] In one embodiment, the dextran polymer contained in the hydrogel is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i The product is a radical derived from a linear or branched thiol polyethylene glycol containing at least 2 sulfur atoms and up to 8 arms, wherein:

[1527] - Mn is 1000 to 25 000 g / mol (1000 ≤ Mn ≤ 25 000 g / mol) or

[1528] - The degree of polymerization (DP) is from 15 to 600 (15≤DP≤600).

[1529] In one embodiment, the dextran polymer contained in the hydrogel is not a dextran polymer in which the at least divalent group L(-) covalently bound to the dextran polymer backbone having i W groups ... i A radical not derived from a linear or branched mercaptopolyethylene glycol containing at least 2 sulfur atoms and containing up to 8 arms, wherein:

[1530] - a number average molecular weight (Mn) of 500 to 40 000 g / mol (500 ≤ Mn ≤ 40 000 g / mol), or

[1531] - The degree of polymerization (DP) is from 8 to 1000 (8≤DP≤1000).

[1532] The cross-linked dextran polymer contained in the hydrogel according to the present invention is a dextran polymer Dx with anionic groups, wherein at least the divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1533] -L(-) i Is a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly ( oxazoline),

[1534] -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),

[1535] --W- is a group comprising at least a linear or branched alkyl group, and optionally comprising heteroatoms (such as oxygen, nitrogen or sulfur), an aromatic ring, a polyether or a poly( and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

[1536] The cross-linked dextran polymer contained in the hydrogel according to the present invention is a dextran polymer Dx with anionic groups, wherein at least the divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1537] -L(-) i It is a linear or branched polyether.

[1538] -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),

[1539] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1540] The cross-linked dextran polymer contained in the hydrogel according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bound to a dextran polymer backbone having i W groups, wherein,

[1541] -L is a straight chain or branched poly ( oxazoline),

[1542] -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),

[1543] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1544] In one embodiment, the hydrogel comprises hyaluronic acid or the sodium salt or potassium salt of hyaluronic acid.

[1545] The cross-linked dextran polymer contained in the hydrogel according to the present invention is a dextran polymer Dx with anionic groups, wherein at least the divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1546] -L(-) i Is a linear or branched polyether, or L(-) i Is a straight chain or branched chain poly ( oxazoline)

[1547] -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),

[1548] --W- is a group comprising at least a linear or branched alkyl group, and optionally comprising heteroatoms (such as oxygen, nitrogen or sulfur), an aromatic ring, a polyether or a poly( and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

[1549] The cross-linked dextran polymer contained in the hydrogel according to the present invention is a dextran polymer Dx with anionic groups, wherein at least the divalent group L(-) i covalently bound to a dextran polymer backbone having i -W- groups, wherein

[1550] -L(-) i Is a linear or branched polyether

[1551] -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),

[1552] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1553] The cross-linked dextran polymer contained in the hydrogel according to the present invention is a dextran polymer Dx- with anionic groups, wherein at least a divalent group L is covalently bound to a dextran polymer backbone having i W groups, wherein,

[1554] -L is a linear or branched poly( oxazoline)

[1555] -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),

[1556] --W- is a group comprising at least a 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 α amino acid residues, in particular α amino acid residues linked by a peptide bond.

[1557] In one embodiment, the hydrogel according to the invention is characterized by a Tan δ of less than 1.

[1558] In one embodiment, the hydrogel is transparent.

[1559] In one embodiment, the hydrogel is translucent.

[1560] In one embodiment, the hydrogel according to the invention is characterized in that, after swelling in water, the concentration of the cross-linked dextran polymer is between 0.01 and 0.2 g / g.

[1561] In one embodiment, the hydrogel has a Young's modulus of 1 to 200 kPa.

[1562] In one embodiment, the hydrogel has a G' of 0.5 to 70 kPa.

[1563] In one embodiment, the hydrogel has a compression set at break greater than or equal to 10%.

[1564] In one embodiment, the hydrogel has a swelling ratio greater than 0.7.

[1565] In one embodiment, the water content of the hydrogel is at least 80 wt%.In one embodiment, the hydrogel according to the invention is characterized in that it further comprises biological cells.

[1566] In one embodiment, the cell is a protein, hormone or peptide secreting cell.

[1567] In one embodiment, the cell is selected from the group consisting of:

[1568] -Insulin-secreting cells for diabetes treatment

[1569] - Factor VIII- or Factor IX-secreting cells for the treatment of hemophilia and β-glucocerebrosidase-secreting cells for Gaucher disease.

[1570] In one embodiment, the hydrogel according to the invention is characterized in that the biological cells are pseudo-islets.

[1571] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention in the form of a hydrogel, which comprises the following steps:

[1572] g) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-,

[1573] h) Preparation of precursor L(-) i of sterile solution,

[1574] i) adding the sterile solution obtained from step b) to the solution obtained from step a),

[1575] j) Addition is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1576] k) crosslinking and gelling, for example at room temperature (20° C. to 25° C.) or at 37° C.,

[1577] l) Demolding and swelling are performed to obtain a hydrogel.

[1578] The present invention also relates to a method for synthesizing the cross-linked dextran polymer according to the present invention in the form of a hydrogel, which comprises the following steps:

[1579] g) 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,

[1580] h) Preparation of precursor L(-) i of sterile solution,

[1581] i) adding the sterile solution obtained from step b) to the solution obtained from step a),

[1582] j) Addition is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1583] k) crosslinking and gelling, for example at room temperature (20° C. to 25° C.) or at 37° C.,

[1584] l) Demolding and swelling are performed to obtain a hydrogel.

[1585] In one embodiment, steps c) and d) are performed simultaneously.

[1586] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[1587] Dextran with anionic groups of formula II is prepared by grafting or substituting on the hydroxyl groups carried by dextran. In one embodiment, dextran with anionic groups of formula II is prepared by grafting or substituting on the carboxymethyl groups carried by dextran carboxylic acid methyl ester / salt.

[1588] The cross-linking step is the gelling step leading to the formation of the hydrogel according to the invention.

[1589] The present invention also relates to a method for preparing a hydrogel containing biological cells, comprising the following steps:

[1590] i) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-,

[1591] j) Preparation of precursor L(-) i of sterile solution,

[1592] k) preparing a biological cell suspension,

[1593] 1) mixing the biological cell suspension obtained from step c) with the solution obtained from step b) or a),

[1594] m) adding the sterile solution obtained from step a) or b) not used in step d) to the solution obtained from step d),

[1595] n) the addition of step e) is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1596] o) performing cross-linking and gelation reactions at room temperature (20° C. to 25° C.),

[1597] p) performing demoulding and swelling to obtain a hydrogel containing biological cells.

[1598] The present invention also relates to a method for preparing a hydrogel containing biological cells, comprising the following steps:

[1599] i) 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,

[1600] j) Preparation of precursor L(-) i of sterile solution,

[1601] k) preparing a biological cell suspension,

[1602] 1) mixing the biological cell suspension obtained from step c) with the solution obtained from step b) or a),

[1603] m) adding the sterile solution obtained from step a) or b) not used in step d) to the solution obtained from step d),

[1604] n) the addition of step e) is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1605] o) performing cross-linking and gelation reactions at room temperature (20° C. to 25° C.),

[1606] p) performing demoulding and swelling to obtain a hydrogel containing biological cells.

[1607] The present invention also relates to a method for preparing a hydrogel containing biological cells, comprising the following steps:

[1608] 1) preparing a sterile solution comprising a dextran having an anionic group of formula II and at least two precursors -W-,

[1609] m) preparing a precursor L(-) selected from the group consisting of i Sterile solution of: mercaptan polyethylene glycol, mercapto poly (ethylene oxide), pentaerythritol poly (ethylene oxide) azide or pentaerythritol poly (dibenzocyclooctyne) polyoxyethylene,

[1610] n) preparing a sterile solution of sodium hyaluronate,

[1611] o) preparing a sterile suspension of biological cells,

[1612] p) mixing the sodium hyaluronate solution obtained from step c) with the precursor solution from step b),

[1613] q) mixing the biological cell suspension obtained from step d) with the solution obtained from step e) or from step a),

[1614] r) mixing the solution obtained from step f) with the solution obtained from step e),

[1615] s) adding the sterile solution obtained from step a) or e) not used in step g) to the solution obtained from step f),

[1616] t) the addition of step g) is carried out directly in the mold, or the solution is introduced into the mold after mixing,

[1617] u) performing cross-linking and gelation reactions at room temperature (20° C. to 25° C.),

[1618] v) performing demoulding and swelling to obtain a hydrogel containing biological cells.

[1619] According to one embodiment, during the molding step, the solution comprises an osmotic agent, which is a non-ionic osmotic agent, such as trehalose.

[1620] According to one embodiment, during the molding step, the solution comprises a weight ratio of nonionic osmotic agent to NaCl greater than 2, in particular greater than 5, more in particular greater than 10.

[1621] According to one embodiment, during the molding step, the solution comprises 5 to 50 mg / ml of a non-ionic osmotic agent, in particular trehalose.

[1622] In one embodiment, the die is an endless mesh.

[1623] In one embodiment, the cross-linking and gelling reactions are carried out at room temperature (20°C to 25°C).

[1624] The present invention also relates to a kit comprising:

[1625] - A solution of the dextran polymer of formula VIII before undergoing a cross-linking reaction:

[1626]

[1627] in

[1628] If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to formula IV,

[1629] ·and

[1630] x is equal to 0 or 1,

[1631] 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,

[1632] If one of b' and c is not equal to 0, then -A' is A as defined above,

[1633] If b', b and c are equal to 0, then a is equal to 0, and A' is the precursor A before undergoing the cross-linking reaction.

[1634] If c is not equal to 0, R'1 is R1 as defined above and G'1 is the precursor G1.

[1635] If c is equal to 0, then b is equal to 0 and R'1 is the precursor R1 before undergoing the cross-linking reaction.

[1636] - a solution of thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene.

[1637] -Biological cells.

[1638] The present invention also relates to a kit comprising:

[1639] - A solution of the dextran polymer of formula VIII before undergoing a cross-linking reaction:

[1640]

[1641] in

[1642] If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to formula IV,

[1643] ·and

[1644] x is equal to 0 or 1,

[1645] 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,

[1646] If one of b' and c is not equal to 0, then -A' is A as defined above,

[1647] If b', b and c are equal to 0, then a is equal to 0, and A' is the precursor A before undergoing the cross-linking reaction.

[1648] If c is not equal to 0, R'1 is R1 as defined above and G'1 is the precursor G1.

[1649] If c is equal to 0, then b is equal to 0 and R'1 is the precursor R1 before undergoing the cross-linking reaction.

[1650] - a solution of thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene.

[1651] -Biological cells.

[1652] -Non-cross-linked sodium hyaluronate solution.

[1653] The present invention also relates to a kit comprising:

[1654] - A solution of the dextran polymer of formula VIII before undergoing a cross-linking reaction:

[1655]

[1656] in

[1657] If a, a', b and b' are all equal to 0, then f1, f2, f3, f4, Dx are defined according to formula IV,

[1658] ·and

[1659] x is equal to 0 or 1,

[1660] 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,

[1661] If one of b' and c is not equal to 0, then -A' is A as defined above,

[1662] If b', b and c are equal to 0, then a is equal to 0, and A' is the precursor A before undergoing the cross-linking reaction.

[1663] If c is not equal to 0, R'1 is R1 as defined above and G'1 is the precursor G1.

[1664] If c is equal to 0, then b is equal to 0 and R'1 is the precursor R1 before undergoing the cross-linking reaction.

[1665] - a solution of thiol polyethylene glycol, thiol poly(ethylene oxide), pentaerythritol poly(ethylene oxide) azide or pentaerythritol poly(dibenzocyclooctyne)polyoxyethylene.

[1666] -Biological cells.

[1667] The present invention also relates to the use of the cross-linked dextran copolymer according to the present invention in the form of a hydrogel to prepare a cell composition.

[1668] 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 a mammal.

[1669] The present invention also relates to the therapeutic use of the hydrogel according to the invention for treating a disorder or disease in a mammal, wherein the disorder or disease is due to an endocrine deficiency or dysfunction of the pancreatic organ.

[1670] The present invention also relates to a hydrogel for use as a medicament.

[1671] The present invention also relates to hydrogels for use in treating diseases such as diabetes.

[1672] The present invention also relates to an implant comprising the hydrogel according to the invention.

[1673] In one embodiment, the present invention relates to an implant comprising a mesh, a ring, a hydrogel according to the present invention and cells.

[1674] The ring and mesh structure makes the hydrogel easy to handle and has good resistance to manipulation, including implantation. This is true even for hydrogels with larger mesh sizes (e.g., -W- with lower DS and lower concentration of reactive groups during cross-linking).

[1675] The ring and mesh structure makes the hydrogel easy to handle and has good resistance to manipulation including implantation. This is even for hydrogels with larger mesh sizes (e.g., -(A-f2) with lower DS during cross-linking). a The same is true for hydrogels with -G1- and lower concentrations of reactive groups.

[1676] The ring and mesh structures allow for easy manipulation of the hydrogel, providing good resistance to manipulation, including implantation, given that the hydrogel is very thin but has a relatively large planar surface.

[1677] In one embodiment, the implant is a parallelepiped rectangle with rounded corners.

[1678] In one embodiment, the thickness of the implant is less than 3 000 μm.

[1679] In one embodiment, the total surface area of ​​the implant is 10 cm 2 Up to 200cm 2 .

[1680] In one embodiment, the implant comprises 0.5 to 20 ml of hydrogel.

[1681] In one embodiment, the inner diameter of the ring is from 10 to 100 mm.

[1682] In one embodiment, the ring is a parallelepiped rectangle with rounded corners.

[1683] In one embodiment, the material of the ring is a bioinert material.

[1684] In one embodiment, the material of the ring is a biocompatible elastomer.

[1685] In one embodiment, the material of the ring is selected from silicone, in particular PDMS, polyurethane, polyether, polyether polyester copolymer and polypropylene oxide.

[1686] In one embodiment, the net is non-biodegradable.

[1687] In one embodiment, the mesh is biocompatible.

[1688] In one embodiment, the mesh is non-absorbable.

[1689] In one embodiment, the mesh is a surgical mesh.

[1690] In one embodiment, the filament material in the material of the mesh is selected from polypropylene, polyethylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride) and ePVDF (elongated PVDF).

[1691] In one embodiment, the mesh has a thickness in the range of 50 to 500 μm.

[1692] In one embodiment, the mesh has a pore size ranging from 0.4 to 4 mm.

[1693] In one embodiment, the mesh has pores with side dimensions ranging from 0.4 to 4 mm.

[1694] In one embodiment, the fabric of the web is selected from the group consisting of knitted fabric, warp knitted fabric, woven fabric, nonwoven fabric.

[1695] In one embodiment, the implant may be obtained by the following process:

[1696] - incorporating the hydrogel composition into a 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,

[1697] - cross-linking leading to gelation is performed, and then the ring network + hydrogel composition is introduced into a solution of Tris 150 mM / NaCl 30 mM / Cysteine ​​10 mM at pH 8 or into PBS at pH 7.4,

[1698] - The hydrogels were rinsed with cysteine-free PBS solution and further immersed in the PBS solution at 37°C overnight, and then the hydrogel pieces were stored in PBS solution at 4°C until use.

[1699] Hydrogels for Cell Therapy - F116 / F118 Case Studies Example

[1700] Section A - Chemistry

[1701] Example A1: Synthesis of substituted dextran

[1702] Table 1: List of synthesized polysaccharides

[1703]

[1704]

[1705]

[1706]

[1707]

[1708]

[1709]

[1710]

[1711] Polysaccharide 1-dextran carboxylic acid methyl ester / salt sulfate ester / salt and maleimide

[1712] Polysaccharide 1.1-dextran carboxylic acid methyl ester / salt

[1713] 50 g (0.31 mol glucoside units, 0.93 mol hydroxyl groups) of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol were dissolved in water (225 g / L) at 30°C, followed by addition of NaBH4 (2 x 58 mg, 2 x 1.54 mmol) 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. 10 N NaOH (103 mL, 1.03 mol) was then 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 through a PES membrane (MWCO 5 kDa) using a pH 7 phosphate buffer followed by water. The concentration of polysaccharide 1.1 in the final solution was determined by dry extraction and subsequent acid / base assay to determine the degree of substitution with the methyl carboxylate.

[1714] Based on dry extract: [Polysaccharide 1.1] = 46.3 mg / g.

[1715] According to the acid / base determination, the degree of substitution of the methyl carboxylate (DS2) = 0.8.

[1716] Polysaccharide 1,2-methylcarboxylate dextran

[1717] 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 units) was protonated with an acidified sulfonic acid resin (Purolite C100H, 2.0 eq / 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

[1718] 10.0 g (48.0 mmol glucoside units) 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. SO 3 DMF complex (58.8 g, 383.9 mmol) was quickly added and the reaction mixture was stirred at 30° C. for 3 hours. The mixture was neutralized by slowly adding 5% NaHCO 3 aqueous solution (800 mL) and purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using 30% v:v EtOH in NaCl (9 g / L in water), NaCl (9 g / L in water), and then water. The polysaccharide 1.3 concentration of the final solution was determined by dry extract, and the degree of substitution of sulfate was determined by liquid chromatography after complete sulfate hydrolysis of a representative sample.

[1719] Based on dry extract: [Polysaccharide 1.3] = 39.6 mg / g.

[1720] According to LC analysis, the degree of substitution of sulfate (DS3) = 1.5.

[1721] Polysaccharide 1-dextran carboxylic acid methyl ester / salt sulfate ester / salt and maleimide

[1722] 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 units) was added 2-hydroxypyridine 1-oxide (HOPO) (1.76 g, 15.82 mmol), and the mixture was cooled to 4° C. 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) were added to the solution, and the reaction mixture was stirred for 2 hours at 4° C. Two additional EDC additions (3.03 g, 15.82 mmol) were made every 2 hours. The mixture was diluted with pH 7 phosphate buffer and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using pH 7 phosphate buffer, NaCl (9 g / L in water) and then water. The concentration of polysaccharide 1 in the final solution was determined by dry extract and the degree of substitution of maleimide was determined by filtration in D2O. 1 The final solution was stored at -20 °C.

[1723] Based on dry extract: [Polysaccharide 1] = 22.2 mg / g.

[1724] according to 1H NMR (D2O), degree of substitution of maleimide (DS1) = 0.25.

[1725] Polysaccharide 2-dextran carboxylic acid methyl ester / salt sulfate ester / salt and maleimide

[1726] Polysaccharide 2.1-dextran carboxylic acid methyl ester / salt

[1727] Using a method similar to that used for the preparation of polysaccharide 1.1, starting from dextran with a weight-average molar mass of 40 kg / mol (0.25 mol of glucoside units, 0.75 mol of hydroxyl groups, degree of polymerization n=205), and at 60° C. with sodium chloroacetate (35.9 g, 0.31 mol) and 10 N NaOH (82 mL, 0.82 mol), polysaccharide 2.1 was obtained.

[1728] Based on dry extract: [Polysaccharide 2.1] = 44.2 mg / g.

[1729] According to the acid / base determination, the degree of substitution of the carboxylic acid methyl ester / salt (DS2) = 0.5.

[1730] Polysaccharide 2,2-methylcarboxylate dextran

[1731] Polysaccharide 2.2 was obtained using a method analogous to that used for the preparation of polysaccharide 1.2 starting from polysaccharide 2.1 (44.2 mg / g, DS2=0.5, 12.0 g, 59.4 mmol glucoside units).

[1732] Polysaccharide 2.3-dextran carboxylic acid methyl ester / salt sulfate / salt

[1733] Polysaccharide 2.3 was obtained using a method analogous to that used for the preparation of polysaccharide 1.3 starting from polysaccharide 2.2 (4.0 g, 20.9 mmol glucoside units).

[1734] Based on dry extract: [Polysaccharide 2.3] = 22.0 mg / g.

[1735] According to LC analysis, the degree of substitution of sulfate (DS3) = 1.9.

[1736] Polysaccharide 2-dextran carboxylic acid methyl ester / salt sulfate ester / salt and maleimide

[1737] Polysaccharide 2 was obtained using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 2.3 (22.0 mg / g, DS2=0.5, DS3=1.9, 4.6 g, 11.6 mmol glucoside units) and N-(2-aminoethyl)maleimide hydrochloride (615 mg, 3.48 mmol).

[1738] Based on dry extract: [Polysaccharide 2] = 12.3 mg / g.

[1739] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.23.

[1740] Polysaccharide 3-dextran carboxylic acid methyl ester / salt sulfate ester / salt and vinyl sulfone

[1741] To a 280 g solution of polysaccharide 1.3 (35.7 mg / g, DS3=1.5, DS2=0.8, 10.0 g, 26.37 mmol glucoside units) was added HOPO (1.46 g, 13.18 mmol), and the mixture was cooled to 4° C. 2-[[2-(vinylsulfonyl)ethyl]thio]ethanamine hydrochloride (VS) (1.83 g, 7.91 mmol) (synthesized according to: SA Stewart 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 EDC additions (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 on a PES membrane (MWCO 5 kDa) with 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 dry extract, and the degree of substitution of vinyl sulfone was determined by ultrafiltration in D2O. 1 The final solution was stored at -20 °C.

[1742] Based on dry extract: [Polysaccharide 3] = 21.8 mg / g.

[1743] according to 1 H NMR (D2O), degree of substitution of vinyl sulfone (DS1) = 0.28.

[1744] Polysaccharide 4-dextran carboxylic acid methyl ester / salt sulfonate / salt and maleimide

[1745] Polysaccharide 4.1-dextran carboxylic acid methyl ester / salt

[1746] 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 were dissolved in water (285 g / L) at 30° C., followed by the addition of NaBH 4 (74 mg, 1.95 mmol) 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. 10 N NaOH (200 mL, 2 mol) was then 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 through a PES membrane (MWCO 5 kDa) using a pH 7 phosphate buffer followed by water. The intermediate polysaccharide (polysaccharide 4.1.1) was then freeze-dried.

[1747] At 65° C., 90 g (0.35 mol glycoside units) of freeze-dried polysaccharide 4.1.1 were dissolved in water (260 g / L), followed by the addition of sodium chloroacetate (204 g, 1.75 mol) and the mixture maintained at 65° C. for 1 hour. 10 N NaOH (175 mL, 1.75 mol) was then slowly added over 1 hour, and the mixture was stirred at 65° C. for another hour. Another portion of sodium chloroacetate (122 g, 1.05 mol) was then added, and the mixture was maintained at 65° C. for 0.5 hour. 10 N NaOH (105 mL, 1.05 mol) was then 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 through a PES membrane (MWCO 5 kDa) using a pH 7 phosphate buffer, followed by water. The polysaccharide 4.1 concentration in the final solution was determined by dry extraction and subsequent acid / base assay to determine the degree of substitution with the carboxylic acid methyl ester.

[1748] Based on dry extract: [Polysaccharide 4.1] = 48.4 mg / g.

[1749] According to the acid / base determination, the degree of substitution of the carboxylic acid methyl ester / salt (DS2) = 2.1.

[1750] Polysaccharide 4.2-dextran carboxylic acid methyl ester / salt sulfonate / salt

[1751] 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) was added HOPO (9.77 g, 87.95 mmol), 3-amino-1-propanesulfonic acid (homotaurine) (9.79 g, 70.36 mmol), Et3N (9.81 mL, 70.36 mmol), and EDC (16.86 g, 87.95 mmol), and the reaction mixture was stirred at 25°C for 2 hours. Two additional EDC additions (16.86 g, 87.95 mmol) were made every 2 hours. The mixture was diluted with pH 7 phosphate buffer and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using pH 7 phosphate buffer, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 4.2 in the final solution was determined by dry extract and the degree of substitution of high taurine was determined by filtration in D2O. 1 The final solution was stored at 4 °C.

[1752] Based on dry extract: [Polysaccharide 4.2] = 40.9 mg / g.

[1753] according to 1 H NMR (D2O), degree of substitution of homotaurine (DS3) = 1.15.

[1754] Polysaccharide 4-dextran carboxylic acid methyl ester / salt sulfonate / salt and maleimide

[1755] Polysaccharide 4 was obtained using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 4.2 (40.9 mg / g, DS2=2.1, DS3=1.15, 9.4 g, 20.0 mmol glucoside units) and N-(2-aminoethyl)maleimide hydrochloride (1.06 g, 6.0 mmol).

[1756] Based on dry extract: [Polysaccharide 4] = 19.9 mg / g.

[1757] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.25.

[1758] Polysaccharide 5-dextran carboxylic acid methyl ester / salt ammonium sulfonate and maleimide

[1759] Polysaccharide 5.1-Dextran Carboxylic Acid Methyl Ester / Ammonium Sulfonate

[1760] Polysaccharide 5.1 was obtained using a method similar to that used to prepare polysaccharide 4.2, starting from polysaccharide 4.1 (48.4 mg / g, DS2 = 2.1, 7.3 g, 22.11 mmol glucoside units) and HOPO (2.95 g, 26.53 mmol), 3-((2-aminoethyl)-dimethylammonium)propane-1-sulfonate / salt (SB) (7.51 g, 26.53 mmol) (synthesized according to: 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).

[1761] Based on dry extract: [Polysaccharide 5.1] = 34.6 mg / g.

[1762] according to 1 H NMR (D2O), degree of substitution (DS3) of SB = 0.7.

[1763] Polysaccharide 5-dextran carboxylic acid methyl ester / salt ammonium sulfonate and maleimide

[1764] Polysaccharide 5 was obtained using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 5.1 (34.6 mg / g, DS2=2.1, DS3=0.7, 7.5 g, 16.7 mmol glucoside units) and N-(2-aminoethyl)maleimide hydrochloride (885 mg, 5.01 mmol).

[1765] Based on dry extract: [Polysaccharide 5] = 15.0 mg / g.

[1766] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.22.

[1767] Polysaccharide 6-dextran carboxylic acid methyl ester / salt and cyclooctyne (DBCO)

[1768] To a 30 g solution of polysaccharide 4.1 (48.4 mg / g, DS2 = 2.1, 1.45 g, 4.39 mmol glucoside units) was added HOPO (244 mg, 2.20 mmol) and DMF (25 mL), and the mixture was cooled to 4° C. To this solution was added 3-amino-1-(11,12-didehydrodibenzo[b,f]azo-5(6H)-yl)-1-propanone (DBCO-NH2) (365 mg, 1.32 mmol) and EDC (422 mg, 2.20 mmol) in DMF (5 mL), and the reaction mixture was stirred at 4° C. to 25° C. for 2 hours. Two additional EDC additions (422 mg, 2.20 mmol) were made every 2 hours. The mixture was diluted with pH 7 phosphate buffer and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) with pH 7 phosphate buffer, NaCl (9 g / L in water) and then water. The concentration of polysaccharide 6 in the final solution was determined by dry extract and the degree of substitution of DBCO-NH2 was determined by filtration in D2O. 1 The final solution was stored at -20 °C.

[1769] Based on dry extract: [Polysaccharide 6] = 6.1 mg / g.

[1770] according to 1 H NMR (D2O), degree of substitution (DS1) of DBCO-NH2 = 0.28.

[1771] Polysaccharide 7-dextran carboxylic acid methyl ester / salt and azide

[1772] Using a method similar to the last step for the preparation of polysaccharide 1 (step of polysaccharide 1.3 to polysaccharide 1), starting from polysaccharide 4.1 (48.4 mg / g, DS2=2.1, 6.2 g, 18.8 mmol glucoside units) and using 11-azido-3,6,9-trioxaundecane-1-amine (N3-PEG3-NH2) (1.23 g, 5.64 mmol), polysaccharide 7 was obtained.

[1773] Based on dry extract: [Polysaccharide 7] = 24.6 mg / g.

[1774] according to 1 H NMR (D2O), degree of substitution (DS1) of N3-PEG3-NH2 = 0.28.

[1775] Polysaccharide 8-dextran carboxylic acid methyl ester / salt phosphonate / salt and maleimide

[1776] Polysaccharide 8.1-Dextran carboxylic acid methyl ester / salt phosphonate / salt

[1777] Polysaccharide 8.1 was obtained using a procedure analogous to that used to prepare polysaccharide 4.2, starting from polysaccharide 4.1 (41.3 mg / g, DS2 = 2.1, 4.13 g, 12.51 mmol glucoside units) and 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).

[1778] Based on dry extract: [Polysaccharide 8.1] = 16.0 mg / g.

[1779] according to 1 H NMR (D2O), degree of substitution of 3-aminopropylphosphonic acid (DS3) = 0.05.

[1780] Polysaccharide 8-dextran carboxylic acid methyl ester / salt phosphonate / salt and maleimide

[1781] Polysaccharide 8 was obtained using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 8.1 (16.0 mg / g, DS2=2.1, DS3=0.05, 2.64 g, 7.83 mmol glucoside units) and N-(2-aminoethyl)maleimide hydrochloride (415 mg, 2.35 mmol).

[1782] Based on dry extract: [Polysaccharide 8] = 8.0 mg / g.

[1783] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.25.

[1784] Polysaccharide 9-dextran carboxylic acid methyl ester / salt phosphonate / salt and maleimide

[1785] Polysaccharide 9.1-Dextran carboxylic acid methyl ester / salt phosphonate / salt

[1786] A method similar to that used to prepare polysaccharide 4.2 was used, starting from polysaccharide 4.1 (41.3 mg / g, DS2=2.1, 4.13 g, 12.51 mmol glucoside units) and using 3-aminopropylphosphonic acid (2.09 g, 15.01 mmol) to obtain a solution. The pH of the 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 on a PES membrane (MWCO 5 kDa) with NaCl (9 g / L in water) and then water.

[1787] Based on dry extract: [Polysaccharide 9.1] = 13.3 mg / g.

[1788] according to 1 H NMR (D2O), degree of substitution of 3-aminopropylphosphonic acid (DS3) = 0.75.

[1789] Polysaccharide 9-dextran carboxylic acid methyl ester / salt phosphonate and maleimide

[1790] Polysaccharide 9 was obtained using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 9.1 (13.3 mg / g, DS2=2.1, DS3=0.75, 3.32 g, 7.59 mmol glucoside units) and N-(2-aminoethyl)maleimide hydrochloride (402 mg, 2.28 mmol).

[1791] Based on dry extract: [Polysaccharide 9] = 11.5 mg / g.

[1792] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.21.

[1793] Polysaccharide 10-Dextran Carboxylic Acid Methyl Ester / Salt Sulfate Ester / Salt and Cyclooctyne (DBCO)

[1794] Using a method similar to that used to prepare polysaccharide 6, starting from polysaccharide 1.3 (33.3 mg / g, DS2=0.8, DS3=1.5, 10.0 g, 26.37 mmol glucoside units) and using DBCO-NH2 (2.19 g, 7.91 mmol), polysaccharide 10 was obtained.

[1795] Based on dry extract: [Polysaccharide 10] = 23.3 mg / g.

[1796] according to 1 H NMR (D2O), degree of substitution (DS1) of DBCO-NH2 = 0.23.

[1797] Polysaccharide 11-dextran carboxylic acid methyl ester / salt and cyclooctyne (DBCO)

[1798] Polysaccharide 11.1-dextran carboxylic acid methyl ester / salt

[1799] Using a method analogous to that used for the preparation of polysaccharide 4.1, starting from dextran having a weight-average molar mass of 250 kg / mol, polysaccharide 11.1 was obtained.

[1800] Based on dry extract: [Polysaccharide 11.1] = 44.6 mg / g.

[1801] According to the acid / base determination, the degree of substitution of the methyl carboxylate (DS2) = 2.0.

[1802] Polysaccharide 11-dextran carboxylic acid methyl ester / salt and cyclooctyne (DBCO)

[1803] Using a method similar to that used to prepare polysaccharide 6, starting from polysaccharide 11.1 (44.6 mg / g, DS2=2.0, 15.6 g, 48.42 mmol glucoside units) and using DBCO-NH2 (803 mg, 2.91 mmol), polysaccharide 11 was obtained.

[1804] Based on dry extract: [Polysaccharide 11] = 20.4 mg / g.

[1805] According to 1H NMR (D 2 O), the degree of substitution (DS1) of DBCO-NH 2 = 0.06.

[1806] Polysaccharide 12-dextran carboxylic acid methyl ester / salt and cyclooctyne (DBCO)

[1807] Polysaccharide 12.1-dextran carboxylic acid methyl ester / salt

[1808] Using a method analogous to that used for the preparation of polysaccharide 4.1, starting from dextran having a weight-average molar mass of 500 kg / mol, polysaccharide 12.1 was obtained.

[1809] Based on dry extract: [Polysaccharide 12.1] = 45.3 mg / g.

[1810] According to the acid / base determination, the degree of substitution of the methyl carboxylate (DS2) = 2.0.

[1811] Polysaccharide 12-dextran carboxylic acid methyl ester / salt and cyclooctyne (DBCO)

[1812] Using a method similar to that used to prepare polysaccharide 6, starting from polysaccharide 12.1 (45.3 mg / g, DS2=2.0, 9.06 g, 28.12 mmol glucoside units) and using DBCO-NH2 (466 mg, 1.69 mmol), polysaccharide 12 was obtained.

[1813] Based on dry extract: [Polysaccharide 12] = 21.7 mg / g.

[1814] According to 1H NMR (D 2 O), the degree of substitution (DS1) of DBCO-NH 2 = 0.06.

[1815] Polysaccharide 14-dextran carboxylic acid methyl ester / salt and vinyl sulfone

[1816] 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 units) was rapidly added divinyl sulfone (38.8 mL, 387.3 mmol). After 4 minutes at room temperature, the reaction was stopped by adjusting the pH to 6 with 1 M HCl. The mixture was diluted with pH 7 phosphate buffer and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using pH 7 phosphate buffer, NaCl (9 g / L in water) and then water. The concentration of polysaccharide 14 in the final solution was determined by dry extracts, and the degree of substitution of vinyl sulfone was determined by filtration in D2O. 1 The final solution was stored at -20 °C.

[1817] Based on dry extract: [Polysaccharide 14] = 20.7 mg / g.

[1818] according to 1 H NMR (D2O), degree of substitution of vinyl sulfone (DS1) = 0.17.

[1819] Polysaccharide 15-dextran carboxylic acid methyl ester / salt and triazole-PEG-azide

[1820] Polysaccharide 15.1-dextran carboxylic acid methyl ester / salt and propargyl

[1821] 20 g (123.4 mmol glucoside units, 370.1 mmol hydroxyl groups) of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molar mass of 40 kg / mol were dissolved in water (300 g / L) at 30 ° C. NaBH4 (2 x 23 mg, 2 x 0.6 mmol) was then added every 30 minutes, and the mixture was stirred at 30 ° C for 1 hour. The mixture was cooled at 10 ° C., and KOH (5.54 g, 98.7 mmol) and benzyltriethylammonium chloride (1.69 g, 7.4 mmol) were added in one portion. A solution of propargyl bromide (5.87 g, 49.3 mmol) in toluene (43% w:w) was then slowly added over 30 minutes, and the mixture was stirred at 10 ° C for 20 minutes and at 25 ° C for 18 hours. The mixture was heated at 60 ° C., and sodium chloroacetate (71.8 g, 616.8 mmol) was added in one portion. After 1 hour, 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 in one go. After 1 hour, 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. pH 7 phosphate buffer (750 mL) and EtOH (575 mL) were added, the mixture was filtered and subsequently purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using pH 7 phosphate buffer, NaCl (9 g / L in water) and then water. The polysaccharide 15.1 concentration in the final solution was determined by dry extract and the degree of substitution of propargyl groups was determined by filtration in D2O. 1 H NMR was used to determine the degree of substitution of the methyl carboxylate.

[1822] Based on dry extract: [Polysaccharide 15.1] = 40.9 mg / g.

[1823] according to 1 H NMR (D2O), degree of propargyl substitution = 0.24.

[1824] According to the acid / base determination, the degree of substitution of the carboxylic acid methyl ester / salt (DS2) = 1.8.

[1825] Polysaccharide 15-dextran carboxylic acid methyl ester / salt and triazole-PEG-azide

[1826] 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 units) were added 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-diazide-3,6,9,12,15-pentaoxoheptadecane (4.03 g, 12.1 mmol) in succession. The reaction mixture was stirred at room temperature for 18 hours, diluted with pH 7 phosphate buffer, and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using pH 7 phosphate buffer, NaCl (9 g / L in water), and then water. The concentration of polysaccharide 15 in the final solution was determined by dry extract and the degree of azide substitution was determined by filtration in D2O. 1 The final solution was stored at -20 °C.

[1827] Based on dry extract: [Polysaccharide 15] = 7.0 mg / g.

[1828] according to 1 H NMR (D2O), degree of substitution of azide (DS1) = 0.24.

[1829] Polysaccharide 17-dextran carboxylic acid methyl ester / salt and methyl furan

[1830] Using a method similar to the last step for the preparation of polysaccharide 1 (polysaccharide 1.3 to polysaccharide 1 step), starting from polysaccharide 4.1 (50.8 mg / g, DS2=2.1, 17.8 g, 53.9 mmol glucoside units) and with 5-methylfurfurylamine (1.80 g, 16.17 mmol), polysaccharide 17 was obtained.

[1831] Based on dry extract: [Polysaccharide 17] = 30.2 mg / g.

[1832] according to 1 H NMR (D2O), degree of substitution of furan (DS1) = 0.30.

[1833] Polysaccharide 18-dextran carboxylic acid methyl ester / salt and tetrazine

[1834] Using a method similar to that used to prepare polysaccharide 6, starting from polysaccharide 4.1 (50.8 mg / g, DS2=2.1, 5.08 g, 15.38 mmol glucoside units) and using (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (1.1 g, 4.62 mmol), polysaccharide 18 was obtained.

[1835] Based on dry extract: [Polysaccharide 18] = 11.7 mg / g.

[1836] according to 1 H NMR (D2O), degree of substitution of tetrazine (DS1) = 0.25.

[1837] Polysaccharide 24-dextran glycine carbamate and cyclooctyne (DBCO)

[1838] Polysaccharide 24.1-Dextran Glycine Carbamate

[1839] 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 NaBH 4 (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 pH 7 phosphate buffer, cooled to room temperature, and then purified by ultrafiltration on a PES membrane (MWCO 5 kDa) using pH 7 phosphate buffer and then water. The resulting solution was filtered and then freeze-dried to obtain the intermediate polysaccharide 24.1.1.

[1840] 25 g (154.2 mmol glucoside units, 462.6 mmol hydroxyl functional groups) of polysaccharide 24.1.1 were 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 isocyanatoacetate (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. 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 10N NaOH. After 2 hours, the mixture was neutralized with 6M HCl, filtered, and then purified by ultrafiltration on a PES membrane (MWCO 5kDa) with NaCl (9 g / L in water) and then water. The polysaccharide 24.1 concentration in the final solution was determined by dry extract and then subjected to acid / base assay to determine the degree of substitution of glycine carbamate.

[1841] Based on dry extract: [Polysaccharide 24.1] = 47.8 mg / g.

[1842] According to the acid / base assay, the degree of substitution (DS2) of glycine carbamate was 2.4.

[1843] Polysaccharide 24-dextran glycine carbamate and cyclooctyne (DBCO)

[1844] Using a method similar to that used to prepare polysaccharide 6, starting from polysaccharide 24.1 (47.8 mg / g, DS2=2.4, 5.98 g, 13.07 mmol glucoside units) and using DBCO-NH2 (1.08 g, 3.92 mmol), polysaccharide 24 was obtained.

[1845] Based on dry extract: [Polysaccharide 24] = 20.8 mg / g.

[1846] according to 1 H NMR (D2O), degree of substitution (DS1) of DBCO-NH2 = 0.28.

[1847] Polysaccharide 25-dextran glycine carbamate and maleimide

[1848] Using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 24.1 (47.8 mg / g, DS2=2.4, 5.98 g, 13.07 mmol glucoside units) and using N-(2-aminoethyl)maleimide hydrochloride (693 mg, 2.92 mmol), polysaccharide 25 was obtained.

[1849] Based on dry extract: [Polysaccharide 25] = 22.8 mg / g.

[1850] according to 1 H NMR (D2O), degree of substitution of maleimide (DS1) = 0.26.

[1851] Example A2: Polyethylene glycol derivatives comprising at least two reactive functional groups

[1852] Commercial polyethylene glycol (PEG) derivatives functionalized with reactive functional groups were purchased. Reactive functional groups included thiol ("PEG-SH"), azide (PEG-N3), and alkyne (PEG-DBCO). Linear homobifunctional and multi-arm homobifunctional with different molecular weights and carrying different functional groups were used and are shown in Table 2 below.

[1853] Table 2: List of commercial PEG derivatives used

[1854]

[1855]

[1856] These PEG derivatives correspond to the precursors of formula I -L

[1857] Section B - Biology

[1858] Example B1A: Preparation of pseudo-islets

[1859] Min-6 cell lines (Caltag Medsystems) were cultured in an incubator at 37°C and 5% CO2 in the culture medium shown in Table 3. Cells were detached using 0.05% trypsin / EDTA three times a week and subcultured by diluting 5-fold in the culture medium.

[1860] Table 3: Culture medium composition

[1861] Culture medium for Min-6 pseudoislets Dulbecco's modified Eagle's medium (DMEM) 10% FBS 1% penicillin / streptomycin 1 mM pyruvate 0.05 mM β-mercaptoethanol

[1862] Using the Min-6 cell line, pseudo-islets with an average diameter of 150 μm were formed by seeding 500 cells per microwell using a 400 μm micropore Eplasia plate (Corning) and incubating them for 3 days at 37°C and 5% CO. The pseudo-islets were then collected, concentrated by centrifugation, and finally suspended in 0.9% NaCl.

[1863] Example B2A: Isolation of primary human pancreatic islets

[1864] Pancreas was obtained from human brain-dead donors. Islets were generated according to the method described in the pancreas harvesting technique for islet isolation (Pattou et al., Anchir 2005). Briefly, the pancreas was separated from the tissue and perfused into Wirsung tubes for islet isolation with collagenase I and II ( The islets were digested with a mixture of 1% Fibroblast (Roche, France) to ensure the release of islets. The islets were then purified using density gradient centrifugation (EuroFicoll, Sigma-Aldrich). The purified islets were cultured in CMRL medium supplemented with 0.625% BSA and 1% penicillin / streptomycin at 37°C in a 5% CO2 atmosphere in a culture flask. The culture medium was changed every 2 to 3 days.

[1865] Example B2B: Isolation of Primary Rat Pancreatic Islets

[1866] Pancreatic islets were isolated from male Wistar or Lewis rats (approximate weight: 300 g) according to a method similar to that described in A Pratical Guide to Rodent Islet Isolation and Assessment, Carter et al. Biological Procedures Online 2009.

[1867] In brief, the pancreas was perfused by injecting collagenase through the common bile duct. After perfusion, the pancreas was removed and digested at 37°C for 10 minutes. The islets were then purified by density gradient centrifugation. The purified islets were cultured in non-adherent culture flasks in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 2g / L glucose and 1% penicillin / streptomycin, or in CMRL medium at 37°C and 5% CO2. The culture medium was replaced every 2 to 3 days.

[1868] Example 3A: Islet Equivalent Counts

[1869] In order to normalize the amount of islets or pseudo-islets used in each experiment, the islets or pseudo-islets were 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 the counting period, a multiplication factor was applied to each islet according to its size. This mathematical compensation for the different diameters of the islets allows normalization between preparations (see NIH CIT Consortium Chemistry Manufacturing Controls Monitoring Committee; Purified Human Islets: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium. Cell R.4 Repair Replace Regen Reprogram).

[1870] Two 50 μL samples from each islet or sham islet batch were counted on a slide with a 50 μm grid. Islets or sham islets were categorized by size according to Table 4A.

[1871] Table 4A: Multiplication factors of islet size used for islet equivalent determination

[1872] 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

[1873] Islet equivalent counts were determined by averaging the counting results from two independent samples.

[1874] Example B3B: Islet Equivalent Counts

[1875] 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 the count, a multiplication factor was applied to each islet according to its size. This mathematical compensation for the different diameters of the islets allows normalization between islet preparations (see NIH CIT Consortium Chemical Manufacturing Control Monitoring Committee; Purified Human Islets: Qualitative and Quantitative Evaluation of Islets Using Dithizone (DTZ): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium. CellR.4 Repair, Replace, Regen, Reprogram).

[1876] Two 50 μL samples from each islet batch were counted on a slide with a 50 μm grid. Islets were categorized by size according to Table 4B.

[1877] Table 4B: Multiplication factors of islet size used for islet equivalent determination

[1878] 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

[1879] Islet equivalent counts were determined by averaging the counting results from two independent samples.

[1880] Part C - Physical Chemistry

[1881] Example C1A: Preparation of a solution of concentrated polysaccharide functionalized with maleimide (Mal) groups

[1882] Prepare a concentrated polysaccharide solution by weighing the appropriate weight of sterile freeze-dried polysaccharide obtained according to Part A1 and adding the appropriate weight of sterile deionized water. Place the solution on an orbital shaker at 70 rpm overnight to completely dissolve. Adjust the pH of the solution to pH 4 by adding concentrated hydrochloric acid and then sterile filter (0.22 μm). The mass concentration (mg / g) of the polysaccharide solution is determined by dry extract. The volume concentration (mg / mL) of the polysaccharide solution is determined by density measurement, weighing 100 μL of solution three times. Frozen at -20°C until use.

[1883] Example C1B: Preparation of concentrated polysaccharide solutions functionalized with vinyl sulfone (VS), DBCO or azide groups

[1884] A concentrated polysaccharide solution was prepared by weighing the appropriate weight of sterile freeze-dried polysaccharide obtained according to Part A1 and adding the appropriate weight of sterile deionized water. The solution was placed on an orbital shaker at 70 rpm overnight to completely dissolve. 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 dry extract. The volume concentration (mg / mL) of the polysaccharide solution was determined by density measurement, and 100 μL of the solution was weighed three times. The solution was frozen at -20°C until use.

[1885] Example C2: Preparation of concentrated PEG derivative solution

[1886] A concentrated solution of PEG (from the list according to Table 2) was prepared by weighing the appropriate weight of PEG powder and adding the appropriate weight of sterile deionized water. The solution was placed on a roller shaker at 15 rpm for 2 hours before sterile filtration (0.22 μm) to allow complete dissolution.

[1887] The mass concentration (mg / g) of the solution was determined by dry extract. The volume concentration (mg / mL) of the PEG solution was determined by density measurement, and 100 μL of the solution was weighed three times. The sterile solution was frozen at -20°C until use.

[1888] Example C3: Preparation of concentrated sodium hyaluronate solution

[1889] A concentrated solution of sodium hyaluronate (Pharma Grade 150 supplied by Novamatrix) was prepared by weighing the appropriate weight of sodium hyaluronate powder and adding the appropriate weight of sterile deionized water. Alternatively, another hyaluronate salt (Pharma Grade 300 supplied by echelon biosens, HTL Biotechnologies) specifically for use with C4B-16 gels was used. The solution was placed on a roller shaker at 10 rpm overnight to completely dissolve before being sterile filtered (0.22 μm).

[1890] Example C3bis: Preparation of concentrated sodium hyaluronate solution

[1891] A concentrated solution of Trehalose Dihydrate D(+) (Sigma-Aldrich) was prepared by weighing an appropriate mass of Trehalose Dihydrate D(+) powder and adding an appropriate weight of sterile deionized water. The solution was heated at 45°C with mixing until completely dissolved and cooled to ambient temperature before sterile filtration (0.22 μm).

[1892] Example C3A: Hydrogel Preparation

[1893] The hydrogels were prepared under sterile conditions.

[1894] Concentrated sterile solutions of polysaccharides and PEG derivatives prepared according to Example C1A or C1B and Example C2, respectively, 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 at 4° C. Optionally, the concentrated polysaccharide solutions carrying VS or DBCO groups were supplemented with tris buffer at pH 7.4 or pH 8.

[1895] In a 2 mL Eppendorf, a concentrated solution of PEG was added to a concentrated solution of polysaccharide. The volume ratio of PEG solution to polysaccharide solution was 70:30 (%:%) or 80:20 (%:%). The solutions were mixed with 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.

[1896] Table 5A: Molded hydrogel geometry conditions.

[1897]

[1898] The cross-linking process leading to gelation was carried out for 1 hour at room temperature (20-25° C.) or at 37° C. The hydrogel was demolded and introduced into a Tris 150 mM / NaCl 30 mM / cysteine ​​10 mM solution (2 mL) or PBS, pH 7.4, at 37° C. for 1 hour.

[1899] The hydrogels were rinsed with 20 mL of cysteine-free PBS solution and further immersed in 10 mL of the PBS solution overnight at 37° C. The hydrogel pieces were then stored in 10 mL of PBS solution at 4° C. until use.

[1900] Example C3B: Hydrogel Preparation

[1901] The hydrogels were prepared under sterile conditions.

[1902] Concentrated sterile solutions of polysaccharides and PEG derivatives prepared according to Example C1A or C1B and Example C2, respectively, 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 at 4°C. Alternatively, a combination of NaCl and a nonionic agent (e.g., trehalose) can be used to adjust the osmotic pressure, particularly in Example C4B24. Optionally, the concentrated polysaccharide solution bearing VS or DBCO groups was supplemented with a tris buffer at pH 7.4 or pH 8.

[1903] A concentrated solution of PEG / hyaluronate was prepared by mixing the concentrated solution of PEG prepared according to Example C2 with the concentrated solution of sodium hyaluronate prepared according to Example C3.

[1904] A polysaccharide concentrated solution supplemented with pluronic F127 (Sigma-Aldrich) was prepared by mixing the polysaccharide concentrated solution prepared according to Example C1B.

[1905] In 2mL Eppendorf, PEG or PEG / hyaluronate concentrated solution is added to polysaccharide or polysaccharide / poloxamer (particularly polysaccharide / poloxamer F127) concentrated solution.When hyaluronate is added to PEG solution, the volume ratio of PEG solution to polysaccharide solution is 70:30 (%:%) or 80:20 (%:%).Solution is mixed with pipette, and the mixture of controlled volume is introduced into the circular silicone isolator that adheres to slide glass.Prepare different molded hydrogel geometries.

[1906] The addition of a poloxamer-type nonionic surfactant can help improve the wettability of the composition.

[1907] Table 5B: Molded hydrogel geometry conditions.

[1908]

[1909] Crosslinking leading to gelation was performed at room temperature (20-25° C.) or for 1 hour at 37° C. The hydrogel was demolded and introduced into a solution of Tris 150 mM, NaCl 30 mM, cysteine ​​10 mM, pH 8, or PBS, pH 7.4, for 1 hour at 37° C.

[1910] The hydrogels were rinsed with cysteine-free PBS solution and further immersed in the PBS solution overnight at 37° C. The hydrogel pieces were then stored in PBS solution at 4° C. until use.

[1911] Example C4A: Hydrogel Composition

[1912] Different hydrogel compositions were prepared according to the protocol described in Example C3A and are shown in Table 6A. The concentrations of the two reactive groups (maleimide (Mal) or vinyl sulfone (VS) for reaction with thiol (SH) or alkyne (DBCO) for reaction with azide (N3)) and polymers (polysaccharide and PEG derivative) correspond to the final concentrations when the polymer solutions are mixed.

[1913] Table 6A: Compositions of various hydrogels made from polysaccharides and PEG derivatives.

[1914]

[1915]

[1916]

[1917] A solid disc-shaped hydrogel mass was obtained. The hydrogel mass was easily demolded and handled with tweezers for characterization.

[1918] Table 7A: Structure of hydrogels

[1919]

[1920]

[1921]

[1922]

[1923]

[1924]

[1925]

[1926]

[1927]

[1928]

[1929]

[1930]

[1931]

[1932]

[1933] Example C4B: Hydrogel Composition

[1934] Different hydrogel compositions were prepared according to the protocol described in Example C3B (Table 6B). The concentrations of the two reactive groups (maleimide (Mal) or ethyl sulfone (VS) for reaction with thiols (SH), or alkynes (DBCO) for reaction with azides (N3)) and 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, optionally incorporating sodium hyaluronate.

[1935]

[1936]

[1937] *HA 3,000 kDa, all other HAs are 1,500 kg / mol.

[1938] #Add Pluronic F127 (10mg / ml)

[1939] A solid disc-shaped hydrogel mass was obtained. The hydrogel mass was easily demolded and handled with tweezers for characterization.

[1940] Table 7B: Structure of hydrogels

[1941]

[1942]

[1943]

[1944]

[1945]

[1946] Example C5A: Hydrogel Rheological Characterization

[1947] Oscillatory shear tests were performed with a rotational rheometer (AR2000, TAinstrument) equipped with a cone-plate geometry. The cross-linking process leading to gelation was completed "in situ", which means that before starting the oscillation measurement, drops of polysaccharide and PEG concentrated solution were introduced between the cone and the plate and mixed by rotation of the geometry. Oscillatory time sweep 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 measurement (G', G") at 1600 seconds.

[1948] Table 8A: Results of hydrogel rheological characterization.

[1949]

[1950]

[1951]

[1952] The hydrogels exhibited low Tan δ values, meaning that G' was much higher than G", which is a typical property 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, page 97). An increase in the concentration of Mal:SH resulted in an increase in the elastic modulus G' value.

[1953] Increasing temperature and pH are two ways to accelerate the crosslinking process that leads to gelation of hydrogels prepared from polysaccharides with VS groups and PEG-SH. For example, proceeding from neutral pH to pH 8 and / or increasing the temperature from 25°C to 37°C results in faster gelation.

[1954] This can allow fine-tuning of the gelation rate, which can be convenient because fast gelation can be advantageous to avoid cell sedimentation, whereas slow gelation can be advantageous for casting the polymer mixture prior to gelation.

[1955] Example C5B: Hydrogel Rheological Characterization

[1956] Oscillatory shear tests were performed with a rotational rheometer equipped with a cone-plate geometry (AR2000, TA instrument). The cross-linking leading to gelation was done "in situ", meaning that before starting the oscillation measurement, drops of polysaccharide and PEG concentrated solutions were introduced between the cone and the plate and mixed by rotation of the geometry. Oscillatory time sweep tests were performed at 25°C or 37°C with a constant strain of 0.1% and a constant oscillation frequency of 1 Hz. The storage modulus G' (i.e. elastic modulus) and Tanδ (ratio G" / G') values ​​were reported as a function of time at 1600 seconds in the plateau region of the measurement (G", G').

[1957] Table 8B: Results of hydrogel rheological characterization.

[1958]

[1959] The hydrogels exhibited low Tan δ values, meaning that G' was much higher than G", which is a typical property 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, page 97).

[1960] An increase in the concentration of reactive groups leads to an increase in the elastic modulus G' value.

[1961] This can allow fine-tuning of the speed of cross-linking leading to gelation, which can be convenient because fast gelation can be advantageous to avoid cell sedimentation, whereas slow gelation can be advantageous for casting the polymer mixture prior to gelation.

[1962] In the presence of non-reactive sodium hyaluronate, the gelling properties are maintained.

[1963] This may allow further adjustment of the hydrogel composition to avoid cell sedimentation, even for slow-gelling compositions.

[1964] Example C6A: Hydrogel Swelling and Water Content

[1965] The hydrogel pieces were weighed immediately after demolding (w0) and 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 deduced from the mass of the hydrogel in the swollen state and the control of the concentration of the polymer precursors used to synthesize the hydrogel.

[1966] Table 9A: Hydrogel swelling and water content.

[1967]

[1968]

[1969] Hydrogels have a high water content, which varies depending on the structure and concentration of the polymer precursors.

[1970] Example C6B: Hydrogel Swelling and Water Content

[1971] The hydrogel pieces were weighed immediately after demolding (w0) and swelling in PBS solution overnight (w overnight). The swelling ratio was defined as the mass ratio overnight / w0. The water content of the hydrogel was determined by measuring the mass of the hydrogel in the swollen state and controlling the concentration of the polymer precursors used to synthesize the hydrogel.

[1972] Table 9B: Hydrogel swelling and water content.

[1973]

[1974] *HA 3,000 kg / mol, all other examples HA 1,500 kg / mol

[1975] Hydrogels contain a high water content.

[1976] Example C7A: Stability of hydrogel in physiological medium at 37°C

[1977] The hydrogel pieces were stored at 37°C in PBS pH 7.4 or serum (FBS fetal bovine serum) and weighed at different time periods.Disc-shaped hydrogels with a diameter of 9 mm and a thickness of 1.6 mm were tested.

[1978] Table 10A: Stability of hydrogels in physiological media

[1979]

[1980] The hydrogel was recovered intact and after storage in a physiological medium at 37°C, its mass did not change significantly. For example, in the case of a hydrolysis side reaction, the hydrogel would be expected to swell (increase in mass) or dissolve (decrease in mass) if the network structure changes. This indicates that the hydrogel is stable under physiological conditions.

[1981] Example C7B: Stability of hydrogel in physiological medium at 37°C

[1982] The hydrogel pieces were stored at 37°C in PBS pH 7.4 or serum (FBS fetal bovine serum) or 100 mM acetate buffer pH = 4. The pieces were weighed at different time periods. Disc-shaped hydrogels with a diameter of 9 mm and a thickness of 1.6 mm were tested.

[1983] Table 10B: Stability of hydrogels in physiological media

[1984]

[1985]

[1986] The hydrogel was recovered intact and after storage in a physiological medium at 37°C, its mass did not change significantly. For example, in the case of a hydrolysis side reaction, the hydrogel would be expected to swell (increase in mass) or dissolve (decrease in mass) if the network structure changes. This indicates that the hydrogel is stable under physiological conditions.

[1987] At acidic pH, fairly good gel stability was achieved, as no significant mass loss was reported.

[1988] Example C8A: Encapsulation of macromolecular probes within hydrogels

[1989] Commercially available fluorescent dextran-FITC 3 kDa and dextran-FITC 70 kDa were each dissolved in water to obtain concentrated stock solutions.

[1990] Concentrated sterile solutions of polysaccharides DMCMal and PEG-SH prepared according to Examples C1A and C2, respectively, were equilibrated at 4° C. For DMCVS-based hydrogels, concentrated solutions of polysaccharides DMCMal and PEG-SH prepared according to Examples C1B and C2, respectively, were equilibrated at 20-25° C.

[1991] The solution of polysaccharide DMCMal or DMCVS is mixed with fluorescent dextran. 100 μL of concentrated solution of PEG-SH is added to 100 μL of concentrated solution of polysaccharide DMCMal or DMCVS and fluorescent dextran. The solution is mixed with a pipette, and 200 μL of the latter mixture is introduced into a rectangular silicone mold (IBIDI 12×7.75 mm). For DMCMal-based hydrogels, the cross-linking process leading to gelation is carried out at room temperature (20°C to 25°C) for 1 hour, or for DMCVS-based hydrogels, the cross-linking process leading to gelation is carried out at 37°C for 1 hour.

[1992] The hydrogel pieces were demolded, introduced into wells (12-well multiwell plates), and soaked with 1.3 mL of Tris (200 mM) / NaCl (50 mM) buffer solution at pH 8 containing the same concentration of encapsulated fluorescent dextran. The hydrogels were swollen overnight at 37°C, and the supernatant was weighed to estimate the degree of swelling and the amount (mg) in the hydrogel volume.

[1993] Before the release experiment, the hydrogel was quickly washed twice with 1 mL of pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution.

[1994] Table 11A: Composition of hydrogels with encapsulated fluorescent macromolecular probes

[1995]

[1996] Example C8B: Encapsulation of macromolecular probes within hydrogels

[1997] Commercially available fluorescent dextran-FITC 3 kDa and dextran-FITC 70 kDa were each dissolved in water to obtain concentrated stock solutions.

[1998] Concentrated sterile solutions of polysaccharides and PEG or polysaccharides DMCMal and PEG-SH prepared according to Examples C1A, C1B, and C2, respectively, were equilibrated at 4° C. For DMCVS-based hydrogels, concentrated solutions of polysaccharides DMCMal and PEG-SH prepared according to Examples C1B and C2, respectively, were equilibrated at 20° C. to 25° C.

[1999] The solution of polysaccharide DMCMal or DMCVS or DMCDBCO is mixed with fluorescent dextran. 100 μL of concentrated solution of PEG-SH or PEG-N3 is added to 100 μL of concentrated solution of polysaccharide, polysaccharide DMCMal or DMCVS and fluorescent dextran. The solution is mixed with a pipette, and 200 μL of the latter mixture is introduced into a rectangular silicone mold (IBIDI 12×7.75 mm). For the hydrogel based on DMCMal and DMC-DBCO, the crosslinking that causes gelation is carried out at room temperature (20°C to 25°C) for 1 hour, or for the hydrogel based on DMCVS, the crosslinking that causes gelation is carried out at 37°C for 1 hour.

[2000] The hydrogel pieces were demolded, introduced into wells (12-well multiwell plates), and soaked with 1.3 mL of Tris (200 mM) / NaCl (50 mM) buffer solution at pH 8 containing the same concentration of encapsulated fluorescent dextran. The hydrogels were swollen overnight at 37°C, and the supernatant was weighed to estimate the degree of swelling and the amount (mg) in the hydrogel volume.

[2001] Prior to release experiments, the hydrogels were quickly washed twice with 1 mL of Tris (200 mM) / NaCl (50 mM) buffer solution at pH 8 (shown in Example C7B).

[2002] Table 11B: Composition of hydrogels with encapsulated fluorescent macromolecular probes

[2003]

[2004] Example C9A: Release of macromolecular probes within hydrogels

[2005] The hydrogel of the fluorescent probe with sealing produced in embodiment C8A is introduced into hole (12 hole multi-well plate) separately, and soaked with Tris (200mM) / NaCl (50mM) buffer solution of 2mL pH 8.The plate is covered with film, and put in the baking oven under (introduce) 37 ℃.At different time points, 200 μ L buffer solutions are sampled out, and replaced by fresh buffer solution.Calibration curve is used to determine the fluorescent probe concentration in the sample by fluorescence (fluorescence plate reader SAFAS).The cumulative fraction of the fluorescent probe discharged at each time point corresponds to the cumulative amount of the fluorescent probe discharged in swelling hydrogel and the ratio of the initial amount of fluorescent probe.

[2006] Table 12A: Cumulative fraction of macromolecular probes released from hydrogels at different time points.

[2007]

[2008] Increasing the size of the macromolecular probes resulted in slower release kinetics, demonstrating the selective permeability of the hydrogel network structure.

[2009] Example C9B: Release of macromolecular probes from hydrogels

[2010] The hydrogel of the fluorescent probe with sealing that produces among the embodiment C8B is introduced in the hole (12 hole multi-well plate) separately, and soaks with the buffer solution of Tris (200mM) / NaCl (50mM) of 2mL pH 8.The plate is covered with film, and put in the baking oven under 37 ℃.At different time points, 200 μ L buffer solutions are sampled out, and replaced by fresh buffer solution.Use calibration curve to determine the fluorescent probe concentration in the sample by fluorescence (fluorescence plate reader SAFAS).The cumulative fraction of the fluorescent probe that discharges at each time point corresponds to the cumulative amount of the fluorescent probe that discharges in swelling hydrogel and the ratio of the initial amount of fluorescent probe.

[2011] Table 12B: Cumulative fraction of macromolecular probes released from hydrogels at different time points.

[2012]

[2013] Increasing the size of the macromolecular probes resulted in slower release kinetics, demonstrating the selective permeability of the hydrogel network structure.

[2014] Example C10: Molding of thin hydrogel discs

[2015] The concentrated polymer solution was equilibrated at 4° C. according to the protocol described in Example C3A. The solution was mixed with a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator (10 mm diameter and 0.5 mm, 432 μm, or 356 μm thick) adhered to a glass slide. A second glass slide was placed over the drop to facilitate its spreading across the entire diameter of the mold.

[2016] After a cross-linking process leading to gelation at 20-25° C. for 1 hour, the hydrogel discs were demoulded. The thickness was deduced from the diameter and volume of the gel.

[2017] Table 13: Thickness of hydrogel compositions

[2018]

[2019] This method allows obtaining hydrogel discs of controlled diameter and thickness by adjusting the diameter of the mold and the volume of the hydrogel.

[2020] Example C11A: Determination of the mechanical resistance of hydrogels.

[2021] For compression, swollen rectangular hydrogel blocks as described in Example C3A were introduced into a flat glass crystallizer and immersed in PBS. Uniaxial compression was performed at 20°C to 25°C in 0.9% NaCl at a rate of 0.2 mm / min using a universal mechanical testing apparatus (Zwickroell) equipped with a flat compression plate. When the force began to increase, the initial thickness of the sample was determined from the contact of the plate with the hydrogel. The deformation was defined by the ratio of the compression displacement (mm) to the initial thickness (mm). The deformation at fracture was determined from the force / displacement curve. Fracture was defined when a decrease in force relative to displacement was observed.

[2022] 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 a speed of 3 mm / min in 0.9% NaCl at 20°C to 25°C using a universal mechanical testing apparatus (Zwickroell) equipped with screw clamps. The initial length of the sample was measured between the clamps with a ruler. The deformation was defined by the ratio of the traction displacement (mm) to the initial length (mm). The deformation at fracture was determined by the force / displacement curve. Fracture was defined when a decrease in force relative to displacement was observed. Young's modulus was determined by the slope of the true strain / deformation curve. The true strain (kPa) corresponds to the ratio of the pressure (N) to the instantaneous surface area (mm2) of the sample under compression.

[2023] Table 14A: Mechanical resistance of hydrogels

[2024]

[2025]

[2026] NM means not measured

[2027] The hydrogels according to the invention exhibit very good mechanical resistance characteristics combining deformability and stiffness suitable for surgical implantation.

[2028] Example C11B: Determination of the mechanical resistance of hydrogels.

[2029] For compression, swollen rectangular hydrogel blocks as described in Example C3B were introduced into a flat glass crystallizer and immersed in PBS. Uniaxial compression was performed at 20°C to 25°C in 0.9% NaCl at a rate of 0.2 mm / min using a universal mechanical testing apparatus (Zwickroell) equipped with a flat compression plate. When the force began to increase, the initial thickness of the sample was determined from the contact of the plate with the hydrogel. The deformation was defined by the ratio of the compression displacement (mm) to the initial thickness (mm). The deformation at fracture was determined from the force / displacement curve. Fracture was defined when a decrease in force relative to displacement was observed.

[2030] Young's modulus is determined from the slope of the true strain / deformation curve. True strain (kPa) corresponds to the ratio of pressure (N) to the instantaneous surface area of ​​the sample under compression (mm2).

[2031] Table 14B: Mechanical resistance of hydrogels

[2032]

[2033] The hydrogels according to the invention exhibit very good mechanical resistance characteristics combining deformability and stiffness suitable for surgical implantation.

[2034] Example C12: Surgical net

[2035] Example C12A: Alkali Treatment of Polyester Surgical Mesh

[2036] PETKM3002 warp-knitted polyester multifilament surgical mesh fabric (1×0.9 mm pore size) supplied by SurgicalMesh™ was treated in 1 M NaOH at 70°C for 5 hours and then rinsed with deionized water and 96% ethanol. This treatment increased the hydrophilicity of the mesh fabric, improving its wettability by aqueous solutions of the polymers that comprise the hydrogel.

[2037] Example C12B: PVDF surgical mesh fabric

[2038] Warp knitted PVDF surgical mesh fabric (1 x 1 mm pore size) supplied by Dynamesh is formed from knitted monofilaments having a diameter of 100 μm.

[2039] Example C12C: PTFE Perforated Surgical Membrane

[2040] A 150 μm thick PTFE surgical membrane supplied by Aran Biomedical was perforated in a star pattern (1×2 mm pore size).

[2041] Example C13: Fabrication of constructs

[2042] Example C13A: Fabrication of Ring Network Structure

[2043] Biocompatible PDMS sheets supplied by Grace Biolabs or Interstate Specialty Product or Limitless Shielding were cut using a stainless steel punch in square forms that incorporated into the circular empty disks.

[2044] A portion of the treated polyester surgical mesh or a portion of the surgical mesh described in Example C12 was introduced between two square PDMS sheets: the two PDMS sheets and the surgical mesh were glued together using biocompatible silicone glue (Silbione MED ADH 4200 supplied by Elkem). The circular empty disk was aligned and the surgical mesh was kept taut during gluing.

[2045] Finally, the square construct was cut with a stainless steel punch to obtain a final object consisting of two PDMS rings sandwiching the surgical mesh and glued together.

[2046] Prior to steam or ETO (ethylene oxide) sterilization, the sheets were washed with 1% poloxamer F127 solution and rinsed with water.

[2047] Example C13B: Fabrication of Proportional Rectangular Structures

[2048] For human surgery, to allow easier surgical implantation through a 15 mm internal diameter trocar, the implant may have an elongated form, such as a rectangle with a width of less than 100 mm.

[2049] Biocompatible PDMS sheets supplied by Grace Biolabs or Interstate Specialty Product or Limitless Shielding were cut into hollow frames in various forms, such as elongated rectangles.

[2050] A portion of the surgical mesh described in Example C12 was introduced between the frame PDMS sheets: two PDMS sheets and the surgical mesh were glued together using biocompatible silicone glue (Silbione MED ADH 4200 supplied by Elkem). The empty frame was aligned and the surgical mesh was kept taut during gluing.

[2051] Finally, the excess mesh around the frame was cut to obtain a final object consisting of two PDMS frames sandwiching the surgical mesh and glued together.

[2052] The excess mesh around the frame may be intentionally left uncut to allow for easier fixation to tissue via absorbable or non-absorbable tacks.

[2053] Prior to steam or ETO sterilization, the sheets were washed with 1% Poloxamer F127 solution and rinsed with water.

[2054] Example C14: Constructs of different sizes

[2055] Example C14A: Ring Network Structures of Different Sizes

[2056] According to Example C13A, different ring network constructs were generated by varying parameters such as the inner / outer diameter and thickness of the PDMS rings and the thickness of the glue.

[2057] Table 15A: Dimensions of ring network constructs.

[2058]

[2059] Example C14B: Proportional constructs of different sizes

[2060] According to Example C13B, different constructs were generated by varying parameters such as the inner / outer diameter and thickness of the PDMS ring and the thickness of the glue.

[2061] Table 15B: Dimensions of proportional constructs.

[2062]

[2063] Example C15: Hydrogel / Construct Composite Composition

[2064] Example C15A: Hydrogel / Ring Net Composite Composition

[2065] The hydrogel composition prepared according to Example C3B and described in Example C4B was incorporated into the ring network construct described in Example C 14. The concentrated polymer solution was mixed with a pipette, and a controlled volume of the mixture was introduced into the ring network construct adhered to a glass slide.

[2066] Crosslinking leading to gelation was performed for 1 hour at room temperature (20-25° C.) or at 37° C. The ring network / hydrogel composition was then introduced into a solution of Tris 150 mM / NaCl 30 mM / cysteine ​​10 mM at pH 8 or PBS at pH 7.4 at 37° C. for 1 hour.

[2067] The hydrogels were washed with cysteine-free PBS solution and further soaked in PBS solution overnight at 37° C. The hydrogel pieces were then stored in PBS solution at 4° C. until use.

[2068] Different hydrogel / ring network composite compositions were prepared (Table 16A):

[2069] Table 16A: Hydrogel / ring mesh or proportioned composite compositions.

[2070]

[2071] Therefore hydrogel / ring mesh composite material is easy to be handled with tweezers, and can be folded for the needs of surgical implantation, especially the needs of minimally invasive surgery.In addition, available sutures (sutures) ring is fixed to tissue.

[2072] The volume of the hydrogel can be adjusted by the inner diameter and thickness or 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.

[2073] Example C15B: Hydrogel / Proportional Composite Composition

[2074] To facilitate implantation, ensure the absence of filling defects, and limit islet sedimentation, scaled implants for human assays were fabricated with devices that allow for horizontal or vertical filling with hydrogel:

[2075] The device consists of glass plates with a silicone sheet of 50 to 2000 μm thickness fixed to one side. Two holes with a diameter of 1 to 6 mm are pierced through one of the glass plates and the corresponding sheet. A female Luer lock is glued in the middle of one of the holes. An empty implant is placed between the two sheets of laminated glass. To ensure the desired thickness and seal during hydrogel casting, this assembly is pressed against both sides of the silicone frame of the construct.

[2076] An appropriate volume of the hydrogel composition was injected into the proportioned constructs by two methods:

[2077] a. Reconstitution and injection: After mixing the gel precursor solution, place the gel solution in a syringe with a male Luer lock. Screw the syringe onto the Luer lock on the glass plate and then inject the gel into the implant.

[2078] b. Double syringe injection with mixing chamber (from Dual Syringe Biomaterial Delivery System (M-System): The hydrogel precursor solution is placed in two syringes. The coupling connecting the flow of the two syringes is extended through a mixing chamber with a male Luer lock screwed on the filling device. The clamp pushes both syringes simultaneously, reconstituting the hydrogel and immediately injecting it into the device.

[2079] Table 16B: Hydrogel / ring mesh or proportioned composite compositions.

[2080]

[2081] Example C16A: Encapsulation of Insulin-Producing Pancreatic Islets in Hydrogels

[2082] Islet encapsulation was performed in a sterile environment.

[2083] Concentrated sterile solutions of polysaccharides and PEG prepared according to Examples C1A or C1B and C2, respectively, were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg).The solutions were equilibrated at room temperature (20-25°C or at 4°C).

[2084] A concentrated mixture of PEG and islets was prepared by mixing equal volumes of an isotonic PEG solution (prepared according to Example C2) with an islet or pseudo-islet suspension (prepared according to Example B1A or B2A). The mixture containing PEG and islets was then gently mixed with an isotonic concentrated solution of polysaccharide at a 70:30 volume:volume ratio of islet suspension / PEG:polysaccharide.

[2085] The solution was gently mixed with a pipette and a controlled volume of the mixture was introduced into a round silicone isolator from Grace Biolab adhered to a glass slide.Different hydrogel sizes were prepared depending on the volume and the diameter of the silicone mold.

[2086] Table 17A: Molded Hydrogel Volumes and Dimensions

[2087]

[2088] The cross-linking process that causes gelation is carried out at room temperature (room temperature, RT, 20 ℃ to 25 ℃) or in a controlled temperature, for example, an oven at 20 ℃, 25 ℃ or 37 ℃ for 1 hour. The hydrogel that will be incorporated into the islets is demoulded and introduced into a Tris 150mM / NaCl 30mM / cysteine ​​10mM solution of pH 8 or in a culture medium for 15 minutes at room temperature. The hydrogel is then immersed in a culture medium containing 10mM cysteine ​​at 37 ℃ for 1 hour. After 1 hour, the culture medium containing cysteine ​​is removed and replaced with a culture medium. Before further in vitro testing or in vivo implantation, the sterile hydrogel containing the cells is stored at 37 ℃ and 5% CO 2 .

[2089] According to this method, different hydrogel compositions were prepared using pseudo-islets or primary islets.

[2090] Table 18A: Hydrogel compositions incorporating islets of varying properties at varying densities.

[2091]

[2092] RT means room temperature, 20℃ to 25℃

[2093] Example C16B: Encapsulation of insulin-producing pancreatic islets in hydrogels

[2094] Islet encapsulation was performed in a sterile environment.

[2095] Concentrated sterile solutions of polysaccharides and PEG prepared according to Examples C1A or C1B and C2, respectively, were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg).The solutions were equilibrated at room temperature (20-25°C or at 4°C).

[2096] A concentrated mixture of PEG and islets was prepared by mixing equal volumes of an isotonic PEG solution or a PEG / sodium hyaluronate solution (prepared according to Examples C2, C3, and C3B) with an 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 70:30 volume:volume ratio of islet suspension / PEG:polysaccharide or an 80:20 volume:volume ratio of islet suspension / PEG / hyaluronate:polysaccharide.

[2097] 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) attached to a glass slide or into a ring network construct. Different hydrogel sizes were prepared using different silicone molds (Table 5B) or ring network constructs (Table 16).

[2098] The cross-linking that causes gelation is carried out at room temperature (RT, 20 ℃ to 25 ℃) or in a controlled temperature such as 20 ℃, 25 ℃ or 37 ℃ oven for 1 hour.At room temperature, the hydrogel disc or hydrogel / ring network that is incorporated into pancreatic islets is introduced into Tris 150mM / NaCl 30mM / cysteine ​​10mM solution of pH 8 (Mal or VS:SH cross-linking) or in culture medium for 15 minutes (DBCO:N3 cross-linking).Then the hydrogel is immersed in the culture medium containing 10mM cysteine ​​at 37 ℃ for 1 hour or is immersed in the culture medium that does not contain cysteine.After 1 hour, the culture medium containing cysteine ​​is removed and replaced with culture medium.Before further in vitro testing or in vivo implantation, the sterile hydrogel containing cells is stored at 37 ℃ and 5% CO2.

[2099] Different hydrogel / islet compositions were prepared according to this method.

[2100] Table 18B: Hydrogel compositions incorporating islets of varying properties at varying densities.

[2101]

[2102]

[2103]

[2104]

[2105] *HA 3,000 kg / mol, all other examples HA 1,500 kg / mol

[2106] #10 mg / ml Pluronic F127 was added during the hydrogel preparation process

[2107] Example C17: Spatial uniformity of encapsulated cells within the thickness of the hydrogel

[2108] Hydrogel discs incorporating islets were prepared as described in Example C16B. The hydrogels were photographed from the side to assess the uniformity of the islets by measuring the thickness of the hydrogel. Uniformity was achieved when the islets were located throughout the thickness of the hydrogel and not primarily on one side of the hydrogel.

[2109] Table 17B: Homogeneity of islets in hydrogels

[2110]

[2111] The addition of sodium hyaluronate increased the viscosity of the mixture and allowed obtaining uniformity in the spatial positioning of the islets throughout the thickness of the hydrogel.

[2112] The use of longer hyaluronate chains allows for a higher level of homogeneity to be achieved.

[2113] Example C18: Evaluation and Control of Hydrogel Transparency

[2114] The hydrogel compositions described in Examples C4A and C4B and the other hydrogels disclosed in Example C were visually clear.

[2115] To quantify the transparency properties of the hydrogels, UV absorbance measurements were performed by casting 80 μL of the precursor solution in a 96-well plate (Spectrofluorimeter SAFAS). 100 μL of PBS was added on top of the gel to prevent drying. Absorbance measurements at 400 nm were used to quantify the turbidity level of the sample. Five measurements taken at different locations were averaged. Measurements were taken 3 hours after casting at full swelling equilibrium. The samples in the table below were also observed under standard conditions on a black panel ( Apollo II Liquid Detection Unit).

[2116] A hydrogel of composition C4B-13 was prepared similarly to Example C3B. Concentrated sterile solutions of polysaccharide / Pluronic DMCDBCO and PEG-N3 prepared according to Examples C1 and C2, respectively, were adjusted with concentrated NaCl solution to obtain an isotonic stock solution (300 mOsm / kg) and equilibrated at 4°C. A concentrated sterile solution of PEG-N3 prepared according to Example C2 was adjusted with a combination of trehalose and concentrated NaCl to obtain an isotonic stock solution (300 mOsm / kg) and equilibrated at 4°C.

[2117] Table 17C: Hydrogel Composition

[2118]

[2119] Table 17B: Visual Appearance and UV Absorbance of Hydrogel Compositions

[2120]

[2121] All gels are transparent under "normal" conditions (real life).

[2122] However, since changes in absorbance assess the variable homogeneity of the macroscopic network, we had to use harsh conditions as described above in order to grade the effect of osmolyte changes on transparency. These results show that in the presence of HA, the turbidity of the hydrogel decreased when the osmolyte NaCl was changed to trehalose.

[2123] Other non-ionic osmolytes such as mannitol or glycerol can also be used to improve clarity in the presence of HA.

[2124] Such a feature does not imply a change in the gel properties and no change in permeability. Therefore, the diffusion of small molecules such as insulin is maintained regardless of the degree of turbidity.

[2125] Example C19: Laparoscopic Implantation of Hydrogel / Proportional Composite

[2126] The implantability of the hydrogel / scaled composite was evaluated in pigs via laparoscopic surgery.

[2127] First, the two implants (C15B-1 and C15B-4) were each inserted into a surgical bag and subsequently rolled up for insertion into the peritoneal cavity through a 15 mm trocar. The implants were then removed from the bag and subsequently secured to the peritoneum (C15B-1) or to the level of the peritoneal tear (C15B-4) in contact with the muscle using stiches or non-absorbable surgical staples secured by a silicone frame.

[2128] The implant was intact / undamaged during multiple procedures and at the end of the implantation.

[2129] Section D - Biological Evaluation of Hydrogels

[2130] Example D1A: In vitro evaluation of hydrogel cytotoxicity profile by extract testing

[2131] An extraction method was used to evaluate the cytotoxicity profile of the hydrogels according to ISO 10993-5: Biological evaluation of medical devices recommendations.

[2132] Place the hydrogel 3 cm in a 24-well plate. 2 / ml of culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). It was incubated at 37 ° C and 5% CO2 under orbital stirring (70 rpm) for 24 hours to obtain hydrogel extraction medium. In parallel, 3T3 cells were plated in 96-well plates with 5000 cells / well in culture medium and incubated overnight at 37 ° C and 5% CO2. The next day, the culture medium of the cells was removed and replaced with hydrogel incubation medium. After incubating 3T3 cells with extraction medium at 37 ° C and 5% CO2 for 24 hours, viability was measured by quantifying intracellular ATP concentration using an ATPLite kit (Perkin Elmer) according to the manufacturer's instructions.

[2133] Calculate the percent viability using the following formula:

[2134]

[2135] The cytotoxicity of the hydrogel composition C4A-21 was evaluated by this extract test. The results are shown in Table 19 below. The viability percentage was compared to the untreated control. The standard deviation (SD) of the mean viability of the triplicate wells was calculated (n = 2 hydrogels, each extract was deposited in triplicate wells).

[2136] Table 19A: Viability of hydrogels by the extract test (ISO 10993-5).

[2137]

[2138] N = 2 independent experiments.

[2139] The selected compositions did not show any significant cytotoxicity compared to untreated cells (t-test, p=0.48) (see Table 19).

[2140] Example D1B: In vitro evaluation of the cytotoxicity profile of C16B-18, C16B-19, and C16B-B20 hydrogels in the presence or absence of human pancreatic islets by extract testing

[2141] Following ISO 10993-5: Biological evaluation of medical devices, an extraction method was used to evaluate the cytotoxicity profile of C16B-18, C16B-19, and C16B-20. C16B-18, C16B-19, and C16B-20 were tested in the presence or absence of 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.

[2142] C16B-18, C16B-19, and C16B-20 were placed in a 6-well plate at 6 cm in the presence or absence of islets. 2 / mL of culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). It was incubated at 37°C and 5% CO2 under orbital stirring (70 rpm) for 24 hours to obtain hydrogel extraction medium. In parallel, 3T3 cells were plated in 96-well plates with 5000 cells / well in culture medium and incubated overnight at 37°C and 5% CO2. The next day, the culture medium of the cells was removed and replaced with hydrogel incubation medium. After the 3T3 cells were incubated with extraction medium at 37°C and 5% CO2 for 24 hours, viability was measured by quantifying the intracellular ATP concentration using an ATPLite kit (Perkin Elmer) according to the manufacturer's instructions.

[2143] Calculate the percent viability using the following formula:

[2144]

[2145] The control was untreated 3T3 cells.

[2146] The cytotoxicity of C16B-18, C16B-19 and C16B-20 was evaluated by extract test. The results are shown in Table 19B. The values ​​were normalized relative to untreated 3T3.

[2147] Table 19B: Viability of 3T3 cells measured by the extract test (ISO 10993-5) after treatment with C16B-18, C16B-19 and C16B-20 in the presence or absence of pancreatic islet cells.

[2148]

[2149] Negative control showed a viability of 75.2 ± 24.8% (mean ± SD). SD means standard deviation. N = 2 independent experiments, n = 3 independent hydrogels / experiment.

[2150] The selected compositions did not show any effect on 3T3 cell viability in the presence or absence of encapsulated human islets (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-18 with islets, C16B-19, C16B-19 with islets, C16B-20, and C16B-20 with islets, respectively).

[2151] In conclusion, C16B-18, C16B-19, and C16B-20 were not associated with any cytotoxicity in the presence or absence of encapsulated human islets.

[2152] Example D1C: Short-term in vitro function of encapsulated rat primary pancreatic islets in Example C16B-11

[2153] Two days after isolation, rat primary islets were encapsulated at 40 kIEQ / mL as described in Example C16B-11 and maintained in culture medium in a 37° C. / 5% CO 2 incubator (see Example B2B).

[2154] The function of rat primary islets was evaluated 2 and 8 days after encapsulation. For the first time point, naked islets from the same islet preparation were evaluated in parallel. See Table 19C.

[2155] Table 19C: Function of naked or encapsulated rat primary islets from Example C16B-11.

[2156]

[2157] Error is standard deviation. NA not applicable. N = 1 islet preparation, n = number of replicates. Ex = x days after encapsulation

[2158] After encapsulation, the secretory index was maintained for more than 2 days, up to 8 days, and was no different from the control naked islets (one-way ANOVA, p value = 0.4699). In addition, compared with the control naked islets, the insulin secretion level was not reduced up to 8 days after encapsulation (one-way ANOVA, p value = 0.5632). In summary, these results indicate that the function of rat primary islets is maintained for at least 8 days after encapsulation in C16B-11.

[2159] Example D1D: Short-term in vitro function of encapsulated rat primary islets in Example C16B-13

[2160] To evaluate the in vitro function of naked or encapsulated rat primary islets, GSIS (Glucose Stimulated Insulin Secretion) experiments were performed as described in Example D2C.

[2161] Two days after isolation, rat primary islets were encapsulated at 20 kIEQ / mL as described in Example C16B-13 and maintained in culture medium in a 37°C / 5% CO2 incubator (see Example B2B). After 8 days of in vitro culture, their function was evaluated in parallel with denuded islets from the same islet preparation. See Table 19D.

[2162] Table 19D: Function of naked rat primary islets or encapsulated islets from Example C16B-13.

[2163]

[2164]

[2165] N = 1 islet preparation, n = 2 hydrogel / bare islet samples. Errors are standard deviations. Evaluations were performed 8 days after encapsulation.

[2166] After encapsulation, the secretory index remained above 2 for up to 8 days and was not different from control naked islets (t-test, p-value=0.07), indicating that rat primary islet function was maintained for up to 8 days after encapsulation in C16B-13.

[2167] Example D1D: Short-term in vitro function of rat primary islets encapsulated in Example C16B-16 a / quantification of total insulin content

[2168] After functional evaluation, exposed or encapsulated islets were lysed on ice in RIPA buffer for 10 minutes. The samples were then sonicated for 1 minute and stored at -80°C. A qualified ELISA assay specific for insulin was used to determine the insulin levels in these samples. Insulin levels were normalized to the IEQ content of the sample (see Example B3A). The encapsulation method in b / Example C16B-16 did not affect insulin synthesis or the islet function of rat primary islets.

[2169] Three days after isolation, rat primary pancreatic islets were encapsulated at 15 kIEQ / mL as described in Example C16B-16 and maintained in culture medium in a 37°C / 5% CO2 incubator as described in Example B2B. Their function was evaluated after 1, 2, and 8 days of in vitro culture (see Example D2C). In parallel, the samples were analyzed for total insulin content. See Table 19E.

[2170] Table 19E: Function of rat primary islets encapsulated in Example C16B-16.

[2171]

[2172] Error is standard deviation. N = 1 islet preparation, n = number of replicates. Ex = x days after encapsulation

[2173] Rat islets encapsulated in C16B-16 still contained insulin 8 days after encapsulation. Furthermore, they were able to secrete insulin in response to glucose, as indicated by a mean secretion index greater than 3.

[2174] Taken together, these results demonstrate that both insul...

Claims

1. A cross-linked dextran polymer with anionic groups, wherein at least two dextran sugar units belonging to two different polymer chains are covalently cross-linked via at least one central linker group L(-)i, wherein at least one of the groups is a linear, branched or cyclic alkyl radical containing at least two valencies of a polyethylene glycol chain, or wherein at least one of the groups is a linear, branched or cyclic alkyl radical containing at least two valencies of a polyethylene glycol chain. An at least divalent linear, branched or cyclic alkyl group of the oxazoline (POx) chain.

2. The cross-linked dextran polymer according to claim 1, wherein the cross-linked dextran polymer is selected from a dextran polymer Dx with anionic groups, wherein the at least 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, or L(-) i It 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 poly(alkylene) ring 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(alkylene) ring. The invention relates to a group of oxazoline (oxazoline) derivatives and does not comprise two or more α-amino acid residues, in particular α-amino acid residues linked by a peptide bond.

3. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the central linker L(-) i is a linear or branched polyethylene glycol (PEG) group.

4. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the central linker L(-) i is a PEG selected from the group consisting of PEGs of Formula I: in: i is an integer from 2 to 8 (2≤i≤8), p is an integer equal to 0 or 1, and if i=2, then p=0, q is an integer from 8 to 1000 (8≤q≤1000), r is an integer equal to 0 or 1, Q is a carbon atom, or a linear, branched or cyclic alkyl chain containing from 2 to 10 carbon atoms and which may contain heteroatoms such as nitrogen, oxygen or sulfur, or an aromatic radical, * 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 cross-linking process is performed by native chemical ligation (NCL).

5. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the central linker L is a linear or branched POx group.

6. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the POx central linker is a 2-arm POx selected from linkers of formula XII: in: The group -R is a straight chain -(CH2) n1 -CH3, branched or cyclic alkyl derivatives, wherein n1 is an integer from 0 to 4 (0≤n1≤4), * represents the site of f3, which is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

7. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the POx central linker is a 2-arm POx selected from linkers of formula XIIbis: in: The group -R is a straight chain -(CH2) n1 -CH3, branched or cyclic alkyl derivatives, wherein n1 is an integer from 0 to 4 (0≤n1≤4), * indicates the site of f3, which is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

8. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the POx central linker is a 4-arm POx selected from linkers of formula XIII, in: The group -R is a straight chain -(CH2) n1 -CH3, branched or cyclic alkyl derivatives, wherein n1 is an integer from 0 to 4 (0≤n1≤4), * represents the site of f3, which is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

9. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the POx central linker is a 4-arm POx selected from linkers of formula XIV: in: The group -R1 is a straight chain -(CH2) n1 -CH3, branched or cyclic alkyl derivatives, wherein n1 is an integer from 0 to 4 (0≤n1≤4), The divalent group -R2- is a straight chain -(CH2) n2 -, wherein n2 is an integer from 2 to 6 (2≤n2≤6), * indicates the site of f3, which is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

10. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the POx central linker is a 4-arm POx selected from linkers of formula XV: in: The group -R is a straight chain -(CH2) n1 -CH3, branched or cyclic alkyl derivatives, wherein n1 is an integer from 0 to 4 (0≤n1≤4), In one embodiment, R1 = -CH2-CH2-, and R2 is a linear -(CH2) n2 -, wherein n2 is an integer from 2 to 6 (2≤n2≤6), In another embodiment, R2=-CH2-CH2-, and R1 is a linear *-(CH2) n2 -*, wherein n2 is an integer from 2 to 6 (2≤n2≤6), * represents the site of f3, which is an amine functional group, an ether functional group, a thioether functional group, an amide functional group, a carbamate functional group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

11. The cross-linked dextran hydrogel according to one of the preceding claims, wherein -W- is selected from a group of formula IV: in * indicates the site of f1, and ° indicates the site of connection with L, a is an integer equal to 0 or 1, b is an integer equal to 0 or 1, c is an integer equal to 0 or 1, In one embodiment, a=0, f1 is an ether functional group or a carbamate functional group, In one embodiment, a=1, ○ The divalent group -A- is a straight chain -(CH2) n1 -, branched or cyclic alkyl derivatives, wherein n1 is an integer from 1 to 7 (1≤n1≤7); the divalent group -A- may also be branched by at least one hydroxyl group, -CH2-CH(OH)-(CH2) n2 -, wherein n2 is an integer from 1 to 5 (1≤n2≤5); f1 is an ether functional group or a carbamate functional group, and f2 is an amide functional group, or, o the divalent group -A- is a linear polyether (PEG) derivative; f1 is an ether functional group or a carbamate functional group, and f2 is an amide functional group, or, o 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, or, o 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, the divalent group -R1- is a linear, 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, ○ If b = 0, then f1 is an ether functional group or a carbamate functional group, o 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 cross-linking process is carried out by native chemical ligation (NCL), the divalent group -G1- is a linear, branched or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, for example: up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms or up to one phosphorus atom; 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 an ethylamide derivative, or a 1,4-triazole derivative, or a polycyclic derivative from a Diels-Alder reaction, or an aromatic phosphine derivative produced by a Staudinger ligation, or a cysteine ​​derivative from a natural chemical ligation, ○ If c = 0, then f1 is an ether functional group or a carbamate functional group, ○ If c=1, 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 cross-linking process is carried out by native chemical ligation (NCL).

12. The cross-linked dextran hydrogel according to claim 1, wherein the at least divalent group L(-) i covalently bonded to a dextran polymer backbone having i groups selected from a dextran of formula X, in: a is an integer equal to 0 or 1, i is an integer from 2 to 8 (2≤i≤8), ·L can be the same as [Dx-f1-(A-f2) a -G1-f3] group, or with different [Dx-f1-(A-f2) a -G1-f3] group connection, Dx- is a dextran moiety which may be substituted with a specific anionic group in salt form and optionally with an alkyl carboxylic acid derivative in salt form, f1 is an ether functional group, The divalent group -A- is a straight chain -(CH2) n1 -, branched or cyclic alkyl derivatives, wherein n1 is an integer from 1 to 7 (1≤n1≤7), the divalent group -A- may also be branched by at least one hydroxyl group, -CH2-CH(OH)-(CH2) n2 -, wherein n2 is an integer from 1 to 5 (1≤n2≤5), f2 is an amide functional group, The divalent group -G1- is a linear, branched or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms, for example: up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms; in a preferred embodiment, -G1- is a succinimide derivative, or an alkylsulfone derivative which may contain one heteroatom, such as oxygen or sulfur, or a 1,4-triazole derivative, The integer i is the valence of the central linker L, and the same or different [Dx-f1-(A-f2) a -G1-f3] groups, 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, ·The central joint L is poly( Oxazoline) (POx) derivatives, which may be linear or branched.

13. The cross-linked dextran hydrogel according to one of the preceding claims, wherein it is selected from the dextran polymers of formula V, in f1, f2, f3, f4, -A-, -R1-, -G1- are defined according to the above formula IV, and Dx- is a dextran moiety which, as defined above, may be substituted with a specific anionic group in salt form and optionally with an alkyl carboxylic acid derivative in salt form, The integer i is the valence of the central linker L, and the same or different [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ] the number of groups, The central linker L is a polyether (PEG) derivative, which can be linear or branched, In one embodiment, if b=0 and c=1, the central linker L can be poly( Oxazoline) (POx) derivatives, which may be linear or branched.

14. The cross-linked dextran hydrogel according to one of the preceding claims, wherein it is selected from the dextran polymers of formula V, in f1, f2, f3, f4, -A-, -R1-, -G1- are defined according to the above formula IV, and Dx- is a dextran moiety which, as defined above, may be substituted with a specific anionic group in salt form and optionally with an alkyl carboxylic acid derivative in salt form, The integer i is the valence of the central linker L, and the same or different [Dx-f1-(A-f2) a -(R1-f3) b -(G1-f4) c ] the number of groups, The central linker L is a polyether (PEG) derivative, which can be linear or branched.

15. The cross-linked dextran hydrogel according to claim 1, wherein the hydroxyl functional groups of the dextran polymer Dx- can be functionalized with at least one specific anionic group, such as: an alkyl carboxylate, a sulfate anion, or a sulfonate anion, or a phosphate anion, or a phosphonate anion.

16. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the cross-linked dextran polymer carrying anionic groups according to the invention is a dextran polymer wherein the dextran polymer backbone conforms to formula III, Where R is selected from --H, an anionic group of formula II, or a group with L(-) i -W- group of the cross-linking group, -i is 20 to 5000 (20≤i≤5000), --W- and L(-) i The radicals have the meanings defined above.

17. The cross-linked dextran hydrogel according to one of the preceding claims, wherein the cross-linked dextran polymer carrying anionic groups according to the invention is a dextran polymer wherein the dextran polymer backbone conforms to formula XI, Where R is selected from --H, an anionic group of formula II, or a group with L(-) i Cross-linked -(A-f2) a -G1- group, -I is 20 to 5000 (20≤I≤5000), --(A-f2) a -G1- and L(-) I The radicals have the meanings defined above.

18. The cross-linked dextran hydrogel according to one of the preceding claims, wherein it is selected from the dextran polymers of formula V, in: i is an integer from 2 to 8 (2≤i≤8), ·a＝1， ·b＝1， ·c＝1， Dx is a dextran derivative as described in formula III, L is the PEG central linker described in Formula I, f1 is an ether functional group or a carbamate functional group, The divalent group -A- is a straight chain -(CH2) n1 -, branched or cyclic alkyl derivatives, wherein n1 is an integer from 1 to 7 (1≤n1≤7), f2 is an amide functional group, The divalent group -R1- is a straight chain -(CH2) n1 -, branched or cyclic alkyl derivatives, wherein n1 is an integer from 1 to 7 (1≤n1≤7), f3 is an amide functional group, The divalent group -G1- is a 1,4-triazole derivative, f4 is a carbon-nitrogen covalent bond, particularly a carbon-nitrogen covalent bond wherein the nitrogen atom is within a triazole ring.

19. The cross-linked dextran hydrogel according to one of the preceding claims, wherein it is selected from the dextran polymers of formula V, in: i is an integer from 2 to 8 (2≤i≤8), ·a＝1， ·b＝0， ·c＝1， Dx is a dextran derivative as described in formula III, L is the PEG central linker described in Formula I, The divalent group -A- is a straight chain -(CH2) n1 -, branched or cyclic alkyl derivatives, wherein n1 is an integer from 1 to 7 (1≤n1≤7), f1 is an ether functional group or a carbamate functional group, the divalent group -G1- is a 1,4-triazole derivative which is a cyclooctenyl derivative from a strained cyclooctyne which may contain one heteroatom, such as nitrogen, oxygen or sulfur, and which is optionally functionalized with a linear, branched or cyclic alkyl derivative containing 2 to 20 carbon atoms, or with an aromatic derivative, or with a heteroatom such as nitrogen, oxygen or sulfur, or with a halogen, in particular with fluorine, The 1,4-triazole derivative carries nitrogen, which is covalently linked to -CORa- to form an amide functional group, Ra is an alkyl group containing 1 to 4 carbon atoms, and Ra is linked to f2 as an amide functional group, and f4 is a carbon-nitrogen covalent bond, wherein the nitrogen atom is within the triazole ring.

20. The dextran polymer of formula VIII before the cross-linking reaction, in If none of a, a', b and b' is equal to 0, then f1, f2, f3, Dx are defined as above, ·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.

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