Hydrogels for the immobilization of one or more enzymes and methods for preparing same - Patents.com

Abstract

The present invention provides a method for preparing a biocompatible hydrogel, a hydrogel obtainable by the method, a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, and a composition comprising any of the hydrogels.The present invention further provides a method for encapsulating one or more enzymes in the hydrogel described herein, and the use of any of the hydrogels for non-covalent immobilization of one or more enzymes in the hydrogel, or the use of any of the hydrogels in a biosensor.In addition, the present invention provides a kit comprising the composition or hydrogel of the present invention.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention provides a method for preparing a biocompatible hydrogel, a hydrogel obtainable by the method, a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, and a composition comprising any of the hydrogels of the present invention.The present invention further provides a method for encapsulating one or more enzymes in the hydrogel described herein, and the use of any of the hydrogels for non-covalent immobilization of one or more enzymes in the hydrogel, or the use of any of the hydrogels in a biosensor.In addition, the present invention provides a kit comprising the composition or hydrogel of the present invention. [Background technology]

[0002] Enzymes in solution usually have a limited life span, since they can decompose quickly at ambient temperature. Moreover, they also have limited tolerance to organic solvents. The aim of the present invention is in particular to provide an effective method for the stabilization of enzymes. Encapsulation of enzymes for various purposes usually requires modification by covalent bonds, which leads to a loss of activity. Furthermore, the bonds used for hydrogel formation are often not stable, for example when using Schiff bases, leading to early degradation of the gel and subsequent leaching of the payload, or they may contain toxic and carcinogenic, for example hydrazone functional groups.

[0003] Various methods have been developed to immobilize enzymes within hydrogel networks, for example using glutaraldehyde. However, for example, BSA mixtures offer only limited enzyme protection and suffer from short life spans and toxicity. Other methods using wired enzymes require chemistries with toxic components, limiting their application in implantable devices [1].

[0004] Other methods in the prior art use polymers modified with furan / furan derivatives (methylfuran) reacting with poly(ethylene glycol) [2]. Other methods in the prior art use the formation of Schiff bases (between amino and aldehyde groups), leading to the formation of imine bonds, for the preparation of hydrogels [3]. For example, Ma et al. use the formation of Schiff bases (aldehyde and hydrazide groups) to construct injectable hydrogels [4].

[0005] Other possibilities include chemically specific "click" reactions with oxime bridges. Oxime bonds are more stable than hydrazines, but this has the disadvantage that the hydrogels may be reversible in some biological environments [5].

[0006] Instead, Peng et al. provided stabilization of the collagen sponge with glutaraldehyde and used vapor crosslinking.[6] Jia et al.[7] taught the use of an enzyme solution and BSA stabilizer in acetic acid with chitosan, but the final crosslinking of the hydrogel was performed with glutaraldehyde vapor.

[0007] However, all of these methods described in the prior art have several drawbacks that make them inapplicable to the encapsulation of enzymes. For example, the formation of Schiff bases is problematic as it is reversible, and hydrazones are undesirable due to their toxicity. Furthermore, the hydrogels provided in the prior art have limited reproducibility, for example with regard to layer thickness. Further drawbacks are that the coating with imines / hydrazones is biodegradable, the product of decomposed hydrazones (hydrazine) is toxic, the glutaraldehyde cannot be spun, and there is insufficient quality control, plus any steam process is random and not reproducible [6].

[0008] Furthermore, for example, with regard to the encapsulation of enzymes within hydrogels, a major drawback of the prior art is that cross-linking with glutaraldehyde can result in covalent modification of amino groups in the enzyme, which causes changes in the structure and conformation of the enzyme, possibly resulting in undesired inactivation of the enzyme.

[0009] Also, the gelation process may begin immediately after mixing the individual components, leading to incomplete filling of the form factors and resulting in a non-uniform hydrogel.

[0010] The present invention is directed to and addresses these aforementioned needs. Summary of the Invention

[0011] The aforementioned problems are solved by the subject matter as defined in the claims and as defined herein.

[0012] The present invention describes a novel strategy for immobilizing enzymes in biocompatible hydrogels that does not require covalent attachment of one or more enzymes. The hydrogels of the present invention provide a perfect stabilizing environment for enzymes, resulting in better longevity and enzyme stability. The hydrogels of the present invention protect the enzymes from biofouling and the body's immune response.

[0013] In a first aspect, the present invention provides a method for preparing a biocompatible hydrogel, comprising the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, hyaluronan, and dextran; n is the number of monomer repeat units of the first and / or second polysaccharide, and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-; where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000001.tif28128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0014] In one aspect, the present invention provides a method for preparing a biocompatible hydrogel, comprising the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide, and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-; where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000002.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0015] In a further aspect, the present invention is directed to a hydrogel obtainable by the method for preparing a biocompatible hydrogel described herein.

[0016] In one aspect, the present invention provides a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, comprising a cross-linked polymer comprising the following structure: a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, Here, B is TIFF2024537390000003.tif20128, wherein R' is selected from the group consisting of -CF3, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units.

[0017] The invention further provides a composition comprising a hydrogel of any aspect of the invention described herein.

[0018] The invention further provides methods for encapsulating one or more enzymes in a hydrogel of any aspect of the invention described herein.

[0019] The present invention further comprises: a) for the non-covalent immobilization of one or more enzymes in the hydrogel, b) In a biosensor, Use of a hydrogel according to any aspect described herein is provided.

[0020] The invention further provides kits comprising a composition or hydrogel of any aspect of the invention described herein. [Brief description of the drawings]

[0021] [Figure 1] 1 shows an exemplary structure of a biocompatible hydrogel of the present invention. [Diagram 2] 1 illustrates an exemplary gelation principle for forming a biocompatible hydrogel according to the method of the present invention. [Figure 3A-1] The micromorphology of the 3D-hydrogel network was analyzed by scanning electron microscopy (SEM) using forced ion beam (FIB) in Figure 3. Figure 3A shows the pullulan hydrogel polymerized in PBS buffer, while Figures 3B and 3C show the pullulan hydrogel polymerized in dH2O. [Figure 3A-2] See legend to Figure 3A-1. [Figure 3B-1] See legend to Figure 3A-1. [Figure 3B-2]See legend to Figure 3A-1. [Figure 3C-1] See legend to Figure 3A-1. [Figure 3C-2] See legend to Figure 3A-1. [Figure 4-1] Figure 4 shows the surface layer, roughness and pores of the outer surface analyzed by atomic force microscopy (AFM). The higher the degree of modification, the denser the layer is and the fewer microcavities and pores it contains. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 4-5] See description of Figure 4-1. [Figure 4-6] See description of Figure 4-1. [Diagram 5] Figure 1 shows the degree of substitution of pullulan biopolymers with different amounts of carboxymethyl groups introduced. It shows different fingerprint regions in the FT-IR spectrum to characterize the degree of carboxymethylation of pullulan (PCM1, PCM3 and PCM5). The intensity of the band between 1600 and 1000 cm-1 increases with the degree of substitution. [Figure 6] 1H-NMR spectroscopy shows the increase in the amount of introduced carboxymethyl groups in the biopolymers. The signal intensity of the anomeric protons of the polysaccharide (squares) decreases with increasing degree of modification, while the signal of the polysaccharide backbone becomes broader (dashed line) and the signal intensity of the introduced carboxymethyl groups (dotted line) increases. [Figure 7] Stepwise modification process of polysaccharides (e.g. pullulan) by 1H-NMR spectroscopy. This compares carboxymethylated polysaccharides with unmodified polysaccharides. In this case, the signal intensity of the anomeric protons is reduced (see c and d). In the NMR spectrum of the "PCM3 linker" the signals of the oxanorbornadiene linker units introduced into the polysaccharide chain by copper-free cycloaddition reaction are visible (see a and b). [Figure 8]Figure 1 shows a direct correlation between the degree of substitution of polysaccharides by 1H-NMR spectroscopy and the introduced oxanorbornadiene linker units. c and d show the decrease in the signal intensity of the anomeric protons with increasing degree of substitution. a and b show the increase in the signal intensity of the introduced linker with increasing number of carboxymethyl groups in the polysaccharide. [Figure 9] Figure 1 shows the increasing degree of substitution with repeated carboxymethylation reactions as measured by conductometric titration. Data are shown for low molecular weight dextran (10 kDa) and high molecular weight pullulan (100 kDa). [Figure 10] Figure 1 shows the IR spectra of five different batches of 5-times carboxymethylated pullulan chains (PCM5), which contain azido linker units ((-NH-(CH2CH2O)s-CH2CH2N3, s=3). Data is shown for molecular weight pullulan (100 kDa). [Figure 11] 1 shows the time course of the storage module in the rheological measurements of three modified pullulan hydrogels with different degrees of carboxymethylation. [Figure 12] Figure 1 shows the increase in signal intensity with increasing degree of substitution and number of introduced linker units in the 19F-NMR spectra of linkers introduced into the pullulan chain relative to an internal standard. [Figure 13A-1] 13A and 13B show the results of swelling ratio determined for pullulan hydrogels with low and high modified pullulan (PCM1 and PCM8) obtained according to the procedure in Example 6. W=dH2O; P3=PBS buffer; pH 3.1, P7=PBS buffer; pH 7.4; W-P3 refers to swelling in PBS buffer, pH 3.1 after gelation in dH2O and lyophilization. [Figure 13A-2] See legend to Figure 13A-1. [Figure 13A-3] See legend to Figure 13A-1. [Figure 13B-1] See legend to Figure 13A-1. [Figure 13B-2] See legend to Figure 13A-1. [Figure 14A]FIG. 14 shows the enzyme saturation curve of glucose oxidase (50 mU / mL) in solution (FIG. 14A) and two curves of lowly (PCM2, FIG. 14B) and highly modified (PCM8, FIG. 14C) enzyme-immobilized pullulan hydrogel samples. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15A] Figure 15 details the lifetime of glucose oxidase (25 mU / mL) in hydrogels after immobilization on PCM9 and for the enzyme in solution. The kinetics of glucose oxidase in solution and immobilized in hydrogels are shown after 10 days (see Figure 15A) and 43 days (see Figure 15B). [Figure 15B] See legend to Figure 15A. [Figure 16A] Figure 16 details the lifetime of glucose oxidase (100 mU / mL) in hydrogels after immobilization on PCM5 and for the enzyme in solution. The kinetics of glucose oxidase in solution and immobilized in hydrogels are shown after 10 days (see Figure 16A) and 15 days (see Figure 16B). [Figure 16B] See legend to Figure 16A. [Figure 17] FIG. 1 shows lifetime data for the enzyme uricase (100 mU / mL), specifically the time course kinetics of uricase immobilized in hydrogel (PCM1). [Figure 18] Degree of substitution of dextran biopolymers (10 kDa) with different amounts of introduced carboxymethyl groups. It shows different fingerprint regions in the FT-IR spectrum to characterize different degrees of carboxymethylation (CM1, CM3 and CM5). The intensity of the band between 1600 and 1000 cm-1 increases with the degree of substitution. [Figure 19]The difference between unmodified lentinan and carboxymethylated lentinan (CM1) is shown. It visualizes the different fingerprint regions in the FT-IR spectrum to confirm the degree of carboxymethylation (lentinan and lentinan CM1). The intensity of the band between 1600 and 1000 cm-1 increases with the degree of substitution. [Figure 20] The difference between hyaluronan and hyaluronan with linker units (azide and oxanorbornadiene units) is shown. The wavenumber of the bands in the FT-IR spectrum varies between 1600 and 1000 cm-1 due to the different use of linker units. [Figure 21] FIG. 1 shows the 1H-NMR spectrum of a five-times carboxymethylated pullulan biopolymer with an azido linker unit (-NH-(CH2)r-N3, r=6). [Figure 22] Figure 1 shows the 1H-NMR spectrum of five-times carboxymethylated pullulan with oxanorbornadiene units (-NH-(CH2)rQ, r=6, Q=Q-1(CF3) and M / M'=H). [Figure 23] The 1H-NMR spectrum of five-times carboxymethylated pullulan having an azide unit (-NH-(CH2CH2O)s-CH2CH2N3, s=8) is shown. [Figure 24] 1H-NMR spectra of hyaluronan (A) compared with hyaluronan (B) having oxanorbornadiene units (-NH-(CH2)rQ, r=2, Q=Q-1(CF3) and M / M'=H) and hyaluronan (C) having azide units (-NH-(CH2CHO)s-CH2CH2N3, s=3). [Diagram 25] 1H-NMR spectra of dextran (10 kDa) with different amounts of carboxymethyl groups introduced. [Figure 26A]Figure 26 shows the viscosity of various biopolymers at a shear rate of 100 s-1. Different polysaccharides (A), pullulan with various carboxymethyl groups (B), viscosity difference due to pullulan modification (unmodified pullulan, pullulan carboxymethylated six times and pullulan mixture (mixture: pullulan with azide linker and pullulan with oxanorbornadiene units, before gelation) (C), viscosity difference due to alginate modification (unmodified alginate and alginate mixture (mixture: alginate with azide linker and alginate with oxanorbornadiene units, before gelation) (D). [Figure 26B] See legend to Figure 26A. [Figure 26C] See legend to Figure 26A. [Figure 26D] See legend to Figure 26A. [Figure 27] Figure 2 shows the enzyme kinetics of immobilized GOx in pullulan hydrogels (PCM1 and PCM9) after different washing steps. [Figure 28A] FIG. 28 shows the enzyme kinetics of immobilized UOx in unwashed and washed pullulan hydrogels (PCM1) (A) and supernatants (B). [Figure 28B] See legend to Figure 28A. [Figure 29] 4 shows the enzyme kinetics of immobilized UOx in pullulan hydrogel (PCM5). [Figure 30A] Figure 30 shows the enzymatic kinetics of immobilized GOx in various hydrogels. Dextran hydrogel (A), hyaluronan and alginate hydrogels (B), alginate-pullulan hydrogel (C) and pullulan hydrogels consisting of two linker units before gelation: 5-times carboxymethylated pullulan with oxanorbornadiene units (-NH-(CH2)rQ, r=6, Q=Q-1(CF3) and M / M'=H) and 5-times carboxymethylated pullulan with azide units (-NH-(CH2CHO)s-CH2CH2N3, s=8) (D). [Figure 30B] See legend to Figure 30A. [Figure 30C]See legend to Figure 30A. [Figure 30D] See legend to Figure 30A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention In a first aspect, the present invention provides a method for preparing a biocompatible hydrogel, comprising the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, hyaluronan, and dextran; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the first polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000004.tif28128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0023] In one aspect, the present invention provides a method for preparing a biocompatible hydrogel, comprising the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the first polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000005.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0024] In one embodiment, the method of preparing a biocompatible hydrogel comprises the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s-CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000006.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0025] In one embodiment, the method of preparing a biocompatible hydrogel comprises the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000007.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0026] In one embodiment, the method of preparing a biocompatible hydrogel comprises the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000008.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0027] In one embodiment, the method of preparing a biocompatible hydrogel comprises the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000009.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 30° C. to 60° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours.

[0028] In one embodiment, the method for preparing the biocompatible hydrogel of the present invention comprises the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the first polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000010.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature ranging from 30° C. to 60° C. for 1 to 10 hours.

[0029] The term "biocompatible" as used herein and in the context of the present invention, particularly in relation to hydrogels, means that each material that is called or evaluated as being biocompatible has the property of not having a toxic or injurious effect on a living system, does not induce any undesirable local or systemic effects in the recipient, but has the ability to generate the most appropriate beneficial response in that particular situation to perform its desired function, or has the ability to exist in harmony with tissues without causing adverse changes. The preferred properties of a biocompatible material are reduced inflammation and immunological responses, and / or low / limited fibrotic encapsulation.

[0030] The term "hydrogel" as used herein and in the context of the present invention is a term well known to those skilled in the art and includes any network of hydrophilic covalently crosslinked polymer chains. It usually builds a three-dimensional solid, consisting of hydrophilic polymer chains, held together by specific crosslinkers. Due to the inherent crosslinkers, the structural integrity of the hydrogel network is not dissolved in water. Hydrogels are highly absorbent (can contain more than 90% water) natural or synthetic polymer networks.

[0031] The "first polysaccharide" and "second polysaccharide" used in the context of the present invention may be any polysaccharide known to those skilled in the art. However, it is preferred that the first polysaccharide and / or the second polysaccharide may be selected, independently of each other, from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof, more preferably from the group consisting of pullulan, alginate, hyaluronan, and dextran. In this context, the "monomer repeat unit" of each of these representative examples of the first and / or second polysaccharide may be defined herein as follows:

[0032] Pullulan is a polysaccharide polymer composed of maltotriose units. The three glucose units of maltotriose are linked by α-1,4-glycosidic bonds, whereas consecutive maltotriose units are linked to each other by α-1,6-glycosidic bonds. Pullulan can be produced from starch by the fungus Aureobasidium pullulans. It can be utilized by cells primarily to resist desiccation and feeding. The presence of this polysaccharide also facilitates the diffusion of molecules into and out of the cell. In the context of the present invention, each monomeric repeating unit of pullulan is TIFF2024537390000011.tif28128 structure, where n is the number of monomer repeating units of pullulan and n is an integer between 10 and 10,000. In the context of the present invention, the degree of carboxymethylation of pullulan is represented by the expression "PCM" followed by a number (e.g., PCM1, PCM3 and PCM5). The number characterizes the respective degree of carboxymethylation and refers to the carboxymethyl groups introduced into pullulan by repeatedly applying the carboxymethylation reaction cycle a specified number of times. As used herein, CM1, CM2, CM3 etc. generally describe the respective degree of carboxymethylation of polysaccharides (without specifically relating to pullulan).

[0033] Alginic acid, also called algin, is a polysaccharide widely distributed in the cell walls of hydrophilic brown algae that forms a viscous gum upon hydration. Alginic acid is a linear copolymer in which homopolymeric blocks of (1-4)-linked β-D-mannuronic acid (M) and its C-5 epimer, α-L-guluronic acid (G) residues are covalently linked together in different sequences or blocks. The monomers may occur in homopolymeric blocks of consecutive G residues (G blocks), consecutive M residues (M blocks) or alternating M and G residues (MG blocks). Its salts with metals such as sodium and calcium are known as alginates. In the context of the present invention, each monomeric repeating unit of alginate is TIFF2024537390000012.tif31128, where n and m are the number of monomer repeat units of alginate, and n and m are each, independently of the other, integers in the range of 10 to 10,000.

[0034] Hyaluronic acid (abbreviated HA; conjugate base: hyaluronate), also called hyaluronan, is an anionic, non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial and neural tissues. It is unique among glycosaminoglycans in that it is non-sulfated, occurs in cell membranes instead of the Golgi apparatus, and can be very large. Hyaluronic acid is a polymer of disaccharides, the disaccharides themselves consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked via alternating β-(1→4) and β(1→3) glycosidic bonds. In the context of the present invention, each monomeric repeating unit of hyaluronan is It has the structure of TIFF2024537390000013.tif31128, where n is the number of monomer repeating units of hyaluronan and n is an integer between 10 and 10,000.

[0035] Dextran is a complex branched glucan (a polysaccharide derived from the condensation of glucose). IUPAC defines dextran as "a branched poly-α-D-glucoside of microbial origin, with predominantly C-1 → C-6 glycosidic linkages". Dextran chains vary in length (3-2000 kilodaltons). The polymer backbone consists of α-1,6-glycosidic bonds between glucose monomers, with random branching from α-1,3-linkages. This characteristic branching distinguishes dextran from dextrins, which are linear glucose polymers linked by α-1,4- or α-1,6-linkages. In the context of the present invention, each monomeric repeating unit of dextran is It has the structure of TIFF2024537390000014.tif39128, where n is the number of monomer repeat units of dextran and n is an integer between 10 and 10,000.

[0036] The first and / or second polysaccharide of the method of the invention may optionally be carboxymethylated in step b), in which at least one OH-group of the first and / or second polysaccharide as defined herein above is carboxymethylated. In one embodiment of the method of the invention, carboxymethylation is preferably carried out when the first and / or second polysaccharide is pullulan or dextran.

[0037] The functionalization of the first polysaccharide as defined above in step c) of the method of the invention may be carried out by one or more linker units having the structure -AX, if the monomeric repeating units of the first polysaccharide do not contain carboxylic acid residues, or by one or more linker units having the structure -X, if the monomeric repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s-CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15, and t is an integer from 1 to 15.

[0038] If the monomeric repeating units of the second polysaccharide do not contain a carboxylic acid residue, the functionalization of the second polysaccharide as defined above in step c) of the method of the invention may be carried out by one or more linker units having the structure -A'-Y, or if the monomeric repeating units of the second polysaccharide contain a carboxylic acid residue, the functionalization of the second polysaccharide as defined above in step c) of the method of the invention may be carried out by one or more linker units having the structure -Y, where A' is -(CH2). d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3, and where Y is -NH-(CH2) r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000015.tif29128, where M, M'=H or Me, and where W=OMe, OEt, OH, NH2 or NHMe.

[0039] As used herein, Q is TIFF2024537390000016.tif29128, where M, M' = H or Me, and where W = OMe, OEt, OH, NH2 or NHMe. Regarding TIFF2024537390000017.tif29128, the structural formula When TIFF2024537390000018.tif28128 is used, this does not mean that for the latter structural formula, both M must be the same (either H or Me). Rather, the invention also includes that in one embodiment, both M can be H, in one embodiment, both M can be Me, and in one embodiment, one M can be H and the other M can be Me (the two possibilities, independent of the position of M, that one M is H and the other M is Me). Thus, in the context of the present invention, two structural formulas TIFF2024537390000019.tif29128 may be used interchangeably herein.

[0040] Furthermore, in the context of the present invention, there is provided one or more linker units having the structure -A'-Y, Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000020.tif29128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; Regarding the functionalization of the second polysaccharide with the linker unit, -NH-(CH2) r -Q, -NH-(CH2CH2O) s -CH2CH2Q or -NH-(CH2-CH2-C(O)) t The linkage Q in -CH2-CH2-Q is represented by the structural formula The dashed lines shown in TIFF2024537390000021.tif28128 are completely clear to those skilled in the art.

[0041] The term "functionalization" or "functionalized" as used herein and in the context of the present invention generally refers to the addition of specific functional groups to confer new desirable properties to a compound, for example, in the present invention, the addition of linker units as defined above to an existing polysaccharide structure.

[0042] In one embodiment, step d) of the method of the invention can optionally be carried out by adding one or more enzymes to the mixture formed by steps a) to c). In this embodiment, any enzyme is in principle possible for the method of the invention.

[0043] In step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 25°C to 70°C for at least 1 hour. More preferably, in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 25°C to 70°C for 1 to 15 hours. Even more preferably, in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 25°C to 70°C for 1 to 10 hours.

[0044] In step e) of the method of the present invention, it is preferred that the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 30°C to 60°C for at least 1 hour. It is more preferred that the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 30°C to 60°C for 1 to 15 hours. It is even more preferred that the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 30°C to 60°C for 1 to 10 hours.

[0045] It is further preferred that in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 35°C to 50°C for at least 1 hour. It is further preferred that in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 35°C to 50°C for 1 to 15 hours. It is further preferred that in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 35°C to 50°C for 1 to 10 hours.

[0046] It is further preferred that in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 35°C to 45°C for at least 1 hour. It is further preferred that in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 35°C to 45°C for 1 to 15 hours. It is further preferred that in step e) of the method of the present invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature in the range of 35°C to 45°C for 1 to 10 hours.

[0047] More preferably, in step e) of the method of the invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature of about 40° C. for at least 1 hour. Even more preferably, in step e) of the method of the invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature of about 40° C. for 1 to 15 hours. Even more preferably, in step e) of the method of the invention, the incubation of the mixture formed by steps a) to d) is carried out in an aqueous medium at a temperature of about 40° C. for 1 to 10 hours.

[0048] In step e) of the method of the present invention, it is more preferred to carry out the incubation of the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 30°C to 60°C for 4 to 10 hours. In step e) of the method of the present invention, it is more preferred to carry out the incubation of the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 35°C to 50°C for 4 to 10 hours. In step e) of the method of the present invention, it is more preferred to carry out the incubation of the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 35°C to 45°C for 4 to 10 hours. In step e) of the method of the present invention, it is more preferred to carry out the incubation of the mixture formed by steps a) to d) in an aqueous medium at a temperature of about 40°C for 4 to 10 hours.

[0049] In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature in the range of 30°C to 60°C for 4 to 6 hours. In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature in the range of 35°C to 50°C for 4 to 6 hours. In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature in the range of 35°C to 45°C for 4 to 6 hours. In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature of about 40°C for 4 to 6 hours.

[0050] In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature in the range of 30°C to 60°C for 6 to 8 hours. In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature in the range of 35°C to 50°C for 6 to 8 hours. In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature in the range of 35°C to 45°C for 6 to 8 hours. In step e) of the method of the present invention, it is more preferred that the mixture formed by steps a) to d) is incubated in an aqueous medium at a temperature of about 40°C for 6 to 8 hours.

[0051] This method of preparing biocompatible hydrogels may include, together with step e), a step involving thermally induced gelation, which allows the formation of a specific shape factor, for example by complete bubble-free filling of a suitably formed mold, or by dropping a mixture of two non-viscous solutions of the individual components into a lipophilic organic medium to form droplets of a defined diameter. The formation of a covalent, non-degradable network of the biocompatible hydrogel can be induced by heating to the aforementioned warm temperatures compatible with maintaining the enzyme activity, if an enzyme is encapsulated therein. The method may also involve crosslinking via a 1,3-dipolar cycloaddition reaction, thermal gelation under very mild reaction conditions (for example, 40° C. in an aqueous medium) without side reactions and toxic reagents (for example, glutaraldehyde). Optimization of the pore size of the biocompatible hydrogel is possible by feasible adjustment of the parameters and any degree of carboxymethylation. When enzymes are added as described in step d), one or more enzymes are immobilized in the resulting biocompatible hydrogel, where the one or more enzymes are significantly longer stable and active than the free enzymes in solution at ambient or elevated temperatures, e.g., 37°C, which is body temperature. Unstable sensitive enzymes, such as alcohol oxidase or glucose oxidase, have better life performance under these conditions. This embodiment is also applicable to other sensitive enzymes. The biocompatible hydrogels so produced can be stored dry without losing more enzyme activity. Furthermore, leaching of the enzymes can be completely or almost completely prevented. Such hydrogels prepared according to this method of the invention can be suspended in aqueous or organic solvents (e.g., acetone) while maintaining the enzyme activity. The viscosity of the individual components, as well as the mixture, can be easily adjusted.

[0052] Thus, enzyme stability can be significantly improved by encapsulation as described herein, resulting in long-lasting enzymes.

[0053] In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof. In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof. In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan, and mixtures thereof. In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan, and mixtures thereof. In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan, and mixtures thereof. The polysaccharides pullulan, alginate, hyaluronan, dextran are defined herein above.

[0054] Cellulose has the formula (CH 10 O5) n Cellulose is an organic compound of the 1 to 4 carbon chain, a polysaccharide consisting of linear chains of hundreds to thousands of β(1→4)-linked D-glucose units. It is an important structural component of the primary cell wall of green plants, many forms of algae and oomycetes. Some species of bacteria secrete cellulose to form biofilms. In the context of the present invention, each monomeric repeating unit of cellulose is It has the structure TIFF2024537390000022.tif34128, where n is the number of monomer repeat units of cellulose, and n is an integer from 10 to 10,000.

[0055] Lichenin, also known as lichenan or lichen starch, is a complex glucan present in certain species of lichens. Chemically, it is a mixed-linkage glycan consisting of repeating glucose units linked by β-1,3- and β-1,4-glycosidic bonds. In the context of the present invention, each monomeric repeating unit of lichenin is It has the structure of TIFF2024537390000023.tif39128, where n is the number of monomer repeat units of lichenin, and n is an integer from 10 to 10,000.

[0056] Lentinan is a polysaccharide isolated from the fruiting body of the shiitake mushroom. Chemically, lentinan is a β-1,3 β-glucan with β-1,6 branches. In the context of the present invention, each monomer repeat unit of lentinan is TIFF2024537390000024.tif54157, where n is the number of monomer repeat units of lentinan, and n is an integer from 10 to 10,000.

[0057] In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin and lentinan. In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin and lentinan. In one embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran and lentinan. In a further embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran and lentinan. The monomer repeating units of each of these polysaccharides are as defined herein above.

[0058] In one preferred embodiment of the method for preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are each pullulan.

[0059] In one embodiment of the method for preparing a biocompatible hydrogel, the first and / or second polysaccharide is dextran or pullulan, and carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b). For this embodiment, it is preferred that carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b) at C6 of dextran and / or pullulan. For this embodiment, it is preferred that carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b) at C2 of dextran and / or pullulan. For this embodiment, it is preferred that carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b) at C3 of dextran and / or pullulan. For this embodiment, it is preferred that carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b) at C4 of dextran and / or pullulan.

[0060] In a further embodiment of the method for preparing a biocompatible hydrogel, in step c), the first polysaccharide is functionalized with an A of 0.01 to 1.5 per monomeric repeat unit of the first polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel, in step c), the first polysaccharide is functionalized with an A of 0.05 to 1.5 per monomeric repeat unit of the first polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel, in step c), the first polysaccharide is functionalized with an A of 0.05 to 1.0 per monomeric repeat unit of the first polysaccharide.

[0061] For the method for preparing a biocompatible hydrogel of the invention, it is preferred that in step c) the second polysaccharide is functionalized with an A' of 0.01 to 1.5 per monomeric repeat unit of the second polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel of the invention, in step c) the second polysaccharide is functionalized with an A' of 0.05 to 1.5 per monomeric repeat unit of the second polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel of the invention, in step c) the second polysaccharide is functionalized with an A' of 0.05 to 1.0 per monomeric repeat unit of the second polysaccharide.

[0062] In a further embodiment of the method for preparing a biocompatible hydrogel, in step c), the first polysaccharide is functionalized with an X of 0.01 to 1.5 per monomeric repeat unit of the first polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel of the invention, in step c), the first polysaccharide is functionalized with an X of 0.05 to 1.5 per monomeric repeat unit of the first polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel of the invention, in step c), the first polysaccharide is functionalized with an X of 0.05 to 1.0 per monomeric repeat unit of the first polysaccharide.

[0063] For the method for preparing a biocompatible hydrogel of the invention, it is preferred that in step c) the second polysaccharide is functionalized with a Y of 0.01 to 1.5 per monomeric repeat unit of the second polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel of the invention, in step c) the second polysaccharide is functionalized with a Y of 0.05 to 1.5 per monomeric repeat unit of the second polysaccharide. In a further preferred embodiment of the method for preparing a biocompatible hydrogel of the invention, in step c) the second polysaccharide is functionalized with a Y of 0.05 to 1.0 per monomeric repeat unit of the second polysaccharide.

[0064] In one embodiment of the method for preparing the biocompatible hydrogel of the present invention, d is 1.

[0065] For the preparation method of the biocompatible hydrogel of the present invention, it is further preferred that step e) is a thermally induced cycloaddition reaction between X and Y to form a crosslinked polymer.

[0066] In one embodiment of the method for preparing a biocompatible hydrogel of the present invention, the content of N3 is 0.01 to 1.5 N3 per monomer repeat unit of the first polysaccharide. In one preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the content of N3 is 0.01 to 1.0 N3 per monomer repeat unit of the first polysaccharide. In one further preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the content of N3 is 0.1 to 1.0 N3 per monomer repeat unit of the first polysaccharide.

[0067] In one embodiment of the method for preparing a biocompatible hydrogel of the present invention, the content of Q is 0.01 to 1.5 per monomer repeat unit of the second polysaccharide. In one preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the content of Q is 0.01 to 1.0 per monomer repeat unit of the second polysaccharide. In one preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the content of Q is 0.1 to 1.0 per monomer repeat unit of the second polysaccharide.

[0068] In one preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, in step c), -AX is linked to at least one primary or secondary OH-group of the first polysaccharide, preferably via at least one of C2, C3, C4 or C6 of the monomeric repeating unit of the first polysaccharide, more preferably via C6 of the monomeric repeating unit of the first polysaccharide.

[0069] In one preferred embodiment of the method for preparing a biocompatible hydrogel according to any one of the preceding claims, in step c) -A'-Y is linked to at least one primary or secondary OH-group of the second polysaccharide, preferably via at least one of C2, C3, C4 or C6 of the monomeric repeating unit of the second polysaccharide, more preferably via C6 of the monomeric repeating unit of the second polysaccharide.

[0070] It is further preferred that for the method of preparation of the biocompatible hydrogel of the present invention, no toxic reagents are used, preferably no glutaraldehyde is used.

[0071] In one embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the non-functionalized first polysaccharide is in the range of 5 to 2000 kDa. In one embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized first polysaccharide is in the range of 5 to 2500 kDa. In one preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized first polysaccharide is in the range of 5 to 2000 kDa. In one more preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized first polysaccharide is in the range of 5 to 1500 kDa. In an even more preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized first polysaccharide is in the range of 10 to 1500 kDa.

[0072] In one embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the non-functionalized second polysaccharide is in the range of 5 to 2000 kDa. In one embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized second polysaccharide is in the range of 5 to 2500 kDa. In one preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized second polysaccharide is in the range of 5 to 2000 kDa. In one more preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized second polysaccharide is in the range of 5 to 1500 kDa. In an even more preferred embodiment of the method for preparing a biocompatible hydrogel of the present invention, the molecular weight of the functionalized second polysaccharide is in the range of 10 to 1500 kDa.

[0073] The present invention further provides a hydrogel obtainable by any of the methods for preparing a biocompatible hydrogel described above, the hydrogel preferably comprising one or more encapsulated enzymes.More preferably, the hydrogel is a swellable or swollen hydrogel matrix.

[0074] In a further aspect, the present invention provides a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, comprising a cross-linked polymer comprising the following structure: a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, Here, B is TIFF2024537390000025.tif19128, wherein R' is selected from the group consisting of -CF3-C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units.

[0075] In a further aspect, the present invention provides a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, comprising a cross-linked polymer comprising the following structure: a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, hyaluronan, and dextran; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O))t -CH2-CH2-, Here, B is TIFF2024537390000026.tif19128, wherein R' is selected from the group consisting of -CF3, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units.

[0076] In a further aspect, the present invention provides a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, comprising a cross-linked polymer comprising the following structure: a first polysaccharide and a second polysaccharide, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, Here, B is TIFF2024537390000027.tif19128, wherein R' is selected from the group consisting of -CF3, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units.

[0077] In a further aspect, the present invention provides a biocompatible hydrogel for non-covalent immobilization of one or more enzymes, comprising a cross-linked polymer comprising the following structure: a first polysaccharide and a second polysaccharide, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, and mixtures thereof; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, Here, B is TIFF2024537390000028.tif19128, wherein R' is selected from the group consisting of -CF3, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units.

[0078] The above definitions relating to the preparation method of a biocompatible hydrogel also apply to the biocompatible hydrogel when the same terms are used. As used herein for the biocompatible hydrogel of the present invention, the parameters r, s and t, defined herein as r is an integer between 2 and 20, s is an integer between 1 and 15, and t is an integer between 1 and 15, apply to both Z1 and Z2.

[0079] In a preferred embodiment, the biocompatible hydrogel comprises a cross-linked polymer comprising the following structure for non-covalent immobilization of one or more enzymes: a first polysaccharide and a second polysaccharide, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, hyaluronan, and dextran; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, Here, B is TIFF2024537390000029.tif19128, wherein R' is selected from the group consisting of -CF3, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units.

[0080] In one embodiment of the biocompatible hydrogel of the present invention, the hydrogel is a swellable or swelling hydrogel matrix.

[0081] In one further embodiment of the biocompatible hydrogel of the present invention, the non-covalent immobilization of one or more enzymes includes encapsulation of one or more enzymes and non-covalent binding of one or more enzymes in the hydrogel. The term "non-covalent" as used herein and in the context of the present invention refers to interactions that are different from covalent bonds in that they do not involve sharing of bonds, but rather dispersive changes in inter- or intra-molecular electromagnetic interactions, such as dipole-dipole or charge-charge interactions. The chemical energy released in the formation of non-covalent interactions is typically about 1-5 kcal / mol. Non-covalent interactions can be classified into different categories such as electrostatic effects, π effects, van der Waals forces, and hydrophobic effects. Non-covalent interactions are important in maintaining the three-dimensional structure of large molecules such as proteins and nucleic acids. In addition, they are also involved in many biological processes in which large molecules bind to each other in a specific but temporary manner.

[0082] In one embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharides are, independently of each other, selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof.

[0083] In a further embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharides are independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof. In a further embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharides are independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan, and mixtures thereof. In a further embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharides are independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan, and mixtures thereof. In a further embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharides are independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, and mixtures thereof.

[0084] In one embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharide is pullulan.

[0085] In a further preferred embodiment of the biocompatible hydrogel of the present invention, -A-Z1- is represented by the formula -CH2-CO-NH-(CH2-CH2-O) n It has -CH2-CH2-, where n is preferably 1 to 5.

[0086] In one embodiment of the biocompatible hydrogel of the present invention, the first and / or second polysaccharide has a concentration of 5 to 120 mg / ml, preferably 10 to 80 mg / ml, more preferably 20 to 60 mg / ml, relative to the total hydrogel.

[0087] In a further embodiment of the biocompatible hydrogel of the present invention, the one or more enzymes for non-covalent immobilization may be selected from the group consisting of lipases and oxidases, preferably glucose oxidase, lactate oxidase, uricase, glutamate oxidase, cortisol oxidase, xanthine oxidase, cholesterol oxidase, sarcosine oxidase, and alcohol oxidase.

[0088] The present invention further provides a composition comprising a biocompatible hydrogel of the present invention and as defined herein.

[0089] The present invention further provides a method for encapsulating one or more enzymes in a biocompatible hydrogel according to the invention and as defined herein, the method comprising the step of contacting the biocompatible hydrogel with one or more enzymes and then incubating it in an aqueous medium at a temperature in the range of 25°C to 70°C, preferably about 40°C, for at least 1 hour, preferably 1 to 10 hours. The method preferably comprises the step of contacting the biocompatible hydrogel with one or more enzymes and then incubating it in an aqueous medium at a temperature in the range of 25°C to 70°C for 1 to 15 hours. The method more preferably comprises the step of contacting the biocompatible hydrogel with one or more enzymes and then incubating it in an aqueous medium at a temperature in the range of 25°C to 70°C for 1 to 10 hours.

[0090] In a further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) for the non-covalent immobilization of one or more enzymes in a hydrogel, or b) In a biosensor, The present invention provides a use of the hydrogel as defined herein. As used herein, the term "biosensor" refers to a self-contained integrated system that is distinct from a biological sensor or biotest and that provides specific quantitative or semi-quantitative analytical information, consisting of a biological recognition element (biochemical receptor or enzyme) and a transducer (e.g., an electrode) in direct spatial contact.

[0091] The present invention also provides a kit comprising a composition or hydrogel of the present invention and as defined herein.

[0092] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein.

[0093] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0094] The term "and / or" as used anywhere in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0095] The terms "less than" or, by extension, "more than," do not include a specific number.

[0096] For example, "less than 20" means less than a stated number. Similarly, "more" or "greater than" means more than or greater than a stated number, for example, "more than 80%" means more than or greater than 80% of a stated number.

[0097] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are understood to imply the inclusion of a recited integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including", or, as sometimes used herein, the term "having". As used herein, "consisting of" excludes any element, step, or ingredient not specified.

[0098] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0099] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0100] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference is inconsistent or inconsistent with this specification, the present specification will take precedence over any such material.

[0101] The contents of all publications and patent documents cited herein are incorporated herein by reference in their entirety.

[0102] A better understanding of the present invention and its advantages will be gained from the following examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention in any way. EXAMPLES

[0103] material and method: Synthesis of the oxanorbornadiene linker: TIFF2024537390000030.tif107153 Scheme 1: Oxanorbornadiene Linker NH2-(CH2) r Synthesis of -Q (r=2 / 3 / 6, Q=Q-1(CF3), M=H or Me and M'=H or Me) as TFA salt

[0104] 4,4,4-Trifluoro-3-oxo-2-(triphenyl-λ 5 -Phosphaneilidene)-ethyl butanoate (Compound 1) At 0°C, (2-ethoxy-2-oxoethyl)triphenylphosphonium bromide (100g, 0.233mol) was dissolved in anhydrous THF (400mL) under argon atmosphere. Triethylamine (65mL, 0.47mol) was slowly added to the reaction mixture over 20 minutes. Trifluoroacetic anhydride (36mL, 0.26mol) was then added dropwise. The reaction mixture was stirred for 24 hours while slowly warming to room temperature. The precipitate was filtered and washed with cold THF. The solvent of the filtrate was removed on a rotary evaporator and H2O / THF (3:1, 400mL) was added to the residue. The precipitated light yellow solid was then filtered, dried under reduced pressure at 40°C, and recrystallized several times from methanol / H2O. The product (90.9g, 0.205mol, 88%) was obtained as a crystalline solid. TIFF2024537390000031.tif46165

[0105] Ethyl 4,4,4-trifluorobut-2-ynoate (Compound 2) 4,4,4-Trifluoro-3-oxo-2-(triphenyl-λ 5 Ethyl (-phosphaneilidene)butanoate (10 g, 23 mmol) was distilled in a pyrolyzer at 220 °C and 100-160 mbar for 4-5 h. The product (3.30 g, 19.9 mmol, 89%) was obtained as a clear, colorless liquid. TIFF2024537390000032.tif26165

[0106] Ethyl 1,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylate (Compound 3) Ethyl 4,4,4-trifluorobut-2-ynoate (3.30 g, 19.9 mmol) was mixed with 2,5-dimethylfuran (3.15 mL, 29.8 mmol) and heated in a microwave reactor at 60° C. for 60 min. The reaction mixture was concentrated by azeotroping with toluene (3×). The product was obtained as a brown oily residue (2.91 g, 0.11 mol, 56%). TIFF2024537390000033.tif52165

[0107] 3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (compound 4) In a round-bottom flask, 4,4,4-trifluoro-3-oxo-2-(triphenyl-λ 5 Ethyl 4,4,4-trifluorobut-2-ynoate (38.8 g, 87.4 mmol) was distilled in a pyrolyzer at 210 °C and 100-160 mbar for 4-5 h. Furan (38.0 mL, 52.4 mmol) was then added to the colorless distillate (ethyl 4,4,4-trifluorobut-2-ynoate) and reacted in a microwave reactor at 60 °C for 60 min. The reaction mixture was concentrated under reduced pressure by azeotropy with toluene (3x). The ester could be obtained as an oily residue, which was dissolved without further purification in H2O / THF (7:1, 100 mL), mixed with 1 M aqueous LiOH (100 mL, 100 mM) and stirred at room temperature for 3 days. The aqueous phase was then adjusted to pH 1-2 with 2 M HCl and extracted with diethyl ether (3x). The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure. The oily residue was dissolved in dichloromethane and petroleum ether was added. The precipitated solid was filtered and washed with petroleum ether. This gave the product (6.16 g, 29.9 mmol, 35% over three steps) as a crystalline solid. TIFF2024537390000034.tif45165

[0108] 1,4-Dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (compound 5) Ethyl 1,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylate (191 mg, 0.73 mmol) was dissolved in HO / THF (7:1, 2 mL), mixed with 1 M aqueous LiOH (2 mL, 2 mM) and stirred at room temperature for 1.5 days. The aqueous phase was then brought to pH 1-2 with 2 M HCl and extracted with diethyl ether (3x). The combined organic phases were dried over NaSO and the solvent was removed under reduced pressure. The product (127 mg, 0.54 mmol, 75%) was obtained as a brown oil. TIFF2024537390000035.tif39165

[0109] tert-Butyl (2-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)ethyl)carbamate (Compound 6) 3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (1.18 g, 5.73 mmol) was dissolved in anhydrous dichloromethane (16 mL) under argon atmosphere. After stepwise addition of 4-DMAP (1.4 g, 11 mmol), EDC-HCl (1.6 g, 8.6 mmol), and Boc-ethylenediamine (1.1 mL, 6.9 mmol), the mixture was stirred at room temperature for 22 h. The reaction mixture was washed with saturated aqueous NaCl, the organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure. After purification of the crude product by column chromatography (eluent: PE / EtOAc 1:1), the product was obtained as a white solid (1.01 g, 2.91 mmol, 51%). TIFF2024537390000036.tif66165

[0110] tert-Butyl (2-(1,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)ethyl)carbamate (Compound 7) 1,4-Dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (0.68 g, 2.90 mmol) was dissolved in anhydrous dichloromethane (12 mL) under Ar atmosphere. After stepwise addition of 4-DMAP (0.71 g, 5.8 mmol), EDC-HCl (0.83 g, 4.3 mmol), and Boc-ethylenediamine (0.55 mL, 3.5 mmol), the mixture was stirred at room temperature for 20 h. The reaction mixture was washed with saturated aqueous NaCl, the organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure. After purification of the crude product by column chromatography (eluent: PE / EtOAc 3:1; 1:1), the product was obtained as an oil (0.23 g, 0.60 mmol, 21%). TIFF2024537390000037.tif66165

[0111] (3-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)propyl)-λ 2 -tert-Butyl azanecarboxylate (compound 8) 3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (512 mg, 2.49 mmol) was dissolved in anhydrous dichloromethane (6 mL) under Ar atmosphere. After stepwise addition of 4-DMAP (607 mg, 4.97 mmol), EDC-HCl (715 mg, 3.73 mmol), and N-Boc-1,3 diaminopropane (0.52 mL, 3.0 mmol), the mixture was stirred at room temperature for 22 h. The reaction mixture was washed with saturated aqueous NaCl, the organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure. After purification of the crude product by column chromatography (eluent: PE / EtOAc 1:1), the product was obtained as an oil (528 mg, 1.46 mmol, 55%). TIFF2024537390000038.tif66165

[0112] (6-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)hexyl)-λ 2 -tert-Butyl azanecarboxylate (compound 9) 3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (0.50 g, 2.4 mmol) was dissolved in anhydrous dichloromethane (10 mL) under Ar atmosphere. After stepwise addition of 4-DMAP (597 mg, 4.88 mmol), EDC-HCl (702 mg, 3.66 mmol), and N-Boc-1,6-diaminohexane (657 μL, 2.93 mmol), the mixture was stirred at room temperature for 22 h. The reaction mixture was washed with saturated aqueous NaCl, the organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure. After purification of the crude product by column chromatography (eluent: PE / EtOAc 7:1, 3:1, 1:1), the product was obtained as an oil (610 mg, 1.51 mmol, 62% yield). TIFF2024537390000039.tif72165

[0113] 2-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamide)ethane-1-ammonium trifluoroacetate (compound 10) tert-Butyl (2-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)ethyl)carbamate (1.00 g, 2.87 mmol) was dissolved in dichloromethane (10 mL). At 0° C., TFA (8.8 mL, 0.12 mol) was added dropwise. After stirring for 30 min at the same temperature, no starting material was observed by TLC and the reaction mixture was concentrated by aceotropic distillation with toluene (3×). The brown residue was then treated with ethyl acetate and crystallized at 4° C. overnight. The product was obtained after filtration as a crystalline solid (0.98 g, 2.7 mmol, 95%). TIFF2024537390000040.tif59166

[0114] 2-(1,4-Dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamide)ethane-1-ammonium trifluoroacetate (Compound 11) tert-Butyl (2-(1,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)ethyl)carbamate (88.6 mg, 0.235 mmol) was dissolved in dichloromethane (2 mL). At 0° C., TFA (0.72 mL, 9.4 mmol) was added dropwise. After stirring for 30 min at the same temperature, no starting material was observed by TLC and the reaction mixture was concentrated by azeotropic distillation with toluene (3×). The product was obtained as an oil (88.9 mg, 0.228 mmol, 97%). TIFF2024537390000041.tif53166

[0115] 3-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)propane-1-ammonium trifluoroacetate (compound 12) (3-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)propyl)-λ 2tert-Butyl-1-azanecarboxylate (100 mg, 0.28 mmol) was dissolved in dichloromethane (2 mL). At 0° C., TFA (0.85 mL, 0.011 mol) was added dropwise. After stirring for 30 min at the same temperature, no starting material was observed by TLC and the reaction mixture was concentrated by azeotropic distillation with toluene (3×). The residue was then treated with ethyl acetate and diethyl ether and decanted. The product was obtained as an oil (66 mg, 0.18 mmol, 67%). TIFF2024537390000042.tif66166

[0116] 6-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)hexane-1-ammonium trifluoroacetate (compound 13) (6-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)hexyl)-λ 2 tert-Butyl-1-azanecarboxylate (205 mg, 0.507 mmol) was dissolved in dichloromethane (4 mL). At 0° C., TFA (1.55 mL, 0.020 mol) was added dropwise. After stirring at the same temperature for 30 min, no starting material was observed by TLC and the reaction mixture was concentrated by azeotropic distillation with toluene (3×). The residue was subsequently treated with diethyl ether and petroleum ether and decanted. The product was obtained as an oil (215 mg, 0.051 mmol, quantitative). TIFF2024537390000043.tif72166

[0117] (Table 1) The chemical formulas of the above compounds are shown in this table. TIFF2024537390000044.tif144128TIFF2024537390000045.tif237119TIFF2024537390000046.tif45128

[0118] General steps: Carboxymethylation of biopolymers: The desired biopolymer (e.g., pullulan (25 g)) was dissolved in deionized water (375 mL). Subsequently, 8 M aqueous NaOH (125 mL) and chloroacetic acid (50.1 g, 0.53 mol) were added and the reaction mixture was heated to 62° C. After 90 min, the solution was adjusted to pH 6.5 with 6 M aqueous HCl and added to distilled methanol (3 L). The resulting precipitate was filtered and the residue was dried at 40° C. and 20 mbar. The carboxymethylated biopolymer was obtained as a white solid.

[0119] Higher degrees of substitution were achieved by subjecting the resulting products to repeated carboxymethylation reactions, and the number of repeated cycles is noted using the suffix CM (e.g., CM3 corresponds to three repeated carboxymethylation cycles).

[0120] Two-component systems (addition of a linker unit to a carboxymethylated biopolymer): The desired biopolymer (dextran 250 kDa CM6, 100 mg, 0.50 mmol) was dissolved in 0.025 M MES buffer (50 mL). The desired linker unit NH2-(CH2CH2O) s -CH2CH2N3(s=3) (98.15 μL, 0.50 mmol), EDC-HCl (0.948 g, 4.95 mmol) and NHS (56.93 mg, 0.50 mmol) were added successively. The reaction mixture was stirred at room temperature for 2.5 days and then transferred to a dialysis tube (cut-off: 14 kDa). The latter was placed in a 5 L beaker containing a deionized aqueous solution of NaCl and dialyzed for 4 days with gradually decreasing NaCl concentrations (day 1: 20 g / L, day 2: 10 g / L, and days 3-4: 0 g / L). The deionized aqueous NaCl solution was replaced three times each day. The dialyzed solution was subsequently filtered through absorbent cotton and lyophilized. The modified biopolymer could be isolated in a raw cotton-like solid (87.80 mg).

[0121] Hydrogel gelation reaction: Modified biopolymer chains (e.g., pullulan, dextran, alginate, and hyaluronan) with different functional groups (variable degree of substitution) were dissolved in an appropriate solvent (e.g., deionized water, buffer, organic solvent mixture) and the two components (according to Figure 2) were mixed. Gelation proceeded under mild conditions (40 °C, overnight) via a thermally induced copper-free "click" reaction to irreversibly form a covalently crosslinked hydrogel, which contains triazole units connecting the two biopolymer chains.

[0122] NMR analysis: 1 H and 13 C and 19 F NMR spectra were measured on Bruker AVANCE-400 / 500 / 600 and DPX-200 / 400 spectrometers at room temperature in the indicated deuterated solvents. Residual proton signals of the solvent (CDCl3: δ( 1 H-NMR) = 7.26 ppm, DO: δ( 1 H-NMR) = 4.79 ppm and DMSO: δ( 1 H-NMR) = 2.50 ppm 1 It served as a standard and for calibration of the H-NMR spectra. 13 C-NMR spectra were recorded with broad-band decoupling and calibrated to the solvent signal (CDCl3:( 13 C-NMR) = 77.2 ppm, and DMSO: ( 13 C-NMR) = 39.5 ppm. 19 For F-NMR-based quantification, trifluoroacetic acid methyl ester was used as an internal standard. Samples in deuterium oxide were measured using the water suppression method.

[0123] Coupling constants J are expressed in Hz and chemical shifts in ppm. Signal multiplicities were abbreviated for simplicity as follows: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m).

[0124] High resolution mass spectrometry (HR-MS): High-resolution mass spectra were measured on a Water Aquity UPLC system (ESI and APCI-MS / MS) equipped with a QTof Premier detector or a Water Allicance 2695 equipped with a Micromass LCT Premier detector. Samples were dissolved in water, acetonitrile, or methanol and injected using either the HPLC system or a direct inlet. Measurements are given as mass / charge (m / z).

[0125] Column chromatography: Manual column chromatography was carried out using overpressure. For this, Macherey-Nagel silica gel (particle size: 40-63 μm, normal phase) and the indicated elution medium mixtures (e.g., PE / EtOAc 1:1) were used. Products were detected using Merck thin-layer chromatography (DC silica gel 60 F254 glass plates) (pore size: 60 Å, layer thickness: 210-270 μm, fluorescent indicator UV254).

[0126] SEM: Morphological investigation of the hydrogel network was carried out using scanning electron microscopes (FEI Nova 600 FEG and FEI NOVA 200 NanoLAB) supplemented with focused ion beam. For this, liquid hydrogel samples were drop-coated and spin-coated on a small silicon chip surface and polymerized overnight at 40 °C to obtain the hydrogel. The samples were then coated with a 5 nm thick elemental carbon layer using a LEICA EMACE600 and then observed under SEM.

[0127] Swelling Ability: After successful gelation, the hydrogel was frozen at -20°C and then lyophilized. Deionized water / buffer was added to the lyophilized hydrogel (m0) to allow for maximal swelling. Excess liquid was removed and the swollen hydrogel was weighed (m1).

[0128] The swelling ratio was calculated as follows: TIFF2024537390000047.tif9128

[0129] Rheological measurements of polysaccharides: The rheological properties of the polysaccharides were tested using an Anton Paar rheometer MCR302. The gelation kinetics of the hydrogels at 40 °C was investigated using a PP20-SN33813 system at a normal force of 0 N, a frequency of 1 Hz, an amplitude γ = 0.1% and a gap size of 1 mm. The samples were prepared as follows: modified biopolymer chains (e.g. pullulan, dextran, alginate and hyaluronan) with different functional groups (variable degree of substitution) were dissolved in appropriate solvents (e.g. deionized water, buffer, organic solvent mixtures) and the two components (according to Figure 2) were mixed. The gelation reaction occurred during the measurements as indicated by an increase in the storage modulus.

[0130] Viscosity measurement of polysaccharides: The viscosity properties of the polysaccharides were tested using an Anton Paar rheometer MCR302. Viscosity measurements were performed using a PP20-SN33813 system with a normal force of 0 N, amplitude γ=0.1, 100% log, [slope]=10 points / decade, 10 s -1 ~10,000s -1 A given shear rate up to TIFF2024537390000048.tif5128, temperature 19.3 °C, and gap size 1 mm. Samples were prepared as follows: modified biopolymer chains (e.g., pullulan, dextran, alginate, and hyaluronan) with different functional groups (variable degree of substitution) were dissolved in appropriate solvents (e.g., deionized water, buffer, organic solvent mixtures) and analyzed with a rheometer.

[0131] Measurement of enzyme activity by colorimetric assay: Modified biopolymer chains (e.g., pullulan, dextran, alginate, and hyaluronan) with different functional groups (variable degree of substitution) were dissolved in an appropriate solvent (e.g., deionized water, buffer, organic solvent mixture) and the two components (according to Figure 2) were mixed.

[0132] To this mixture, a fixed concentration of enzyme was added. Gelation was performed in a microtiter plate with a volume of 25 μl by overnight incubation at 40° C. Afterwards, the hydrogel samples were washed with deionized water by shaking at 50 rpm for 30 min to remove excess deionized water. All standards, blanks, and samples were spiked with the indicated reaction mixture (see Table 1) and fluorescence (excitation: 530 / 13, emission: 590 / 18; AmplexRed®) was measured on a BioTek Cytation5. Measurements were performed at a constant temperature of 37° C.

[0133] Table 2. Composition of the reaction mixture for the AmplexRed® assay TIFF2024537390000049.tif41158*Final concentration in well is divided by 2 (25 μl sample, 25 μl assay reaction mix)

[0134] Fourier transform infrared spectroscopy: Infrared spectra (IR) were recorded on a Shimadzu ATR-FT-IR spectrometer. All biopolymer samples were measured as lyophilizates.

[0135] Atomic Force Microscopy: The surface topography of the hydrogels was analyzed using a Bruker Dimension ICON.

[0136] Conductive titration: The degree of substitution of the biopolymers was determined by conductometric titration on a TitroLine® 7000.

[0137] According to FIG. 1, a two-component system (cross-linked polysaccharide with three degrees of variability) is provided: two modified biopolymer chains (e.g., pullulan, dextran, lentinan, alginate, and hyaluronan) with reactive functional groups (one biopolymer has an azide residue and the other biopolymer has an oxanorbornadiene derivative; variable degree of substitution) form a triazole via a thermally induced, copper-free "click" reaction, as described in more detail in the specific examples below.

[0138] Example 1: Carboxymethylation of Dextran Dextran (500 kDa, 10.00 g, 0.06 mol) was dissolved in deionized water (150 mL). Subsequently, 8 M aqueous NaOH (50 mL) and chloroacetic acid (20.40 g, 0.22 mol) were added and the reaction mixture was heated to 62 °C. After 90 min, the solution was adjusted to pH 6.5 with 6 M aqueous HCl and added to distilled methanol (1.4 L). The resulting precipitate was filtered and the residue was dried at 40 °C and 20 mbar. The carboxymethylated biopolymer was obtained as a white solid (12.34 g).

[0139] The resulting product was subjected to repeated carboxymethyl (CM) reactions to achieve higher degrees of substitution.

[0140] The following examples were obtained in a similar manner as described above for Example 1.

[0141] Table 3. Carboxymethylated biopolymers TIFF2024537390000050.tif65170

[0142] Carboxymethylated lentinan Lentinan (400-800 kDa) CM1: Scale: 8 g; Yield: 3.49 g.

[0143] Table 4 Carboxymethylated pullulan TIFF2024537390000051.tif78128

[0144] The degree of substitution of the obtained polysaccharide was determined by FT-IR spectroscopy (see FIG. 5 ). 1 It was characterized by H-NMR spectroscopy (see FIG. 6) and conductometric titration (see FIG. 9).

[0145] Both biopolymer chains are attached to a functionalized spacer, e.g., a PEG-linker unit. The length of the PEG-linker is variable and should be short (PEG(3) to PEG(25)). Different PEG-linker units (shown in Figure 2, see "R-N3") and carboxymethylated biopolymers result in different pore sizes. The pore size affects the activity of the enzyme and the diffusion behavior of substrates and other molecules (see Figure 1). In this context, the term "functionalized" refers to a spacer bearing either an azide moiety or an oxanorbornadiene derivative Q as previously described (see Scheme 1). The attachment is achieved via amide coupling between the carboxy group of the biopolymer and the amine group of the linker.

[0146] Example 2: Azide functionalization of dextran The desired biopolymer (dextran, 250 kDa, CM4, 100 mg, 0.45 mmol) was dissolved in 0.025 M MES buffer (50 mL). The desired linker NH2-(CH2CH2O) s -CH2CH2N3(s=3) (90.12 μL, 0.45 mmol), EDC-HCl (0.87 g, 4.54 mmol) and NHS (52.27 mg, 0.45 mmol) were added successively. The reaction mixture was stirred at room temperature for 2.5 days and then transferred to a dialysis tube (cut-off: 14 kDa). The latter was placed in a 5 L beaker containing a deionized aqueous solution of NaCl and dialyzed for 4 days with gradually decreasing NaCl concentrations (day 1: 20 g / L, day 2: 10 g / L, and days 3-4: 0 g / L). The deionized aqueous NaCl solution was replaced three times each day. The dialyzed solution was subsequently filtered through absorbent cotton and lyophilized. The modified biopolymer could be isolated as a raw cotton-like solid (95 mg).

[0147] The following examples were obtained as described above.

[0148] Table 5. Azide units (-NH-(CH2CH2O)) on polysaccharide chains s -CH2CH2N3, s=3) TIFF2024537390000052.tif39168 Alginate (120-190 kDa): Scale: 200 mg; Yield: 172 mg. Hyaluronan (70-80 kDa): Scale: 100 mg; Yield: 80 mg. Lentinan CM1 (400-800 kDa): Scale: 100 mg; Yield: 74 mg.

[0149] Table 6 Azide units (-NH-(CH2CH2O)) on pullulan s -CH2CH2N3, s=3) TIFF2024537390000053.tif71128

[0150] Azide unit (-NH-(CH2CH2O) s -CH2CH2N3, s=8) Pullulan (100 kDa) PCM5: Scale: 50 mg; Yield: 63 mg.

[0151] Addition of azide units (-NH-(CH2)) to pullulan r -Introduction of N3, r=6) Pullulan (100 kDa) PCM1: Scale: 150 mg; Yield: 122 mg. Pullulan (100 kDa) PCM5: Scale: 150 mg; Yield: 147 mg. Alginate (120-190 kDa): Scale: 200 mg; Yield: 89 mg.

[0152] Example 3: Oxanorbornadiene functionalization of dextran The desired biopolymer (dextran, 250 kDa, CM4, 100 mg, 0.45 mmol) was dissolved in 0.025 M MES buffer (50 mL). The desired linker NH2-(CH2) r-Q(r=2, Q-1(CF3) and M / M'=H) (112.74 mg, 0.45 mmol), EDC-HCl (0.87 g, 4.54 mmol) and NHS (52.27 mg, 0.45 mmol) were added successively. The reaction mixture was stirred at room temperature for 2.5 days and then transferred to a dialysis tube (cut-off: 14 kDa). The latter was placed in a 5 L beaker containing a deionized aqueous solution of NaCl and dialyzed for 4 days with gradually decreasing NaCl concentrations (day 1: 20 g / L, day 2: 10 g / L, and days 3-4: 0 g / L). The deionized aqueous NaCl solution was replaced three times each day. The dialyzed solution was subsequently filtered through absorbent cotton and lyophilized. The modified biopolymer could be isolated as a raw cotton-like solid (88 mg).

[0153] The following examples were obtained as described above.

[0154] Table 7. Oxanorbornadiene units (-NH-(CH)) on polysaccharide chains r -Q, r=2, Q-1(CF3) and M / M'=H) TIFF2024537390000054.tif25128 Alginate (120-190 kDa): Scale: 200 mg; Yield: 234 mg. Hyaluronan (70-80 kDa): Scale: 100 mg; Yield: 99 mg. Lentinan CM1 (400-800 kDa): Scale: 100 mg; Yield: 65 mg.

[0155] Table 8 Oxanorbornadiene units (-NH-(CH)) on pullulan r -Q, r=2, Q-1(CF3) and M / M'=H) TIFF2024537390000055.tif71128

[0156] Addition of oxanorbornadiene units (-NH-(CH2)) to pullulan r -Q, r=6, Q-1(CF3) and M / M'=H) Pullulan (100 kDa) PCM5: Scale: 100 mg; Yield: 122 mg.

[0157] The resulting product is 1 H- and 19 The derivatives can be analyzed by F-NMR spectroscopy (see, for example, Figures 7, 8 and 12). Particular IR spectra of samples from different batches of one derivative are shown in Figure 10.

[0158] Example 4: Bioorthogonal 1,3-dipolar cycloaddition reaction The reaction (see FIG. 2) involves crosslinking by bioorthogonal 1,3-dipolar cycloaddition, a copper-free "click" reaction. To enable this reaction, linker units linked to the biopolymers of the previous examples (azide-linked biopolymer and oxanorbornadiene-linked biopolymer, respectively) were mixed. Specific thermogelation occurs under mild conditions (e.g., in aqueous media, <40° C.). The mild reaction conditions are suitable for sensitive enzymes. The thus obtained irreversible network of glycans with immobilized enzymes provides high specificity and selectivity, as well as a bioorthogonal material in homogeneous solution. Furthermore, adjustment of the viscosity can be easily achieved. In addition, the uncrosslinked biopolymer and enzyme can be washed. Gelation occurs after a certain time (approximately 1 h to 10 h). Processing into different form factors is possible, for example by printing or spin-coating the material. Thus, the hydrogels of the present invention allow the immobilization of enzymes in a biocompatible network without the need for covalent binding of the enzyme (see Figure 2, where a = 1-10 h, depending on the material, the warm reaction temperature, and the aqueous medium).

[0159] No side reactions occur and no toxic reagents (e.g., glutaraldehyde) are used. Very mild reaction conditions (40 °C in aqueous medium) are applied as described above, but the gelation time can be controlled by the choice of material (degree of substitution) and the concentration of the biopolymer (e.g., 10-40 mg / ml). More highly modified biopolymers (PCM5-PCM9) have short gelation times (2-5 h; 40 mg / ml of material). Low modified biopolymers (PCM1-3) have gelation times of 3-10 h (40 mg / ml of material).

[0160] Example 5: The biocompatible hydrogels of the present invention are modular systems whose synthesis can be analyzed using a variety of methods and can be manufactured in a well-defined manner.

[0161] The carboxymethylation of the polysaccharide repeats can be analyzed by conductometric titration (see Figure 7), and the degree of substitution increases with increasing carboxymethylation steps. Furthermore, the increase in substitution can be confirmed by FT-IR and 1 It can be measured by H-NMR. In the FT-IR spectrum, a specific band in the fingerprint region becomes stronger as the degree of substitution increases and the number of functional groups increases (see FIG. 5). 1 In the H-NMR spectrum, a decrease in the signal intensity of the anomeric proton was observed with an increase in the number of introduced carboxymethyl groups (see FIG. 6). 19 Quantification by F-NMR showed an increase in the signal intensity of the introduced linker in the different modified polysaccharides. The number of introduced linker units correlates with the carboxymethyl groups present in the polysaccharide (see Figure 10). Rheological properties in relation to the gelation rate showed that the more highly modified polysaccharides gel faster due to the higher number of crosslinkable functional groups. A specific increase in the storage modulus over time was recorded for the different materials (see Figure 11). Moreover, five different batches of 5-fold modified pullulan with azide linkers showed similar FT-IR spectra (see Figure 8).

[0162] Example 6: Investigation of the swelling ratio of hydrogels (low and high modified pullulan hydrogels): Investigations: Determine the effect of the solvent used (dH2O or PBS buffer) on gelation and network formation as well as the resulting pore size, and determine the change in swelling ratio when freeze-dried hydrogels are redissolved in the presence of salt at different pH values.

[0163] Results (see FIG. 13): All the hydrogels shown have the ability to swell more than 1000% in dH2O. Less modified pullulan contains more free OH-groups and a highly porous structure. Water can easily penetrate and interact with the hydrophilic groups of the polysaccharide structure. PCM8: Higher crosslinking degree results in denser hydrogels and lower water absorption.

[0164] For the low-modification hydrogel (PCM1), the swelling ratio increased with decreasing salt concentration at physiological pH values. The highest swelling ratio was observed when the hydrogel was polymerized in dH2O. In contrast, hydrogels gelled in PBS buffer at pH 3 and redissolved in PBS buffer at pH 3 (PCM1 and PCM8) showed very low swelling.

[0165] Example 7: The enzyme can be non-covalently immobilized in the hydrogel. After complete gelation, the non-immobilized enzyme can be removed by washing.

[0166] Preparation: Different reactive functional groups (azide units -NH-(CH2CH2O) with s=3 for pullulan s -CH2CH2N3 and -NH-(CH2) with r=2, Q=Q-1(CF3) and M / M'=H for pullulan r The resulting modified biopolymer chains (e.g., pullulan CM5) with 1,2-diamino-3-pyridine (-Q) were dissolved in phosphate buffer pH 7.4 (4 mg / 100 μL for each polysaccharide chain individually) and the two resulting components (according to Figure 2) were mixed.

[0167] To this mixture, a fixed concentration of enzyme (e.g., glucose oxidase final concentration in well 100 mU / mL) was added. Gelation was performed in a microtiter plate in a volume of 25 μL (for each sample) by overnight incubation at 40°C. Afterwards, the hydrogel samples were washed with deionized water (25 μL) by shaking at 50 rpm for 30 min to remove excess deionized water. All standards, blanks, and samples were spiked with the specified reaction mixture (see Table 1) and fluorescence was measured.

[0168] An example of immobilized uricase was obtained as described above. The reaction mixture was adjusted according to Table 1.

[0169] Results: Enzyme saturation curves could be recorded for different modified pullulan hydrogels (e.g., PCM2 and PCM8, i.e. pullulan biopolymers obtained by performing 2 and 8 carboxymethylation cycles, respectively). As a control, the enzyme was used in solution (see Figure 14).

[0170] Long-term data (FIGS. 15 and 16) of different hydrogel samples with immobilized glucose oxidase was performed and the enzyme activity was plotted on different days compared to the enzyme in solution as a control.

[0171] Long-term data for the less modified hydrogel (PCM1) with immobilized uricase was performed and the enzyme activity was plotted over a period of 22 days (Figure 17).

[0172] The present invention is further characterized by the following:

[0173] item: 1. A method for preparing a biocompatible hydrogel, comprising the steps of: a) providing a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are selected, independently of each other, from the group consisting of pullulan, alginate, hyaluronan, and dextran; n is the number of monomer repeat units of the first and / or second polysaccharide, and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here A is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH2) r -N3, -NH-(CH2CH2O) s -CH2CH2N3, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-N3, r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; process, And functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or functionalizing the second polysaccharide with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide contain carboxylic acid residues, Here A' is -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Y is -NH-(CH2). r -Q, -NH-(CH2CH2O) s -CH2CH2Q, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-Q; Here, Q is TIFF2024537390000056.tif28128, where M, M'=H or Me, and Wherein W=OMe, OEt, OH, NH2 or NHMe; process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); e) incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature in the range of 25° C. to 70° C., preferably 40° C., for at least 1 hour, preferably 1 to 10 hours. 2. The method for preparing a biocompatible hydrogel according to item 1, wherein the first polysaccharide and the second polysaccharide are independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof. 3. The method for preparing a biocompatible hydrogel according to item 1 or 2, wherein the first polysaccharide and the second polysaccharide are independently selected from the group consisting of pullulan, alginate, hyaluronan, dextran, and mixtures thereof. 4. A method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein the first polysaccharide and the second polysaccharide are pullulan. 5. A method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein the first and / or second polysaccharide is dextran or pullulan, and carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b). 6. The method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein in step c), the first polysaccharide is functionalized with 0.01 to 1.5 A or X per monomer repeat unit of the first polysaccharide. 7. The method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein in step c) the second polysaccharide is functionalized with 0.01 to 1.5 A' or Y per monomer repeat unit of the second polysaccharide. 8. A method for preparing a biocompatible hydrogel described in any one of the preceding items, wherein d is 1. 9. The method for preparing a biocompatible hydrogel according to any one of the preceding paragraphs, wherein step e) is a thermally induced cycloaddition reaction between X and Y to form a crosslinked polymer. 10. A method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein the content of N3 is 0.01 to 1.5 N3 per monomer repeat unit of the first polysaccharide. 11. The method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein the content of Q is 0.01 to 1.5 per monomer repeat unit of the first and / or second polysaccharide. 12. The method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein in step c), -AX is linked to at least one primary or secondary OH-group of the first polysaccharide, preferably via at least one of C2, C3, C4 or C6 of the monomeric repeating unit of the first polysaccharide, more preferably via C6 of the monomeric repeating unit of the first polysaccharide. 13. The method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein in step c), -A'-Y is linked to at least one primary or secondary OH-group of the second polysaccharide, preferably via at least one of C2, C3, C4 or C6 of the monomeric repeating unit of the second polysaccharide, more preferably via C6 of the monomeric repeating unit of the second polysaccharide. 14. A method for preparing a biocompatible hydrogel according to any one of the preceding paragraphs, which does not use toxic reagents, preferably does not use glutaraldehyde. 15. A method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein the molecular weight of the non-functionalized first polysaccharide is in the range of 5 to 2000 kDa. 16. A method for preparing a biocompatible hydrogel according to any one of the preceding items, wherein the molecular weight of the non-functionalized second polysaccharide is in the range of 5 to 2000 kDa. 17. A hydrogel obtainable by the method according to any one of items 1 to 16. 18. The hydrogel according to item 17, comprising one or more encapsulated enzymes. 19. The hydrogel according to item 17 or 18, which is a swellable or swelling hydrogel matrix. 20. A biocompatible hydrogel comprising a crosslinked polymer comprising the following structure for non-covalent immobilization of one or more enzymes: a first polysaccharide and a second polysaccharide, Preferably, wherein the first polysaccharide and / or the second polysaccharide are independently selected from the group consisting of pullulan, alginate, hyaluronan, and dextran; n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; A first polysaccharide and a second polysaccharide, - one or more linker units linking the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z1-B-Z2-A'-, wherein A and A' are each independently -(CH2) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here, Z1 is -O-, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, d is an integer from 1 to 4; r is an integer from 2 to 20; s is an integer from 1 to 15; and t is an integer from 1 to 15; and, Here, Z2 is -OH, -(CH2) r -, -NH-(CH2CH2O) s -CH2CH2-, and -NH-(CH2-CH2-C(O)) t -CH2-CH2-, Here, B is TIFF2024537390000057.tif19128, wherein R' is selected from the group consisting of -CF3, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH2, and -C(O)-NHMe; One or more linker units. 21. The hydrogel according to item 20, which is a swellable or swelling hydrogel matrix. 22. The hydrogel according to item 20 or 21, wherein the non-covalent immobilization of the one or more enzymes comprises encapsulation of the one or more enzymes and non-covalent binding of the one or more enzymes in the hydrogel. 23. The hydrogel according to any one of items 20 to 22, wherein the first and / or second polysaccharide is, independently of each other, selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof. 24. The hydrogel according to any one of items 20 to 23, wherein the first and / or second polysaccharide is, independently of each other, selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof. 25. The hydrogel according to any one of items 20 to 24, wherein the first and / or second polysaccharide is pullulan. 26. -A-Z1- is a compound of the formula -CH2-CO-NH-(CH2-CH2-O) n 26. The hydrogel according to any one of items 20 to 25, having -CH2-CH2-, where n is preferably 1 to 5. 27. The hydrogel according to any one of items 20 to 26, wherein the first and / or second polysaccharide has a concentration, relative to the total hydrogel, of 5 to 120 mg / ml, preferably 10 to 80 mg / ml, more preferably 20 to 60 mg / ml. 28. The hydrogel according to any one of items 20 to 27, wherein the one or more enzymes for non-covalent immobilization are selected from the group consisting of lipases or oxidases, preferably glucose oxidase, lactate oxidase, uricase, glutamate oxidase, cortisol oxidase, xanthine oxidase, cholesterol oxidase, sarcosine oxidase, and alcohol oxidase. 29. A composition comprising the hydrogel described in any one of items 17 to 28. 30. A method for encapsulating one or more enzymes in a hydrogel according to any one of items 17 to 28. 31. a) for the non-covalent immobilization of one or more enzymes in a hydrogel, or b) In a biosensor, 29. Use of the hydrogel according to any one of items 17 to 28. 32. A kit comprising the composition or hydrogel described in any one of items 17 to 29.

[0174] References TIFF2024537390000058.tif165166

Claims

1. A method for preparing a biocompatible hydrogel, comprising the steps of: a) providing a first polysaccharide and a second polysaccharide, n is the number of monomer repeat units of the first and / or second polysaccharide, and n is an integer from 10 to 10,000; process, b) optionally carboxymethylating at least one OH-group of the first and / or second polysaccharide; c) functionalizing the first polysaccharide with one or more linker units having the structure -AX if the monomer repeating units of the first polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -X if the monomer repeating units of the first polysaccharide do contain carboxylic acid residues, Here, A is -(CH 2 ) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where X is -NH-(CH 2 ) r -N 3 , -NH-(CH 2 CH 2 O) s -CH 2 CH 2 N 3 , and -NH-(CH 2 -CH 2 -C(O)) t -CH 2 -CH 2 -N 3 selected from the group consisting of r is an integer from 2 to 20; s is an integer from 1 to 15, and t is an integer from 1 to 15; process, and functionalizing the second polysaccharide with one or more linker units having the structure -A'-Y when the monomer repeat units of the second polysaccharide do not contain carboxylic acid residues, or with one or more linker units having the structure -Y when the monomer repeat units of the second polysaccharide do contain carboxylic acid residues; Here, A' is -(CH 2 ) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; where Y is -NH-(CH 2 ) r -Q, -NH-(CH 2 CH 2 O) s -CH 2 CH 2 Q, and -NH-(CH 2 -CH 2 -C(O)) t -CH 2 -CH 2 -Q, Here, Q is and where M, M' = H or Me, and Where W = OMe, OEt, OH, NH 2 or NHMe, process, d) optionally adding one or more enzymes to the mixture formed by steps a) to c); and e) Incubating the mixture formed by steps a) to d) in an aqueous medium at a temperature ranging from 25°C to 70°C for at least 1 hour.

2. 2. The method for preparing a biocompatible hydrogel according to claim 1, wherein the first polysaccharide and the second polysaccharide are independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof.

3. 2. The method for preparing a biocompatible hydrogel according to claim 1, wherein the first and / or second polysaccharide is dextran or pullulan, and carboxymethylation of at least one OH-group of dextran or pullulan is carried out in step b).

4. 2. The method for preparing a biocompatible hydrogel according to claim 1, wherein in step c) the first polysaccharide is functionalized with 0.01 to 1.5 A or X per monomer repeat unit of the first polysaccharide, and / or in step c) the second polysaccharide is functionalized with 0.01 to 1.5 A' or Y per monomer repeat unit of the second polysaccharide.

5. 2. The method for preparing a biocompatible hydrogel according to claim 1, wherein step e) is a thermally induced cycloaddition reaction between X and Y to form a crosslinked polymer.

6. N 3 per monomer repeat unit of the first polysaccharide is 0.01 to 1.5 N 3 and / or the content of Q is 0.01 to 1.5 per monomer repeat unit of the first and / or second polysaccharide.

7. 2. The method for preparing a biocompatible hydrogel according to claim 1, wherein in step c) -AX is linked to at least one primary or secondary OH-group of the first polysaccharide, and / or in step c) -A'-Y is linked to at least one primary or secondary OH-group of the second polysaccharide.

8. A method for preparing a biocompatible hydrogel as described in claim 1, wherein in step c), -AX is linked to at least one primary or secondary OH-group of the first polysaccharide via at least one of C2, C3, C4 or C6 of the monomer repeating unit of the first polysaccharide, and / or in step c), -A'-Y is linked to at least one primary or secondary OH-group of the second polysaccharide via at least one of C2, C3, C4 or C6 of the monomer repeating unit of the second polysaccharide.

9. 2. The method for preparing a biocompatible hydrogel according to claim 1, wherein the molecular weight of the unfunctionalized first polysaccharide is in the range of 5 to 2000 kDa and / or the molecular weight of the unfunctionalized second polysaccharide is in the range of 5 to 2000 kDa.

10. A hydrogel obtainable by the method of claim 1.

11. The hydrogel of claim 10, comprising one or more encapsulated enzymes.

12. A biocompatible hydrogel for non-covalent immobilization of one or more enzymes, comprising a cross-linked polymer comprising the following structure: a first polysaccharide and a second polysaccharide, n is the number of monomer repeat units of the first and / or second polysaccharide; and n is an integer from 10 to 10,000; a first polysaccharide and a second polysaccharide, - one or more linker units that connect the first polysaccharide to the second polysaccharide, wherein the structure of the one or more linker units is -A-Z 1 -BZ 2 -A'-, wherein A and A' are independently -(CH 2 ) d -C(O)- or -C(O)-NH-, where d is an integer from 1 to 3; Here Z 1 -O-, -(CH 2 ) r -, -NH-(CH 2 CH 2 O) s -CH 2 CH 2 -, and -NH-(CH 2 -CH 2 -C(O)) t -CH 2 -CH 2 - selected from the group consisting of d is an integer from 1 to 4, r is an integer from 2 to 20; s is an integer from 1 to 15, and t is an integer from 1 to 15, and, Here Z 2 -O-, -(CH 2 ) r -, -NH-(CH 2 CH 2 O) s -CH 2 CH 2 -, and -NH-(CH 2 -CH 2 -C(O)) t -CH 2 -CH 2 - selected from the group consisting of Here, B and where R' is -CF 3 , -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH 2 and —C(O)—NHMe, One or more linker units.

13. 13. The hydrogel of claim 12, wherein the non-covalent immobilization of the one or more enzymes comprises encapsulation of the one or more enzymes and non-covalent bonding of the one or more enzymes in the hydrogel.

14. 13. The hydrogel of claim 12, wherein the first and / or second polysaccharides are independently selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan, and mixtures thereof.

15. -A-Z 1 - is the formula -CH 2 -CO-NH-(CH 2 -CH 2 -O) n -CH 2 -CH 2 where n is 1 to 5, and / or the first and / or second polysaccharide has a concentration of 5 to 120 mg / ml of the total hydrogel; and / or The enzyme for non-covalent immobilization is selected from the group consisting of lipases and oxidases; 13. The hydrogel of claim 12.

16. 13. A composition comprising the hydrogel of claim 10 or 12.

17. 13. A method for encapsulating one or more enzymes in a hydrogel according to claim 10 or 12.

18. A biosensor comprising the hydrogel of claim 10 or 12.

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