Hydrogel for cell therapy

Abstract

The invention relates to hydrogels comprising the crosslinked dextran polymers of the invention and / or hydrogels further comprising biological cells. The invention also relates to therapeutic uses of the hydrogels of the invention for treating disorders or diseases in mammals, the disorders or diseases resulting from lack of or insufficient endocrine function of the pancreatic organ, and to implants comprising the hydrogels of the invention. The invention also relates to implants comprising the hydrogels of the invention and further comprising rings and nets.

Description

[Technical Field]

[0001] The field of the invention is therapy, particularly cell therapy. More particularly, the invention relates to an implant comprising a hydrogel incorporating: active ingredients such as peptides, hormones or proteins, or Secretory cells: Secretory cells may be cells that secrete peptides or hormones.

[0002] The goal is to prevent, treat or cure diseases. In particular, it may be possible to prevent and / or treat chronic diseases by completely or partially replacing the function of defective naturally occurring cells in a patient. The invention also relates to crosslinked polymers, precursors thereof, methods for obtaining crosslinked polymers, and methods for obtaining hydrogels, in particular cell-laden hydrogels.

[0003] The cells may be isolated or aggregated, and may be of one type or a variety of types.

[0004] Hydrogels can be used in a variety of systems, including: scaffolds as controlled drug or active pharmaceutical ingredient release systems, or A scaffold used as an implantable device containing cells.

[0005] Hydrogels contain or consist of polymers that are cross-linked in a three-dimensional network. They can be natural or synthetic, and can be homopolymers or copolymers. Hydrogels have the ability to absorb and retain large amounts of water. This is known as the swelling of the hydrogel.

[0006] A number of features must be achieved in order to obtain a system that is a potential implant capable of delivering an active ingredient for an extended period of time.

[0007] These features include the following: Low degradability, particularly low biodegradability, or no biodegradability, or good in vivo stability, so as to prevent the embedded cells from escaping and invading the patient's body or the host's cells from invading the graft; Good permselectivity, defined as the selective permeability to biological elements due to their size or molecular weight, which allows the passage of active ingredients, such as hormones, peptides or proteins, while isolating the incorporated cells completely or partially from the host's immune system, thereby reducing or even eliminating the immune response. Good alleviation of foreign body reactions or good biocompatibility, especially low cytotoxicity, and good local tolerance; The cells have a high survival rate, such as good vascularization near the cells and sufficient flow of nutrients to the cells. Cells can have good functionality within the hydrogel.

[0008] To be used as a controlled release system or as a scaffold for cells, hydrogels must have special characteristics so as to exhibit all or some of the desired properties mentioned above, as well as good mechanical and rheological properties.

[0009] Important rheological and mechanical properties for hydrogels include: Good uniformity; this is related to good transparency or translucency. Adequate resistance to compression or tension techniques and flexibility (especially when handling and implanting, e.g., in laparoscopic surgery) Defined mesh size to maximize oxygen and nutrient exchange, controlled transport properties, and permselectivity; Good in vivo stability; e.g., resistance to hydrolytic, enzymatic or oxidative degradation.

[0010] Parameters that provide an indication of the desired rheological and mechanical properties include: tan δ (called loss tangent); this gives an index to the mechanical properties. G': This gives an indication of the elastic modulus (stiffness) and mesh size. Compressive and / or tensile deformation at break; this provides an indication of the elasticity and resistance of the hydrogel. Swellability: This gives an indication of moisture content, dimensional and mechanical properties.

[0011] The problem to be solved is to obtain a hydrogel with properties that allow: manipulation to implant the hydrogel, such as by laparoscopy, without tearing; and / or The gel remains in place after implantation, eg, the hydrogel does not collapse and / or becomes immobilized relative to the tissue into which it is implanted.

[0012] A very difficult problem to solve is to obtain an implant that has a small thickness (to allow cells to be in close proximity to the tissue), a large surface area (to allow for a relatively large volume), excellent mechanical characteristics (to allow for minimally invasive surgery) and good biocompatibility.

[0013] Another problem to be addressed concerns the settling of cells or islets during crosslinking, which leads to gelation.

[0014] Prior art for the following hydrogels: 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, pp5580-5585, Hanwell Zhang et al.,''In situ gelable interpenetrating double network hydrogel formulation from binary components: thiolated chitosan and oxidized dextran'', Biomacromolecules, 2011, Vol.12, n°5, pp1428-1437, Rongsheng Zhang et al., ''A novel pH and ionic strength sensitive carboxymethyl dextran hydrogsel, Biomaterials, 2005, Vol.26, n°22, pp4677-4683, and Taichi Ito et al., "Dextran-based in situ cross-linked injectable hydrogels to prevent peritoneal adhesions," Biomaterials, 2007, Vol. 28, No. 23, pp. 3418-3426, discloses hydrogels that do not solve the technical problems that the hydrogels of the present invention solve.

[0015] In the prior art, cell-containing, crosslinked hydrogels are often intended to allow the growth of cellular objects, e.g., three-dimensional cell culture. This type of application requires that the hydrogel be able to embed the starting cells while simultaneously creating space for new cells acquired by growth and / or proliferation. To address these two conflicting characteristics, the solution is to have a hydrogel that is strong enough to embed cells and degradable, e.g., via cleavable bonds, that creates sufficient space for new cells upon degradation. One method of choice for achieving this degradation is to have peptidic structures in the crosslinked hydrogel, particularly at the crosslinker stage between the polymerized backbones.

[0016] This kind of practice is completely incompatible with the objective of the present invention, which is to obtain a long-lasting crosslinked hydrogel that encapsulates / embeds cells, in which case the hydrogel needs to be almost non-degradable, or better still, not degrade at all, since this key feature allows the cells to remain hidden from the immune system.

[0017] The underlying problem is solved by providing a gel that exhibits physicochemical properties that allow the fabrication of implantable devices and biocompatibility that allows cell survival.

[0018] Furthermore, contrary to much of the prior art, the invention allows for the preparation of hydrogels with tunable characteristics, taking into account the precursors used and the method of performing crosslinking, resulting in hydrogels with controlled incorporation and release of specific entities from the hydrogel.

[0019] The compatibility of a hydrogel is determined by its bulk structure, therefore the key parameters used to characterize the network structure of the hydrogels of the invention are the polymer volume fraction in the swollen state, the molecular weight of the polymer chain between two adjacent crosslink points, and the associated mesh size.

[0020] The problem is solved by providing new cross-linked dextran polymers, which have anionic groups and at least two sugar units of dextran belonging to two different polymer chains are linked by at least one central linker radical L(-). i wherein the at least one radical is at least a divalent linear, branched, or cyclic alkyl radical comprising at least a polyethylene glycol chain, or at least a divalent linear, branched, or cyclic alkyl radical comprising at least a poly(oxazoline) (POx) chain.

[0021] The problem is solved by providing new cross-linked dextran polymers, which have anionic groups and at least two sugar units of dextran belonging to two different polymer chains are linked by at least one central linker radical L(-). i wherein at least the radical is at least a divalent linear, branched, or cyclic alkyl radical comprising at least a polyethylene glycol chain.

[0022] The problem is solved by providing new cross-linked dextran polymers, which have anionic groups and at least two sugar units of dextran belonging to two different polymer chains are linked by at least one central linker radical L(-). i wherein at least one radical is at least a divalent linear, branched, or cyclic alkyl radical comprising at least a poly(oxazoline) (POx) chain.

[0023] In one embodiment, in the crosslinked dextran polymer Dx having anionic groups, at least a divalent radical L(-) i is covalently attached to a dextran polymer backbone bearing i W radicals, L(-) i is a linear or branched polyether, i is the valence of L and the number of W radicals attached to the dextran polymer, and is an integer between 2 and 8 (2≦i≦8); W is a radical containing at least one straight or branched chain alkyl radical, optionally containing heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0024] In one embodiment, the crosslinked dextran polymer of the invention is not a dextran polymer having carboxylate groups as disclosed and described in International Patent Application PCT / EP2022 / 050466, filed January 11, 2022.

[0025] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, L(-) i is a linear or branched polyether having a heteroatom such as oxygen, nitrogen, or sulfur at the end, i is the valence of L, and -(R1) m G1 - the number of radicals, which is an integer from 2 to 8 (2≦i≦8), m is an integer of 0 or 1, W is -(R1) m G1 - radical, -R1- is a divalent linear or branched alkyl radical having from 1 to 6 carbon atoms and optionally containing a heteroatom such as oxygen, nitrogen, or sulfur; -G1- is a divalent linear or branched or cyclic alkyl radical containing from 1 to 6 carbon atoms and optionally containing heteroatoms such as oxygen, nitrogen, or sulfur.

[0026] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, W is -(R1) m G1 - radical, -G1- is a sulfone derivative represented by the formula [ka] (In the formula, n1 is an integer between 0 and 7 (0≦n1≦7), X is a sulfur atom, * denotes the dextran backbone and the divalent radical L(-) i (represents the binding site of · or ·-G1- is a succinimide derivative represented by the following formula: [ka] (In the formula, X is a linear chain*-(CH2) where n1 is an integer between 1 and 7 (1≦n1≦7). n1 -* and * denotes the dextran backbone and the divalent radical L(-) i represents the binding site of

[0027] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, W is -(R1) m G1 - radical, -(R1) m G1- is a divalent linear or branched alkyl radical containing fewer than 13 carbon atoms and optionally containing oxygen, nitrogen, or sulfur atoms.

[0028] In one embodiment, the dextran polymer comprises at least a divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, Mn is between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 15 and 600 (15≦DP≦600).

[0029] In one embodiment, the dextran polymer comprises at least one divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is not a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and at most eight arms, The number average molecular weight (Mn) is between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 8 and 1000 (8≦DP≦1000).

[0030] A characteristic of this family of hydrogels is that they can be tuned and tailored for application by selecting and adapting the crosslinking reaction conditions, degree of substitution, and molecular weight of the dextran and crosslinker.

[0031] The problem is solved by providing a new hydrogel comprising: Living cells a non-crosslinked hyaluronic acid salt in the form of a solution, and Cross-linked dextran polymer Dx having anionic groups, In the crosslinked dextran polymer Dx having anionic groups, at least a divalent radical L(-) i is covalently attached to a dextran polymer backbone bearing i W radicals, L(-) i is a linear or branched polyether, i is the valence of L and the number of W radicals attached to the dextran polymer, and is an integer between 2 and 8 (2≦i≦8); W is a radical containing at least one straight or branched chain alkyl radical, optionally containing heteroatoms such as oxygen, nitrogen or sulfur, aromatic rings, polyether derivatives, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0032] In one embodiment, the cross-linked dextran polymers contained in the hydrogel of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least two radicals L(-) i is covalently attached to the dextran polymer backbone with i W radicals, L(-) iis a linear or branched polyether having a heteroatom such as oxygen, nitrogen, or sulfur at the end, i is the valence of L, and -(R1) m G1 - the number of radicals, which is an integer from 2 to 8 (2≦i≦8), m is an integer of 0 or 1, W is -(R1) m G1 - radical, -R1- is a divalent linear or branched alkyl radical having from 1 to 6 carbon atoms and optionally containing a heteroatom such as oxygen, nitrogen, or sulfur; -G1- is a divalent linear or branched or cyclic alkyl radical containing from 1 to 6 carbon atoms and optionally containing heteroatoms such as oxygen, nitrogen, or sulfur.

[0033] In one embodiment, the dextran polymer contained in the hydrogel comprises at least one divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, Mn is between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 15 and 600 (15≦DP≦600).

[0034] In one embodiment, the dextran polymer contained in the hydrogel comprises at least one divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is not a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and at most eight arms, The number average molecular weight (Mn) is between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol), or It is not a dextran polymer with a degree of polymerization (DP) between 8 and 1000 (8≦DP≦1000).

[0035] The cross-linked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having an anionic group, and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether or a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, a polyether or poly(oxazoline) derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0036] The cross-linked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having an anionic group, and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0037] The crosslinked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having anionic groups, in which at least divalent radicals L are covalently bonded to the dextran polymer backbone having i -W- radicals, L is a linear or branched poly(oxazoline); i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0038] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, L(-) i is a linear or branched polyether having a heteroatom such as oxygen, nitrogen, or sulfur at the end, i is the valence of L, and -(R1) m G1 - the number of radicals, which is an integer from 2 to 8 (2≦i≦8), m is an integer of 0 or 1, W is -(R1) m G1 - radical, -R1- is a divalent linear or branched alkyl radical having from 1 to 6 carbon atoms and optionally containing a heteroatom such as oxygen, nitrogen, or sulfur; -G1- is a divalent linear or branched or cyclic alkyl radical containing from 1 to 6 carbon atoms and optionally containing heteroatoms such as oxygen, nitrogen, or sulfur.

[0039] In one embodiment, the dextran polymer comprises at least a divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals.i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, the number average molecular weight Mn is between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 15 and 600 (15≦DP≦600).

[0040] In one embodiment, the dextran polymer comprises at least a divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is not a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and at most eight arms, The number average molecular weight (Mn) is between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 8 and 1000 (8≦DP≦1000).

[0041] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, W is -(R1) m G1 - radical, -G1- is a sulfone derivative represented by the formula [ka] (In the formula, n1 is an integer between 0 and 7 (0≦n1≦7), X is a sulfur atom, * denotes the dextran backbone and the divalent radical L(-) i (representing the binding site of · or ·-G1- is a succinimide derivative represented by the following formula: [ka] (In the formula, X is a linear chain*-(CH2) where n1 is an integer between 1 and 7 (1≦n1≦7). n1 -* and * denotes the dextran backbone and the divalent radical L(-) i represents the binding site of

[0042] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, W is -(R1) m G1 - radical, -(R1) m G1- is a divalent linear or branched alkyl radical containing fewer than 13 carbon atoms and optionally containing oxygen, nitrogen, or sulfur atoms.

[0043] The invention also relates to implants comprising the hydrogels of the invention.

[0044] Applicants have surprisingly found that the presence of hyaluronic acid or sodium or potassium hyaluronate in the crosslinking mixture helps to improve the uniform distribution of cells or islets in the hydrogel, which in turn reduces the effects of sedimentation of the cells or islets.

[0045] In one embodiment, the hydrogel comprises hyaluronic acid or sodium hyaluronate or potassium hyaluronate.

[0046] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 100 to 2500 kg / mol.

[0047] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 250 to 2500 kg / mol.

[0048] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 500 to 2250 kg / mol.

[0049] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 750 to 2000 kg / mol.

[0050] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 1000 to 1500 kg / mol.

[0051] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 250 to 4000 kg / mol.

[0052] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 500 to 3750 kg / mol.

[0053] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 750 to 3500 kg / mol.

[0054] In one embodiment, the weight average molecular weight (Mw) of the hyaluronic acid or sodium or potassium hyaluronate ranges from 1000 to 3250 kg / mol.

[0055]

[0056] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 0.5 to 30 mg / ml.

[0057] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 0.5 to 20 mg / ml.

[0058] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 0.5 to 10 mg / ml.

[0059] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 0.5 to 5 mg / ml.

[0060] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 0.75 to 2.5 mg / ml.

[0061] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 1.0 to 1.5 mg / ml.

[0062] In one embodiment, the concentration of hyaluronic acid or sodium hyaluronate or potassium hyaluronate in the hydrogel ranges from 0.8 to 1.2 mg / ml.

[0063] In one embodiment, hyaluronic acid with a Mw in the range of 2000 to 4000 kg / mol, particularly about 3000 kg / mol, is present at a concentration in the range of 0.5 to 1.5 mg / ml.

[0064] In one embodiment, hyaluronic acid with a Mw in the range of 2000 to 4000 kg / mol, particularly about 3000 kg / mol, is present at a concentration in the range of 0.7 to 1.2 mg / ml.

[0065]

[0066] In one embodiment, hyaluronic acid or sodium or potassium hyaluronate with a Mw in the range of 1000 to 2000 kg / mol, particularly about 1500 kg / mol, is present at a concentration in the range of 0.5 to 2 mg / ml.

[0067] In one embodiment, hyaluronic acid or sodium or potassium hyaluronate with a Mw in the range of 1000 to 2000 kg / mol, particularly about 1500 kg / mol, is present at a concentration in the range of 1.0 to 1.5 mg / ml.

[0068] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether or a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, a polyether or poly(oxazoline) derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0069] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0070] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0071] In one embodiment, -W- contains up to 60 carbon atoms.

[0072] In one embodiment, -W- contains up to 60 carbon atoms, not counting the -CH2-CH2O- radical.

[0073] In one embodiment, -W- contains up to 50 carbon atoms.

[0074] In one embodiment, -W- contains up to 50 carbon atoms, not counting the -CH2-CH2O- radical.

[0075] In one embodiment, -W- contains up to 40 carbon atoms.

[0076] In one embodiment, -W- contains up to 40 carbon atoms, not counting the -CH2-CH2O- radical.

[0077] In one embodiment, -W- contains up to 30 carbon atoms.

[0078] In one embodiment, -W- contains up to 30 carbon atoms, not counting the -CH2-CH2O- radical.

[0079] In one embodiment, -W- contains up to 20 carbon atoms.

[0080] In one embodiment, -W- contains up to 20 carbon atoms, not counting the -CH2-CH2O- radical.

[0081] In one embodiment, -W- contains up to 10 carbon atoms.

[0082] In one embodiment, -W- contains up to 10 carbon atoms, not counting the -CH2-CH2O- radical.

[0083] In one embodiment, -W- contains up to 10 oxygen atoms.

[0084] In one embodiment, -W- contains up to 10 oxygen atoms, not counting the -CH2-CH2O- radical.

[0085] In one embodiment, -W- contains up to 5 oxygen atoms.

[0086] In one embodiment, -W- contains up to 5 oxygen atoms, not counting the -CH2-CH2O- radical.

[0087] The crosslinked dextran hydrogel of the invention has a central linker L(-) i is a dextran polymer in which is a linear or branched polyethylene glycol (PEG) radical.

[0088] Branched PEG refers to various PEG arms linked by linear, branched, or cyclic alkyl, or aromatic groups containing between 2 and 20 carbon atoms and which may contain heteroatoms such as nitrogen, oxygen, or sulfur.

[0089] In one embodiment, the crosslinked dextran hydrogel of the invention comprises a central linker L(-) i is a branched PEG radical with up to eight arms, a dextran polymer.

[0090] In one embodiment, the central linker L(-) i is selected from PEG of Formula I [ka] (In formula I, i is an integer between 2 and 8 (2≦i≦8); p is an integer of 0 or 1, and when i=2, p=0; q is an integer between 8 and 1000 (8≦q≦1000), r is an integer of 0 or 1; Q is a carbon atom or a linear, branched or cyclic alkyl chain containing 2 to 10 carbon atoms and optionally containing heteroatoms such as nitrogen, oxygen or sulfur, or aromatic; * represents the moiety f4, which is an amine group, or an ether group, or a thioether group, or an amide group, or a carbamate group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond if the crosslinking step is performed by native chemical ligation (NCL).

[0091] In one embodiment, q is an integer between 80 and 500 inclusive (80≦q≦500).

[0092] In one embodiment, q is an integer between 100 and 300 inclusive (100≦q≦300).

[0093] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, The number average molecular weight (Mn) is between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol), or It is a dextran polymer with a degree of polymerization (DP) ranging from 8 to 1000 (8≦DP≦1000).

[0094] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, The number average molecular weight (Mn) is between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol), or It is a dextran polymer with a degree of polymerization (DP) ranging from 15 to 600 (15≦DP≦600).

[0095] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a dextran polymer, which is a radical according to formula I derived from thiol polyethylene glycols or mercaptopoly(oxyethylenes) as set out in the table below. [Table 1]

[0096] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a radical according to formula I, derived from pentaerythritol tetra(mercaptoethyl) polyoxyethylene, CAS# 188492-68-4, dextran polymer.

[0097] In one embodiment, the crosslinked dextran polymers of the invention are L(-) iis a radical according to formula I, derived from linear (mercaptoethyl) polyoxyethylene, CAS#68865-60-1, dextran polymer.

[0098] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a dextran polymer, which is a radical according to formula I derived from pentaerythritol poly(oxyethylene) azide as set forth in the table below. [Table 2]

[0099] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a radical according to formula I, derived from pentaerythritol 4-arm PEG azide, CAS#225531-50-0, dextran polymer.

[0100] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a dextran polymer derived from pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene, a radical according to formula I, as set forth in the table below. [Table 3]

[0101] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a pentaerythritol 4-arm PEG DBCO-derived radical according to formula I, a dextran polymer.

[0102] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a dextran polymer, where R is a radical according to formula I, derived from maleimide polyethylene glycols as set out in the table below. [Table 4]

[0103] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a 4-arm poly(ethylene glycol) maleimide-derived radical according to formula I, a dextran polymer.

[0104] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a dextran polymer derived from norbornene polyethylene glycols, radicals according to formula I, as set forth in the table below. [Table 5]

[0105] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i is a 4-arm poly(ethylene glycol) norbornene-derived radical according to formula I, a dextran polymer.

[0106] In one embodiment, the crosslinked dextran hydrogel of the invention is a dextran polymer in which the central linker L is a linear or branched POx radical.

[0107] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer in which L is a linear or branched POx radical having a number average molecular weight (Mn) comprised between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol) and containing up to 8 arms.

[0108] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer in which L is a linear or branched POx radical having a number average molecular weight (Mn) comprised between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol) and containing up to 8 arms.

[0109] In one embodiment, the POx core linker is a two-arm POx selected from the linkers of formula XII. [ka] (In formula XII, The radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), or a branched or cyclic alkyl derivative; * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0110] In one embodiment, the POx core linker is a 2-arm POx selected from the linkers of formula XIIbis. [ka] (In formula XIIbis, Radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer from 0 to 4 (0≦n1≦4)), branched chain, or cyclic alkyl derivatives; * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0111] In one embodiment, the POx core linker is a 4-arm POx selected from the linkers of formula XIII. [ka] (In formula XIII, Radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer from 0 to 4 (0≦n1≦4)), branched chain, or cyclic alkyl derivatives; * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0112] In another embodiment, the POx core linker is a 4-arm POx selected from the linkers of formula XIV. [ka] (In formula XIV, Radical -R1 is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), branched chain, or cyclic alkyl derivatives. The divalent radical -R2- is a straight chain -(CH2) n2 -(n2 is an integer between 2 and 6 (2≦n2≦6)). * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0113] In another embodiment, the POx core linker is a 4-arm POx selected from the linkers of formula XV. [ka] (In formula XV, Radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦2)), branched chain, or cyclic alkyl derivatives. In one embodiment, R1 = -CH2-CH2- and R2 is linear, -(CH2) n2 -(n2 is an integer between 2 and 6 (2≦n2≦6)). In another embodiment, R2 = -CH2-CH2- and R1 is linear, *-(CH2) n2 -*(n2 is an integer between 2 and 6 (2≦n2≦6)). * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0114] The hydroxy groups of the dextran polymer Dx- can be functionalized with at least one specific anionic group such as alkyl carboxylate, sulfate anion, sulfonate anion, phosphate anion, or phosphonate anion.

[0115] In one embodiment, the hydroxy groups of the dextran polymer Dx- may be functionalized with sulfate anions in salified form and, optionally, with alkyl carboxylic acid ester derivatives in salified form.

[0116] In another embodiment, the hydroxy groups of the dextran polymer Dx- may be functionalized with sulfonate anions in salified form and, optionally, with alkyl carboxylic acid ester derivatives in salified form.

[0117] In another embodiment, the hydroxy groups of the dextran polymer backbone Dx- may be functionalized with phosphate anions in salified form and, optionally, with alkyl carboxylic acid ester derivatives in salified form.

[0118] In another embodiment, the hydroxy groups of the dextran polymer Dx- may be functionalized with phosphonate anions in salified form and, optionally, with alkylcarboxylic acid ester derivatives in salified form.

[0119] In another embodiment, the hydroxy groups of the dextran polymer Dx- may be functionalized with alkyl carboxylic acid ester derivatives in salified form.

[0120] In one embodiment, the hydroxy groups of the dextran polymer Dx- may be functionalized with one particular type of anionic group: an alkyl carboxylic acid ester anion.

[0121] In one embodiment, the hydroxy groups of the dextran polymer Dx- are functionalized only with one specific type of anionic group: an alkyl carboxylic acid ester anion.

[0122]

[0123] The particular anionic group defined above is selected from the group of formula II. [ka] (in formula II * represents a bond with an O atom of dextrin, forming an ether group. y=2 or 3 When y=2, alkyl carboxylic acid ester derivatives, Y=C and a=1 k=1, l=0 and m=0 R2 = alkyl When y=3, an anionic group Y=S and a=1, or Y=P and a=2 k=0 or 1 l=0 or 1 m=0 or 1 n=1 or 2, especially n=1 o=0 or 1 If l=1, then m=1 R3 = linear, branched, or cyclic alkyl, optionally containing one heteroatom such as nitrogen, or aromatic, or PEG R2 = alkyl and Z is a counterion, which can be an alkali metal and z=1, or an alkaline earth metal and z=2.

[0124] In a preferred embodiment, the dextran backbone, Dx-, may be functionalized with sulfate anions in salified form and, optionally, with alkyl carboxylic acid ester derivatives in salified form.

[0125] In another preferred embodiment, the dextran backbone, Dx-, may be functionalized with alkylsulfonate anions in salified form and, optionally, with alkylcarboxylic acid ester derivatives in salified form.

[0126] In another preferred embodiment, the dextran backbone, Dx-, is supported by alkyl chains containing dimethyl-ammonium cations and can be functionalized with sulfonate anions in salified form and, optionally, alkyl carboxylic acid ester derivatives in salified form.

[0127] In another preferred embodiment, the dextran backbone, Dx-, may be functionalized with alkyl carboxylic acid ester derivatives in salified form.

[0128] In one embodiment, the crosslinked dextran polymers having anionic groups of the invention are dextran polymers in which the dextran polymer backbone conforms to Formula III. [ka] (In formula III, R is H, an anionic group of formula II, or L(-) i -W- radicals having a cross-linker, i is between 20 and 5000 (20≦i≦5000), -W- and L(-) i The radicals have the meanings defined above.

[0129] In one embodiment, the crosslinked dextran polymers having anionic groups of the invention are dextran polymers in which the dextran polymer backbone conforms to Formula XI. [ka] (In formula XI, R is H, an anionic group of formula II, or L(-) i (A-f2) with a cross-linker a -G1- radicals, l is in the range from 20 to 5000 (20≦l≦5000), -(A-f2) a -G1- and L(-) i has the meaning defined above.)

[0130] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 5 and 1000 kDa.

[0131] In other words, the crosslinked dextran polymers of the invention are obtained after substitution and crosslinking of native dextran polymers having a weight average molecular weight (Mw) comprised between 5 and 1000 kDa.

[0132] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 5 and 250 kDa.

[0133] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 5 and 100 kDa.

[0134] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 5 and 50 kDa.

[0135] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 5 and 25 kDa.

[0136] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 250 and 1000 kDa.

[0137] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 10 and 500 kDa.

[0138] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 20 and 500 kDa.

[0139] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 20 and 100 kDa.

[0140] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 20 and 50 kDa.

[0141] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 40 and 250 kDa.

[0142] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 40 and 100 kDa.

[0143] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0144] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having a cross-linking linker in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0145] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0146] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.01 to 0.4 (0.01≦DS1≦0.4).

[0147] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having a cross-linking linker in the range of 0.01 to 0.4 (0.01≦DS1≦0.4).

[0148] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.01 to 0.4 (0.01≦DS1≦0.4).

[0149] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i-W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.05 to 0.4 (0.05≦DS1≦0.4).

[0150] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.05 to 0.4 (0.05≦DS1≦0.4).

[0151] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.05 to 0.4 (0.05≦DS1≦0.4).

[0152] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0153] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0154] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i (A-f2) with a cross-linker aThe crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0155] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0156] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution, and L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0157] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 5 to 250 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0158] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) aThe crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.2 to 0.4 (0.2≦DS1≦0.4).

[0159] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.2 to 0.4 (0.2≦DS1≦0.4).

[0160] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.2 to 0.4 (0.2≦DS1≦0.4).

[0161] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.2 to 0.3 (0.2≦DS1≦0.3).

[0162] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and L(-) iThe cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.2 to 0.3 (0.2≦DS1≦0.3).

[0163] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 20 to 100 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.2 to 0.3 (0.2≦DS1≦0.3).

[0164] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0165] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having a cross-linking linker in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0166] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker aThe crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0167] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.01 to 0.4 (0.01≦DS1≦0.4).

[0168] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having a cross-linking linker in the range of 0.01 to 0.4 (0.01≦DS1≦0.4).

[0169] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.01 to 0.4 (0.01≦DS1≦0.4).

[0170] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) aThe crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.05 to 0.4 (0.05≦DS1≦0.4).

[0171] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i The cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.05 to 0.4 (0.05≦DS1≦0.4).

[0172] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.05 to 0.4 (0.05≦DS1≦0.4).

[0173] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i -W- radical with a bridging linker or -(A-f2) a The crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0174] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) iThe cross-linked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -W- radicals having cross-linking linkers in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0175] In one embodiment, the crosslinked dextran polymer of the invention has a dextran polymer backbone with a weight average molecular weight (Mw) of 250 to 1000 kDa before crosslinking and substitution, and L(-) i (A-f2) with a cross-linker a The crosslinked dextran polymer has a degree of substitution of the dextran backbone with -G1- radicals (DS1) in the range of 0.1 to 0.4 (0.1≦DS1≦0.4).

[0176] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution (DS2) of the dextran backbone with total methyl carboxylates grafted to the dextran in the range of 0.3 to 2.5 (0.3≦DS2≦2.5).

[0177] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution of the dextran backbone with total methyl carboxylates grafted to the dextran (DS2) in the range of 0.5 to 2.3 (0.5≦DS2≦2.3).

[0178] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution of the dextran backbone with total methyl carboxylates grafted to the dextran (DS2) in the range of 1.5 to 2.5 (1.5≦DS2≦2.5).

[0179] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution (DS2) of the dextran backbone with total methyl carboxylates grafted to the dextran in the range of 1.7 to 2.3 (1.7≦DS2≦2.3).

[0180] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution (DS2) of the dextran backbone with total methyl carboxylates grafted to the dextran in the range of 1.8 to 2.2 (1.8≦DS2≦2.5).

[0181] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution (DS2) of the dextran backbone with total methyl carboxylates grafted to the dextran in the range of 0.3 to 1.5 (0.3≦DS2≦1.5).

[0182] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer having a degree of substitution (DS2) of the dextran backbone with total methyl carboxylates grafted to the dextran in the range of 0.3 to 0.8 (0.3≦DS2≦0.8).

[0183] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I or radicals of formula XII, XIIbis, XIII or XIV is in the range of 0.5 to 3 (0.5≦DS4≦3).

[0184] In one embodiment, the crosslinked dextran polymers of the invention are crosslinked dextran polymers in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I is comprised within the range of from 0.5 to 3 (0.5≦DS4≦3).

[0185] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula XII, XIIbis, XIII or XIV is in the range of 0.5 to 3 (0.5≦DS4≦3).

[0186] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I or radicals of formula XII, XIIbis, XIII or XIV is in the range of from 1 to 2.75 (1≦DS4≦2.75).

[0187] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I is comprised within the range of from 1 to 2.75 (1≦DS4≦2.75).

[0188] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula XII, XIIbis, XIII or XIV is in the range of 1 to 2.75 (1≦DS4≦2.75).

[0189] In one embodiment, the crosslinked dextran polymers of the invention are crosslinked dextran polymers in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I or radicals of formula XII, XIIbis, XIII or XIV is in the range of from 1.5 to 2.5 (1.5≦DS4≦2.5).

[0190] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I is comprised within the range of from 1.5 to 2.5 (1.5≦DS4≦2.5).

[0191] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula XII, XIIbis, XIII or XIV is in the range of from 1.5 to 2.5 (1.5≦DS4≦2.5).

[0192] In one embodiment, the crosslinked dextran polymers of the invention are crosslinked dextran polymers in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I or radicals of formula XII, XIIbis, XIII or XIV is in the range of from 1.75 to 2.25 (1.75≦DS4≦2.25).

[0193] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula I is comprised within the range of from 1.75 to 2.25 (1.75≦DS4≦2.25).

[0194] In one embodiment, the crosslinked dextran polymer of the invention is a crosslinked dextran polymer in which the degree of substitution (DS4) of the dextran backbone with radicals of formula XII, XIIbis, XIII or XIV is in the range of 1.75 to 2.25 (1.75≦DS4≦2.25).

[0195] In one embodiment, the crosslinked dextran polymers of the invention have a degree of carboxylate substitution (DS) of the dextran backbone. C ) is between 0.2 and 3 (0.2≦DS C ≦3).

[0196] In one embodiment, the crosslinked dextran polymers of the invention have a degree of carboxylate substitution (DS) of the dextran backbone. C ) is between 0.3 and 2.5 (0.3≦DS C ≦2.5).

[0197] In one embodiment, the crosslinked dextran polymers of the invention are crosslinked dextran polymers having a degree of sulfate, sulfonate, phosphate, or phosphonate substitution (DS3) of the dextran backbone in the range of 0.2 to 2.5 (0.2≦DS3≦2.5).

[0198] In one embodiment, the crosslinked dextran polymers of the invention are crosslinked dextran polymers having a degree of sulfate, sulfonate, phosphate, or phosphonate substitution (DS3) of the dextran backbone in the range of 0.3 to 2.0 (0.3≦DS3≦2.0).

[0199] In one embodiment, the crosslinked dextran polymers of the invention are provided with a -W- radical or -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the cross-linker to the molar concentration of the reactive functional groups in the polymer, which is in the range of 0.5 to 1.5 (0.5≦DC≦1.5).

[0200] In one embodiment, the crosslinked dextran polymers of the invention are prepared by mixing the molar concentrations of -W- radicals and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the reactive functional group of the cross-linker in the range of 0.5 to 1.5 (0.5≦DC≦1.5).

[0201] In one embodiment, the crosslinked dextran polymer of the invention is -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the cross-linker to the molar concentration of the reactive functional groups in the polymer, which is in the range of 0.5 to 1.5 (0.5≦DC≦1.5).

[0202] In one embodiment, the crosslinked dextran polymers of the invention are provided with a -W- radical or -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the reactive functional groups of the cross-linker to the molar concentration of the reactive functional groups in the cross-linked dextran polymer, which is in the range of 0.8 to 1.2 (0.8≦DC≦1.2).

[0203] In one embodiment, the crosslinked dextran polymers of the invention are prepared by mixing the molar concentrations of -W- radicals and L(-) iThe cross-linked dextran polymer has a molar ratio (DC) of the reactive functional groups of the cross-linker to the molar concentration of the reactive functional groups in the cross-linked dextran polymer, which is in the range of 0.8 to 1.2 (0.8≦DC≦1.2).

[0204] In one embodiment, the crosslinked dextran polymer of the invention is -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the reactive functional groups of the cross-linker to the molar concentration of the reactive functional groups in the cross-linked dextran polymer, which is in the range of 0.8 to 1.2 (0.8≦DC≦1.2).

[0205] In one embodiment, the crosslinked dextran polymers of the invention are provided with a -W- radical or -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the reactive functional groups of the cross-linker to the molar concentration of the reactive functional groups in the cross-linked dextran polymer, which is in the range of 0.9 to 1.1 (0.9≦DC≦1.1).

[0206] In one embodiment, the crosslinked dextran polymers of the invention are prepared by mixing the molar concentrations of -W- radicals and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the reactive functional groups of the cross-linker to the molar concentration of the reactive functional groups in the cross-linked dextran polymer, which is in the range of 0.9 to 1.1 (0.9≦DC≦1.1).

[0207] In one embodiment, the crosslinked dextran polymer of the invention is -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the reactive functional groups of the cross-linker to the molar concentration of the reactive functional groups in the cross-linked dextran polymer, which is in the range of 0.9 to 1.1 (0.9≦DC≦1.1).

[0208] In one embodiment, the crosslinked dextran polymers of the invention are provided with a -W- radical or -(A-f2) a Molar concentration of -G1- radical and L(-) iThe cross-linked dextran polymer has a molar ratio (DC) of the cross-linker to the molar concentration of the reactive functional groups in the polymer in the range of 0.95 to 1.05 (0.95≦DC≦1.05).

[0209] In one embodiment, the crosslinked dextran polymers of the invention are prepared by mixing the molar concentrations of -W- radicals and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the cross-linker to the molar concentration of the reactive functional groups in the polymer in the range of 0.95 to 1.05 (0.95≦DC≦1.05).

[0210] In one embodiment, the crosslinked dextran polymer of the invention is -(A-f2) a Molar concentration of -G1- radical and L(-) i The cross-linked dextran polymer has a molar ratio (DC) of the cross-linker to the molar concentration of the reactive functional groups in the polymer in the range of 0.95 to 1.05 (0.95≦DC≦1.05).

[0211] In one embodiment, the crosslinked dextran polymers of the invention are provided with a -W- radical or -(A-f2) a Molar concentration of -G1- radical and L(-) i It is a cross-linked dextran polymer in which the molar ratio of reactive functional groups of the cross-linker to the molar concentration is 1 (DC=1).

[0212] In one embodiment, the crosslinked dextran polymers of the invention are prepared by mixing the molar concentrations of -W- radicals and L(-) i It is a cross-linked dextran polymer in which the molar ratio of reactive functional groups of the cross-linker to the molar concentration is 1 (DC=1).

[0213] In one embodiment, the crosslinked dextran polymer of the invention is -(A-f2) a Molar concentration of -G1- radical and L(-) i It is a cross-linked dextran polymer in which the molar ratio of reactive functional groups of the cross-linker to the molar concentration is 1 (DC=1).

[0214] In one embodiment, the crosslinked dextran polymer of the invention is formed by combining the reactive functional groups of the -W- precursor with the L(-) i Crosslinked dextran polymers are obtained by reaction of precursors with reactive functional groups, which are present in the reaction at the same concentration (DC=1), ranging from 5 to 25 mM.

[0215] In one embodiment, the reactive functional group is in the range of 5 to 10 mM.

[0216] In one embodiment, the reactive functional group is in the range of 10 to 15 mM.

[0217] In one embodiment, the reactive functional group is in the range of 15 to 20 mM.

[0218] In one embodiment, the reactive functional group is in the range of 20 to 25 mM.

[0219] In one embodiment, -W- is selected from the radical of formula IV. [ka] (in formula IV * represents the site of f1, and ° represents the binding site with L. a is an integer of 0 or 1; b is an integer of 0 or 1; c is an integer of 0 or 1; In one embodiment, a=0 and f1 is an ether group or a carbamate group. In one embodiment, a=1, The divalent radical -A- is linear, -(CH2) n1 - (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, and at least one hydroxy group, -CH2-CH(OH)-(CH2) n2 -(n2 is an integer of 1 to 5 (1≦n2≦5)) which may be branched; f1 is an ether group or a carbamate group, and f2 is an amide group; or The divalent radical -A- is a linear polyether (PEG) derivative; f1 is an ether or carbamate group, and f2 is an amide group; or In another embodiment, the divalent radical -A- is a 4-alkyl-1,4-triazole derivative or a 4-PEG-1,4-triazole derivative; f1 is an ether group or a carbamate group, and f2 is a carbon-nitrogen covalent bond. or In another embodiment, the divalent radical -A- is a 1-alkyl-1,4-triazole derivative or a 1-PEG-1,4-triazole derivative; f1 is an ether group or a carbamate group, and f2 is a carbon-aromatic carbon covalent bond. the divalent radical -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, When b=0, f1 is an ether group or a carbamate group. When b=1, f1 is an ether group or a carbamate group, and f3 is an amide group, or an amine group, or an ether group, or a thioether group, or a carbamate group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond if the crosslinking step is performed by native chemical ligation (NCL); the divalent radical -G1- is a linear, branched, or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms such as up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms, or up to 1 phosphorus atom; in a preferred embodiment, -G1- is a succinimide derivative, or an alkylsulfone derivative which may contain one heteroatom such as oxygen or sulfur, or an ethylamide derivative, or a 1,4-triazole derivative, or a heterocyclic derivative from a Diels-Alder reaction, or an aromatic phosphine derivative produced by Staudinger ligation, or a cysteine derivative produced by native chemical ligation (NCL); When c=0, f1 is an ether group or a carbamate group; When c=1, f1 is an ether group or a carbamate group, and f4 is an amine group, or an amide group, or a carbamate group, or a thioether group, or an ether group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond if the cross-linking step is by native chemical ligation (NCL).

[0220] The crosslinked dextran polymer of the invention is a dextran polymer Dx- having anionic groups, which has at least a divalent radical L(-) i is a dextran polymer Dx- covalently attached to a dextran polymer backbone having a radical of i and selected from dextran of formula X. [ka] (In formula X, a is an integer of 0 or 1; i is an integer between 2 and 8 (2≦i≦8), L may be linked to the same [Dx-f1-(A-f2)a-G1-f3] radical or to different radicals, Dx- is a part of dextran, which can be substituted by anionic groups in specific salt form and optionally by alkylcarboxylate derivatives in salt form; f1 is an ether group, The divalent radical -A- is a straight chain, -(CH2) n1 - (n1 is an integer from 1 to 7 (1≦n1≦7)), branched, or cyclic alkyl derivatives, and also contains at least one hydroxy group, -CH2-CH(OH)-(CH2) n2 - (n2 is an integer from 1 to 5 (1≦n2≦5)) may be branched, f2 is an amide group, the divalent radical -G1- is a linear, branched or cyclic alkyl derivative or an aromatic derivative, which may contain heteroatoms such as up to 5 nitrogen atoms, up to 10 oxygen atoms or up to 5 sulfur atoms; in a preferred embodiment, -G1- is a succinimide derivative or an 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 number of identical or different [Dx-f1-(A-f2)a-G1-f3] radicals attached to L; f3 is an amine group, or a thioether group, or an ether group, or an amide group, or a carbamate group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond; The central linker L is a poly(oxazoline) (POx) derivative and can be linear or branched.

[0221] In one embodiment, the crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In formula V, f1, f2, f3, f4, -A-, -R1-, -G1- are defined as in formula IV above, and Dx- is a part of dextran, as defined above, which may be substituted by specific anionic groups in the form of salts and optionally by alkylcarboxylate derivatives in the form of salts; 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 ] is the number of radicals, the central linker L is a polyether (PEG) derivative and can be linear or branched; In one embodiment, when b=0 and c=1, the central linker L can be a poly(oxazoline) (POx) derivative, which can be linear or branched.

[0222] In one embodiment, the crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In formula V, f1, f2, f3, f4, -A-, -R1-, -G1- are defined as in formula IV above, and Dx- is a part of dextran, as defined above, which may be substituted by specific anionic groups in the form of salts and optionally by alkylcarboxylate derivatives in the form of salts; 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 ] is the number of radicals, The central linker L is a polyether (PEG) derivative and can be linear or branched.

[0223] In one embodiment, L is the same as [Dx-f1-(A-f2)a-(R1-f3) b -(G1-f4) c ] linking with the radical.

[0224] In one embodiment, L is different [Dx-f1-(A-f2)a-(R1-f3) b -(G1-f4) c ] linking with the radical.

[0225] When a=0, In one embodiment, f1 is an ether group. In another embodiment, f1 is a carbamate group.

[0226] When a=1, In one embodiment, f1 is an ether group. In another embodiment, f1 is a carbamate group.

[0227] When a=0 and b=0, In one embodiment, f1 is an ether group. In another embodiment, f1 is a carbamate group.

[0228] When a=0 and b=1, In one embodiment, f1 is an ether group. In another embodiment, f1 is a carbamate group.

[0229] If a=b=c=0, In one embodiment, f1 is an ether group. In another embodiment, f1 is a carbamate group.

[0230] In one embodiment, with respect to Formula V, the divalent radical -A- is a linear polyester (PEG) derivative selected from PEGs of the formula: [ka] (In the formula, n1 is an integer of 0 or 1; n2 is an integer between 1 and 7 (1≦n1≦7), * represents the f1 and f2 sites, In a preferred embodiment, * represents the moieties f1 and f2, which are an ether group and an amide group, respectively.

[0231] In another embodiment, with respect to formula V, the divalent radical -A- is a 1-alkyl-1,4-triazole derivative or a 1-PEG-1,4-triazole derivative selected from triazole derivatives of the formula: [ka] (In the formula, ·X is a linear chain*-(CH2) n1 -* (n1 is an integer between 1 and 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, or X is a PEG derivative; The * represents the site of f1, and the dotted bond represents f2.)

[0232] In another embodiment, with respect to formula V, the divalent radical -A- is a 4-alkyl-1,4-triazole derivative selected from triazole derivatives of the formula: or a 4-PEG-1,4-triazole derivative: [ka] (In the formula, ·X is a linear chain*-(CH2) n1 -* (n1 is an integer between 1 and 7 (1≦n1≦7)), branched, or cyclic alkyl derivatives, or X is a PEG derivative; The * represents the site of f1, and the dotted bond represents f2.)

[0233] When a=1, In one embodiment, f2 is an amide group. In another embodiment, f2 is a carbon-nitrogen covalent bond. In another embodiment, f2 is a carbon-aromatic carbon covalent bond.

[0234] In one embodiment, with respect to formula X, -A- is linear, -(CH) n1 - (n1 is an integer from 1 to 7 (1≦n1≦7)), branched, or cyclic alkyl derivatives, and also contains at least one hydroxy group, -CH2-CH(OH)-(CH2) n2 - (n2 is an integer between 1 and 5 (1≦n2≦5)) may be branched.

[0235] In one embodiment, with respect to formula V, the divalent radical -R1- is a straight chain alkyl derivative according to the formula: [ka] (In the formula, n1 is an integer between 1 and 7 (1≦n1≦7), In one embodiment, when a=1, * represents the moieties f2 and f3; In another embodiment, when a=0, * represents the positions f1 and f3.

[0236] In another embodiment, with respect to Formula V, the divalent radical -R1- is a polyether (PEG) derivative according to the following formula: [ka] (In the formula, n1 is an integer of 0 or 1; n2 is an integer between 1 and 7 (1≦n2≦7), In one embodiment, when a=1, * represents the moieties f2 and f3; In another embodiment, when a=0, * represents the positions f1 and f3.

[0237] In another embodiment, with respect to formula V, when a=0, the divalent radical -R1- can be a branched alkyl with at least one hydroxy group attached from f1 to the alkyl chain at the β-position, which is an ether group. [ka] (In the formula: n2 is an integer between 1 and 5 (1≦n2≦75), In another embodiment, when a=0, * represents the positions f1 and f3.

[0238] In a preferred embodiment, with respect to formula V, the divalent radical -R1- is a straight chain alkyl derivative according to the following formula: [ka] (In the formula: * is an amide group, f1, and 、 f3 part.)

[0239] In a preferred embodiment, with respect to formula V, the divalent radical -R1- is a PEG derivative according to the following formula: [ka] (In the formula: n1 is an integer between 1 and 7 (1≦n1≦7), * represents the moieties f2 and f3, which are two amide groups.

[0240] When b=1, In one embodiment, f3 is an amine group. In another embodiment, f3 is an ether group. In another embodiment, f3 is a thioether group. In another embodiment, f3 is an amide group. In another embodiment, f3 is a carbamate group. In another embodiment, f3 is a carbon-nitrogen covalent bond. In another embodiment, f3 is a carbon-aromatic carbon covalent bond. In another embodiment, f3 is a carbon-carbon covalent bond when the cross-linking step is performed by native chemical ligation (NCL).

[0241] The nature of the radical G1 is determined by the crosslinking process. The various crosslinking processes along with the G1 radical are described below.

[0242] In one embodiment, the cross-linking step is achieved by Michael addition using maleimide derivatives, or vinyl sulfone derivatives, or acrylamide derivatives.

[0243] In one embodiment, for formula X, the integer a=1 and the cross-linking step is achieved by Michael addition using maleimide derivatives or vinyl sulfone derivatives.

[0244] In one embodiment, with respect to formula V, the integers a=c=1 and L are POx derivatives, and the cross-linking step is achieved by Michael addition using maleimide derivatives or vinyl sulfone derivatives.

[0245] In one embodiment, with respect to formula V, the divalent radical -G1- is a succinimide derivative according to the formula: [ka] During the ceremony: ·X is a linear chain*-(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, or X is aromatic, or X is a PEG derivative; In one embodiment, when b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when a=1 and b=0, * represents the moieties f2 and f4. In a preferred embodiment, X is an ethyl group and * represents the moiety f2, which is an amide group, and f4, which is a thioether group.

[0246] In one embodiment, with respect to formula X, the divalent radical -G1- is a succinimide derivative according to the formula: [ka] (In the formula: R is a linear, branched, or cyclic alkyl derivative, or R is aromatic, or R is a PEG derivative.

[0247] * represents the moiety of f2, which is an amide group, and f3, which is an amine group, an ether group, or a thioether group.

[0248] In one embodiment, with respect to formula V, when b=0, c=1 and L is a POx derivative, the divalent radical -G1- is a succinimide derivative according to the following formula: [ka] (In the formula: R is a linear, branched or cyclic alkyl derivative, or R is aromatic, or R is a PEG derivative, * represents the moiety f2, which is an amide group, and f3, which is an amine group, an ether group, or a thioether group.

[0249] In another embodiment, with respect to formula X, the divalent radical -G1- is a succinimide derivative according to the formula: [ka] (In the formula: X is an oxygen atom, a sulfur atom, or a nitrogen atom, R is a linear, branched, or cyclic alkyl derivative, or R is a PEG derivative. The * represents the moiety f2, which is an amide group, and the dotted bond represents f3, which is a carbon-nitrogen covalent bond.

[0250] In another embodiment, with respect to formula V, when b=0, c=1 and L is a POx derivative, the divalent radical -G1- is a succinimide derivative according to the following formula: [ka] (In the formula: X is an oxygen atom, a sulfur atom, or a nitrogen atom, R is a linear, branched, or cyclic alkyl derivative, or R is a PEG derivative. The * represents the moiety f2, which is an amide group, and the dotted bond represents f3, which is a carbon-nitrogen covalent bond.

[0251] In another embodiment, with respect to formula V, the divalent radical -G1- is a diethylsulfone derivative according to the formula: [ka] (In the formula: In one embodiment, when b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when b=0, * represents the moieties f2 and f4. In a preferred embodiment, * represents the moieties f3 and f4, which are thioether groups.

[0252] In another embodiment, with respect to formula V, the divalent radical -G1- is a sulfone derivative according to the formula: [ka] (In the formula: n1 is an integer between 0 and 7 (0≦n1≦7), X can be an oxygen atom, a sulfur atom, or a CH2 group; In one embodiment, when b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when a=1 and b=0, * represents the moieties f2 and f4. In a preferred embodiment, a=1, b=0, X is a sulfur atom, n1=2, f2 is an amide group, and f4 is a thioether group.

[0253] In another embodiment, with respect to formula X, the divalent radical -G1- is a sulfone derivative according to the formula: [ka] (In the formula: n1 is an integer between 0 and 7 (0≦n1≦7), X is an oxygen atom, a sulfur atom, or a CH2 group; * represents the moiety f2, which is an amide group, and f3, which is an amine group, an ether group, or a thioether group.

[0254] In another embodiment, with respect to formula V, when b=0, c=1 and L is a POx derivative, the divalent radical -G1- is a sulfone derivative according to the following formula: [ka] (In the formula: n1 is an integer between 0 and 7 (0≦n1≦7), X is an oxygen atom, a sulfur atom, or a CH2 group; * represents the moiety f2, which is an amide group, and f3, which is an amine group, an ether group, or a thioether group.

[0255] In another embodiment, with respect to formula V, the divalent radical -G1- is an acrylamide derivative according to the formula: [ka] (In the formula: In one embodiment, * represents a moiety of f3 that is an amine group, or an ether group, or a thioether group, and the dotted bond represents f4 that is a carbon-nitrogen covalent bond. In another embodiment, the dotted bond represents f3, which is a carbon-nitrogen covalent bond, and * represents the moiety of f4, which is an amine group, an ether group, or a thioether group.

[0256] In one embodiment, the crosslinking step is achieved by a 1,3-cycloaddition reaction between an alkyne and an azide derivative, known as the 1,3-dipolar cycloaddition reaction or Huisgen reaction.

[0257] In one embodiment, with respect to formula V, the divalent radical -G1- is a 1,4-triazole derivative according to the formula: [ka] (wherein the two dotted bonds represent f3 and f4, which are covalent bonds or chemical groups as defined above and hereinafter).

[0258] In one embodiment, for formula X, the integer a=1 and the divalent radical -G1- is a 1,4-triazole derivative according to the formula: [ka] (In the formula: 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. * represents the moiety f2, which is an amide group, and the dotted bond represents f3, which may be either a carbon-nitrogen covalent bond or a carbon-aromatic carbon covalent bond.

[0259] In one embodiment, with respect to formula V, the integers a=c=1 and L are POx derivatives and the divalent radical -G1- is a 1,4-triazole derivative according to the formula: [ka] (In the formula: X1 is a linear, branched, or cyclic alkyl derivative, which may contain heteroatoms such as oxygen; X1 is an aromatic derivative; or X1 is a PEG derivative. * represents the moiety f2, which is an amide group, and the dotted bond represents f4, which may be either a carbon-nitrogen covalent bond or a carbon-aromatic carbon covalent bond.

[0260] In another embodiment, for formula X, the integer a=0 and the divalent radical -G1- is a 1,4-triazole derivative according to the formula: [ka] (In the formula: The * represents the moiety of f1, which is an ether group, and the dotted bond represents f3, which is a carbon-nitrogen covalent bond.

[0261] In another embodiment, with respect to formula V, the integers a=0, b=0, c=1 and L is a POx derivative and the divalent radical -G1- is a 1,4-triazole derivative according to the following formula: [ka] (In the formula: The * represents the moiety f1, which is an ether group, and the dotted bond represents f4, which is a carbon-nitrogen covalent bond.

[0262] In one embodiment, the cross-linking step is achieved by a 1,3-cycloaddition reaction between strained alkynes and azide derivatives, known as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0263] In one embodiment, for formula X, the integer a=1, the cross-linking step is achieved by a 1,3-cycloaddition reaction between a strained alkyne and an azide derivative, known as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0264] In one embodiment, with respect to Formula V, the integers a=c=1, b=0 and L are POx derivatives, and the cross-linking step is achieved by a 1,3-cycloaddition reaction between a strained alkyne and an azide derivative, known as strain-promoted azide-alkyne cycloaddition or SPAAC.

[0265] In one embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the formula: [ka] (In the formula: the dotted circle represents a cyclooctene derivative, which is derived from a strained cyclooctyne and may contain one heteroatom such as nitrogen, oxygen or sulfur, optionally functionalized with a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine; The two dotted bonds represent f3 and f4, which are covalent bonds or chemical bonds as defined above and below; In another embodiment, when a=b=0, the two dotted bonds represent f1 and f4; In another embodiment, when a=1 and b=0, the two dotted bonds represent f2 and f4; In a preferred embodiment, the dotted bond represents f2, an amide group, and f4, a carbon-nitrogen covalent bond.

[0266] In another embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the formula: [ka] (In the formula: the dotted circle represents a cyclooctene derivative, which is derived from a strained cyclooctyne and may contain one heteroatom such as nitrogen, oxygen or sulfur, optionally functionalized with a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine; ·X is a linear chain*-(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched, or cyclic alkyl derivatives, or X is a PEG derivative; The two dotted bonds represent f3 and f4, which are covalent bonds or chemical bonds as defined above and below; In another embodiment, when a=b=0, * represents the position of f1 and the dotted bond represents f4; In another embodiment, when a=1 and b=0, * represents the position of f1 and the dotted bond represents f4; In a preferred embodiment, X is a PEG derivative and the dotted bonds represent amide groups, f2 and f4.

[0267] In another embodiment, with respect to formula X, the divalent radical -G1- is a triazole derivative according to the formula: [ka] (In the formula: the dotted circle represents a cyclooctene derivative, which is derived from a strained cyclooctyne and may contain one heteroatom such as nitrogen, oxygen or sulfur, optionally functionalized with a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine; 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; 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; * represents the moiety of f2, which is an amide group, and the dotted bond represents f3, which is a carbon-nitrogen covalent bond, or an amide group, or a carbamate group.

[0268] In another embodiment, with respect to formula V, b=0 and c=1 and the divalent radical -G1- is a triazole derivative according to the formula: [ka] (In the formula: the dotted circle represents a cyclooctene derivative, which is derived from a strained cyclooctyne and may contain one heteroatom such as nitrogen, oxygen or sulfur, optionally functionalized with a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine; 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; 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; The * represents the moiety of f2, which is an amide group, and the dotted bond represents f4, which is a carbon-nitrogen covalent bond, or an amide group, or a carbamate group.

[0269] In a preferred embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the formula: [ka] (wherein * represents the moiety of f2, which is an amide group, and the dotted bond represents f4, which is a carbon-nitrogen covalent bond.)

[0270] In a preferred embodiment, with respect to formula X, the divalent radical -G1- is a triazole derivative according to the following formula: [ka] (wherein * represents the moiety of f2, which is an amide group, and the dotted bond represents f3, which is a carbon-nitrogen covalent bond.)

[0271] In another preferred embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the following formula: [ka] (In the formula: X is a PEG derivative, * represents the moieties f2 and f4, which are amide groups.

[0272] In another preferred embodiment, with respect to formula X, the divalent radical -G1- is a triazole derivative according to the following formula: [ka] (In the formula: 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; * represents the moieties f2 and f3, which are amide groups.

[0273] In another preferred embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the following formula: [ka] (In the formula: 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; * represents the moieties f2 and f4, which are amide groups.

[0274] In another embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the formula: [ka] (wherein * represents the site of f3, and the dotted bond represents f4.)

[0275] In another embodiment, with respect to formula V, the divalent radical -G1- is a triazole derivative according to the formula: [ka] (wherein the dotted bond represents f3, and * represents the site of f4.)

[0276] In one embodiment, the cross-linking step is achieved by a Diels-Alder cycloaddition reaction between a maleimide and a furan derivative.

[0277] In one embodiment, with respect to formula V, the divalent radical -G1- is a multi-ring derivative consisting of one succinimide moiety according to the formula: [ka] (In the formula: ·X is a linear chain*-(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched, or cyclic alkyl derivatives, or X is a PEG derivative; X1 is a straight chain*-(CH2) n1 -* (n1 is an integer from 0 to 7 (0≦n1≦7)), may be either a branched or cyclic alkyl derivative, X2 may be either -H or -Me; In one embodiment, when a=b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when a=1 and b=0, * represents the moieties f2 and f4.

[0278] In one embodiment, the crosslinking step is achieved by an inverse electron demand Diels-Alder reaction or IEDDA between a tetrazine and a norbornene derivative.

[0279] In one embodiment, with respect to formula V, the divalent radical -G1- is a multi-ring derivative consisting of one pyridazine moiety according to the formula: [ka] (In the formula: ·X is a linear chain*-(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, or X is an aromatic derivative, or X is a PEG derivative; In one embodiment, when a=b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when a=1 and b=0, * represents the moieties f2 and f4.

[0280] In another embodiment, with respect to formula V, the divalent radical -G1- is a multi-ring derivative consisting of one pyridazine moiety according to the formula: [ka] (In the formula: ·X is a linear chain*-(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, or X is an aromatic derivative, or X is a PEG derivative; In one embodiment, when a=b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when a=1 and b=0, * represents the moieties f2 and f4.

[0281] In one embodiment, the cross-linking step is achieved by Staudinger ligation between an aromatic phosphine and an azide derivative.

[0282] In one embodiment, with respect to formula V, the divalent radical -G1- is an aromatic derivative according to the formula: [ka] (In the formula: In one embodiment, when a=b=1, * represents the positions f3 and f4, In another embodiment, when a=b=0, * represents the positions f1 and f4. In another embodiment, when a=1 and b=0, * represents the moieties f2 and f4.

[0283] In one embodiment, with respect to Formula V, the cross-linking step is achieved by native chemical ligation (NCL) between a thioether and an N-terminal cysteine derivative.

[0284] In one embodiment, the divalent radical -G1- is formulated according to the following formula: [ka] (In the formula: The dotted line represents a carbon-nitrogen covalent bond. In one embodiment, when a=b=1, the * and dotted lines represent the positions f3 and f4. In another embodiment, when a=b=0, the * and dotted lines represent the positions f1 and f4. In another embodiment, when a=1 and b=0, the * and dotted lines represent the positions f2 and f4. For c=1: In one embodiment, f4 is an amine group. In another embodiment, f4 is an ether group. In another embodiment, f4 is a thioether group. In another embodiment, f4 is an amide group. In another embodiment, f4 is a carbamate group. In another embodiment, f4 is a carbon-nitrogen covalent bond. In another embodiment, f4 is a carbon-aromatic carbon covalent bond. In another embodiment, when the cross-linking step is performed by native chemical ligation (NCL), f4 is a carbon-carbon covalent bond.

[0285] In one embodiment, f3 is an amine group.

[0286] In another embodiment, f3 is an ether group.

[0287] In another embodiment, f3 is a thioether group.

[0288] In another embodiment, f3 is an amide group.

[0289] In another embodiment, f3 is a carbamate group.

[0290] In another embodiment, f3 is a carbon-nitrogen covalent bond.

[0291] In another embodiment, f3 is a carbon-aromatic carbon covalent bond.

[0292] The crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In the formula: i is an integer between 2 and 8 (2≦i≦8), a=1, · b=1, ·c=1, Dx is a dextran derivative according to formula III, L is a PEG central linker according to formula I, f1 is an ether group or a carbamate group; The divalent radical -A- is a straight-chain -(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, f2 is an amide group, The divalent radical -R1- is a straight-chain -(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, f3 is an amide group, The divalent radical -G1- is a 1,4-triazole derivative, f4 is a carbon-nitrogen covalent bond, especially a carbon-nitrogen covalent bond where the nitrogen atom is in the triazole ring.

[0293] In a preferred embodiment, the integer i is 4, i=4.

[0294] According to the above embodiment, the triazole derivatives include cyclooctene derivatives derived from strained cyclooctynes, which may contain one heteroatom such as nitrogen, oxygen or sulfur, and are optionally functionalized with a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine.

[0295] According to the two above embodiments, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains from 4 to 6 nitrogen atoms.

[0296] According to one embodiment, the 1,4-triazole derivatives are obtained via a copper-catalyzed reaction.

[0297] The crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In the formula: i is an integer between 2 and 8 (2≦i≦8), a=1, · b=0, ·c=1, Dx is a dextran derivative according to formula III, L is a PEG central linker according to formula I, The divalent radical -A- is a straight-chain -(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, f1 is an ether group or a carbamate group; the divalent radical -G1- is a cyclooctene derivative derived from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur, and which is optionally a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or a 1,4-triazole derivative functionalized with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine; The 1,4-triazole derivative has a nitrogen atom, which is linked to -CORa-, where Ra is an alkyl group containing 1 to 4 carbon atoms covalently bonded, thus forming an amide group, and Ra is linked to f2, which is an amide group, where f4 is a carbon-nitrogen covalent bond, and the nitrogen atom is in the triazole ring.

[0298] In a preferred embodiment, the integer i is four (i=4).

[0299] According to one embodiment, the 1,4-triazole is a multi-ring group containing an acyl group linked by an amide group to a nitrogen in one of the rings, but not from the triazole ring.

[0300] According to one embodiment, the 1,4-triazole comprises a cyclooctyne having a nitrogen in the cyclooctyne ring.

[0301] According to one embodiment, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains from 4 to 6 nitrogen atoms.

[0302] In one embodiment, the divalent radical -G1- is a 1,4-triazole derivative as described by the formula: [ka] (In the formula: * represents the site of f2, which is an amide group, and the dotted bond represents f4, which is a carbon-nitrogen covalent bond. f4 is a carbon-nitrogen covalent bond, and the nitrogen atom is in the triazole ring.

[0303] According to one embodiment, the 1,4-triazole is obtained via a copper-catalyzed reaction.

[0304] According to one embodiment, Dx is a dextran derivative according to formula III. [ka] wherein R is selected from the following: -H, an anionic group of formula II, or L(-) i -W- radicals with bridging linkers, i is between 20 and 5000 (20≦i≦5000), -W- and L(-) i The radicals have the meanings defined above.

[0305] According to one embodiment, L is a PEG central linker according to formula I: [ka] (In the formula: i is an integer equal to 4, p is an integer equal to 1, q is an integer between 8 and 1000 (8≦q≦1000), r is an integer of 0 or 1; Q is a branched alkyl chain containing from 2 to 10 carbon atoms; * represents the carbon-nitrogen covalent bond, f4, where the nitrogen atom is in the triazole ring.

[0306] The 1,4-triazoles are obtained via a copper-free reaction.

[0307] The invention also relates to the dextran polymer of formula VIII prior to the cross-linking reaction. [ka] During the ceremony, f1, f2, f3, Dx are defined as above when a, a', b, and b' are not 0. · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R1- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction.

[0308] In one embodiment, with respect 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) proparogyl derivative, or a 2-hydroxyalkyl carboxylate, or a 2-hydroxyalkylamine.

[0309] The invention also relates to the dextran polymer of formula XVIII prior to the cross-linking reaction. [ka] (In the formula, f1, f2, Dx are defined as above when a is not 0. · and When a is 0, f1 is an ether group and G'1 is a propargyl derivative; If a is not 0, then -A' is A as defined above.

[0310] In one embodiment, with respect to Formula XVIII, when a=1, A′ is an alkyl carboxylate derivative, or a 2-hydroxyalkyl carboxylate derivative, or a 2-hydroxyalkyl amine.

[0311] In one embodiment, with respect to Formula XVIII, when a=1, b=0 and L is a POx derivative, A is an alkyl carboxylate derivative, or a 2-hydroxyalkyl carboxylate derivative, or a 2-hydroxyalkylamine.

[0312] A' as a carboxylate derivative can be formalized as follows: [ka]

[0313] A' as an azide derivative can be formulated as follows, with reference to formula VIII: [ka]

[0314] A' as a propargyl derivative can be formulated as follows, with reference to formula VIII: [ka]

[0315] A' as a 2-hydroxyalkyl carboxylate derivative can be formulated as follows: [ka]

[0316] A' as a 2-hydroxyalkylamine derivative can be formulated as follows: [ka] (In the formula: n is an integer between 1 and 7 (1≦n≦7); m is an integer between 1 and 5 (1≦m≦5); f1 is defined as above.)

[0317] In one embodiment, with respect to Formula VIII, when a=1, R′1 is an alkyl radical or poly(oxyethylene) radical having a terminal amine, or terminal hydroxy, or terminal thiol, or terminal carboxylate, or terminal azide, or terminal alkyne.

[0318] R'1 can be formalized as follows: [ka]

[0319] R'1 as a carboxylate derivative can be formulated as follows: [ka]

[0320] R'1 as an azide derivative can be formulated as follows: [ka] (In the formula: n is an integer between 1 and 7 (1≦n≦7); X=-NH2, or -OH, or -SH, f2 is defined as above.)

[0321] Or, in another embodiment, with respect to Formula VIII, when a=0, R′1 is a branched alkyl with at least one hydroxy group attached to the alkyl chain in the β position from f1, and has a terminal hydroxy, or a terminal thiol, or a terminal azide, or a terminal alkyne.

[0322] R'1 can be formalized as follows: [ka] (In the formula: n is an integer between 1 and 5 (1≦n≦5); X=-NH2, or -OH, or -SH, or -N3, or -C≡CH; f1 is defined as above.)

[0323] Or, in another embodiment, R'1 is a propargyl derivative, which can be formulated as follows: [ka] (In the formula: n is an integer between 1 and 7 (1≦n≦7); f1 is defined as above.)

[0324] In one embodiment, with respect to Formula VIII, -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 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 hydroxy derivative.

[0325] In one embodiment, with respect to Formula XVIII, when a=1, 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.

[0326] In one embodiment, with respect to Formula VIII, when a=1, b=0, and L is a POx derivative, 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.

[0327] -G'1 as a thiol, amine, or hydroxy derivative can be formalized with respect to Formula VIII as follows: [ka]

[0328] G'1 as a thiol, amine, or hydroxy derivative can be formalized with respect to formula XVIII as follows: [ka]

[0329] G'1 as a maleimide can be formalized in terms of Formula VIII as follows: [ka]

[0330] G'1 as a maleimide can be formalized in terms of formula XVIII as follows: [ka]

[0331] G'1 as a vinyl sulfone can be formalized in terms of Formula VIII as follows: [ka]

[0332] G'1 as a vinyl sulfone can be formalized in terms of formula XVIII as follows: [ka]

[0333] G'1 as a strained cyclooctyne can be formalized in terms of formula VIII as follows: [ka]

[0334] G'1 as an azide derivative can be formulated as follows, with reference to formula VIII: [ka]

[0335] G'1 as an azide derivative can be formulated as follows, with reference to formula XVIII: [ka]

[0336] G'1 as a propargyl derivative can be formulated as follows, with reference to formula VIII: [ka]

[0337] G'1 as a propargyl derivative can be formulated as follows, with reference to formula XVIII: [ka] (In the formula: n is an integer between 0 and 7 (0≦n1≦7), R is a linear, branched, or cyclic alkyl derivative, or R is a PEG derivative; X=-NH2, or -OH, or -SH, X1 is an oxygen atom, a sulfur atom, or a CH2 group, f2 is defined as above.)

[0338] In one embodiment, with respect to Formula XVIII, when a=0, G′1 is a propargyl derivative.

[0339] G'1 as a propargyl derivative can be formalized with respect to formula XVIII as follows: [ka] (In the formula: f1 is defined as above.)

[0340] G'1 as a furan derivative can be formulated as follows, with respect to Formula VIII: [ka]

[0341] G'1 as an acrylamide derivative can be formulated as follows, with respect to Formula VIII: [ka]

[0342] G'1 as a norbornene derivative can be formulated as follows, with respect to formula VIII: [ka]

[0343] G'1 as a trans-cyclooctene derivative can be formulated as follows, with respect to Formula VIII: [ka]

[0344] G'1 as a tetrazine derivative can be formulated as follows, with respect to Formula VIII: [ka]

[0345] G'1 as an aromatic phosphine derivative can be formulated as follows, with respect to Formula VIII: [ka]

[0346] G'1 as a cysteine derivative can be formulated as follows, with reference to formula VIII: [ka]

[0347] G'1 as a thioester derivative can be formulated in terms of formula VIII as follows: [ka] (In the formula: X=-NH2, or -OH, or -SH, X1=-O- or -S-, n is an integer of 0 or 1, R1 = alkyl ·X2=-CH2-, or aromatic, R2=-H or -CH3, f3 is defined as above, The dotted bond represents f3, which is a carbon-nitrogen covalent bond or a carbon-carbon covalent bond.

[0348] The invention also relates to hydrogels comprising the crosslinked dextran polymers of the invention.

[0349] In one embodiment, the hydrogel is transparent.

[0350] By "transparent" is meant that an observer considers the hydrogel to be transparent compared to a standard 2 (6 NTU) as disclosed for visual inspection in Example C21 of patent application PCT / EP2022 / 050466 and / or the UV absorbance of the hydrogel is less than 0.06 (absorbance) as measured in Example C21 of patent application PCT / EP2022 / 050466.

[0351] In one embodiment, the hydrogel is visually clear and has a UV absorbance of less than 0.06 (absorbance).

[0352] In one embodiment, the hydrogels of the invention are characterized by a Tan δ of less than 1.

[0353] As used herein, Tan δ is the ratio of loss modulus G'' to storage modulus (also called elastic modulus) G' (Tan δ=G'' / G').

[0354] In one embodiment, the hydrogels of the invention are characterized by a Tan δ of 0.5 or less.

[0355] In one embodiment, the hydrogels of the invention are characterized by a Tan δ of 0.1 or less.

[0356] In one embodiment, the hydrogels of the invention are characterized by a Tan δ of 0.05 or less.

[0357] In one embodiment, the hydrogels of the invention are characterized by a Tan δ of 0.01 or less.

[0358] In one embodiment, the hydrogel of the invention is characterized in that after swelling in water it contains a concentration of cross-linked dextran polymers ranging from 0.01 to 0.2 g / g.

[0359] In one embodiment, the hydrogel of the invention is characterized in that after swelling in water, the concentration of cross-linked dextran polymer is comprised between 0.03 and 0.1 g / g.

[0360] In one embodiment, the hydrogel of the invention is characterized in that after swelling in water it contains a concentration of cross-linked dextran polymers ranging from 0.05 to 0.1 g / g.

[0361] In one embodiment, the hydrogel is translucent.

[0362] In another embodiment, the hydrogel is transparent.

[0363] In one embodiment, the hydrogel has a Young's modulus between 1 and 200 kPa.

[0364] In one embodiment, the hydrogel has a Young's modulus between 5 and 200 kPa.

[0365] In one embodiment, the hydrogel has a Young's modulus between 20 and 200 kPa.

[0366] In one embodiment, the hydrogel has a Young's modulus between 30 and 200 kPa.

[0367] In one embodiment, the hydrogel has a Young's modulus between 50 and 200 kPa.

[0368] In one embodiment, the hydrogel has a Young's modulus between 30 and 180 kPa.

[0369] In one embodiment, the hydrogel has a Young's modulus between 50 and 150 kPa.

[0370] In one embodiment, the hydrogel has a Young's modulus between 5 and 100 kPa.

[0371] In one embodiment, the hydrogel has a Young's modulus between 10 and 90 kPa.

[0372] In one embodiment, the hydrogel has a Young's modulus between 10 and 75 kPa.

[0373] In one embodiment, the hydrogel has a G' comprised between 0.5 and 70 kPa.

[0374] In one embodiment, the hydrogel has a compressive deformation at break of 10% or more.

[0375] In one embodiment, the hydrogel has a compressive deformation at break of 15% or greater.

[0376] In one embodiment, the hydrogel has a compressive deformation at break of 20% or more.

[0377] In one embodiment, the hydrogel has a compressive deformation at break of 25% or more.

[0378] In one embodiment, the hydrogel has a compressive deformation at break of 30% or greater.

[0379] In one embodiment, the hydrogel has a compressive deformation at break of 35% or greater.

[0380] In one embodiment, the hydrogel has a compressive deformation at break of 40% or greater.

[0381] In one embodiment, the hydrogel has a compressive deformation at break of 45% or greater.

[0382] In one embodiment, the hydrogel has a compressive deformation at break of 50% or more.

[0383] In one embodiment, the hydrogel has a compressive deformation at break of 55% or greater.

[0384] In one embodiment, the hydrogel has a compressive deformation at break of 60% or greater.

[0385] In one embodiment, the hydrogel has a tensile deformation at break of 10% or more.

[0386] In one embodiment, the hydrogel has a tensile deformation at break of 15% or greater.

[0387] In one embodiment, the hydrogel has a tensile deformation at break of 20% or more.

[0388] In one embodiment, the hydrogel has a tensile deformation at break of 25% or more.

[0389] In one embodiment, the hydrogel has a tensile deformation at break of 30% or greater.

[0390] In one embodiment, the hydrogel has a tensile deformation at break of 35% or greater.

[0391] In one embodiment, the hydrogel has a tensile deformation at break of 40% or greater.

[0392] In one embodiment, the hydrogel has a swelling ratio greater than 0.7.

[0393] In one embodiment, the hydrogel has a swelling ratio greater than 0.8.

[0394] In one embodiment, the hydrogel has a swelling ratio greater than 0.9.

[0395] In one embodiment, the hydrogel has a swelling ratio of greater than one.

[0396] In one embodiment, the hydrogel has a swelling ratio greater than 1.1.

[0397] In one embodiment, the hydrogel has a swelling ratio of 1.2 or greater.

[0398] In one embodiment, the hydrogel has a swelling ratio of 1.3 or greater.

[0399] In one embodiment, the hydrogel has a swelling ratio of 1.4 or greater.

[0400] In one embodiment, the hydrogel has a swelling ratio of 1.5 or greater.

[0401] In one embodiment, the hydrogel has a swelling ratio of 1.6 or greater.

[0402] In one embodiment, the hydrogel has a swelling ratio of 5 or less.

[0403] In one embodiment, the hydrogel has a swelling ratio of 4 or less.

[0404] In one embodiment, the hydrogel has a swelling ratio of 3 or less.

[0405] In one embodiment, the hydrogel has a swelling ratio of 2.8 or less.

[0406] In one embodiment, the hydrogel has a swelling ratio of 2.5 or less.

[0407] In one embodiment, the hydrogel has a swelling ratio of 2.3 or less.

[0408] In one embodiment, the moisture content is at least 80 wt%.

[0409] In one embodiment, the moisture content is at least 85 wt%.

[0410] In one embodiment, the moisture content is at least 90 wt%.

[0411] In one embodiment, the moisture content is at least 97 wt%.

[0412] In one embodiment, the moisture content is at least 96 wt%.

[0413] In one embodiment, the moisture content is at least 95 wt%.

[0414] In one embodiment, the moisture content is at least 94 wt%.

[0415] In one embodiment, the moisture content is at least 93 wt%.

[0416] In one embodiment, the moisture content is up to 99 wt%.

[0417] In one embodiment, the moisture content is up to 98 wt%.

[0418] In one embodiment, the hydrogel of the invention is further characterized by comprising biological cells.

[0419] In one embodiment, the cells are of human or animal origin.

[0420] In one embodiment, the cell is a cell line.

[0421] In one embodiment, the cells are stem cells.

[0422] In one embodiment, the stem cells are selected from embryonic stem cells, from induced pluripotent stem cells, or from mesenchymal stem cells.

[0423] In one embodiment, the cells are primary cells.

[0424] In one embodiment, the cell is a protein, hormone, or peptide secreting cell.

[0425] In one embodiment, the cells are selected from: insulin-secreting cells for diabetes treatment, Factor VIII or IX secreting cells for the treatment of hemophilia, and β-Glucocerebrosidase-secreting cells for Gaucher disease.

[0426] In one embodiment, the cells are selected from the group consisting of cells that secrete: growth hormone human growth hormone (hGH), Recombinant human growth hormone (rhGH), growth hormone-releasing hormone (GHRH), Thyroid-stimulating hormone (TSH), Thyrotropin-releasing hormone (TRH), Adrenocorticotropic hormone (ACTH), and Parathyroid hormone (PTH).

[0427] In one embodiment, the cells are selected from the group consisting of cells that secrete: glucagon, insulin, and GLP-1.

[0428] In one embodiment, the cells are selected from the group consisting of insulin-secreting cells.

[0429] In one embodiment, the cells are selected from the group consisting of insulin-secreting cells.

[0430] In one embodiment, the cells are selected from the group consisting of cells that secrete the following growth factors: vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), Transforming growth factors (TGFs), and Insulin-like growth factors-I and -II (IGF-I and IGF-II).

[0431] In one embodiment, the cells are selected from the group consisting of cells that secrete the following blood clotting factors: Factor I (e.g., fibrinogen), Factor II (e.g., prothrombin), Factor III (e.g., tissue factor), Factor V (e.g., proaccelerin, labile factor), factor VI, Factor VII (e.g., stabilizing factor, proconvertin), Factor VIII (e.g., antihemophilic factor A), factor VIIIC, Factor IX (e.g., antihemophilic factor B), Factor X (e.g., Stewart factor), Factor XI (e.g., plasma thromboplastin precursor), Factor XII (e.g., Hagman factor), Factor XIII (e.g., fibrin-stabilizing factor), von Willebrand factor (vWF), Prekallikrein, Heparin cofactor II, high molecular weight kininogens (e.g., Fitzgerald factor), Antithrombin III, and Fibronectin.

[0432] In one embodiment, the cell is selected from the group consisting of cells that secrete an immunoglobulin chain (long or short) or a fragment thereof comprising at least one immunoglobulin variable domain sequence, and optionally comprising an immunoglobulin Fc region.

[0433] In one embodiment, the cells are selected from the group consisting of cells that secrete a cytokine or cytokine receptor, or a chimeric protein comprising cytokines or their receptors.

[0434] In one embodiment, the cells are selected from the group consisting of cells that secrete erythropoietin.

[0435] In one embodiment, the cells are selected from the group consisting of cells that secrete the following interleukins (ILs): IL-1, IL-2 to IL-10.

[0436] In one embodiment, the cells are selected from the group consisting of cells that secrete the following replacement enzymes: Alpha-galactosidase A (GLA), alpha-L-iduronidase (IDVA), Arylsulfatase B (ARSB), Glucocerubrosidase, and N-sulfoglucosamine sulfohydrolase (SGSH).

[0437] In one embodiment, the hydrogel of the invention is characterized in that insulin-secreting cells are selected from the population of cells of the pancreas.

[0438] In one embodiment, the hydrogel of the invention is characterized in that the insulin-secreting cells are islets of Langerhans.

[0439] In one embodiment, the hydrogel of the invention is characterized in that the biological cells are pseudo-pancreatic islets.

[0440] The invention also relates to the use of the cross-linked dextran polymers of the invention for preparing cell compositions in the form of hydrogels.

[0441] In one embodiment, the cells are selected from one or more types of cells, may be isolated or aggregated, and may secrete an active ingredient.

[0442] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i -W- radicals, where i is 2, 4, or 8.

[0443] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to a dextran polymer backbone bearing a radical of i, where i is 2, 4, or 8.

[0444] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i -W- radicals, where i is 2.

[0445] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to a dextran polymer backbone bearing a radical of i, where i is 2.

[0446] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, where i is 4.

[0447] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to a dextran polymer backbone bearing a radical of i, where i is 4.

[0448] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, where i is 8.

[0449] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is covalently attached to a dextran polymer backbone bearing a radical of i, where i is 8.

[0450] The cross-linking step is the gelation step that leads to the formation of the hydrogel of the invention.

[0451] The kinetics of hydrogel formation is a function of temperature and can be controlled by reactant concentration, pH, and temperature.

[0452] In one embodiment, the time to obtain the inventive hydrogel is comprised between 1 minute and 6 hours.

[0453] In one embodiment, the cross-linking step is carried out for 1 hour.

[0454] In one embodiment, the temperature of the crosslinking step is comprised between 4° C. and room temperature (20-25° C.) and may vary between the mixing step and the gelling and molding step.

[0455] In one embodiment, mixing is carried out at 4° C. and gelation is carried out at room temperature (20-25° C.) for 1 hour.

[0456] In one embodiment, the mixing is carried out at room temperature (20-25° C.).

[0457] In one embodiment, mixing is carried out at 4° C. or room temperature (20-25° C.), and gelation is carried out at room temperature (20-25° C.) for 1 hour.

[0458] In one embodiment, the gelation is carried out at 37°C.

[0459] In one embodiment, after crosslinking or gelation, the hydrogel is swollen with a buffer solution, the pH of which is comprised between 5 and 8, preferably between 6 and 8, more preferably between 6.8 and 7.5.

[0460] In one embodiment, the buffer solution is a PBS solution at pH 7.4.

[0461] In one embodiment, the buffer solution is a Tris solution at pH 7.4.

[0462] In one embodiment, the buffer solution is a Tris solution at pH 8.

[0463] In one embodiment, swelling causes the mass of the hydrogel to increase by 1, 2, 3, or 4 times its initial mass.

[0464] The invention also relates to a method for synthesizing the crosslinked dextran polymer of the invention in the form of a hydrogel, the method comprising the steps of: a) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; b) L(-) i preparing a sterile solution of the precursor of c) adding the sterile solution obtained in step b) to the solution obtained in step a); d) Addition is either done directly to the mold or after mixing the solution is introduced into the mold; e) Crosslinking and gelling, for example at room temperature (20-25°C) or 37°C; f) Remove from the mold and allow to swell to obtain a hydrogel.

[0465] The invention also relates to a method for synthesizing the crosslinked dextran polymer of the invention in the form of a hydrogel, the method comprising the steps of: a) an anionic group of formula II and at least two -(A-f2) a -G1-, -(A'-f2) a preparing a sterile solution containing dextran with a precursor of -G'1-; b) L(-) i preparing a sterile solution of the precursor of c) adding the sterile solution obtained in step b) to the solution obtained in step a); d) Addition is either done directly to the mold or after mixing the solution is introduced into the mold; e) Crosslinking and gelling, for example at room temperature (20-25°C) or 37°C; f) Remove from the mold and allow to swell to obtain a hydrogel.

[0466] In one embodiment, steps c) and d) are performed simultaneously.

[0467] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[0468] Dextran having an anion of Formula II is prepared by grafting or substituting onto the hydroxyl groups of dextran. In one embodiment, dextran having an anion of Formula II is prepared by grafting or substituting onto the carboxymethyl groups of carboxymethyl dextran.

[0469] In one embodiment of the method of the invention, the active pharmaceutical ingredient (API) is entrapped in a hydrogel.

[0470] The invention also relates to the therapeutic use of the hydrogels of the invention as therapeutic implants for administering APIs to mammals.

[0471] The invention also relates to a method for preparing a hydrogel containing biological cells, the method comprising the steps of: a) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; b) L(-) i preparing a sterile solution of the precursor of c) preparing a suspension of biological cells; d) mixing the biological cell suspension obtained in step c) with the solution obtained in step b) or a); e) adding the sterile solution from step a) or b) not used in step d) to the solution from step d); f) adding step e) directly to the mold or introducing the solution into the mold after mixing; g) Crosslinking and gelling at room temperature (20-25°C); h) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

[0472] The invention also relates to a method for preparing a hydrogel containing biological cells, the method comprising the steps of: a) an anionic group of formula II and at least two -(A-f2) a -G1-, -(A'-f2) a preparing a sterile solution containing dextran with a precursor of -G'1-; b) L(-) i preparing a sterile solution of the precursor of c) preparing a suspension of biological cells; d) mixing the biological cell suspension obtained in step c) with the solution obtained in step b) or a); e) adding the sterile solution from step a) or b) not used in step d) to the solution from step d); f) adding step e) directly to the mold or introducing the solution into the mold after mixing; g) Crosslinking and gelling at room temperature (20-25°C); h) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

[0473] The invention also relates to a method for preparing a hydrogel containing biological cells, the method comprising the steps of: a) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; b) L(-) selected from thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene i preparing a sterile solution of the precursor of c) preparing a sterile solution of sodium hyaluronate; d) preparing a sterile suspension of biological cells; e) mixing the sodium hyaluronate solution obtained in step c) with the precursor solution from step b); f) mixing the biological cell suspension obtained in step d) with the liquid obtained in step e) or step a); g) mixing the liquid obtained in step f) with the liquid obtained in step e); h) adding the sterile solution from step a) or step e) not used in step g) to the solution from step f); i) The addition of step g) can be direct molding, in which the solution is introduced into a mold after mixing; j) crosslinking and gelling at room temperature (20-25°C); k) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

[0474] In one embodiment, the mold is a ring net.

[0475] The ring is an outer ring and consists of an upper and lower section that sandwich the net. The two sections of the ring and the net are glued together.

[0476] In one embodiment, the crosslinking and gelling reaction is carried out at room temperature (20-25° C.).

[0477] In one embodiment, the cross-linking and gelling reaction is carried out at a controlled temperature comprised between 15°C and 37°C.

[0478] In one embodiment, swelling is carried out in a PBS solution at pH 7.4.

[0479] In one embodiment, the hydrogel of the invention is characterized in that it further comprises biological cells.

[0480] In one embodiment, the cells are of human or animal origin.

[0481] In one embodiment, the cell is a cell line.

[0482] In one embodiment, the cells are stem cells.

[0483] In one embodiment, the stem cells are selected from embryonic stem cells, from induced pluripotent stem cells, or from mesenchymal stem cells.

[0484] In one embodiment, the cells are primary cells.

[0485] In one embodiment, the cell is a protein, hormone, or peptide secreting cell.

[0486] In one embodiment, the cells are selected from: insulin-secreting cells for diabetes treatment, Factor VIII or IX secreting cells for the treatment of hemophilia, and β-Glucocerebrosidase-secreting cells for Gaucher disease

[0487] In one embodiment, the hydrogel of the invention is characterized in that insulin-secreting cells are selected from the population of cells of the pancreas.

[0488] In one embodiment, the hydrogel of the invention is characterized in that the insulin-secreting cells are islets of Langerhans.

[0489] In one embodiment, the hydrogel of the invention is characterized in that the biological cells are pseudo-pancreatic islets.

[0490] The invention also relates to the therapeutic use of the hydrogels of the invention for treating a disorder or disease in a mammal, the disorder or disease resulting from a lack or malfunction of the endocrine function of the pancreatic organ.

[0491] The invention also relates to hydrogels for use as medicaments.

[0492] The invention also relates to hydrogels for use in the treatment of diseases such as diabetes.

[0493] The invention also relates to an implantable device comprising at least the inventive hydrogel and obtainable by the inventive method.

[0494] The invention also relates to implants comprising the hydrogels of the invention.

[0495] The invention also relates to implants comprising the hydrogels of the invention.

[0496] The invention also relates to an implant comprising the hydrogel of the invention and cells or pancreatic islets.

[0497] The invention also relates to a kit comprising: a solution of a dextran polymer of formula VIII prior to the crosslinking reaction; [ka] (In the formula, f1, f2, f3, f4, and Dx are defined as in formula IV when a, a', b, and b' are not 0; · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R1- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction), a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene, and Living cells.

[0498] The invention also relates to a kit comprising: a solution of a dextran polymer of formula VIII prior to the crosslinking reaction; [ka] (In the formula, f1, f2, f3, f4, and Dx are defined as in formula IV when a, a', b, and b' are not 0; · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R1- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction), a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells, A solution of non-crosslinked sodium hyaluronate.

[0499] The invention also relates to a kit comprising: a solution of a dextran polymer of formula VIII prior to the crosslinking reaction; [ka] (In the formula, f1, f2, f3, f4, and Dx are defined as in formula IV when a, a', b, and b' are not 0; · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction), a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells.

[0500] In one embodiment, at least 50% of the surface of the hydrogel is in direct contact with the implanted medium.

[0501] In one embodiment, at least 75% of the surface of the hydrogel is in direct contact with the implanted medium.

[0502] In one embodiment, at least 90% of the surface of the hydrogel is in direct contact with the implanted medium.

[0503] In one embodiment, at least 95% of the surface of the hydrogel is in direct contact with the implanted medium.

[0504] In one embodiment, 99% of the surface of the hydrogel is in direct contact with the implanted medium.

[0505] In one embodiment, at least 50% of the surface of the hydrogel is in direct contact with the exterior of the device or implant.

[0506] By "directly contacting the exterior" it is meant that there is no barrier between the hydrogel and the exterior, eg, no barrier of non-hydrogel material between the hydrogel and the exterior of the device or implant.

[0507] In one embodiment, at least 75% of the surface of the hydrogel is in direct contact with the exterior of the device or implant.

[0508] In one embodiment, at least 90% of the surface of the hydrogel is in direct contact with the exterior of the device or implant.

[0509] In one embodiment, at least 95% of the surface of the hydrogel is in direct contact with the exterior of the device or implant.

[0510] In one embodiment, at least 99% of the surface of the hydrogel is in direct contact with the exterior of the device or implant.

[0511] In one embodiment, 100% of the surface of the hydrogel is in direct contact with the exterior of the device or implant.

[0512] The cells or API are trapped within a labyrinth of cross-linked dextran hydrogel.

[0513] As used herein, the word "enclosed" is synonymous with "encapsulated" or "encapsulation."

[0514] The hydrogel matrix allows the passage of small molecules, such as nutrients and APIs, which are either entrapped in the hydrogel or secreted by entrapped cells.

[0515] Typically, APIs are hormone and peptide drugs selected from PTH protein, insulin, and clotting factors.

[0516] In one embodiment, the mesh size of the matrix is immunoisolatory and stops T lymphocytes to protect the cells.

[0517] In one embodiment, the mesh size is less than 1 μm.

[0518] In another embodiment, it is less than 100 nanometers, preferably less than 10 nanometers, and more preferably about 5 nanometers.

[0519] In one embodiment, the invention relates to an implant comprising a ring, a net, an inventive hydrogel, and cells.

[0520] In one embodiment, the implant is less than 3000 μm thick.

[0521] In one embodiment, the implant is less than 2000 μm thick.

[0522] In one embodiment, the implant is less than 1000 μm thick.

[0523] In one embodiment, the implant is less than 900 μm thick.

[0524] In one embodiment, the implant is greater than 300 μm thick.

[0525] In one embodiment, the implant is greater than 400 μm thick.

[0526] In one embodiment, the implant is greater than 500 μm thick.

[0527] In one embodiment, the implant has a total surface area of 10 cm 2 and 200cm 2 It is between.

[0528] In one embodiment, the implant has a total surface area of 15 cm 2 and 100cm 2 It is between.

[0529] In one embodiment, the implant contains from 0.5 to 20 ml of hydrogel.

[0530] In one embodiment, the implant contains 0.75 to 10 ml of hydrogel.

[0531] In one embodiment, the implant contains 0.8 to 5 ml of hydrogel.

[0532]

[0533] In one embodiment, the rings and implants are rectangular prisms, particularly rectangular prisms with rounded corners.

[0534]

[0535] The ring and net structure allows the hydrogels to be easily handled and tolerant to manipulations, including implantation, even for larger mesh-sized hydrogels (e.g., with lower DS of -W- during crosslinking and lower concentration of reactive groups).

[0536] The ring and net structure of the hydrogel makes it easy to handle and has good resistance to manipulation, including implantation. This is because the hydrogel with larger mesh size (e.g., -(A-f2) during crosslinking) a This is true even for -G1- (which has a lower DS and a lower concentration of reactive groups).

[0537] In one embodiment, the ring has an inner diameter of 10 to 100 mm.

[0538] In one embodiment, the ring has an inner diameter of 15 to 50 mm.

[0539] In one embodiment, the ring is 0.5 to 5 mm in diameter.

[0540] In one embodiment, the ring is 0.5 to 10 mm in diameter.

[0541] In one embodiment, the ring is 0.5 to 5 mm in diameter.

[0542] In one embodiment, the ring has a total thickness (bottom portion, top portion, and adhesive) of between 100 and 3000 μm.

[0543] In one embodiment, the ring has a total thickness (bottom portion, top portion, and adhesive) of 150 to 2000 μm.

[0544] In one embodiment, the ring has a total thickness of between 200 and 5000 μm.

[0545] In one embodiment, the ring has a total thickness of between 500 and 3000 μm.

[0546] In one embodiment, the ring has a rectangular, square, or circular cross section.

[0547] In one embodiment, the material of the ring is a bioinert material.

[0548] In one embodiment, the material of the ring is a biocompatible elastomer.

[0549] In one embodiment, the material of the ring is selected from the group consisting of silicones, in particular PDMS, polyurethanes, polyethers, polyether polyester copolymers, and polypropylene oxide.

[0550] In one embodiment, the ring material is silicone.

[0551] In one embodiment, the material of the ring is PDMS.

[0552] In one embodiment, the netting is non-biodegradable.

[0553] In one embodiment, the netting is biocompatible.

[0554] In one embodiment, the netting is non-absorbent.

[0555] In one embodiment, the net is a surgical mesh.

[0556] In one embodiment, the fibrous material of the netting material is selected from the group consisting of polypropylene, polyethylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride), and ePVDF (expanded PVDF).

[0557] In a preferred embodiment, the fiber material of the netting material is selected from the group consisting of PTFE, PVDF, and ePVDF.

[0558] In one embodiment, the fiber material of the netting is selected from the group consisting of polypropylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride).

[0559] In one embodiment, the fibrous material of the netting is selected from the group consisting of polypropylene and polyester, in particular PET.

[0560] In one embodiment, the fibrous material of the netting is selected from the group consisting of polypropylene.

[0561] In one embodiment, the fiber material of the netting is selected from the group consisting of polyesters, in particular PET.

[0562] In one embodiment, the netting has a thickness in the range of 50 to 500 μm.

[0563] In one embodiment, the netting has a thickness in the range of 100 to 300 μm.

[0564] In one embodiment, the diameter of the fibers ranges from 0.08 to 0.2 mm.

[0565] In one embodiment, the size of the holes in the net ranges from 0.4 to 4 mm.

[0566] In one embodiment, the size of the holes in the net ranges from 0.6 to 2 mm.

[0567] In one embodiment, the netting has a plurality of holes with a side size of 0.4 to 4 mm.

[0568] In one embodiment, the netting has a plurality of holes with a side size of 0.6 to 3 mm.

[0569] In one embodiment, the fabric of the netting is selected from the group consisting of knitted fabrics, warp knitted fabrics, woven fabrics, and nonwoven fabrics.

[0570] In one embodiment, the fabric of the net is selected from the group consisting of warp knit fabrics, in particular multifilament warp knit fabrics.

[0571] In one embodiment, the netting is treated to make it more hydrophilic.

[0572] In one embodiment, the netting is treated with a base, particularly for polyester, more particularly PET.

[0573] In one embodiment, this treatment is functionalization of the surface from reactive functional groups such as -OH, -COOH, and reactive molecules or polymers.

[0574] The grafted polymer may therefore expose reactive functional groups, such as thiol groups, for further reaction with hydrogels or precursors to hydrogels.

[0575] In one embodiment, this treatment is carried out by chemical or physical treatment followed by absorption of synthetic polymers such as poloxamer or polyvinylpyrrolidone (PVP), or natural polymers such as collagen, or surfactants.

[0576] In one embodiment, the netting remains below the outer edge of the ring.

[0577] In one embodiment, the net does not contact the exterior of the implant, which comprises the ring, net, and hydrogel.

[0578] In one embodiment, the adhesive is biocompatible.

[0579] In one embodiment, the adhesive is a biocompatible silicone adhesive such as Silbione MED ADH 4200 supplied by Elkem.

[0580] In one embodiment, the adhesive remains below the outer edge of the ring.

[0581] For example, warp knit polyester surgical netting fabric type PETKM3002 (pore size 1x0.9mm) supplied by the trademark SurgicalNet Inc. was treated in 1M NaOH at 70°C for 5 hours and rinsed with deionized water and 96% ethanol. The treatment increased the hydrophilicity of the netting fabric, which led to improved wetting by aqueous solutions.

[0582] For example, a ring net structure may be obtained according to the following method: Biocompatible PDMS sheets supplied by Grace Biolabs or Interstate Specialty Products are cut into square shapes with blank discs using a stainless steel punch. The treated polyester surgical net described above is introduced between two square PDMS pieces; the two PDMS pieces and the surgical net are glued together with a biocompatible silicone adhesive (Silbione MED ADH 4200, supplied by Elkem), with the blank discs aligned and the surgical net held taut during the bonding process. The square construct is then cut with a stainless steel punch, sandwiching the surgical net to obtain the final object consisting of two PDMS rings glued together, and Before steam sterilization, wash with 1% poloxamer F127 solution and rinse with water.

[0583] In one embodiment, the graft is obtained by: Incorporating the hydrogel composition into a ring mesh structure, mixing the concentrated polymer solution with a pipette, and introducing a controlled volume of the mixture into the ring mesh structure attached to a glass slide; Cross-linking is carried out, leading to gelation, and then the ring mesh structure and hydrogel composition are introduced into a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 or PBS at pH 7.4. The hydrogels were rinsed with cysteine-free PBS and further immersed in PBS solution at 37°C overnight, and the hydrogel pieces were stored in PBS solution at 4°C until use.

[0584] The hydrogel / ring mesh implant can be easily manipulated with forceps and folded as required for surgical implantation. The ring can be further secured with sutures.

[0585] The volume of the hydrogel can be adjusted by the inner diameter and thickness of the ring mesh structure. For the same ring mesh structure, the volume of the hydrogel can be adjusted to control the convex / concave surface of the hydrogel above the ring height.

[0586] 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.

[0587] Such a structure may be obtained by the method described in the present application, but by adding the hydrogel precursors in two steps, in the first step adding a hydrogel precursor that does not contain cells or pancreatic islets as a first layer, and in the second step adding a hydrogel precursor that contains cells or pancreatic islets as a second layer while the gelation of the first step is not yet complete, in particular at a time corresponding to 5 to 25% of the gelation time of the first hydrogel.

[0588] Figure 1 shows a graft (1) comprising a hydrogel (11) containing cells or pancreatic islets (not shown), a ring (12), and a net (13). The upper portion of the ring, the lower portion of the ring, and the net are glued together (not shown).

[0589] FIG. 2 shows a graft (1) comprising a hydrogel (11) containing cells or islets (not shown), a ring (12) and a net (13), where the hydrogel is concave.

[0590] FIG. 3 shows a top view of the implant (1) comprising a hydrogel (11) containing cells or islets (not shown), a ring (12), and a net (13).

[0591] Figure 4 shows an implant (1) comprising a cell-free hydrogel (20) sandwiching a cell-containing hydrogel (21). The upper portion of 20 is optional.

[0592] Figure 5 shows nonfasting blood glucose levels measured in control rats (Rats 1, 2, and 3) and rats transplanted with C16B-18 (Rat 4) or C16B-3A / C16B-3B (Rat 338). Rat 4 received 6200 IEQ, and Rat 338 received 4000 IEQ. The vertical dotted lines indicate the transplant, the removal of Rat 4, and the removal of Rat 338, respectively. The gray area with the dotted line indicates the blood glucose levels (minimum, maximum, and average) measured in the animals before diabetes was induced with streptozotocin. Blood glucose levels above 6 g / L were not measurable by the glucose meter (measured as High by the device) and were randomly assigned to 6.5 g / L.

[0593] The invention relates to all of the embodiments described below.

[0594] At least two saccharide units of dextran having an anionic group and belonging to two different polymer chains are linked to at least one central linker radical L(-) i wherein at least one radical is an at least divalent linear, branched, or cyclic alkyl radical comprising at least a polyethylene glycol chain or an at least divalent linear, branched, or cyclic alkyl radical comprising at least a poly(oxazoline) (POx) chain.

[0595] The problem is that at least two saccharide units of dextran, which have an anionic group and belong to two different polymer chains, are linked by at least one central linker radical L(-) i The problem is solved by providing a new cross-linked dextran polymer, which is covalently cross-linked by at least one radical, which is at least a divalent linear, branched, or cyclic alkyl radical containing at least a polyethylene glycol chain.

[0596] The problem is that at least two saccharide units of dextran, which have an anionic group and belong to two different polymer chains, are linked by at least one central linker radical L(-) iThe solution is provided by providing new cross-linked dextran polymers that are covalently cross-linked by at least one radical, which is at least a divalent linear, branched, or cyclic alkyl radical containing at least a poly(oxazoline) (POx) chain.

[0597] In one embodiment, the crosslinked dextran polymers of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with i W radicals, L(-) i is a linear or branched polyether having a heteroatom such as oxygen, nitrogen, or sulfur at the end, i is the valence of L, and -(R1) m G1 - the number of radicals, which is an integer from 2 to 8 (2≦i≦8), m is an integer of 0 or 1, W is -(R1) m G1 - radical, -R1- is a divalent linear or branched alkyl radical having from 1 to 6 carbon atoms and optionally containing a heteroatom such as oxygen, nitrogen, or sulfur; -G1- is a divalent linear or branched or cyclic alkyl radical containing from 1 to 6 carbon atoms and optionally containing heteroatoms such as oxygen, nitrogen, or sulfur.

[0598] In one embodiment, the dextran polymer comprises at least a divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, Mn is between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 15 and 600 (15≦DP≦600).

[0599] In one embodiment, the dextran polymer comprises at least one divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is not a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and at most eight arms, The number average molecular weight (Mn) is between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 8 and 1000 (8≦DP≦1000).

[0600] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether or a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, a polyether or poly(oxazoline) derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0601] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0602] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0603] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether or a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, a polyether or poly(oxazoline) derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0604] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0605] The crosslinked dextran polymer of the invention is a dextran polymer Dx having anionic groups, which has at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0606] In one embodiment, the crosslinked dextran hydrogel of the invention comprises a central linker L(-) i is a dextran polymer in which is a linear or branched polyethylene glycol (PEG) radical.

[0607] In one embodiment, the central linker L(-) i is selected from PEG of Formula I [ka] (In formula I, ·i is an integer between 2 and 8 (2≦i≦8). ·p is an integer of 0 or 1, and when i=2, p=0. ·q is an integer between 8 and 1000 (8≦q≦1000). r is an integer of 0 or 1. Q is a carbon atom or a straight, branched, or cyclic alkyl chain containing from 2 to 10 carbon atoms, and optionally containing heteroatoms such as nitrogen, oxygen, or sulfur, or may be aromatic. * represents the moiety f4, which is an amine group, or an ether group, or a thioether group, or an amide group, or a carbamate group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond if the crosslinking step is performed by native chemical ligation (NCL).

[0608] In one embodiment, the crosslinked dextran hydrogel of the invention is a dextran polymer in which the central linker L is a linear or branched POx radical.

[0609]

[0610] In one embodiment, the POx core linker is a two-arm POx selected from the linkers of formula XII. [ka] (In formula XII, The radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), branched chain, or cyclic alkyl derivatives. * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0611] In one embodiment, the POx central linker is a two-arm POx selected from the linkers of formula XIIbis. [ka] (In formula XIIbis, The radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), branched chain, or cyclic alkyl derivatives. * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0612] In one embodiment, the POx core linker is a 4-arm POx selected from the linkers of formula XIII. [ka] (In formula XIII, The radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), branched chain, or cyclic alkyl derivatives. * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0613] In another embodiment, the POx core linker is a 4-arm POx selected from the linkers of formula XIV. [ka] (In formula XIV, Radical -R1 is a straight chain -(CH2) n1-CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), or a branched or cyclic alkyl derivative. The divalent radical -R2- is a straight chain -(CH2) n2 -(n2 is an integer between 2 and 6 (2≦n2≦6)). * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0614] In another embodiment, the POx core linker is a 4-arm POx selected from the linkers of formula XV. [ka] (In formula XV, Radical -R is a straight chain -(CH2) n1 -CH3 (n1 is an integer between 0 and 4 (0≦n1≦4)), or a branched or cyclic alkyl derivative. In one embodiment, R1 = -CH2-CH2- and R2 is linear, -(CH2) n2 -(n2 is an integer between 2 and 6 (2≦n2≦6)). In another embodiment, R2 = -CH2-CH2- and R1 is linear, *-(CH2) n2 -*(n2 is an integer between 2 and 6 (2≦n2≦6)). * represents the moiety f3, which is an amine group, an ether group, a thioether group, an amide group, a carbamate group, a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond.

[0615] In one embodiment, -W- is selected from the radical of formula IV. [ka] (in formula IV * represents the site of f1, and ° represents the binding site with L. a is an integer of 0 or 1; b is an integer of 0 or 1; c is an integer of 0 or 1; In one embodiment, a=0 and f1 is an ether group or a carbamate group. In one embodiment, a=1, The divalent radical -A- is linear, -(CH2) n1 - (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, and at least one hydroxy group, -CH2-CH(OH)-(CH2) n2 -(n2 is an integer of 1 to 5 (1≦n2≦5)) which may be branched; f1 is an ether group or a carbamate group, and f2 is an amide group; or The divalent radical -A- is a linear polyether (PEG) derivative; f1 is an ether or carbamate group, and f2 is an amide group; or the divalent radical -A- is a 4-alkyl-1,4-triazole derivative or a 4-PEG-1,4-triazole derivative; f1 is an ether group or a carbamate group, and f2 is a carbon-nitrogen covalent bond; or the divalent radical -A- is a 1-alkyl-1,4-triazole derivative or a 1-PEG-1,4-triazole derivative; f1 is an ether group or a carbamate group, and f2 is a carbon-aromatic carbon covalent bond; the divalent radical -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, When b=0, f1 is an ether group or a carbamate group. When b=1, f1 is an ether group or a carbamate group, and f3 is an amide group, or an amine group, or an ether group, or a thioether group, or a carbamate group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond if the crosslinking step is performed by native chemical ligation (NCL); the divalent radical -G1- is a linear, branched, or cyclic alkyl derivative, or an aromatic derivative, which may contain heteroatoms such as up to 5 nitrogen atoms, up to 10 oxygen atoms, up to 5 sulfur atoms, or up to 1 phosphorus atom; in a preferred embodiment, -G1- is a succinimide derivative, or an alkylsulfone derivative which may contain one heteroatom such as oxygen or sulfur, or an ethylamide derivative, or a 1,4-triazole derivative, or a heterocyclic derivative from a Diels-Alder reaction, or an aromatic phosphine derivative produced by Staudinger ligation, or a cysteine derivative produced by native chemical ligation (NCL); When c=0, f1 is an ether group or a carbamate group; When c=1, f1 is an ether group or a carbamate group, and f4 is an amine group, or an amide group, or a carbamate group, or a thioether group, or an ether group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond, or a carbon-carbon covalent bond if the cross-linking step is by native chemical ligation (NCL).

[0616] The crosslinked dextran polymers of the invention are dextran polymers Dx- with anionic groups selected from dextran of formula X, which contain at least a divalent radical L(-) i The dextran polymer Dx- is covalently attached to the dextran polymer backbone with an i radical. [ka] (In formula X, a is an integer of 0 or 1; i is an integer between 2 and 8 (2≦i≦8), L may be linked to the same [Dx-f1-(A-f2)a-G1-f3] radical or to different radicals, Dx- is a part of dextran, which can be substituted by anionic groups in specific salt form and optionally by alkylcarboxylate derivatives in salt form; f1 is an ether group, The divalent radical -A- is a straight chain, -(CH2) n1 - (n1 is an integer from 1 to 7 (1≦n1≦7)), branched, or cyclic alkyl derivatives, and also contains at least one hydroxy group, -CH2-CH(OH)-(CH2) n2 - (n2 is an integer from 1 to 5 (1≦n2≦5)) may be branched, f2 is an amide group, the divalent radical -G1- is a linear, branched or cyclic alkyl derivative or an aromatic derivative, which may contain heteroatoms such as up to 5 nitrogen atoms, up to 10 oxygen atoms or up to 5 sulfur atoms; in a preferred embodiment, -G1- is a succinimide derivative or an 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 number of identical or different [Dx-f1-(A-f2)a-G1-f3] radicals attached to L; f3 is an amine group, or a thioether group, or an ether group, or an amide group, or a carbamate group, or a carbon-nitrogen covalent bond, or a carbon-aromatic carbon covalent bond; The central linker L is a poly(oxazoline) (POx) derivative and can be linear or branched.

[0617] In one embodiment, the crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] In formula V, f1, f2, f3, f4, -A-, -R1-, -G1- are defined as in formula IV above, and Dx- is a part of dextran, which may be substituted by specific anionic groups in the form of salts as defined above and optionally by alkylcarboxylate derivatives in the form of salts; 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 ] is the number of radicals, the central linker L is a polyether (PEG) derivative and can be linear or branched; In one embodiment, when b=0 and c=1, the central linker L can be a poly(oxazoline) (POx) derivative, which can be linear or branched.

[0618] In one embodiment, the crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In formula V, f1, f2, f3, f4, -A-, -R1-, -G1- are defined as in formula IV above, and Dx- is a moiety of dextran, as defined above, which may be substituted by specific anionic groups in salified form and optionally by alkylcarboxylate derivatives in salified form; 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 ] is the number of radicals, The central linker L is a polyether (PEG) derivative and can be linear or branched.

[0619] The hydroxy groups of the dextran polymer Dx- can be functionalized with at least one specific anionic group such as alkyl carboxylate, sulfate anion, sulfonate anion, phosphate anion, or phosphonate anion.

[0620] In one embodiment, the hydroxy groups of the dextran polymer Dx- are functionalized with one particular type of anionic group: an alkyl carboxylic acid ester anion.

[0621]

[0622] In one embodiment, the crosslinked dextran polymers having anionic groups of the invention are dextran polymers in which the dextran polymer backbone conforms to Formula III. [ka] (In formula III, R is H, an anionic group of formula II, or L(-) i -W- radicals having a cross-linker, i is between 20 and 5000 (20≦i≦5000), -W- and L(-) i The radicals have the meanings defined above.

[0623] In one embodiment, the crosslinked dextran polymers having anionic groups of the invention are dextran polymers in which the dextran polymer backbone conforms to Formula XI. [ka] In formula XI, R is H, an anionic group of formula II, or L(-) i (A-f2) with a cross-linker a -G1- radicals, l is in the range of 20 to 5000 (20≦l≦5000), -(A-f2) a -G1- and L(-) i has the meaning defined above.

[0624] In one embodiment, the crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In the formula: i is an integer between 2 and 8 (2≦i≦8), a=1, · b=1, ·c=1, Dx is a dextran derivative according to formula III, L is a PEG central linker according to formula I, f1 is an ether group or a carbamate group; The divalent radical -A- is a straight-chain -(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, f2 is an amide group, The divalent radical -R1- is a straight-chain -(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, f3 is an amide group, The divalent radical -G1- is a 1,4-triazole derivative, f4 is a carbon-nitrogen covalent bond, especially a carbon-nitrogen covalent bond where the nitrogen atom is in the triazole ring.

[0625] In a preferred embodiment, the integer i is 4, i=4.

[0626]

[0627] According to the above embodiment, the triazole derivatives include cyclooctene derivatives derived from strained cyclooctynes, which may contain one heteroatom such as nitrogen, oxygen or sulfur, and are optionally functionalized with a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine.

[0628] According to the two above embodiments, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains from 4 to 6 nitrogen atoms.

[0629] According to one embodiment, the 1,4-triazole derivatives are obtained by a copper-catalyzed reaction.

[0630] In one embodiment, the crosslinked dextran polymers of the invention are selected from dextran polymers of formula V: [ka] (In the formula: i is an integer between 2 and 8 (2≦i≦8), a=1, · b=0, ·c=1, Dx is a dextran derivative according to formula III, L is a PEG central linker according to formula I, The divalent radical -A- is a straight-chain -(CH2) n1 -* (n1 is an integer from 1 to 7 (1≦n1≦7)), branched or cyclic alkyl derivatives, f1 is an ether group or a carbamate group; the divalent radical -G1- is a cyclooctene derivative derived from a strained cyclooctyne, which may contain one heteroatom such as nitrogen, oxygen or sulfur, and which is optionally a linear, branched or cyclic alkyl derivative containing between 2 and 20 carbon atoms, or an aromatic derivative, or a 1,4-triazole derivative functionalized with a heteroatom such as nitrogen, oxygen or sulfur, or a halogen, in particular fluorine; The 1,4-triazole derivative has a nitrogen atom, which is linked to -CORa-, where Ra is an alkyl group containing 1 to 4 carbon atoms covalently bonded, thus forming an amide group, and Ra is linked to f2, which is an amide group, where f4 is a carbon-nitrogen covalent bond, and the nitrogen atom is in the triazole ring.

[0631] In a preferred embodiment, the integer i is four (i=4).

[0632]

[0633] According to one embodiment, the 1,4-triazole is a multi-ring group containing an acyl group linked by an amide group to a nitrogen in one of the rings, but not from the triazole ring.

[0634] According to one embodiment, the 1,4-triazole comprises a cyclooctyne having a nitrogen in the cyclooctyne ring.

[0635]

[0636] According to one embodiment, the triazole derivative contains more than 10 and less than 30 carbon atoms, and optionally contains from 4 to 6 nitrogen atoms.

[0637] In one embodiment, the divalent radical -G1- is a 1,4-triazole derivative as described by the formula: [ka] During the ceremony: * represents the site of f2, which is an amide group, and the dotted bond represents f4, which is a carbon-nitrogen covalent bond. ·f4 is a carbon-nitrogen covalent bond, with the nitrogen atom in the triazole ring.

[0638]

[0639]

[0640] According to one embodiment, the 1,4-triazole is obtained by a reaction that does not involve a copper catalyst.

[0641]

[0642] According to one embodiment, Dx is a dextran derivative according to formula III. [ka] wherein R is selected from the following: -H, an anionic group of formula II, or L(-) i -W- radicals with bridging linkers, -i is between 20 and 5000 (20≦i≦5000), -W- and L(-) i The radicals have the meanings defined above.

[0643] According to one embodiment, L is a PEG core linker according to formula I. [ka] During the ceremony: i is an integer equal to 4, p is an integer equal to 1, q is an integer between 8 and 1000 (8≦q≦1000), r is an integer of 0 or 1; Q is a branched alkyl chain containing from 2 to 10 carbon atoms; The * represents the f4 moiety, which is a carbon-nitrogen covalent bond, and the nitrogen atom is in the triazole ring.

[0644]

[0645] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to the dextran polymer backbone with i -W- radicals, where i is 2, 4, or 8.

[0646] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to the dextran polymer backbone having a radical of i, where i is 2, 4, or 8.

[0647] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to the dextran polymer backbone with i -W- radicals, where i is 2.

[0648] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) iis attached to a dextran polymer backbone bearing a radical of i, where i is 2.

[0649] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to the dextran polymer backbone with -W- radicals of i, where i is 4.

[0650] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to a dextran polymer backbone bearing a radical of i, where i is 4.

[0651] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to the dextran polymer backbone with -W- radicals of i, where i is 8.

[0652] In one embodiment, the crosslinked dextran polymer of the invention is a dextran polymer having an anionic group of formula II, wherein the dextran polymer has at least a divalent radical L(-) i is attached to the dextran polymer backbone with a radical of i, where i is 8.

[0653] In one embodiment, the crosslinked dextran polymers of the invention are dextran polymers in which the dextran polymer backbone is a dextran having a weight average molecular weight (Mw) before crosslinking and substitution comprised between 5 and 1000 kDa.

[0654] In one embodiment, the crosslinked dextran polymers of the invention are L(-) i -W- radical with a bridging linker or -(A-f2) aThe crosslinked dextran polymer has a degree of substitution (DS1) of the dextran backbone with -G1- radicals in the range of 0.001 to 0.4 (0.001≦DS1≦0.4).

[0655] In one embodiment, the crosslinked dextran polymers of the invention are crosslinked dextran polymers having a degree of sulfate, sulfonate, phosphate, or phosphonate substitution (DS3) of the dextran backbone in the range of 0.2 to 2.5 (0.2≦DS3≦2.5).

[0656] The invention also relates to the dextran polymer of formula VIII prior to the cross-linking reaction. [ka] (In the formula, f1, f2, f3, Dx are defined as above when a, a', b, and b' are not 0. · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R1- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction.

[0657] The invention relates to a hydrogel comprising: An anionic group and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone bearing the W radical of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of W radicals attached to the dextran polymer, and is an integer between 2 and 8 (2≦i≦8); A crosslinked dextran polymer Dx, wherein -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, or an aromatic ring, or a polyether derivative, and does not contain two or more alpha amino acid residues, particularly two or more alpha amino acid residues linked by peptide bonds.

[0658] In one embodiment, the hydrogel comprises non-crosslinked hyaluronate in the form of a solution.

[0659] The invention relates to a hydrogel comprising: Living cells, a non-crosslinked hyaluronic acid salt in the form of a solution, and It has an anionic group and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone bearing the W radical of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of W radicals attached to the dextran polymer, and is an integer between 2 and 8 (2≦i≦8); A crosslinked dextran polymer Dx, wherein -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, or an aromatic ring, or a polyether derivative, and does not contain two or more alpha amino acid residues, particularly two or more alpha amino acid residues linked by peptide bonds.

[0660] In one embodiment, the cross-linked dextran polymers contained in the hydrogel of the invention are not dextran polymers having carboxylate groups, but rather dextran polymers having at least two radicals L(-) i is covalently attached to the dextran polymer backbone with i W radicals, L(-) i is a linear or branched polyether having a heteroatom such as oxygen, nitrogen, or sulfur at the end, i is the valence of L, and -(R1) m G1 - the number of radicals, which is an integer from 2 to 8 (2≦i≦8), m is an integer of 0 or 1, W is -(R1) m G1 - radical, -R1- is a divalent linear or branched alkyl radical containing from 1 to 6 carbon atoms and optionally containing a heteroatom such as oxygen, nitrogen, or sulfur; -G1- is a divalent linear or branched or cyclic alkyl radical containing from 1 to 6 carbon atoms and optionally containing heteroatoms such as oxygen, nitrogen, or sulfur.

[0661] In one embodiment, the dextran polymer contained in the hydrogel comprises at least one divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and up to eight arms, Mn is between 1000 and 25000 g / mol (1000≦Mn≦25000 g / mol), or It is not a dextran polymer, with a degree of polymerization (DP) between 15 and 600 (15≦DP≦600).

[0662] In one embodiment, the dextran polymer contained in the hydrogel comprises at least one divalent radical L(-) covalently attached to the dextran polymer backbone having i W radicals. i is not a radical derived from a linear or branched mercaptopolyethylene glycol containing at least two sulfur atoms and at most eight arms, The number average molecular weight (Mn) is between 500 and 40,000 g / mol (500≦Mn≦40,000 g / mol), or It is not a dextran polymer with a degree of polymerization (DP) between 8 and 1000 (8≦DP≦1000).

[0663] The cross-linked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having an anionic group, and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether or a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, a polyether or poly(oxazoline) derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0664] The cross-linked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having an anionic group, and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0665] The crosslinked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having anionic groups, in which at least divalent radicals L are covalently bonded to the dextran polymer backbone having i -W- radicals, L is a linear or branched poly(oxazoline); i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0666] In one embodiment, the hydrogel comprises hyaluronic acid or sodium hyaluronate or potassium hyaluronate.

[0667] The cross-linked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having an anionic group, and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether or a linear or branched poly(oxazoline), i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one straight or branched chain alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, a polyether or poly(oxazoline) derivative, and not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0668] The cross-linked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having an anionic group, and at least a divalent radical L(-) i is covalently attached to the dextran polymer backbone with -W- radicals of i, L(-) i is a linear or branched polyether, i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen, or sulfur, an aromatic ring, or a polyether derivative, but not containing two or more alpha amino acid residues, particularly not two or more alpha amino acid residues linked by peptide bonds.

[0669] The crosslinked dextran polymer contained in the hydrogel of the invention is a dextran polymer Dx having anionic groups, in which at least divalent radicals L are covalently bonded to the dextran polymer backbone having i -W- radicals, L is a linear or branched poly(oxazoline); i is the valence of L and the number of -W- radicals attached to the dextran polymer, and is an integer between 2 and 8, inclusive (2≦i≦8); -W- is a radical containing at least one linear or branched alkyl radical, optionally containing heteroatoms such as oxygen, nitrogen, or sulfur, aromatic rings, polyether derivatives, and not containing two or more alpha amino acid residues, in particular not two or more alpha amino acid residues linked by peptide bonds

[0670] In one embodiment, the hydrogels of the invention are characterized by a Tan δ of less than 1.

[0671] In one embodiment, the hydrogel is transparent.

[0672] In one embodiment, the hydrogel is translucent.

[0673] In one embodiment, the hydrogel of the invention is characterized by a concentration of cross-linked dextran polymers after swelling in water of from 0.01 to 0.2 g / g.

[0674] In one embodiment, the hydrogel has a Young's modulus between 1 and 200 kPa.

[0675] In one embodiment, the hydrogel has a G' comprised between 0.5 and 70 kPa.

[0676] In one embodiment, the hydrogel has a compressive deformation at break of 10% or more.

[0677] In one embodiment, the hydrogel has a swelling ratio greater than 0.7.

[0678] In one embodiment, the moisture content is at least 80 wt%. In one embodiment, the hydrogel of the invention is further characterized by comprising biological cells.

[0679] In one embodiment, the cell is a protein, hormone, or peptide secreting cell.

[0680] In one embodiment, the cells are selected from: insulin-secreting cells for diabetes treatment, Factor VIII or IX secreting cells for the treatment of hemophilia, and β-Glucocerebrosidase-secreting cells for Gaucher disease.

[0681] In one embodiment, the hydrogel of the invention is characterized in that the biological cells are pseudo-pancreatic islets.

[0682] The invention also relates to a method for synthesizing the crosslinked dextran polymers of the invention in the form of a hydrogel, the method comprising the steps of: g) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; h) L(-) i preparing a sterile solution of the precursor of i) adding the sterile solution obtained in step b) to the solution obtained in step a); j) Addition is either directly to the mold or after mixing the solution is introduced into the mold; k) crosslinking and gelling, for example at room temperature (20-25°C) or 37°C; l) Remove from the mold and allow to swell to obtain a hydrogel.

[0683]

[0684] The invention also relates to a method for synthesizing the crosslinked dextran polymers of the invention in the form of a hydrogel, the method comprising the steps of: g) an anionic group of formula II and at least two -(A-f2) a -G1-, -(A'-f2) a preparing a sterile solution containing dextran with a precursor of -G'1-; h) L(-) i preparing a sterile solution of the precursor of i) adding the sterile solution obtained in step b) to the solution obtained in step a); j) Addition is either directly to the mold or after mixing the solution is introduced into the mold; k) crosslinking and gelling, for example at room temperature (20-25°C) or 37°C; l) Remove from the mold and allow to swell to obtain a hydrogel.

[0685] In one embodiment, steps c) and d) are performed simultaneously.

[0686] In one embodiment, swelling is performed in a PBS solution at pH 7.4.

[0687] Dextran having an anion of Formula II is prepared by grafting or substituting onto the hydroxyl groups of dextran. In one embodiment, dextran having an anion of Formula II is prepared by grafting or substituting onto the carboxymethyl groups of carboxymethyl dextran.

[0688] The cross-linking step is the gelation step that leads to the formation of the hydrogel of the invention.

[0689] The invention also relates to a method for preparing a hydrogel containing biological cells, the method comprising the following steps: i) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; j) L(-) i preparing a sterile solution of the precursor of k) preparing a suspension of biological cells; l) mixing the biological cell suspension obtained in step c) with the solution obtained in step b) or a); m) adding the sterile solution from step a) or b) not used in step d) to the solution from step d); n) adding step e) directly to the mold or introducing the solution into the mold after mixing; o) crosslinking and gelling at room temperature (20-25°C); p) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

[0690] The invention also relates to a method for preparing a hydrogel containing biological cells, the method comprising the following steps: i) an anionic group of formula II and at least two -(A-f2) a -G1-, -(A'-f2) a preparing a sterile solution containing dextran with a precursor of -G'1-; j) L(-) i preparing a sterile solution of the precursor of k) preparing a suspension of biological cells; l) mixing the biological cell suspension obtained in step c) with the solution obtained in step b) or a); m) adding the sterile solution from step a) or b) not used in step d) to the solution from step d); n) adding step e) directly to the mold or introducing the solution into the mold after mixing; o) crosslinking and gelling at room temperature (20-25°C); p) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

[0691] The invention also relates to a method for preparing a hydrogel containing biological cells, the method comprising the following steps: l) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; m) L(-) selected from thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene i preparing a sterile solution of the precursor of n) preparing a sterile solution of sodium hyaluronate; o) preparing a sterile suspension of biological cells; p) mixing the sodium hyaluronate solution obtained in step c) with the precursor solution from step b); q) mixing the biological cell suspension obtained in step d) with the liquid obtained in step e) or step a); r) mixing the liquid obtained in step f) with the liquid obtained in step e); s) adding the sterile solution from step a) or step e) that was not used in step g) to the solution from step f); t) The addition of step g) can be direct molding, in which the solution is introduced into the mold after mixing; u) crosslinks and gels at room temperature (20-25°C); v) The mixture is removed from the mold and allowed to swell to obtain a hydrogel containing living cells.

[0692] According to one embodiment, in the forming step, the solution comprises an osmotic agent that is a non-ionic osmotic agent, such as trehalose.

[0693] According to one embodiment, in the forming step the solution has a weight ratio of non-ionic osmotic agent to NaCl of greater than 2, in particular greater than 5, more in particular greater than 10.

[0694] According to one embodiment, in the forming step, the solution contains a non-ionic osmotic agent, in particular trehalose, at 5 to 50 mg / ml.

[0695]

[0696] In one embodiment, the mold is a ring net.

[0697] In one embodiment, the crosslinking and gelling reaction is carried out at room temperature (20-25° C.).

[0698] The invention also relates to a kit comprising: A solution of dextran polymer of formula VIII before the cross-linking reaction. [ka] (In the formula, f1, f2, f3, f4, and Dx are defined as in formula IV when a, a', b, and b' are not 0; · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction), a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells.

[0699] The invention also relates to a kit comprising: a solution of a dextran polymer of formula VIII prior to the crosslinking reaction; [ka] (In the formula, f1, f2, f3, f4, and Dx are defined as in formula IV when a, a', b, and b' are not 0; · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R1- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction. a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells, A solution of non-crosslinked sodium hyaluronate.

[0700] The invention also relates to a kit comprising: a solution of a dextran polymer of formula VIII prior to the crosslinking reaction; [ka] (In the formula, f1, f2, f3, f4, and Dx are defined as in formula IV when a, a', b, and b' are not 0; · and x is 0 or 1, When a, a', b, and b' are 0, x is 0, Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, -A'- is -A- as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; when c is not 0, -R'1- is -R1- as defined above and -G'1- is a precursor of -G1-, When c is 0, b is 0 and R'1 is a precursor of R1 before the crosslinking reaction), a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells.

[0701] The invention also relates to the use of the cross-linked dextran polymers of the invention for preparing cell compositions in the form of hydrogels.

[0702] The invention also relates to the therapeutic use of the hydrogels of the invention as therapeutic implants for administering APIs to mammals.

[0703] The invention also relates to the therapeutic use of the hydrogels of the invention for treating a disorder or disease in a mammal, the disorder or disease resulting from a lack or malfunction of the endocrine function of the pancreatic organ.

[0704] The invention also relates to hydrogels for use as medicaments.

[0705] The invention also relates to hydrogels for use in the treatment of diseases such as diabetes.

[0706] The invention also relates to implants comprising the hydrogels of the invention.

[0707] In one embodiment, the invention relates to a ring, a net, an implant comprising the inventive hydrogel and cells.

[0708] The ring and net structure allows the hydrogels to be easily handled and tolerant to manipulations, including implantation, even for larger mesh-sized hydrogels (e.g., with lower DS of -W- during crosslinking and lower concentration of reactive groups).

[0709] The ring and net structure of the hydrogel makes it easy to handle and has good resistance to manipulation, including implantation. This is because the hydrogel with larger mesh size (e.g., -(A-f2) during crosslinking) a This is true even for -G1- (which has a lower DS and a lower concentration of reactive groups).

[0710] The ring and net structure makes the hydrogel easy to handle and well resistant to manipulations, including implantation, where the hydrogel is very thin and has a fairly large flat surface.

[0711] In one embodiment, the implant is a rectangular prism with rounded corners.

[0712] In one embodiment, the implant is less than 3000 μm thick.

[0713] In one embodiment, the implant has a total surface area of 10 cm 2 and 200cm 2 It is between.

[0714] In one embodiment, the implant contains from 0.5 to 20 ml of hydrogel.

[0715] In one embodiment, the ring has an inner diameter of 10 to 100 mm.

[0716] In one embodiment, the ring is a rectangular parallelepiped with rounded corners.

[0717] In one embodiment, the material of the ring is a bioinert material.

[0718] In one embodiment, the material of the ring is a biocompatible elastomer.

[0719] In one embodiment, the material of the ring is selected from the group consisting of silicones, in particular PDMS, polyurethanes, polyethers, polyether polyester copolymers, and polypropylene oxide.

[0720] In one embodiment, the netting is non-biodegradable.

[0721] In one embodiment, the netting is biocompatible.

[0722] In one embodiment, the netting is non-absorbent.

[0723] In one embodiment, the net is a surgical mesh.

[0724] In one embodiment, the fibrous material of the netting material is selected from the group consisting of polypropylene, polyethylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride), and ePVDF (expanded PVDF).

[0725] In one embodiment, the thickness of the net is in the range of 50 to 500 μm.

[0726] In one embodiment, the size of the holes in the net ranges from 0.4 to 4 mm.

[0727] In one embodiment, the netting has a plurality of holes with side sizes ranging from 0.4 to 4 mm.

[0728] In one embodiment, the fabric of the netting is selected from the group consisting of knitted fabrics, warp knitted fabrics, woven fabrics, and nonwoven fabrics.

[0729] In one embodiment, the graft is obtained by: Incorporating the hydrogel composition into a ring mesh structure, mixing the concentrated polymer solution with a pipette, and introducing a controlled volume of the mixture into the ring mesh structure attached to a glass slide; Cross-linking is carried out, leading to gelation, and then the ring mesh structure and hydrogel composition are introduced into a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 or PBS at pH 7.4. The hydrogel is rinsed with cysteine-free PBS and further immersed in PBS solution at 37°C overnight, and the hydrogel pieces are stored in PBS solution at 4°C until use. Example Part A - Chemistry Example A1: Synthesis of substituted dextrans [Table 1] TIFF2025527131000124.tif178166TIFF2025527131000125.tif190166TIFF2025527131000126.tif173166TIFF2025527131000127.tif195166TIFF2025527131000128.tif230147TIFF2025527131000129.tif197139TIFF2025527131000130.tif150139Polysaccharide 1-dextran methylcarboxylate sulfate and maleimide Polysaccharide 1,1-dextran methylcarboxylate

[0730] Fifty grams of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molecular weight of 40 kg / mol (0.31 mol of glycoside units, 0.93 mol of hydroxyl groups) was dissolved in water (225 g / L) at 30 °C. NaBH4 (2 × 58 mg, 2 × 1.54 mmol) was then added every 30 min, and the mixture was stirred at 30 °C for 1 h. Sodium chloroacetate (72 g, 0.62 mol) was added to the solution, and the mixture was heated at 65 °C for 1 h. 10 N NaOH (103 mL, 1.03 mol) was then slowly added over 1.5 h, and the mixture was stirred at 65 °C for 1 h. The mixture was diluted with water (85 mL), cooled to room temperature, neutralized with acetic acid, and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer and then against water. The concentration of polysaccharide 1.1 in the final solution was determined by dry concentration, and an acidity / baseness analysis was performed to determine the degree of methyl carboxylate substitution.

[0731] From the dry concentrate: [Polysaccharide 1.1] = 46.3 m / g

[0732] Acidity / baseness analysis revealed that the degree of substitution of methyl carboxylate (DS2) was 0.8. Polysaccharide 1,2-dextran methyl carboxylic acid

[0733] 550 g of the polysaccharide 1.1 solution obtained above (46.3 mg / g, DS = 0.8, 25.5 g, 112.6 mmol of glucoside units) was protonated with sulfonated resin (Purolite C100H, 2.0 eq / L, 200 mL, 400 mmol) for 2 h. The resulting solution was filtered and lyophilized. Polysaccharide 1,3-dextran methylcarboxylate sulfate

[0734] Lyophilized polysaccharide 1.2 (10.0 g, 48.0 mmol of glucoside units) was dissolved in a mixture of DMF (320 mL) and formamide (80 mL). After complete dissolution, 2-methyl-2-butene (80 mL, 755.2 mmol) was slowly added. SO3-DMF complex (58.5 g, 383.9 mmol) was rapidly added, and the reaction mixture was stirred at 30 °C for 3 h. The mixture was neutralized by slowly adding 5% aqueous NaHCO3 (800 mL) and purified by ultrafiltration through a PES membrane (MWCO 5 kDa) against 30% v / v ethanol, 9 g / L aqueous NaCl, and then water. The concentration of polysaccharide 1.3 in the final solution was determined from the dried concentrate. The degree of sulfate substitution was determined by liquid chromatography after complete sulfate hydrolysis of a representative sample.

[0735] From the dried concentrate, [polysaccharide 1.3] = 39.6 mg / g

[0736] LC analysis revealed that the degree of sulfate substitution (DS3) was 1.5. Polysaccharide 1-dextran methylcarboxylate sulfate and maleimide

[0737] To 303 g of the solution of polysaccharide 1.3 obtained above (39.6 mg / g, DS3 = 1.5, DS2 = 0.8, 12.0 g, 31.64 mmol of glycoside units), 2-hydroxypyridine 1-oxide (HOPO) (1.76 g, 15.82 mmol) was added, and the mixture was cooled to 4 °C. To this solution, 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, and the reaction mixture was stirred at 4 °C for 2 h. Two additional additions of EDC (3.03 g, 15.82 mmol) were made every 2 h. The mixture was diluted with pH 7 phosphate buffer and purified by ultrafiltration through a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer, 9 g / L NaCl aqueous solution, and then water. The concentration of polysaccharide 1 in the final solution was determined from the dried concentrate. The degree of maleimide substitution was determined by the concentration of 1 in D2O. 1 The NMR was determined. The final solution was stored at -20°C.

[0738] From the dried concentrate, [polysaccharide 1] = 22.2 mg / g

[0739] 1 H NMR (DO) showed that the degree of maleimide substitution (DS1) was 0.25. Polysaccharide 2-dextran methylcarboxylate sulfate and maleimide Polysaccharide 2.1-dextran methylcarboxylate

[0740] Using a method similar to that used to prepare polysaccharide 1.1, starting from dextran with a weight-average molecular weight of 40 kg / mol (0.25 mol of glycoside units, 0.75 mol of hydroxyl groups, and a degree of polymerization of n = 205), polysaccharide 2.1 was obtained at 60 °C using sodium chloroacetate (35.9 g, 0.31 mol) and 10 N NaOH (82 mL, 0.82 mol).

[0741] From the dried concentrate, [polysaccharide 2.1] = 44.2 mg / g

[0742] Acidity / basicity analysis showed that the degree of substitution of methyl carboxylate (DS2) was 0.5. Polysaccharide 2.2-dextran methyl carboxylic acid

[0743] Using a method similar to that used to prepare polysaccharide 1.2, polysaccharide 2.2 was obtained starting from polysaccharide 2.1 (44.2 mg / g, DS2 = 0.5, 12.0 g, 59.4 mmol of glycoside units). Polysaccharide 2,3-dextran methylcarboxylate sulfate

[0744] Using a method similar to that used to prepare polysaccharide 1.3, polysaccharide 2.3 was obtained starting from polysaccharide 2.2 (4.0 g, 20.9 mmol of glycoside units).

[0745] From the dried concentrate, [polysaccharide 2.3] = 22.0 mg / g

[0746] LC analysis revealed that the degree of sulfate substitution (DS3) was 1.9. Polysaccharide 2-dextran methylcarboxylate sulfate and maleimide

[0747] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 2.3 (22.0 mg / g, DS2 = 0.5, DS3 = 1.9, 4.6 g, 11.6 mmol of glycoside units) was obtained. Starting from and using N-(2-aminoethyl)maleimide hydrochloride (615 mg, 3.48 mmol), polysaccharide 2 was obtained.

[0748] From the dried concentrate, [polysaccharide 2] = 12.3 mg / g

[0749] 1 H NMR (DO) revealed that the degree of maleimide substitution (DS1) was 0.23. Polysaccharide 3-dextran methylcarboxylate sulfate and vinyl sulfone

[0750] HOPO (1.46 g, 13.18 mmol) was added to 280 g of polysaccharide 1.3 solution (35.7 mg / g, DS3 = 1.5, DS2 = 0.8, 10.0 g, glycoside unit 26.37 mmol), and the mixture was cooled to 4 °C. (Ethenylsulfonyl)ethyl]thio]ethanamine hydrochloride (VS) (1.83 g, 7.91 mmol) (synthesized according to SA Stewart et al., Soft Matter, 2018, 14, 8317), triethylamine (1.10 mL, 7.91 mmol), and EDC (2.53 g, 13.18 mmol) were added, and the reaction mixture was stirred between 4 °C and 25 °C for 2 h. Two additional additions of EDC (2.53 g, 13.18 mmol) were made every 2 h. The mixture was diluted with 9 g / L aqueous NaCl and purified by ultrafiltration through a PES membrane (MWCO 5 kDa) against 9 g / L aqueous NaCl, pH 10 carbonate buffer, 9 g / L aqueous NaCl, pH 7 phosphate buffer, 9 g / L aqueous NaCl, and then water. The concentration of polysaccharide 3 in the final solution was determined from the dry concentrate. The degree of vinyl sulfone substitution was determined by the solubility of VS in DO. 1 The NMR was determined. The final solution was stored at -20°C.

[0751] From the dried concentrate, [polysaccharide 3] = 21.8 mg / g

[0752] 1 From H NMR (DO), the degree of vinyl sulfone substitution (DS1) was 0.28 Polysaccharide 4-dextran methylcarboxylate sulfate and maleimide Polysaccharide 4.1-dextran methylcarboxylate

[0753] Dextran (Pharmacosmos, 65 g, 0.4 mol glycoside units, 1.2 mol hydroxyl groups) with a weight-average molecular weight of 40 kg / mol (degree of polymerization, n = 205) was dissolved in water (285 g / L) at 30 °C. NaBH4 (74 mg, 1.95 mmol) was then added, and the mixture was stirred at 30 °C for 2 h. Sodium chloroacetate (140 g, 1.2 mol) was added to the solution, and the mixture was heated at 65 °C for 1 h. 10 N NaOH (200 mL, 2 mol) was then slowly added over 1.5 h, and the mixture was stirred at 65 °C for 1 h. The mixture was diluted with water (120 mL), cooled to room temperature, neutralized with acetic acid, and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer and then against water. The intermediate polysaccharide (polysaccharide 4.1.1) was lyophilized.

[0754] Ninety grams of lyophilized polysaccharide 4.1.1 (0.35 mol of glycoside units) was dissolved in water at 65 °C (260 g / L). Sodium chloroacetate (204 g, 1.75 mol) was added, and the mixture was maintained at 65 °C for 1 h. 10 N NaOH (175 mL, 1.75 mol) was added slowly over 1 h, and the mixture was stirred at 65 °C for an additional 1 h. Another portion of sodium chloroacetate (122 g, 1.05 mol) was added, and the mixture was maintained at 65 °C for 30 min. 10 N NaOH (105 mL, 1.05 mol) was added slowly over 1 h, and the mixture was stirred at 65 °C for an additional 1 h. The mixture was diluted with water, cooled to room temperature, neutralized with acetic acid, and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer and then against water. The concentration of polysaccharide 4.1 in the final solution was determined by dry concentration, and the degree of methyl carboxylate substitution was determined by acidity / baseness analysis.

[0755] From the dried concentrate, [polysaccharide 4.1] = 48.4 mg / g

[0756] Acidity / baseness analysis revealed that the degree of substitution of methyl carboxylate (DS2) was 2.1. Polysaccharide 4,2-dextran methylcarboxylate sulfate

[0757] To 400 g of the polysaccharide 4.1 solution (48.4 mg / g, DS = 2.1, 19.4 g, glycoside units 58.63 mmol) obtained above, HOPO (9.77 g, 87.95 mmol), 3-amino-1-propanesulfonic acid (homotaurine) (9.79 g, 70.36 mmol), triethylamine (9.81 mL, 70.36 mmol), and EDC (16.86 g, 87.95 mmol) were added, and the reaction mixture was stirred at 25 °C for 2 h. Two additional additions of EDC (16.86 g, 87.95 mmol) were made every 2 h. The mixture was diluted with pH 7 phosphate buffer and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer, 9 g / L NaCl solution, and then water. The concentration of polysaccharide 4.2 in the final solution was determined by dry concentrate in D2O. 1 The degree of homotaurine substitution was determined by H NMR. The final solution was stored at 4 °C.

[0758] From the dried concentrate, [polysaccharide 4.2] = 40.9 mg / g

[0759] 1 H NMR (DO) showed a homotaurine substitution degree (DS3) of 1.15 Polysaccharide 4-dextran methylcarboxylate sulfate and maleimide

[0760] Using a method similar to that used to prepare polysaccharide 1, polysaccharide 4.2 (40.9 mg / g, DS2 = 2.1, DS3 = 1.15, 9.4 g, 20.0 mmol of glycoside units) was prepared using N-(2-aminoethyl)maleimide hydrochloride (1.06 g, 6.0 mmol).

[0761] From the dried concentrate, [polysaccharide 4] = 19.9 mg / g

[0762] 1 H NMR (DO) showed that the degree of maleimide substitution (DS1) was 0.25. Polysaccharide 5-dextran methylcarboxylate ammoniosulfate and maleimide Polysaccharide 5.1-dextran methylcarboxylate ammoniosulfate

[0763] Using a method similar to that used to prepare polysaccharide 4.2, starting from polysaccharide 4.1 (48.4 mg / g, DS = 2.1, 7.3 g, 22.11 mmol of glycoside units), polysaccharide 5.1 was obtained using HOPO (2.95 g, 26.53 mmol), 3-((2-aminoethyl)-dimethylammonio)propane-1-sulfonate (SB) (7.51 g, 26.53 mmol) (synthesized according to L. Yang et al., J. Mater. Chem. B, 2013, 1, 1421), triethylamine (7.4 mL, 53.06 mmol), and EDC (3 × 5.09 g, 3 × 26.53 mmol).

[0764] From the dried concentrate, [polysaccharide 5.1] = 34.6 mg / g

[0765] 1 H NMR (DO) showed SB substitution degree (DS3) = 0.7 Polysaccharide 5-dextran methylcarboxylate ammoniosulfate and maleimide

[0766] Using a method similar to that used to prepare polysaccharide 1, starting with polysaccharide 5.1 (34.6 mg / g, DS2 = 2.1, DS3 = 0.7, 7.5 g, 16.7 mmol of glycoside units), polysaccharide 5 was obtained using N-(2-aminoethyl)maleimide hydrochloride (885 mg, 5.01 mmol).

[0767] From the dried concentrate, [polysaccharide 5] = 15.0 mg / g

[0768] 1 H NMR (DO) revealed that the degree of maleimide substitution (DS1) was 0.22. Polysaccharide 6-dextran methylcarboxylate and cyclooctyne (DBCO)

[0769] To 30 g of polysaccharide 4.1 solution (48.4 mg / g, DS = 2.1, 1.45 g, glycoside unit 4.39 mmol) 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]azocin-5(6H)-yl)-1-propanone (DBCO-NH) (365 mg, 1.32 mmol) and EDC (422 mg, 2.20 mmol) in DMF (5 mL), and the reaction mixture was stirred between 4 °C and 25 °C for 2 h. Two additional additions of EDC (422 mg, 2.20 mmol) were made every 2 h. The mixture was diluted with pH 7 phosphate buffer and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer, 9 g / L NaCl aqueous solution, and then water. The concentration of polysaccharide 6 in the final solution was determined by dry concentration and was calculated as 100% in D2O. 1 The degree of DBCO-NH substitution was determined by H NMR. The final solution was stored at -20 °C.

[0770] From the dried concentrate, [polysaccharide 6] = 6.1 mg / g

[0771] 1 From H NMR (DO), DBCO-NH substitution degree (DS1) = 0.28 Polysaccharide 7-dextran methylcarboxylate and azide

[0772] Using a method similar to that used for the preparation of polysaccharide 1 (steps from polysaccharide 1.3 to polysaccharide 1), starting from polysaccharide 4.1 (48.4 mg / g, DS = 2.1, 6.2 g, 18.8 mmol of glycoside units), polysaccharide 7 was obtained using 11-azido-3,6,9-trioxaundecan-1-amine (N-PEG-NH) (1.23 g, 5.64 mmol).

[0773] From the dried concentrate, [polysaccharide 7] = 24.6 mg / g

[0774] 1 H NMR (DO) showed that the N3-PEG3-NH2 substitution degree (DS1) was 0.28. Polysaccharide 8-dextran methylcarboxylate phosphonate and maleimide Polysaccharide 8.1-dextran methylcarboxylate phosphonate

[0775] Using a method similar to that used to prepare polysaccharide 4.2, starting from polysaccharide 4.1 (41.3 mg / g, DS = 2.1, 4.13 g, 12.51 mmol of glycoside units), polysaccharide 8.1 was obtained using HOPO (695 mg, 6.25 mmol), 3-aminopropylphosphonic acid (261 mg, 1.88 mmol), triethylamine (262 μL, 1.88 mmol), and EDC (3 × 1.20 g, 3 × 6.25 mmol).

[0776] From the dried concentrate, [polysaccharide 8.1] = 16.0 mg / g

[0777] 1 H NMR (DO) showed that the degree of substitution of 3-aminopropylphosphonic acid (DS3) was 0.05. Polysaccharide 8-dextran methylcarboxylate phosphonate and maleimide

[0778] 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 of glycoside units), polysaccharide 8 was obtained using N-(2-aminoethyl)maleimide hydrochloride (415 mg, 2.35 mmol).

[0779] From the dried concentrate, [polysaccharide 8] = 8.0 mg / g

[0780] 1 H NMR (DO) showed that the degree of maleimide substitution (DS1) was 0.25. Polysaccharide 9-dextran methylcarboxylate phosphonate and maleimide

[0781] Polysaccharide 9.1-dextran methylcarboxylate phosphonate

[0782] Using a method similar to that used to prepare polysaccharide 4.2, starting with polysaccharide 4.1 (41.3 mg / g, DS = 2.1, 4.13 g, 12.51 mmol of glycoside units), a solution was obtained using 3-aminopropylphosphonic acid (2.09 g, 15.01 mmol). The pH of this solution was adjusted to pH 2 by adding 1 M HCl. After 16 h, the solution was neutralized with 1 M NaOH and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against a 9 g / L aqueous NaCl solution and then against water.

[0783] From the dried concentrate, [polysaccharide 9.1] = 13.3 mg / g

[0784] 1 H NMR (DO) showed that the degree of substitution of 3-aminopropylphosphonic acid (DS3) was 0.75. Polysaccharide 9-dextran methylcarboxylate phosphonate and maleimide

[0785] Using a method similar to that used to prepare polysaccharide 1, starting with polysaccharide 9.1 (13.3 mg / g, DS2 = 2.1, DS3 = 0.75, 3.32 g, 7.59 mmol of glycoside units), and N-(2-aminoethyl)maleimide hydrochloride (402 mg, 2.28 mmol), polysaccharide 9 was obtained.

[0786] From the dried concentrate, [polysaccharide 9] = 11.5 mg / g

[0787] 1 H NMR (DO) revealed that the degree of maleimide substitution (DS1) was 0.21. Polysaccharide 10-dextran methylcarboxylate sulfate and cyclooctyne (DBCO)

[0788] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 10 was obtained starting from polysaccharide 1.3 (33.3 mg / g, DS2 = 0.8, DS3 = 1.5, 10.0 g, 26.37 mmol of glycoside units) using DBCO-NH2 (2.19 g, 7.91 mmol).

[0789] From the dried concentrate, [polysaccharide 10] = 23.3 mg / g

[0790] 1 From H NMR (DO), DBCO-NH substitution degree (DS1) = 0.23 Polysaccharide 11-dextran methylcarboxylate and cyclooctyne (DBCO)

[0791] Polysaccharide 11.1-Dextran Methylcarboxylate

[0792] Using the same method as used in the preparation of polysaccharide 4.1, a weight-average molecular weight of 250 kg Starting with 11.1 / mol of dextran, polysaccharide 11.1 was obtained.

[0793] From the dried concentrate, [polysaccharide 11.1] = 44.6 mg / g

[0794] Acidity / basicity analysis revealed that the degree of substitution of methyl carboxylate (DS2) was 2.0. Polysaccharide 11-dextran methylcarboxylate and cyclooctyne (DBCO)

[0795] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 11 was obtained starting from polysaccharide 11.1 (44.6 mg / g, DS = 2.0, 15.6 g, 48.42 mmol of glycoside units) and DBCO-NH (803 mg, 2.91 mmol).

[0796] From the dried concentrate, [polysaccharide 11] = 20.4 mg / g

[0797] 1From H NMR (DO), DBCO-NH substitution degree (DS1) = 0.06 Polysaccharide 12-dextran methylcarboxylate and cyclooctyne (DBCO)

[0798] Polysaccharide 12.1-Dextran Methylcarboxylate

[0799] Using the same method as used in the preparation of polysaccharide 4.1, a weight-average molecular weight of 500 kg Starting with 12.1 / mol of dextran, polysaccharide 12.1 was obtained.

[0800] From the dried concentrate, [polysaccharide 12.1] = 45.3 mg / g

[0801] Acidity / basicity analysis revealed that the degree of substitution of methyl carboxylate (DS2) was 2.0. Polysaccharide 12-dextran methylcarboxylate and cyclooctyne (DBCO)

[0802] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 12 was obtained starting from polysaccharide 12.1 (45.3 mg / g, DS = 2.0, 9.06 g, 28.12 mmol of glycoside units) using DBCO-NH (466 mg, 1.69 mmol).

[0803] From the dried concentrate, [polysaccharide 12] = 21.7 mg / g

[0804] 1 From H NMR (DO), DBCO-NH substitution degree (DS1) = 0.06

[0805] Polysaccharide 14-dextran methylcarboxylate and vinyl sulfone

[0806] Divinyl sulfone (38.8 mL, 387.3 mmol) was rapidly added to a 100 mM NaOH solution of polysaccharide 4.1.1 (20.0 mg / mL, DS2 = 1.2, 5.0 g, 19.37 mmol of glycosidic units). After 4 min 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 purified by ultrafiltration through a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer, 9 g / L NaCl aqueous solution, and then water. The concentration of polysaccharide 14 in the final solution was determined by dry concentration and in DO. 1 The degree of vinyl sulfone substitution was determined by H NMR. The final solution was stored at -20 °C.

[0807] From the dried concentrate, [polysaccharide 14] = 20.7 mg / g

[0808] 1 From H NMR (DO), the degree of vinyl sulfone substitution (DS1) was 0.17 Polysaccharide 15-dextran methylcarboxylate and triazole-PEG-azide

[0809] Polysaccharide 15. 1-Dextran methylcarboxylate and propargyl

[0810] Twenty grams of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molecular weight of 40 kg / mol (123.4 mmol of glycoside units, 370.1 mmol of hydroxyl groups) was dissolved in water (300 g / L) at 30°C. NaBH4 (2 × 23 mg, 2 × 0.6 mmol) was added every 30 minutes, and the mixture was stirred at 30°C for 1 hour. The mixture was cooled to 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 43 wt% solution of propargyl bromide (5.87 g, 49.3 mmol) in toluene was 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 to 60 °C, and sodium chloroacetate (71.8 g, 616.8 mmol) was added in one portion. After 1 h, 10 N NaOH (56.7 mL, 567 mmol) was added slowly over 1.5 h, and the mixture was stirred at 60 °C for 1 h. Another portion of sodium chloroacetate (43.1 g, 370.1 mmol) was added in one portion. After 1 h, 10 N NaOH (37.0 mL, 370 mmol) was added slowly over 1.5 h, and the mixture was stirred at 60 °C for 1 h. The mixture was diluted with water (54 mL), cooled to room temperature, and neutralized with acetic acid. pH 7 phosphate buffer (750 mL) and ethanol (575 mL) were added, and the mixture was filtered and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer, 9 g / L NaCl aqueous solution, and then water. The concentration of polysaccharide 15.1 in the final solution was determined by dry concentrate and 1 The degree of propargyl substitution was determined by 1 H NMR, and acidity / baseness analysis was performed to determine the degree of methyl carboxylate substitution.

[0811] From the dried concentrate, [polysaccharide 15.1] = 40.9 mg / g

[0812] 1 H NMR (DO) shows a propargyl substitution degree of 0.24

[0813] Acidity / baseness analysis showed that the degree of substitution of methyl carboxylate (DS2) was 1.8. Polysaccharide 15-dextran methylcarboxylate and triazole-PEG-azide

[0814] To 39 g of polysaccharide 15.1 solution (40.9 mg / g, DS1 = 0.24, DS2 = 1.8, 1.6 g, glycosidic units 5.06 mmol) was 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-diazido-3,6,9,12,15-pentaoxaheptadecane (4.03 g, 12.1 mmol). The reaction mixture was stirred at room temperature for 18 h, diluted with pH 7 phosphate buffer, and purified by ultrafiltration through a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer, 9 g / L NaCl solution, and then water. The concentration of polysaccharide 15 in the final solution was determined by dry concentrate in D2O. 1 The degree of azide substitution was determined by H NMR. The final solution was stored at -20 °C.

[0815] From the dried concentrate, [polysaccharide 15] = 7.0 mg / g

[0816] 1 H NMR (DO) showed that the degree of azide substitution (DS1) was 0.24. Polysaccharide 17-dextran methylcarboxylate and methylfuran

[0817] Using a method similar to the last step used in the preparation of polysaccharide 1 (from polysaccharide 1.3 to polysaccharide 1), starting from polysaccharide 4.1 (50.8 mg / g, DS = 2.1, 17.8 g, 53.9 mmol of glycoside units) and 5-methylfurfurylamine (1.80 g, 16.17 mmol), polysaccharide 17 was obtained.

[0818] From the dried concentrate, [polysaccharide 17] = 30.2 mg / g

[0819] 1From H NMR (DO), the degree of furan substitution (DS1) was 0.30

[0820] Polysaccharide 18-dextran methylcarboxylate and tetrazine

[0821] Using a method similar to that used to prepare polysaccharide 6, starting from polysaccharide 4.1 (50.8 mg / g, DS = 2.1, 5.08 g, 15.38 mmol of glycoside units), polysaccharide 18 was obtained using (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (1.1 g, 4.62 mmol).

[0822] From the dried concentrate, [polysaccharide 18] = 11.7 mg / g

[0823] 1 H NMR (DO) showed a tetrazine substitution degree (DS1) of 0.25

[0824] Polysaccharide 24-dextran glycine carbamate and cyclooctyne (DBCO) Polysaccharide 24.1-dextran glycine carbamate

[0825] 100 g of dextran (Pharmacosmos, degree of polymerization n = 205) with a weight-average molecular weight of 40 kg / mol (0.62 mol of glycoside units, 1.85 mol of hydroxyl groups) was dissolved in water (300 g / L) at 30 °C. NaBH4 (2 × 116 mg, 2 × 3.08 mmol) was added every 30 min, and the mixture was stirred at 30 °C for 1 h. The mixture was diluted with pH 7 phosphate buffer, cooled to room temperature, and purified by ultrafiltration using a PES membrane (MWCO 5 kDa) against pH 7 phosphate buffer and then water. The resulting solution was filtered and lyophilized to obtain intermediate polysaccharide 24.1.1.

[0826] 25 g of polysaccharide 24.1.1 (154.2 mmol of glycoside units, 462.6 mmol of hydroxy groups) was dissolved in 50:50 DMF / DMSO (185 g / L) at 80 °C, and toluene (25 mL) and 1,4-diazabicyclo[2.2.2]octane (6.92 g, 61.7 mmol) were added. 32 g of the reaction mixture was distilled, and ethyl isocyanatoacetate (51.9 mL, 462.6 mmol) was added slowly over 30 min. The mixture was diluted with DMF (150 mL), stirred at 80 °C for 18 h, and cooled to room temperature. Water (900 mL) was added, and the resulting precipitate was filtered off. The precipitate was suspended in 70:30 HO / ethanol (2.5 L), and the pH of the solution was adjusted to pH 13 with 10 N NaOH. After 2 hours, the mixture was neutralized with 6 M HCl, filtered, and purified by ultrafiltration through a PES membrane (MWCO 5 kDa) against a 9 g / L aqueous NaCl solution and then water. The concentration of polysaccharide 24.1 in the final solution was determined by dry concentration, and the degree of glycine carbamate substitution was determined by acidity / baseness analysis.

[0827] From the dried concentrate, [polysaccharide 124.1] = 47.8 mg / g

[0828] Acidity / baseness analysis revealed that the degree of substitution of glycine carbamate (DS2) was 2.4. Polysaccharide 24-dextran glycine carbamate and cyclooctyne (DBCO)

[0829] Using a method similar to that used to prepare polysaccharide 6, polysaccharide 24 was obtained starting from polysaccharide 24.1 (47.8 mg / g, DS = 2.4, 5.98 g, 13.07 mmol of glycoside units) using DBCO-NH (1.08 g, 3.92 mmol).

[0830] From the dried concentrate, [polysaccharide 24] = 20.8 mg / g

[0831] 1 From H NMR (DO), DBCO-NH substitution degree (DS1) = 0.28

[0832] Polysaccharide 25-dextran glycine carbamate and maleimide

[0833] Using a method similar to that used to prepare polysaccharide 1, starting from polysaccharide 24.1 (47.8 mg / g, DS = 2.4, 5.98 g, 13.07 mmol of glycoside units), polysaccharide 25 was obtained using N-(2-aminoethyl)maleimide hydrochloride (693 mg, 2.92 mmol).

[0834] From the dried concentrate, [polysaccharide 25] = 22.8 mg / g

[0835] 1 H NMR (DO) revealed that the degree of maleimide substitution (DS1) was 0.26. Example A2: Polyethylene glycol derivatives having at least two reactive functional groups

[0836] Polyethylene glycol (PEG) derivatives functionalized with commercially available reactive functional groups were purchased. The reactive functional groups include thiols ("PEG-SH"), azides (PEG-N3), and alkynes (PEG-DBCO). Linear homobifunctional and multiarm homofunctional derivatives with different molecular weights and functional groups were used. These are shown in Table 2 below. [Table 2] TIFF2025527131000132.tif125151

[0837] These PEG derivatives correspond to the precursors of -L in Formula I. Part B - Biology Example B1A: Preparation of pseudo-islets

[0838] The Min-6 cell line (Catlag Medsystems) was cultured in the medium shown in Table 3 in an incubator at 37°C and 5% CO. Cells were subcultured three times a week using 0.05% trypsin / EDTA to detach cells and diluted 5-fold with medium. [Table 3]

[0839] Pseudoislets with an average diameter of 150 μm were formed using the Min-6 cell line and 400 μm microwell Eplasia plates (Corning) by seeding 500 cells per microwell and culturing at 37°C and 5% CO for 3 days. The pseudoislets were collected, concentrated by centrifugation, and finally suspended in 0.9% NaCl. Example B2A: Isolation of primary human pancreatic islets

[0840] Pancreases were obtained from human brain-dead donors. Pancreatic islets were generated according to the method described in "Technique of pancreatic procurement for pancreatic islet isolation" (Pattou et al., 2005). Briefly, pancreases were isolated from tissue and digested with a mixture of collagenase I and II (Liberase®, Roche, France) through a Wilsing tube to ensure islet release. Islets were then purified using density gradient centrifugation (EuroFicoll, Sigma-Aldrich). Purified islets were finally cultured in culture flasks at 37°C, 5% CO2, in CMRL medium supplemented with 0.625% BSA and 1% penicillin / streptomycin. The medium was changed every 2–3 days. Example B2B: Isolation of primary rat islets

[0841] Pancreatic islets were isolated from male Wistar or Lewis rats (approximate body weight: 300 g) according to a method similar to that described in A Practical Guide to Rodent Islet Isolation and Assessment, Cater et al., Biological Procedures Online 2009.

[0842] Briefly, the pancreas was perfused with collagenase injected via 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 flasks in DMEM medium (Gibco) or CMRL medium supplemented with 10% fetal bovine serum, 2 g / L glucose, and 1% penicillin / streptomycin at 37°C and 5% CO2. The medium was changed every 2–3 days. Example 3A: Islet Equivalent Counting

[0843] To standardize the amount of islets or pseudoislets in each experiment, islets or pseudoislets were counted to determine islet equivalents (IEQs). One IEQ corresponds to the volume of a perfectly spherical islet / pseudoislet with a diameter of 150 μm. During counting, a multiplying factor was applied to each islet based on its size. This mathematical correction for islets of different diameters allows for standardization between specimens (see NIH CIT Consortium Chemistry Manufacturing Controls Monitoring Committee; Purified Human Pancreatic Islet: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium; CellR4 Repair Replace Regen Reprogram).

[0844] Two 50 μL samples from each islet or pseudoislet batch were counted on a 50 μm gridded glass slide. Islets or pseudoislets were classified according to Table 4A. [Table 4A]

[0845] The number of islet equivalents was determined by averaging the counts of two independent samples. Example B3B: Islet Equivalent Counting

[0846] To standardize the amount of islets or pseudoislets in each experiment, islets were counted to determine islet equivalents (IEQs). 1 IEQ corresponds to the volume of a perfectly spherical islet / pseudoislet with a diameter of 150 μm. During counting, a multiplying factor was applied to each islet based on its size. This mathematical correction for islets of different diameters allows for standardization between islet preparations (see NIH CIT Consortium Chemistry Manufacturing Controls Monitoring Committee; Purified Human Pancreatic Islet: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium; CellR4 Repair Replace Regen Reprogram).

[0847] Two 50 μL samples from each islet batch were counted on a 50 μm gridded glass slide. Islets were classified according to Table 4B. [Table 4B]

[0848] The number of islet equivalents was determined by averaging the counts of two independent samples. Part C - Physical Chemistry Example C1A: Preparation of a concentrated, maleimide (Mal)-functionalized polysaccharide solution

[0849] A concentrated polysaccharide solution was prepared by weighing out the appropriate weight of sterile, lyophilized polysaccharide from Part A1 and adding the appropriate weight of sterile, deionized water. The solution was placed on an orbital shaker at 70 rpm overnight to ensure complete solubilization. The pH of the solution was adjusted to 4 by adding concentrated HCl before sterile filtration (0.22 μm). The mass concentration of the polysaccharide solution (mg / g) was determined from the dry concentrate. The mass-to-volume concentration of the polysaccharide solution (mg / mL) was determined by density measurement after weighing 100 μL of the solution in triplicate. The solution was frozen at -20°C until use. Example C1B: Preparation of concentrated polysaccharide solutions functionalized with vinyl sulfone (VS), DBCO, or azide groups

[0850] A concentrated polysaccharide solution was prepared by weighing out the appropriate weight of sterile, lyophilized polysaccharide from Part A1 and adding the appropriate weight of sterile, deionized water. The solution was placed on an orbital shaker at 70 rpm overnight to ensure complete solubilization. The pH of the solution was adjusted to 7.4 by adding NaOH before sterile filtration (0.22 μm). The mass concentration of the polysaccharide solution (mg / g) was determined from the dry concentrate. The mass-to-volume concentration of the polysaccharide solution (mg / mL) was determined by density measurement after weighing 100 μL of the solution in triplicate. The solution was frozen at -20°C until use. Example C2: Preparation of concentrated REG solution

[0851] Concentrated PEG solutions were prepared by weighing out the appropriate weight of PEG powder (listed in Table 2) and adding the appropriate weight of sterile deionized water. The solution was placed on a roller shaker at 15 rpm for 2 hours for complete solubilization before sterile filtration (0.22 μm). The mass concentration of the PEG solution (mg / g) was determined from the dry concentrate. The mass-to-volume concentration of the PEG solution (mg / mL) was determined by density measurement after weighing 100 μL of the solution in triplicate. The solutions were frozen at -20°C until use. Example C3: Preparation of concentrated sodium hyaluronate solution

[0852] Concentrated sodium hyaluronate solutions were prepared by weighing out the appropriate weight of sodium hyaluronate powder (Pharma Grade 150, Novamatrix) and adding the appropriate weight of sterile deionized water. Alternatively, other hyaluronate salts (Pharma Grade 300, Echelon Biosciences, HTL Biotechnology) were used, specifically for C4B-16 gel. The solution was placed on a roller shaker at 10 rpm overnight for complete solubilization before sterile filtration (0.22 μm). Example C3bis: Preparation of concentrated sodium hyaluronate solution

[0853] Concentrated trehalose dihydrate D(+) solution was prepared by weighing out the appropriate mass of trehalose dihydrate D(+) powder (Sigma-Aldrich) and adding the appropriate weight of sterile deionized water. The solution was heated to 45°C with mixing and cooled to ambient temperature for complete solubilization before sterile filtering (0.22 μm). Example C3A: Hydrogel Preparation

[0854] The preparation of the hydrogel was carried out in a sterile environment.

[0855] Concentrated sterile solutions of polysaccharides and PEG derivatives prepared according to Examples C1A or C1B and C2, respectively, were adjusted with concentrated NaCl solution to obtain isotonic stock solutions (300 mOsm / kg) and equilibrated at either room temperature (20-25° C.) or 4° C. Optionally, concentrated polysaccharide solutions bearing VS or DBCO groups were supplemented with Tris buffer at pH 7.4 or pH 8.

[0856] Concentrated PEG solution was added to concentrated polysaccharide solution in a 2 mL Eppendorf tube. The volume ratio of PEG solution to polysaccharide solution was 70:30 (%:%) or 80:20 (%:%). The solutions were mixed with a pipette, and the adjusted volume of the mixture was introduced into a circular silicone isolator attached to a glass slide. Various molded hydrogel shapes were prepared. [Table 5A]

[0857] The cross-linking process leading to gelation was carried out for 1 h at room temperature (20-25°C) or at 37°C. The hydrogels were removed from the molds and introduced into 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution (2 mL) or PBS, pH 7.4, for 1 h at 37°C.

[0858] The hydrogel was rinsed with 20 mL of cysteine-free PBS solution and then soaked in 10 mL of PBS solution overnight at 37°C. The hydrogel pieces were then stored in 10 mL of PBS solution at 4°C until use. Example C3B: Hydrogel Preparation

[0859] The preparation of the hydrogel was carried out in a sterile environment.

[0860] Concentrated sterile solutions of polysaccharide and PEG derivative prepared according to Examples C1A or C1B and C2, respectively, were adjusted with concentrated NaCl solution to obtain an isotonic stock solution (300 mOsm / kg) and equilibrated at either room temperature (20-25° C.) or 4° C. Alternatively, osmolarity can be adjusted with NaCl and non-ionic agents such as trehalose, particularly in Examples C4B24. Optionally, concentrated polysaccharide solutions bearing VS or DBCO groups were supplemented with Tris buffer at pH 7.4 or pH 8.

[0861] A concentrated PEG / hyaluronate solution was prepared by mixing the concentrated PEG solution prepared according to Example C2 with the concentrated sodium hyaluronate solution prepared according to Example C3.

[0862] A concentrated polysaccharide solution supplemented with Pluronic® F127 (Sigma-Aldrich) was prepared by mixing the concentrated polysaccharide solution prepared according to Example C1B.

[0863] Concentrated PEG or PEG / hyaluronate solutions were added to concentrated polysaccharide or polysaccharide / Pluronic® (specifically Pluronic® F127) solutions in 2 mL Eppendorf tubes. The volume ratio of PEG solution to polysaccharide solution was 70:30 (%:%) or 80:20 (%:%) when hyaluronate was added to the PEG solution. The solutions were mixed with a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator attached to a glass slide. Various molded hydrogel shapes were prepared.

[0864] The addition of a pruonic-type nonionic surfactant may help improve the wetting properties of the composition. [Table 5B]

[0865] The cross-linking process leading to gelation was carried out for 1 h at room temperature (20-25°C) or at 37°C. The hydrogels were removed from the molds and placed in a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 or in PBS at pH 7.4 at 37°C for 1 h.

[0866] The hydrogel was rinsed with cysteine-free PBS solution and further soaked in PBS solution overnight at 37°C. The hydrogel pieces were then stored in 10 mL of PBS solution at 4°C until use. Example C4A: Hydrogel Composition

[0867] Various hydrogel compositions were prepared according to the procedure described in Example C3A. The compositions are shown in Table 6A. The concentrations of the two reactive groups (maleimide (Mal) or vinylsulfone (VS) groups reacting with thiol (SH) groups, or alkyne (DBCO) groups reacting with azide (N) groups) and polymers (polysaccharide and PEG derivative) correspond to the final concentrations of the polymer solutions when mixed. [Table 6A]

[0868] Solid, disk-shaped hydrogel pieces were obtained, which were easily removed from the mold and manipulated with tweezers for characterization. [Table 7A] TIFF2025527131000140.tif238169TIFF2025527131000141.tif242169TIFF2025527131000142.tif193169TIFF2025527 131000143.tif186169TIFF2025527131000144.tif144169TIFF2025527131000145.tif198169TIFF2025527131000146.ti f192169TIFF2025527131000147.tif192169TIFF2025527131000148.tif130169TIFF2025527131000149.tif176169TIFF2025527131000150.tif140169TIFF2025527131000151.tif194169TIFF2025527131000152.tif144169Example C4B: Hydrogel Composition

[0869] Various hydrogel compositions were prepared according to the procedure described in Example C3B (Table 6B). The concentrations of the two reactive groups (maleimide (Mal) or vinylsulfone (VS) groups reacting with thiol (SH) groups, or alkyne (DBCO) groups reacting with azide (N) groups) and polymers (polysaccharide and PEG derivative) correspond to the final concentrations of the polymer solutions when mixed. [Table 6B]

[0870] Solid, disk-shaped hydrogel pieces were obtained, which were easily removed from the mold and manipulated with tweezers for characterization. [Table 7B] TIFF2025527131000155.tif192169TIFF2025527131000156.tif175169TIFF2025527131000157.tif192169TIFF2025527131000158.tif147169Example C5A: Hydrogel Hydrodynamic Characterization

[0871] Oscillatory shear tests were performed on a rotational rheometer (AR2000, TA Instruments) equipped with a cone-plate geometry. The crosslinking process leading to gelation was performed "in situ," meaning that a few drops of the polysaccharide and PEG concentrated solutions were introduced between the cone and plate and mixed by rotating the geometry before the start of the oscillatory measurements. Oscillatory time sweep tests were performed at 25°C or 37°C, a constant strain of 0.1%, and a constant oscillation frequency of 1 Hz. Storage modulus G' (i.e., elastic modulus) and Tan δ (G'' / G'' ratio) values were shown as a function of time at 1600 seconds during the plateau of the (G', G'') measurements. [Table 8A]

[0872] The hydrogels exhibited low values of Tan δ, which means that G' is significantly higher than G'', a typical property of chemically crosslinked hydrogels that behave as solid elastic materials (see Polysaccharide Hydrogels: Characterization and Biomedical Applications, 2016 Pan Stanford Publishing Pte. Ltd.; Chapter 3, p. 97). Increasing the concentration of Mal:SH leads to an increase in the elastic modulus G' value.

[0873] Increasing the temperature and pH were two ways to accelerate the cross-linking process leading to gelation of hydrogels prepared from polysaccharides bearing VS groups and PEG-SH. For example, increasing the pH from neutral to pH 8 and / or increasing the temperature from 25 to 37 °C resulted in faster gelation.

[0874] This allows for fine tuning of the rate of gelation, which is advantageous, as fast gelation is beneficial for preventing cell sedimentation, while slow gelation is beneficial for polymer mixing before gelation. Example C5B: Hydrogel Rheological Characterization

[0875] Oscillatory shear tests were performed on a rotational rheometer (AR2000, TA Instruments) equipped with a cone-plate geometry. Crosslinking leading to gelation was performed "in situ," meaning that a few drops of concentrated polysaccharide and PEG solutions were introduced between the cone and plate and mixed by rotation of the geometry before the start of the oscillatory measurements. Oscillatory time sweep tests were performed at 25°C or 37°C, a constant strain of 0.1%, and a constant oscillation frequency of 1 Hz. Storage modulus G' (i.e., elastic modulus) and Tan δ (G'' / G'' ratio) values were shown as a function of time at 1600 seconds during the plateau of the (G', G'') measurements. [Table 8B]

[0876] The hydrogels exhibited low values of Tan δ, which means that G' was significantly higher than G'', a typical property of chemically crosslinked hydrogels that behave as solid elastic materials (see Polysaccharide Hydrogels: Characterization and Biomedical Applications, 2016 Pan Stanford Publishing Pte. Ltd.; Chapter 3, p. 97). Increasing the concentration of Mal:SH leads to an increase in the elastic modulus G' value.

[0877] Increasing the concentration of reactive groups increases the value of the elastic modulus G'.

[0878] This allows for fine tuning of the rate of cross-linking leading to gelation, which is advantageous, as fast gelation is beneficial for preventing cell sedimentation, while slow gelation is beneficial for polymer mixing before gelation.

[0879] The gelling properties are maintained in the presence of non-reactive sodium hyaluronate.

[0880] This allows for further tailoring of the hydrogel composition to prevent cell sedimentation, even in slow-gelling compositions. Example C6A: Hydrogel Swelling and Water Content

[0881] The hydrogel pieces were weighed immediately after removal from the mold (w0) and after swelling overnight in PBS solution (w overnight). The swelling ratio was defined as the mass ratio of w overnight / w0. The water content of the hydrogel was estimated by measuring the mass of the swollen hydrogel and controlling the concentration of the polymer precursor used in the synthesis of the hydrogel. [Table 9A]

[0882] The hydrogels had high water content, which varied depending on the structure and concentration of the polymer precursor. Example C6B: Hydrogel Swelling and Water Content

[0883] The hydrogel pieces were weighed immediately after removal from the mold (w0) and after swelling overnight in PBS solution (w overnight). The swelling ratio was defined as the mass ratio of w overnight / w0. The water content of the hydrogel was estimated by measuring the mass of the swollen hydrogel and controlling the concentration of the polymer precursor used in the synthesis of the hydrogel. [Table 9B]

[0884] The hydrogel had a high water content. Example C7A: Hydrogel stability in physiological medium at 37°C

[0885] Hydrogel pieces were stored in PBS pH 7.4 or serum (FBS fetal bovine serum) at 37°C and weighed at different time periods. Disc-shaped hydrogels with a diameter of 9 mm and a thickness of 1.6 mm were tested. [Table 10A]

[0886] The hydrogels were recovered intact, and their mass did not significantly evolve upon storage in physiological media at 37 °C. Hydrogel swelling (mass increase) or dissolution (mass decrease) would be expected, for example, in the case of denaturation of the network structure or hydrolytic side reactions, demonstrating that the hydrogels are stable under physiological conditions. Example C7B: Hydrogel stability in physiological medium at 37°C

[0887] The hydrogel pieces were stored at 37°C in PBS or serum (FBS, fetal bovine serum) at pH 7.4 or 100 mM acetate buffer at pH 4. The hydrogel 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. [Table 10B]

[0888] The hydrogels were recovered intact, and their mass did not significantly evolve upon storage in physiological media at 37 °C. Hydrogel swelling (mass increase) or dissolution (mass decrease) would be expected, for example, in the case of denaturation of the network structure or hydrolytic side reactions, demonstrating that the hydrogels are stable under physiological conditions.

[0889] At acidic pH, no significant loss of mass was observed and the gel was quite stable. Example C8A: Encapsulation of polymeric probes in hydrogels

[0890] Commercially available 3 kDa fluorescent dextran-FITC and 70 kDa fluorescent dextran-FITC were each dissolved in water to obtain concentrated stock solutions.

[0891] Polysaccharide DMCMal and PEG-SH concentrated sterile solutions prepared according to Examples C1A and C2, respectively, were equilibrated at 4° C. For DMCVS-based hydrogels, polysaccharide DMCMal and PEG-SH concentrated solutions prepared according to Examples C1B and C2, respectively, were equilibrated at 20-25° C.

[0892] A solution of the polysaccharide DMCMal or DMCVS was mixed with fluorescent dextran. 100 μL of the concentrated PEG-SH solution was added to 100 μL of the concentrated solution of the polysaccharide DMCMal or DMCVS and fluorescent dextran. The solutions were mixed with a pipette, and 200 μL of the latter mixture was introduced into a rectangular silicone mold (IBIDI 12 × 7.75 mm). Crosslinking leading to gelation was carried out for 1 hour at room temperature (20–25°C) for the DMCMal-based hydrogels and for 1 hour at 37°C for the DMCVS-based hydrogels.

[0893] The hydrogel pieces were removed from the molds, placed in wells (12-well multiplates), and soaked with 1.3 mL of a Tris (200 mM) / NaCl (50 mM) buffer solution containing fluorescent dextran at the same concentration as the encapsulated fluorescent dextran, pH 8. The hydrogels were allowed to swell overnight at 37°C, and the supernatant was weighed to estimate the degree of swelling and the amount (mg) in the hydrogel volume.

[0894] Before the release experiment, the hydrogel was quickly rinsed twice with 1 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution. [Table 11A] Example C8B: Encapsulation of polymeric probes in hydrogels

[0895] Commercially available 3 kDa fluorescent dextran-FITC and 70 kDa fluorescent dextran-FITC were each dissolved in water to obtain concentrated stock solutions.

[0896] Concentrated sterile solutions of polysaccharide and PEG, or polysaccharide 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 polysaccharide DMCMal and PEG-SH, prepared according to Examples C1B and C2, respectively, were equilibrated at 20-25° C.

[0897] Solutions of the polysaccharides DMCMal, DMCVS, or DMCDBCO were mixed with fluorescent dextran. 100 μL of concentrated PEG-SH or PEG-N3 solution was added to 100 μL of the concentrated solution of the polysaccharides DMCMal or DMCVS and fluorescent dextran. The solutions were mixed with a pipette, and 200 μL of the latter mixture was introduced into a rectangular silicone mold (IBIDI 12 × 7.75 mm). Crosslinking leading to gelation was carried out for 1 h at room temperature (20–25°C) for DMCMal and DMC-DBCO hydrogels and for 1 h at 37°C for DMCVS hydrogels.

[0898] The hydrogel pieces were removed from the molds, placed in wells (12-well multiplates), and soaked with 1.3 mL of a Tris (200 mM) / NaCl (50 mM) buffer solution containing fluorescent dextran at the same concentration as the encapsulated fluorescent dextran, pH 8. The hydrogels were allowed to swell overnight at 37°C, and the supernatant was weighed to estimate the degree of swelling and the amount (mg) in the hydrogel volume.

[0899] Before the release experiment, the hydrogel was quickly rinsed twice with 1 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution (shown in Example C7B). [Table 11B] Example C9A: Release of polymeric probes in hydrogels

[0900] The hydrogels with encapsulated fluorescent probes prepared in Example C8A were introduced into wells (12-well multiplate) and soaked with 2 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution. The plate was covered with a film and placed in an oven at 37°C. At different time points, 200 μL of the buffer was sampled and replaced with fresh buffer. The concentration of the fluorescent probe in the sample was determined by fluorescence (fluorescence plate reader SAFAS) using a calibration curve. The cumulative fraction of the fluorescent probe released at each time point corresponds to the ratio of the cumulative amount of released fluorescent probe to the initial amount of fluorescent probe in the swollen hydrogel. [Table 12A]

[0901] Increasing the size of the polymeric probes leads to slower release kinetics, demonstrating the permselectivity of the hydrogel network structure. Example C9B: Release of polymeric probes in hydrogels

[0902] The hydrogels with encapsulated fluorescent probes prepared in Example C8B were introduced into individual wells (12-well multiplate) and soaked with 2 mL of a pH 8 Tris (200 mM) / NaCl (50 mM) buffer solution. The plate was covered with a film and placed in an oven at 37°C. At different time points, 200 μL of the buffer was sampled and replaced with fresh buffer. The concentration of the fluorescent probe in the sample was determined by fluorescence (fluorescence plate reader SAFAS) using a calibration curve. The cumulative fraction of the fluorescent probe released at each time point corresponds to the ratio of the cumulative amount of released fluorescent probe to the initial amount of fluorescent probe in the swollen hydrogel. [Table 12B]

[0903] Increasing the size of the polymeric probes leads to slower release kinetics, demonstrating the permselectivity of the hydrogel network structure. Example C10: Forming thin hydrogel discs

[0904] The concentrated polymer solution was equilibrated at 4°C according to the procedure described in Example C4A. 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 or 356 μm thick) attached to a glass slide. A second glass slide was placed on top of the drop to facilitate spreading across the diameter of the mold.

[0905] The hydrogel discs were demolded after a cross-linking process leading to gelation at 20-25° C. for 1 hour. The thickness was estimated from the diameter and volume of the gel. [Table 13]

[0906] The method yields hydrogel discs of tailored diameter and thickness by adjusting the mold diameter and hydrogel volume. Example C11A: Determination of the mechanical resistance of hydrogels

[0907] For compression, rectangular pieces of the swollen hydrogel described in Example C3A were placed in a plate glass crystallizing dish and immersed in PBS. Uniaxial compression was performed at 20-25°C in 0.9% NaCl at a rate of 0.2 mm / min using a universal mechanical testing machine (ZwickRoell) equipped with a flat compression plate. The initial thickness of the sample was determined from the contact between the hydrogel and the plate, when the force began to increase. Deformation was defined as the ratio of the compression displacement (mm) to the initial thickness (mm). Deformation at break was determined from the force / displacement curve. The break point was defined as the point at which a decrease in force versus displacement was observed.

[0908] For tension, dog-bone-shaped hydrogel strips were prepared by forming the hydrogel in a dog-bone-shaped silicone mold. Uniaxial tension was performed at 20-25°C in 0.9% NaCl at a rate of 3 mm / min using a universal mechanical testing machine (ZwickRoell) equipped with screw grips. The initial length of the sample was measured between the grips with a ruler. Deformation was defined as the ratio of the tensile displacement (mm) to the initial length (mm). The deformation at break was determined from the force / displacement curve. The break point was defined as when a decrease in force versus displacement was observed. Young's modulus was determined from the slope of the true strain / deformation curve. The true strain (kPa) was calculated based on the instantaneous surface area (mm) of the sample under compression. 2 ) corresponds to the ratio of force (N) to force (N). [Table 14A]

[0909] The hydrogels of the invention exhibit excellent mechanical resistance combined with deformability and rigidity suitable for surgical implantation. Example C11B: Determination of the mechanical resistance of hydrogels

[0910] For compression, rectangular pieces of the swollen hydrogel described in Example C3B were placed in a glass crystallizing dish and immersed in PBS. Uniaxial compression was performed at 20-25°C in 0.9% NaCl at a rate of 0.2 mm / min using a universal mechanical testing machine (ZwickRoell) equipped with a flat compression plate. The initial thickness of the sample was determined from the contact between the hydrogel and the plate, when the force began to increase. Deformation was defined as the ratio of the compression displacement (mm) to the initial thickness (mm). Deformation at break was determined from the force / displacement curve. The break point was defined as the point at which a decrease in force versus displacement was observed.

[0911] Young's modulus was determined from the slope of the true strain / deformation curve. True strain (kPa) is proportional to the instantaneous surface area (mm 2 ) corresponds to the ratio of force (N) to force (N). [Table 14B]

[0912] The hydrogels of the invention exhibit excellent mechanical resistance combined with deformability and rigidity suitable for surgical implantation. Example C12: Surgical Net Example C12A: Alkaline treatment of polyester surgical netting

[0913] Warp-knit polyester multifilament surgical netting fabric type PETKM3002 (1 x 0.9 mm pore size) supplied by SurgicalMesh, Inc. was treated in 1 M NaOH at 70°C for 5 hours and then rinsed with deionized water and 96% ethanol. The treatment increases the hydrophilicity of the netting fabric, improving its wettability with the aqueous polymer solutions that make up the hydrogel. Example C12B: PVDF surgical netting fabric

[0914] Warp-knitted PVDF surgical netting fabric (1 x 1 mm pore size) made from 100 μm diameter monofilaments supplied by Dynamesh. Example C12C: PTFE Perforated Surgical Film

[0915] A 150 μm thick, star-pattern perforated PTFE surgical film (1 × 2 mm hole size) supplied by Aran Biomedical. Example C13: Assembly of a structure Example C13A: Assembly of ring net structure

[0916] Biocompatible PDMS sheets supplied by Grace Biolabs, Interstate Specialty Products, or Limitless Shielding were cut into square shapes incorporating blank disks using a stainless steel punch.

[0917] The treated polyester surgical net or surgical net described in Example C12 was introduced between two square PDMS pieces. The two PDMS pieces and the net were glued together with a biocompatible silicone adhesive (Silbione MED ADH 4200, supplied by Elkem). The blank disks were aligned, and the surgical net was kept taut during the bonding process.

[0918] Finally, a square construct was cut with a stainless steel punch to obtain the final object, consisting of two PDMS rings glued together with a surgical net sandwiched between them.

[0919] Before steam or ETO (ethylene oxide) sterilization, the plates were washed with 1% poloxamer F127 solution and rinsed with water. Example C13B: Assembly of Scaled Rectangular Structures

[0920] For human surgery, the implant may be of an elongated shape, such as a rectangle less than 100 mm wide, to allow for easy surgical implantation through a trocar with an internal diameter of 15 mm.

[0921] Biocompatible PDMS sheets supplied by Grace Biolabs, Interstate Specialty Products, or Limitless Shielding were cut into blank frames of various shapes, such as vertical rectangles.

[0922] The surgical net described in Example C12 was introduced between the frame-shaped PDMS pieces. The two PDMS pieces and the net were glued together with a biocompatible silicone adhesive (Silbione MED ADH 4200, supplied by Elkem). The empty frames were aligned, and the surgical net was kept taut during the bonding process.

[0923] Finally, the excess netting around the frame was cut off with a stainless steel punch, yielding the final object consisting of two PDMS frames glued together with a surgical net sandwiched between them.

[0924] The remainder of the netting around the frame can be left uncut to facilitate fixation to tissue via absorbable or non-absorbable tacks.

[0925] Before steam or ETO sterilization, the plates were washed with 1% poloxamer F127 solution and rinsed with water. Example C14: Structures of various dimensions Example C14A: Ring net structures of various dimensions

[0926] Following Example C13A, various ring net structures were produced by varying parameters such as inner / outer diameter and thickness of the PDMS rings and thickness of the adhesive. [Table 15A] Example C14B: Sized structures of various dimensions

[0927] Following Example C13B, various structures were produced by varying parameters such as inner / outer diameter and thickness of the PDMS ring and thickness of the adhesive. [Table 15B] Example C15: Hydrogel / Structure Composite Composition Example C15A: Hydrogel / Ring Net Composite Composition

[0928] According to Example C3B, the hydrogel composition prepared as described in Example C4B was introduced into the ring net structure described in Example C14. The concentrated polymer solution was mixed with a pipette, and a controlled volume of the mixture was introduced into the ring net structure attached to a glass slide.

[0929] Crosslinking leading to gelation was carried out for 1 hour at room temperature (20-25°C) or at 37°C. The ring net / hydrogel composition was then introduced into a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 or PBS at pH 7.4 at 37°C for 1 hour.

[0930] The hydrogels were rinsed with cysteine-free PBS solution and further soaked in PBS solution overnight at 37°C, and then the hydrogel pieces were stored in PBS solution at 4°C until use.

[0931] Various hydrogel / ring-net composite compositions were prepared (Table 16A). [Table 16A]

[0932] The hydrogel / ring-net composite can be easily manipulated with tweezers and is foldable, especially for minimally invasive surgery, as required for surgical implantation. Furthermore, the ring can be secured in place with sutures.

[0933] The volume of the hydrogel can be adjusted by the inner diameter and thickness of the ring net structure. For the same ring net structure, the volume of the hydrogel can be adjusted to control the convexity of the hydrogel above the height of the ring. Example C15B: Hydrogel / Sized Composite Composition

[0934] To facilitate engraftment, ensure lack of underfilling, and limit islet settling, sized implants for human analysis were fabricated in a device that could be filled horizontally or vertically with hydrogel.

[0935] The device consists of a glass plate with a 50-2000 μm thick silicone sheet attached to one side. One glass plate and the corresponding silicone sheet are perforated with two holes measuring 1-6 mm in diameter. A female Luer lock is glued in the center of one of the holes. An empty graft is placed between the two laminated glass sheets. The assembly is clamped on both sides against the silicone frame of the construct to ensure the desired thickness and a tight seal during hydrogel injection.

[0936] A suitable volume of the hydrogel composition was injected into the sized constructs in two ways: Reconstitution and injection: After mixing the gel precursor solutions, the gel solution was placed into a syringe with a male luer lock. The syringe was screwed onto the luer lock on the glass plate and the gel was injected into the implant. b. Dual-syringe injection with mixing chamber (Twin-Syringe Biomaterial Delivery System (M-System), MedMix®): Hydrogel precursor solutions were placed separately into two syringes. The flow connector between the two syringes was extended with a mixing chamber with a male Luer lock that was screwed onto the filling device. By simultaneously pressing both syringes with the medmix® clamp, the hydrogel was reconstituted and immediately injected into the device. [Table 16B] Example C16A: Encapsulation of insulin-producing pancreatic islets in hydrogels

[0937] Islet encapsulation was performed under sterile conditions.

[0938] Concentrated sterile solutions of polysaccharide and PEG prepared according to Examples C1A or C1B and C2, respectively, were adjusted with concentrated NaCl solution to obtain an isotonic stock solution (300 mOsm / kg). The solutions were equilibrated at either room temperature (20-25°C) or 4°C.

[0939] An enriched mixture of PEG and islets was prepared by mixing equal volumes of an isotonic PEG solution (prepared according to Example C2) and an islet or pseudoislet suspension (prepared according to Examples B1A or B2A). The PEG and islet-containing mixture was then gently mixed with an isotonic polysaccharide solution at a volume:volume ratio of islet suspension / PEG:polysaccharide of 70:30.

[0940] The solution was gently mixed with a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator (Grace Biolab) attached to a glass slide. Depending on the volume and diameter of the silicone mold, hydrogels of various sizes were produced. [Table 17A]

[0941] The crosslinking process leading to gelation was carried out for 1 hour at room temperature (20-25°C) or in an oven at controlled temperatures such as 20, 25, or 37°C. The islet-incorporated hydrogels were removed from the molds and placed in a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 or in culture medium for 15 minutes at room temperature. The hydrogels were then immersed in culture medium containing 10 mM cysteine for 1 hour at 37°C. After 1 hour, the cysteine-containing medium was removed and replaced with culture medium. The sterile cell-containing hydrogels were stored at 37°C and 5% CO2 before further in vitro studies or in vivo implantation.

[0942] Following this procedure, various hydrogel compositions were prepared using either pseudo islets or primary islets. [Table 18A] Example C16B: Encapsulation of insulin-producing pancreatic islets in hydrogels

[0943] Cellular islet encapsulation was performed under sterile conditions.

[0944] Concentrated sterile solutions of polysaccharide and PEG prepared according to Examples C1A or C1B and C2, respectively, were adjusted with concentrated NaCl solution to obtain an isotonic stock solution (300 mOsm / kg). The solutions were equilibrated at either room temperature (20-25°C) or 4°C.

[0945] An enriched 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) and an islet suspension (prepared according to Example B1B). The PEG and islet-containing mixture was then gently mixed with an isotonic polysaccharide solution at a volume:volume ratio of 70:30 islet suspension / PEG:polysaccharide or 80:20 islet suspension / PEG / hyaluronate:polysaccharide.

[0946] The solution was gently mixed with a pipette, and a controlled volume of the mixture was introduced into a circular silicone isolator (Grace Biolab) or a ring net structure attached to a glass slide. Different silicone molds (Table 5B) or ring net structures (Table 16) were used to generate hydrogels of various sizes.

[0947] The crosslinking process leading to gelation was carried out for 1 hour at room temperature (20-25°C) or in an oven at controlled temperatures such as 20, 25, or 37°C. Islet-incorporated hydrogel disks or hydrogel / ring nets were introduced into a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 (for Mal or VS:SH crosslinking) or medium (for DBCO:N3 crosslinking) for 15 minutes at room temperature. The hydrogels were then immersed in medium containing 10 mM cysteine or medium without cysteine for 1 hour at 37°C. After 1 hour, the cysteine-containing medium was removed and replaced with medium. The sterile cell-containing hydrogels were stored at 37°C and 5% CO2 before further in vitro studies or in vivo implantation.

[0948] Following this procedure, various hydrogel / islet compositions were prepared. [Table 18B] Example C17: Spatial uniformity of encapsulated cells through the thickness of the hydrogel

[0949] Islet-loaded hydrogel discs were prepared as described in Example C16B. Photographs of the hydrogels were taken from the side to assess islet uniformity across the hydrogel thickness. Uniformity is achieved when the islets are located throughout the entire thickness of the hydrogel and are not primarily localized to one side of the hydrogel. [Table 17B]

[0950] The addition of sodium hyaluronate increases the viscosity of the mixture, allowing for uniform spatial location of the islets within the thickness of the hydrogel.

[0951] A higher level of uniformity can be reached by using longer hyaluronate chains. Example C18B: Evaluation and Adjustment of Hide and Gel Transparency

[0952] The hydrogel compositions described in Examples C4A and C4B and the other hydrogels described in Example C are visually transparent.

[0953] To quantify the transparency of the hydrogels, UV absorbance measurements (Spectrofluorometer, Xenius®, SAFAS) were performed on 80 μL of precursor solution in a 96-well plate. 100 μL of PBS was added on top of the gels to prevent drying. Absorbance measurements at 400 nm were used to quantify the turbidity of the samples. Five measurements performed at different positions were averaged. Measurements were performed 3 hours after deposition, in the fully swollen and equilibrated state. The samples in the table below were also visually inspected under standardized conditions in front of a black panel at light levels between 2,000 and 3,750 Lux (Adelphi Apollo II® Liquid Inspection Unit).

[0954] Hydrogel composition C4B-13 was prepared similarly to Example C3B. Polysaccharide / Pluronic® DMCDBCO and PEG-N3 concentrated sterile solutions 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. PEG-N3 concentrated sterile solution 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. [Table 17CB]

[0955] All gels are transparent under "normal" conditions (real life).

[0956] However, because the change in absorbance assesses the variable homogeneity state of the macroscopic network, such harsh conditions had to be used to assess the effect of changing the osmotic agent on transparency. These results show that the turbidity of the hydrogels decreased when the osmotic agent NaCl was replaced by trehalose in the presence of hyaluronan.

[0957] Other non-ionic osmotic agents such as mannitol or glycerin may also be used to enhance transparency in the presence of HA.

[0958] These characteristics do not imply any alteration of the gel properties or any change in permeability, so that the diffusion of small molecules such as insulin is maintained at any turbidity. Example C19: Laparoscopic Implantation of Hydrogel / Sized Composites

[0959] The implantability of the hydrogel / sized composites into domestic pigs via laparoscopic surgery was evaluated.

[0960] Two types of implants (C15B-1 and C15B-4) were first inserted into a surgical pouch, rolled up, and inserted into the abdominal cavity via a 15 mm trocar. The implants were then removed from the pouch and fixed onto the peritoneum (C15B-1) or at the level of the peritoneal discontinuity in contact with the muscle (C15B-4) using non-absorbable surgical tacks secured via stitches or a silicone frame.

[0961] The graft remained intact / undamaged during the various procedures and at the end of the implantation. Part D - Biological evaluation of hydrogels Example D1A: In vitro evaluation of hydrogel cytotoxicity by extractables test

[0962] The cytotoxicity of the hydrogel was evaluated using an extraction method according to ISO10993-5:Biological evaluation of medical devices recommendations.

[0963] Place the hydrogel in a 24-well plate at 3 cm 2 Cells were placed in medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at 0.1% / ml. They were incubated at 37°C, 5% CO2, and 70 rpm on an orbital shaker for 24 hours to obtain hydrogel extraction medium. In parallel, 3T3 cells were plated in medium at 5,000 cells / well in a 96-well plate and cultured overnight at 37°C, 5% CO2. The following day, the cell medium was removed and replaced with hydrogel incubation medium. After 24 hours of incubation of 3T3 cells in extraction medium at 37°C, 5% CO2, viability was measured by quantifying intracellular ATP concentration using an ATPLite kit (PerkinElmer) according to the manufacturer's instructions.

[0964] The survival rate was calculated using the following formula: Viability (%) = Signal テスト項目 / signal 対照 ×100

[0965] The cytotoxicity of hydrogel composition C4A-21 was evaluated in this extract test. The results are shown in Table 19 below. Viability was compared to an untreated control. The standard deviation (SD) of the mean viability of triplicate wells was calculated (n=2 hydrogels, each extract). Triplicate cell wells were loaded. [Table 19A]

[0966] Selected compositions did not exhibit significant cytotoxicity compared to untreated cells (t-test, p=0.48) (see Table 19). Example D1B: In vitro evaluation of the cytotoxicity of C16B-18, C16B-19, and C16B-20 hydrogels by extract test with and without human pancreatic islets

[0967] The cytotoxicity of C16B-18, C16B-19, and C16B-20 was evaluated using an extraction method according to ISO 10993-5: Biological evaluation of medical devices recommendations. Test items were C16B-18, C16B-19, and C16B-20 with or without primary human pancreatic islets. Primary human islets were isolated according to Example B3A and encapsulated as described in Examples C16B-18, C16B-19, and C16B-20.

[0968] C16B-18, C16B-19 and C16B-20 with or without islets were placed in a 6-well plate at 6cm 2 Cells were placed in medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) at 0.1% / ml. They were incubated at 37°C, 5% CO2, and 70 rpm on an orbital shaker for 24 hours to obtain hydrogel extraction medium. In parallel, 3T3 cells were plated in medium at 5,000 cells / well in a 96-well plate and cultured overnight at 37°C, 5% CO2. The following day, the cell medium was removed and replaced with hydrogel incubation medium. After 24 hours of incubation of 3T3 cells in extraction medium at 37°C, 5% CO2, viability was measured by quantifying intracellular ATP concentration using an ATPLite kit (PerkinElmer) according to the manufacturer's instructions.

[0969] The survival rate was calculated using the following formula: Viability (%) = Signal テスト項目 / signal 対照 X100 The control was untreated 3T3 cells.

[0970] The cytotoxicity of hydrogel compositions C16B-18, C16B-19, and C16B-20 was evaluated in this extract assay. The results are shown in Table 19B below. Values were normalized to untreated 3T3. [Table 19B]

[0971] Selected compositions did not affect the viability of 3T3 cells with or without encapsulated human islets (one-way ANOVA, p=0.7868; p=0.7293; p=0.5853; p=0.9105; p=0.8582; p=0.6743, respectively, for C16B-18, C16B-18 with islets, C16B-19, C16B-19 with islets, C16B-20, and C16B-20 with islets).

[0972] In conclusion, C16B-18, C16B-19 and C16B-20, with or without encapsulated human islets, are not associated with cytotoxicity. Example D1C: Short-term in vitro functionality of rat primary islets encapsulated in Example C16B-11

[0973] Two days after isolation, rat primary islets were encapsulated at 40 kIEQ / mL as described in Example C16B-11 and kept in a 37° C. / 5% CO 2 incubator in culture medium (see Example B2B).

[0974] The functionality of rat primary islets was assessed on days 2 and 8 post-encapsulation. Unencapsulated islets from the same islet preparations were assessed in parallel to the first time point. See Table 19C. [Table 19C]

[0975] The secretory index remained above 2 until day 8 after encapsulation and was not different from control unencapsulated islets (one-way ANOVA, p-value = 0.4699). Furthermore, insulin secretion levels did not decrease compared to control unencapsulated islets until day 8 after encapsulation (one-way ANOVA, p-value = 0.5632). Overall, these results indicate that the functionality of rat primary islets is maintained for at least 8 days after encapsulation in C16B-11. Example D1D: Short-term in vitro functionality of rat primary islets encapsulated in Example C16B-13

[0976] To assess the in vitro functionality of unencapsulated and encapsulated rat primary islets, GSIS (glucose stimulated insulin secretion) experiments were performed as described in Example D2C.

[0977] Two days after isolation, rat primary islets were encapsulated at 20 kIEQ / mL as described in Example C16B-13 and maintained in a 37°C / 5% CO2 incubator in culture medium (see Example B2B). Their functionality was assessed in parallel with unencapsulated islets from the same islet preparation after 8 days of in vitro culture. See Table 19D. [Table 19D] The secretory index remained above 2 up to 8 days after encapsulation and was not different from control unencapsulated islets (t-test, p-value = 0.07), indicating that functionality of rat primary islets was maintained up to 8 days after encapsulation in C16B-13. Example D1D: Short-term in vitro functionality of rat primary islets encapsulated in Example C16B-16 a / Total insulin content quantification method After functional assessment, unencapsulated or encapsulated islets were lysed in RIPA buffer on ice for 10 minutes. Samples were then sonicated for 1 minute and stored at -80°C. The insulin concentrations of these samples were quantified using a validated ELISA assay specific for insulin. Insulin concentrations were normalized to the IEQ amount of the sample (see Example B3A). b / The encapsulation process in Example C16B-16 did not affect insulin synthesis or islet functionality in rat primary islets. Three days after isolation, rat primary islets were encapsulated at 15 kIEQ / mL as described in Example C16B-16 and maintained in a 37°C / 5% CO2 incubator in the medium described in Example B2B. Their functionality was assessed after 1, 2, and 8 days of in vitro culture (see Example D2C). In parallel, samples were analyzed for total insulin content. See Table 19E. [Table 19E]

[0978] Rat islets encapsulated in C16B-16 still contained insulin 8 days after encapsulation and were still able to secrete insulin in response to glucose, as indicated by a mean secretory index greater than 3.

[0979] Overall, these results demonstrate that both insulin synthesis and functionality of primary islets are maintained for at least 8 days after encapsulation in C16B-16. Example D2A: In vitro viability and functionality of encapsulated pseudoislets and primary human islets a) Survival rate assessment

[0980] Viability was determined using Live / Dead staining (Thermo Fisher Scientific) on encapsulated or unencapsulated primary or pseudoislets. Samples were washed with phosphate-buffered saline (PBS). They were then incubated for 60 min in PBS supplemented with 2 μM calcein-AM and 8 μM ethidium bromide. Finally, they were washed with PBS and imaged using an epifluorescence microscope.

[0981] Quantification of viability was performed by segmenting the images: the integrated intensity (total pixel area intensity) of the green frequency band, V (live cells), and the integrated intensity of the red frequency band, D (dead cells), were calculated.

[0982] The survival ratio was obtained by calculating V / (V+D). b) Functionality evaluation method

[0983] Perfusion experiments were performed to assess the functionality of primary or pseudo islets (unencapsulated or encapsulated). On selected days after encapsulation, 400 IEQ (see Example B3A) from the batch used for encapsulation (primary or pseudo islets) as well as the encapsulated sample were introduced into the perfusion chamber. The perfusion chambers were simultaneously perfused according to the procedure described in Table 20A. [Table 20A]

[0984] The flow-through buffer was collected and insulin was quantified using a validated sandwich ELISA assay specific for insulin.

[0985] The insulin output for each sample was calculated as the sum of the insulin concentrations measured over the first basal, stimulated, and second basal steps.

[0986] The secretory index for each sample was calculated as the ratio of the mean insulin concentration measured during the stimulated step over the mean insulin concentration measured during the first basal secretory step. c) Encapsulation maintains the viability and functionality of encapsulated Min-6 pseudoislets

[0987] Min-6 pseudo islets were encapsulated as described in Example C16A-1 and maintained in culture medium (see Example B1A) in a 37°C / 5% CO2 incubator. Viability was assessed 4 days after encapsulation as described in Example D2A and compared with unencapsulated pseudo islets from the same batch. See Table 21A. [Table 21A]

[0988] The encapsulation process did not adversely affect the viability and secretory index on day 4 after encapsulation. Insulin output was slightly reduced, but not significantly (Welch's test, p=0.19). In conclusion, the viability and functionality of encapsulated pseudoislets are maintained after encapsulation. d) Encapsulation maintains the viability and functionality of encapsulated primary human islets for at least 22 days after encapsulation

[0989] Primary human islets were encapsulated as described in Examples C16B-2 and C16B-3 and maintained in culture medium (see Example B2A) in a 37°C / 5% CO2 incubator. Viability was assessed at 14 and 22 days post-encapsulation (E14 and E22, respectively), and functionality was assessed at 11 and 22 days post-encapsulation (E11 and E22, respectively). See Table 22A. [Table 22A]

[0990] The encapsulation process results in a survival rate of over 90% after encapsulation. Encapsulation does not significantly adversely affect insulin output or secretory index at 11 and 22 days after encapsulation compared to unencapsulated islets (2-way ANOVA, p=0.46 for insulin output; p=0.39 for secretory index). In conclusion, encapsulation maintains the viability and functionality of encapsulated primary human islets for at least 22 days after encapsulation. c) Encapsulated primary human islets maintain stable functionality for at least 3 months

[0991] Primary human islets were encapsulated at 20 kIEQ / ml as described in Examples C16A-13 and C16A-16 and maintained in culture medium (see Example B2A) in a 37°C / 5% CO2 incubator. Their functionality was assessed at 7 days, 1 month, and 3 to 4 months post-encapsulation and compared to unencapsulated islets, as described in Example D2A. See Table 22B. [Table 22B]

[0992] Encapsulated islets showed no significant differences in basal and stimulated insulin secretion, or secretory index (ratio of stimulated to basal secretion) compared with non-encapsulated islets at 1 week or 1 month after encapsulation (two-way ANOVA, p=0.1071, p=0.5161, and p=0.7642, respectively).

[0993] As expected, unencapsulated islets could not be maintained in culture for more than one month (Marchini A, Ciulla MG, Antonioli B, Agnoli A, Bovio U, Visnoviz V, Bertuzzi F, Gelain F. Long-term cultures of human pancreatic islets in self-assembling peptide hydrogels. Front Bioeng Biotechnol. 2023 Feb 23;11:1105157. doi:10.3389 / fbioe.2023.1105157. PMID:36911193; PMCID:PMC9995881). Encapsulated islets showed no long-term decline in basal and stimulated insulin secretion, or secretory index, at least 3-4 months after encapsulation, using two-way ANOVA (p=0.3394, p=0.6737, and p=0.4482, respectively).

[0994] Overall, these data indicate that encapsulation preserves the viability and functionality of encapsulated primary human islets for at least 3 months after encapsulation. Example D2B: In vitro viability and functionality of rat islets encapsulated in C16B-4A and C16B-4B a) Rat primary islet encapsulation

[0995] Primary rat islets (from Wistar or Lewis rat donors) were isolated as described in Example B2B, encapsulated (17.3 kIEQ / ml) on day 1 or 3 after isolation as described in Examples C16B-4A, C16B-4B, and C16B-14, and maintained in in vitro culture medium. b) In vitro viability assessment

[0996] Viability was determined using Live / Dead staining (Thermo Fisher Scientific) on encapsulated or unencapsulated primary or pseudoislets. Samples were washed with phosphate-buffered saline (PBS). They were then incubated for 60 min in PBS supplemented with 2 μM calcein-AM and 8 μM ethidium bromide. Finally, they were washed with PBS and imaged using an epifluorescence microscope.

[0997] Quantification of viability was performed by segmenting the images: the integrated intensity (total pixel area intensity) of the green frequency band, V (live cells), and the integrated intensity of the red frequency band, D (dead cells), were calculated.

[0998] The survival ratio was obtained by calculating V / (V+D).

[0999] Briefly, samples were first washed in phosphate-buffered saline (PBS) and then incubated for 60 min in PBS supplemented with 2 μM calcein-AM and 8 μM ethidium bromide. Finally, they were washed with PBS and imaged using an epifluorescence microscope. Quantification of viability was performed by segmenting the images. The integrated intensity (total pixel area intensity) of the green frequency band, V (live cells), and the integrated intensity of the red frequency band, D (dead cells), were calculated. The viability ratio was obtained by calculating V / (V + D). c) In vitro functionality evaluation method

[1000] Perfusion experiments were performed to assess the functionality of encapsulated primary rat islets. On selected days after encapsulation, samples were introduced into the perfusion chambers, which were simultaneously perfused according to the procedure described in Table 20B.

[1001] The flow-through buffer was collected and insulin was quantified using a validated ELISA assay specific for insulin.

[1002] The secretory index for each sample was calculated as the ratio of the mean insulin concentration measured during the stimulation step to the mean insulin concentration measured during the first basal secretory step. [Table 20B]

[1003] Perfusion experiments were performed to assess the functionality of primary or pseudo islets (unencapsulated or encapsulated). On selected days after encapsulation, 400 IEQ (see Example B3A) from the batch used for encapsulation (primary or pseudo islets) were introduced into the perfusion chamber, as well as the encapsulated sample. The perfusion chambers were simultaneously perfused according to the procedure described in Table 20C. [Table 20C]

[1004] The flow-through buffer was collected and insulin was quantified using a validated sandwich ELISA assay specific for insulin.

[1005] Basal secretion and stimulated secretion were calculated as the average of insulin concentrations in the first basal secretion-G3 phase and stimulated secretion-G17 phase, respectively.

[1006] The insulin output for each sample was calculated as the sum of the insulin concentrations measured over the first basal, stimulated, and second basal steps.

[1007] The secretory index for each sample was calculated as the ratio of the mean insulin concentration measured during the stimulation step to the mean insulin concentration measured during the first basal secretory step. d) Insulin content in pancreatic islet cells

[1008] After functional assessment, unencapsulated or encapsulated islets were lysed in RIPA buffer on ice for 10 minutes. Samples were then sonicated for 1 minute and stored at -80°C + / - 20°C. The insulin concentration of these samples was quantified using a validated ELISA assay specific for insulin. Insulin concentrations were normalized to the IEQ amount of the sample (see Example B3B). e) Rat primary islet viability and functionality are preserved in C16B-4A and C16B-4B.

[1009] The viability and functionality of primary rat islets encapsulated in C16B-4A and C16B-4B were assessed on days 8 and 9 post-encapsulation, respectively (Table 21A). The results were pooled. [Table 21B]

[1010] The survival rate of primary rat islets encapsulated in C16B-4A and C16B-4B was high at day 9: 87.2% + / - 6.9%. Furthermore, primary rat islets encapsulated in C16B-4A and C16B-4B retained the ability to secrete insulin in response to glucose stimulation: secretory index = 3.45 + / - 1.68. f) The functionality of rat primary islets is preserved in C16B-14.

[1011] The functionality of rat primary islets encapsulated in C16B-14 was assessed 2 days after encapsulation as described in Example D2C, see Table 21C.

[1012] In parallel, samples were analyzed for total insulin content, see Table 21C. [Table 21C]

[1013] Rat islets encapsulated in C16B-14 still contained insulin 2 days after encapsulation and were still able to secrete insulin in response to glucose, as indicated by a mean secretory index of 6.5.

[1014] Overall, these results demonstrate that both insulin synthesis and functionality of primary islets are maintained for at least 2 days after encapsulation in C16B-14. In conclusion, the viability and functionality of rat primary islets is preserved in C16B-4A, C16B-4B, and C16B-14. Example D2C: Long-term in vitro functionality of pancreatic islets encapsulated in C16B-1A Rat primary islet encapsulation

[1015] Primary rat islets were isolated as described in Example B1, encapsulated one day after isolation as described in Example C16B, and maintained in in vitro culture for 28 days. a) In vitro Functionality Evaluation - Method 1

[1016] To assess the in vitro functionality of encapsulated rat primary islets, GSIS (glucose-stimulated insulin secretion) experiments were performed.

[1017] Briefly, encapsulated islets were first washed three times in Krebs buffer containing 0.1% BSA and 3 mM glucose (G3 solution) (Table 23). The encapsulated islets were then incubated twice in G3 solution for 30 minutes at 37°C to equilibrate insulin secretion. The encapsulated islets were further incubated in G3 solution for 60 minutes at 37°C to obtain basal insulin secretion. At the end of this incubation, the medium was collected (basal insulin secretion sample). The encapsulated islets were then stimulated in Krebs buffer containing 0.1% BSA and 17 mM glucose (G17 solution) for 60 minutes at 37°C. At the end of the incubation, the medium was collected (stimulated insulin secretion sample). [Table 23]

[1018] Insulin concentrations in these samples were quantified using an insulin-specific validated ELISA assay, and the secretory index was calculated as the ratio of the stimulated to the basal secretory step of the measured insulin concentrations. b) Long-term functionality of rat primary islets is maintained when encapsulated in C16B-1A.

[1019] The functionality of primary rat islets encapsulated in C16B-1A was evaluated 28 days after encapsulation. The secretory index was 2.9, indicating that the functionality of primary rat islets was maintained in C16B-1A.

[1020] In conclusion, the long-term functionality of rat primary islets is preserved when encapsulated in C16B-1A. Example D2D: In vitro functionality of pancreatic islets encapsulated in C16B-5 a) Rat primary islet encapsulation

[1021] Primary rat islets were isolated as described in Example B and encapsulated one day after isolation as described in Example C16B-5. b) Functionality of primary rat pancreatic islets is preserved when encapsulated in C16B-5

[1022] The in vitro functionality of rat primary islets encapsulated in C16B-5 was assessed 1 day after encapsulation as described in Example D2B (Table 24A). [Table 24A]

[1023] The secretory indices were 3.1 and 3.0, respectively, indicating that the functionality of encapsulated rat primary islets was maintained in C16B-5.

[1024] In conclusion, the functionality of rat primary pancreatic islets is preserved in vitro when encapsulated in C16B-5. Example D2E: Long-term in vitro functionality of human pancreatic islets encapsulated in C16B-18 and C16B-19 a) Human primary islet encapsulation

[1025] Human primary islets from three independent specimens were isolated as described in Example B3, encapsulated in C16B-18 (10 kJ / mL) and C16B-19 (10 kJ / mL) as described in Example C16 on days 6, 7, and 5 after isolation, and maintained in vitro for 33 days. b) In vitro functionality evaluation

[1026] The in vitro functionality of unencapsulated and encapsulated human primary islets was assessed as described in Example D2C. c) Long-term functionality of primary human islets is maintained when encapsulated in C16B-18 and C16B-19

[1027] Unencapsulated and encapsulated human islets were kept in culture medium in a 37°C / 5% CO2 incubator for up to one month. Functionality was assessed and compared at one week and one month after the day of encapsulation, see Table 24B. [Table 24B]

[1028] Unencapsulated human islets and human islets encapsulated in C16B-18 and C16B-19 showed no significant differences in either stimulated insulin secretion or secretory index at 1 week (one-way ANOVA, p=0.7568 and p=0.6142, respectively) or 1 month (one-way ANOVA, p=0.8093 and p=0.5412, respectively) after encapsulation.

[1029] Unencapsulated human islets and human islets encapsulated in C16B-18 and C16B-19 showed no significant decrease in stimulated insulin secretion (paired t-test, p=0.1048, p=0.7533, and p=0.7734, respectively) or secretory index (paired t-test, p=0.4108, p=0.5411, and p=0.0615) over time, at least 1 month after encapsulation.

[1030] In conclusion, long-term in vitro functionality of human primary pancreatic islets is maintained for at least one month when encapsulated in C16B-18 and C16B-19. [Table 25] Example D3: Long-term local tolerance of hydrogels in vivo a) The local tolerance of the hydrogel was evaluated according to the guideline ISO 10993 Part 6 (2016): Testing of local effects after implantation. b) C4A-3 hydrogel and negative control (NC) HDPE discs of the same size as the hydrogel discs were implanted into the dorsal subcutaneous tissue of rats for 13 weeks. The appearance of the hydrogel as well as local tissue effects were evaluated by macroscopic and histopathological analyses (n=5 per article and per site per period) as recommended in ISO 10993 Part 6. After the animals were sacrificed and the hydrogels were removed, the tissue reactivity produced by the implants was assessed by assigning scores to various components on a scale of 0 to 4. The components assessed were polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, and angiogenesis in one area, and fibrosis and fatty infiltration in the second. Various inflammatory cell types or morphological features were reported only if present. A tissue reactivity score was calculated by taking the average score obtained for each test article, minus the average score obtained for the negative control article (HDPE, high-density polyethylene), as recommended by ISO 10993 Part 6. c) Macroscopically, the shape, size, color, and hardness of the C4A-3 hydrogel were well preserved. d) After histopathological examination and according to the tissue reactivity scoring scale from guideline ISO 10993 part 6, C4A-3 was found to induce a "minimal to no reaction" with a me...

Claims

1. A hydrogel comprising a cross-linked dextran polymer Dx having anionic groups, At least a divalent radical L(-) ｉ is covalently attached to a dextran polymer backbone bearing a W radical of i, L(-) ｉ is a linear or branched polyether, i is an integer between 2 and 8, inclusive, where i is the valence of L and the number of W radicals attached to the dextran polymer; 2≦i≦8; -W- is a radical containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether derivative, but not containing two or more alpha amino acid residues, in particular two or more alpha amino acid residues linked by peptide bonds; Hydrogel.

2. The hydrogel of claim 1 , further comprising a non-crosslinked hyaluronic acid salt in the form of a solution.

3. The hydrogel according to claim 1 or 2, further comprising biological cells.

4. At least a divalent radical L(-) ｉ is covalently attached to the dextran polymer backbone with a -W- radical of i, L(-) ｉ is a linear or branched polyether or a linear or branched poly(oxazoline), i is an integer between 2 and 8, inclusive, where i is the valence of L and the number of -W- radicals attached to the dextran polymer; 2≦i≦8; 4. The hydrogel according to claim 1, wherein -W- is selected from anionic dextran polymers Dx, which are radicals containing at least one linear or branched alkyl radical, optionally containing a heteroatom such as oxygen, nitrogen or sulfur, an aromatic ring, a polyether or a poly(oxazoline) derivative, and which do not contain two or more types of α-amino acid residues, in particular two or more types of α-amino acid residues linked by peptide bonds.

5. 5. The hydrogel of claim 1, wherein Tan δ is less than 1.

6. The hydrogel according to any one of claims 1 to 5, which is a transparent hydrogel.

7. The hydrogel according to any one of claims 1 to 6, which is a translucent hydrogel.

8. 8. The hydrogel of claim 1, wherein after swelling in water, the cross-linked dextran polymer concentration is 0.01 to 0.2 g / g.

9. 9. The hydrogel according to claim 1, wherein the Young's modulus is between 1 and 200 kPa.

10. 10. The hydrogel according to claim 1, having a storage modulus G' of 0.5 to 70 kPa.

11. 11. The hydrogel according to claim 1, wherein the compressive deformation at break is 10% or more.

12. 12. The hydrogel of claim 1, having a swelling ratio of greater than 0.

7.

13. 13. The hydrogel of claim 1, having a water content of at least 80% by weight.

14. The hydrogel according to claim 1 , wherein the biological cells are protein-, hormone-, or peptide-secreting cells.

15. The biological cells insulin-secreting cells for diabetes treatment, selected from factor VIII or factor IX secreting cells for the treatment of hemophilia, and β-glucocerebrosidase secreting cells for Gaucher disease; The hydrogel according to any one of claims 1 to 14.

16. The hydrogel according to any one of claims 1 to 15, wherein the biological cells are pseudo-pancreatic islets.

17. 17. A method for synthesizing the crosslinked dextran polymer of any one of claims 1 to 16 in the form of a hydrogel, comprising the steps of: a) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; b) L(-) ｉ preparing a sterile solution of the precursor of c) adding the sterile solution obtained in step b) to the solution obtained in step a); d) the addition is carried out directly in the mold or the solution is introduced into the mold after mixing; e) crosslinking and gelling, for example at room temperature (20-25°C) or 37°C; f) Remove from the mold and allow to swell to obtain a hydrogel.

18. 17. A method for synthesizing the crosslinked dextran polymer of any one of claims 1 to 16 in the form of a hydrogel, comprising the steps of: a) an anionic group of formula II and at least two -(Af ２ ) ａ -G １ -, -(A'-f ２ ) ａ -G' １ - preparing a sterile solution containing dextran having a precursor of b) L(-) ｉ preparing a sterile solution of the precursor of c) adding the sterile solution obtained in step b) to the solution obtained in step a); d) the addition is carried out directly in the mold or the solution is introduced into the mold after mixing; e) crosslinking and gelling, for example at room temperature (20-25°C) or 37°C; f) Remove from the mold and allow to swell to obtain a hydrogel.

19. 19. The method of claim 17 or 18, wherein the cross-linking step is a gelation step leading to the formation of a hydrogel.

20. 17. A method for preparing a hydrogel containing biological cells according to any one of claims 3 to 16, comprising the steps of: a) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; b) L(-) ｉ preparing a sterile solution of the precursor of c) preparing a suspension of biological cells; d) mixing the biological cell suspension obtained in step c) with the solution obtained in step b) or a); e) adding the sterile solution from step a) or b) that was not used in step d) to the solution from step d); f) The addition of step e) is carried out either directly in the mold or by introducing the solution into the mold after mixing; g) Carrying out the crosslinking and gelling reaction at room temperature (20-25°C); h) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

21. 17. A method for preparing a hydrogel containing biological cells according to any one of claims 3 to 16, comprising the steps of: a) an anionic group of formula II and at least two -(Af ２ ) ａ -G １ -, -(A'-f ２ ) ａ -G' １ - preparing a sterile solution containing dextran having a precursor of b) L(-) ｉ preparing a sterile solution of the precursor of c) preparing a suspension of biological cells; d) mixing the biological cell suspension obtained in step c) with the solution obtained in step b) or a); e) adding the sterile solution from step a) or b) that was not used in step d) to the solution from step d); f) The addition of step e) is carried out either directly in the mold or by introducing the solution into the mold after mixing; g) Carrying out the crosslinking and gelling reaction at room temperature (20-25°C); h) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

22. 17. A method for preparing a hydrogel containing biological cells according to any one of claims 3 to 16, comprising the steps of: a) preparing a sterile solution containing a dextran having an anionic group of formula II and at least two precursors of -W-; b) L(-) selected from thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide, or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene ｉ preparing a sterile solution of the precursor of c) preparing a sterile solution of sodium hyaluronate; d) preparing a sterile suspension of biological cells; e) mixing the sodium hyaluronate solution obtained in step c) with the precursor solution of step b); f) mixing the biological cell suspension obtained in step d) with the solution obtained in step e) or a); g) mixing the solution obtained in step f) with the solution obtained in step e); h) adding the sterile solution from step a) or e) not used in step g) to the solution from step f); i) the addition of step g) is carried out either directly in the mold or by introducing the solution into the mold after mixing; j) Crosslinking and gelling reaction is carried out at room temperature (20-25°C); k) Remove from the mold and allow to swell to obtain a hydrogel containing living cells.

23. 17. The method for preparing a hydrogel according to claim 1, wherein in the forming step, the solution contains an osmotic agent that is a non-ionic osmotic agent such as trehalose.

24. 17. A method for preparing a hydrogel according to any one of claims 1 to 16, wherein in the forming step the solution has a weight ratio of non-ionic osmotic agent to NaCl of more than 2, in particular more than 5, more in particular more than 10.

25. The method for preparing a hydrogel according to any one of claims 1 to 16, wherein the mold is a ring net.

26. 17. The method for preparing a hydrogel according to any one of claims 1 to 16, wherein the crosslinking and gelling reaction is carried out at room temperature (20-25°C).

27. Kit includes: a solution of dextran polymer of formula VIII prior to the cross-linking reaction; 【Chemistry 55】 (in formula VIII If none of a, a', b, and b' is 0, then f １ , f ２ , f ３ , f ４ , Dx is defined as in Formula IV; ・and x is 0 or 1, When a, a', b and b' are 0, x is 0 and Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, then -A' is A as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; If c is not 0, then R' １ is R as defined above １ and G' １ is G １ is a precursor of When c is 0, b is 0 and R' １ is R before the crosslinking reaction １ (which is a precursor of a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells.

28. Kit includes: a solution of a pre-crosslinked dextran polymer of formula VIII; 【Chemistry 55】 (in formula VIII If none of a, a', b, and b' is 0, then f １ , f ２ , f ３ , f ４ , Dx is defined as in Formula IV; ・and x is 0 or 1, When a, a', b and b' are 0, x is 0 and Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, then -A' is A as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; If c is not 0, then R' １ is R as defined above １ and G' １ is G １ is a precursor of When c is 0, b is 0 and R' １ is R before the crosslinking reaction １ (which is a precursor of a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells, A solution of non-crosslinked sodium hyaluronate.

29. Kit includes: a solution of a pre-crosslinked dextran polymer of formula VIII; 【Chemistry 55】 (in formula VIII If none of a, a', b, and b' is 0, then f １ , f ２ , f ３ , f ４ , Dx is defined as in Formula IV; ・and x is 0 or 1, When a, a', b and b' are 0, x is 0 and Dx is a dextran polymer backbone according to formula III, and R is selected from -H or an anionic group of formula II; If one of b' and c is not 0, then -A' is A as defined above; When b', b and c are 0, a is 0 and A' is a precursor of A before the crosslinking reaction; If c is not 0, then R' １ is R as defined above １ and G' １ is G １ is a precursor of When c is 0, b is 0 and R' １ is R before the crosslinking reaction １ (which is a precursor of a solution of thiol polyethylene glycol, mercaptopoly(oxyethylenes), pentaerythritol poly(oxyethylene) azide or pentaerythritol poly(dibenzocyclooctyne) polyoxyethylene; Living cells.

30. 17. Use of the cross-linked dextran copolymer of any one of claims 1 to 16 for preparing a cell composition in the form of a hydrogel.

31. 17. Therapeutic use of the hydrogel of any one of claims 1 to 16 as a therapeutic implant for administering at least one API (active pharmaceutical ingredient) to a mammal.

32. Therapeutic use of a hydrogel described in any one of claims 1 to 16 for treating a disorder or disease in a mammal, wherein the disorder or disease is caused by a lack or malfunction of endocrine function of the pancreatic organ.

33. 17. The hydrogel of any one of claims 1 to 16 for use as a medicament.

34. 17. A hydrogel according to any one of claims 1 to 16 for use in the treatment of diseases such as diabetes.

35. 17. An implant comprising the hydrogel of any one of claims 1 to 16.

36. A graft comprising a ring, a net, a hydrogel according to any one of claims 1 to 16 and cells.

37. 37. The implant of claim 35 or 36, which is a rectangular prism with rounded corners.

38. 38. The implant of any one of claims 35 to 37, having a thickness of less than 3000 μm.

39. Total surface area is 10 cm ２ and 200 cm ２ 38. The implant of any one of claims 35 to 37, wherein:

40. 39. The implant of any one of claims 35 to 38, comprising 0.5 to 20 ml of hydrogel.

41. 41. The implant of any one of claims 35 to 40, wherein the inner diameter of the ring is between 10 and 100 mm.

42. 42. The implant of any one of claims 35 to 41, wherein the ring is a rectangular parallelepiped with rounded corners.

43. 43. The implant of any one of claims 35 to 42, wherein the material of the ring is a bioinert material.

44. 44. The implant of any one of claims 35 to 43, wherein the material of the ring is a biocompatible elastomer.

45. 45. An implant according to any one of claims 35 to 44, wherein the material of the ring is selected from the group consisting of silicones, in particular PDMS, polyurethanes, polyethers, polyether polyester copolymers, and polypropylene oxide.

46. 46. The implant of any one of claims 35 to 45, wherein the net is non-biodegradable.

47. 46. The implant of any one of claims 35 to 45, wherein the net is biocompatible.

48. 46. The implant of any one of claims 35 to 45, wherein the netting is non-absorbable.

49. 49. The implant of any one of claims 35 to 48, wherein the net is a surgical mesh.

50. 50. The implant according to any one of claims 35 to 49, wherein the fibrous material of the netting material is selected from the group consisting of polypropylene, polyethylene, polyester, in particular PET, PTFE, PVDF (polyvinylidene fluoride) and ePVDF (extended PVDF).

51. 38. An implant according to any one of claims 35 to 37, wherein the thickness of the netting is in the range of from 50 to 500 μm.

52. 52. The implant of any one of claims 35 to 51, wherein the size of the holes in the net ranges from 0.4 to 4 mm.

53. 38. The implant of any one of claims 35 to 37, wherein the fabric of the net is selected from the group consisting of knitted fabrics, warp knitted fabrics, woven fabrics, and nonwoven fabrics.

54. 54. A graft according to any one of claims 35 to 53, obtained by the following method: Incorporating the hydrogel composition into a ring mesh structure, mixing the concentrated polymer solution with a pipette, and introducing a controlled volume of the mixture into the ring mesh structure attached to a glass slide. Cross-linking is carried out, leading to gelation, and then the ring mesh and hydrogel composition are introduced into a 150 mM Tris / 30 mM NaCl / 10 mM cysteine solution at pH 8 or into PBS at pH 7.

4. The hydrogel is rinsed with cysteine-free PBS solution and further immersed in PBS solution at 37°C overnight, and the hydrogel pieces are stored in PBS solution at 4°C until use.

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