METHOD OF CROSSLINKING GLYCOSAMINOGLYCANS

Abstract

A new hydrogel made of crosslinked glycosaminoglycans, particularly crosslinked hyaluronic acid, chondroitin or chondroitin sulfate, having reversible linkages using boroxole derivatives leading to new benefits. Glycosaminoglycans that are crosslinked via an alkoxyboronate ester anion formed between a backbone diol function of a first glycosaminoglycan and a boronate hemiester grafted to a second glycosaminoglycan.

Claims

1. Crosslinked glycosaminoglycans, wherein said glycosaminoglycans are crosslinked via an alkoxyboronate ester anion formed between a backbone diol function of a first glycosaminoglycan and a boronate hemiester grafted to a second glycosaminoglycan.

2. Crosslinked glycosaminoglycans according to claim 1, wherein said crosslinked glycosaminoglycans has a structure of Formula (III) ##STR00035## wherein R.sup.1 is selected from H, F, Cl, NO.sub.2, C.sub.1-C.sub.3alkyl, C.sub.1-C.sub.3haloalkyl and C.sub.1-C.sub.3alkoxy; R.sup.2, R.sup.3 and R.sup.4 are independently selected from H, F, Cl, C.sub.1-C.sub.3haloalkyl, NO.sub.2, C.sub.1-C.sub.3alkoxy, C.sub.1-C.sub.3alkyl and a linker, said linker binding covalently to said second glycosaminoglycan; X is selected from CHR.sup.7 and a bond; R.sup.5, R.sup.6 and R.sup.7 are independently selected from H, C.sub.1-C.sub.4alkyl, C.sub.3-C.sub.6cycloalkyl, phenyl, and a five- to six-membered heteroaromatic ring comprising 1 to 3 heteroatoms selected from O, N and S; and one of R.sup.2, R.sup.3 and R.sup.4 is a linker.

3. Crosslinked glycosaminoglycans according to claim 1, wherein said glycosaminoglycans are hyaluronic acid.

4. Crosslinked glycosaminoglycans according to claim 2, wherein said linker is NR.sup.9Y or OY and forms an amide bond or an ether bond with said second glycosaminoglycan, wherein R.sup.9 is selected from hydrogen, C.sub.1-C.sub.3alkyl and C.sub.1-C.sub.3fluoroalkyl; Y is selected from a bond and C.sub.1-C.sub.6alkylene in which one or two CH.sub.2 are optionally replaced by a group selected from O, NH and phenylene, said C.sub.1-C.sub.6alkylene being optionally substituted with 1 to 12 R.sup.8; and R.sup.8 is selected from F, Cl, C.sub.1-C.sub.3alkyl, C.sub.1-C.sub.3haloalkyl, phenyl, OH, C.sub.1-C.sub.3hydroxyalkyl, C.sub.1-C.sub.3alkoxy, NH.sub.2, NC.sub.1-C.sub.3alkylamino, N,NC.sub.1-C.sub.4dialkylamino.

5. Crosslinked glycosaminoglycans according to claim 2, wherein R.sup.2 is a linker.

6. Crosslinked glycosaminoglycans according to claim 2, wherein said linker is NR.sup.9Y and forms an amide bond with said second glycosaminoglycan, wherein R.sup.9 is selected from hydrogen, C.sub.1-C.sub.3alkyl and C.sub.1-C.sub.3fluoroalkyl; and wherein Y is a bond or an unsubstituted C.sub.1-C.sub.6alkylene.

7. Crosslinked glycosaminoglycans according to claim 2, wherein R.sup.1, R.sup.3 and R.sup.4 are independently selected from H, F, OCH.sub.3, CF.sub.3 and CH.sub.3; R.sup.2 is a linker; said linker is HNY and forms an amide bond with said second glycosaminoglycan; Y is a bond or an unsubstituted C.sub.1-C.sub.3alkylene; X is a bond or CH.sub.2; and R.sup.5 and R.sup.6 are independently selected from H and C.sub.1-C.sub.3alkyl.

8. Crosslinked glycosaminoglycans according to claim 1, wherein said boronate hemiester is selected from ##STR00036## wherein the boronate hemiester is grafted to said second glycosaminoglycan by that the NH.sub.2 group of the boronate hemiester forms an amide with a backbone carboxylate group of said second glycosaminoglycan.

9. Crosslinked glycosaminoglycans according to claim 1, said crosslinked glycosaminoglycans having a structure of Formula (IV) ##STR00037##

10. A method of cross-linking a first glycosaminoglycan having a backbone diol function and a second glycosaminoglycan being grafted with a boronate hemiester, comprising crosslinking said first glycosaminoglycan with said second glycosaminoglycan by forming an alkoxyboronate ester anion linkage between the boronate hemiester of the second glycosaminoglycan with the backbone diol function of said first glycosaminoglycan.

11. A method according to claim 10, further comprising the step grafting said second glycosaminoglycan with said boronate hemiester, said boronate hemiester being a compound of Formula (I), ##STR00038## wherein R.sup.1 is selected from H, F, Cl, NO.sub.2, C.sub.1-C.sub.3alkyl, C.sub.1-C.sub.3haloalkyl and C.sub.1-C.sub.3alkoxy; R.sup.2, R.sup.3 and R.sup.4 are independently selected from H, F, Cl, C.sub.1-C.sub.3haloalkyl, NO.sub.2, C.sub.r C.sub.3alkoxy, C.sub.1-C.sub.3alkyl and a linker binding covalently to said second glycosaminoglycan; X is selected from CHR.sup.7 and a bond; and R.sup.5, R.sup.6 and R.sup.7 are independently selected from H, C.sub.1-C.sub.4alkyl, C.sub.3-C.sub.6cycloalkyl, phenyl, and a five- to six-membered heteroaromatic ring comprising 1 to 3 heteroatoms selected from O, N and S, wherein one of R.sup.2, R.sup.3 and R.sup.4 is a linker, thereby providing said second glycosaminoglycan being grafted with a boronate hemiester.

12. A method according to claim 10, wherein said first and said second glycosaminoglycans are hyaluronic acid.

13. A method according to claim 11 or claim 12, wherein said linker is H2NY or ##STR00039## and forms an amide bond or an ether bond to said second glycosaminoglycan; Y is selected from a bond and C.sub.1-C.sub.6alkylene in which one or two CH.sub.2 are optionally replaced by a group selected from O, NH and phenylene, said C.sub.1-C.sub.6alkylene being optionally substituted with 1 to 12 R.sup.8; and R.sup.8 is selected from F, Cl, C.sub.1-C.sub.3alkyl, C.sub.1-C.sub.3haloalkyl, phenyl, OH, C.sub.1-C.sub.3hydroxyalkyl, C.sub.3alkoxy, NH.sub.2, NC.sub.1-C.sub.3alkylamino, N,NC.sub.1-C.sub.4dialkylamino.

14. A method according to claim 11, wherein R.sup.2 is a linker.

15. A method according to claim 11, wherein said linker is HR.sup.9NY and forms an amide bond with said second glycosaminoglycan, wherein R.sup.9 is selected from hydrogen, C.sub.1-C.sub.3alkyl and C.sub.1-C.sub.3fluoroalkyl; and Y is a bond or an unsubstituted C.sub.1-C.sub.6alkylene.

16. A method according to claim 11, wherein R.sup.1, R.sup.3 and R.sup.4 are independently selected from H, F, OCH.sub.3, CF.sub.3 and CH.sub.3; R.sup.2 is a linker; said linker is H.sub.2NY and forms an amide bond with said second glycosaminoglycan; Y is a bond or an unsubstituted C.sub.1-C.sub.3alkylene; X is a bond or CH.sub.2; and R.sup.5 and R.sup.6 are independently selected from H and C.sub.1-C.sub.3alkyl.

17. A method according to claim 10, wherein said boronate hemiester is selected from ##STR00040## wherein the boronate hemiester is grafted to said second glycosaminoglycan by that the NH.sub.2 group of the boronate hemiester forms an amide with a backbone carboxylate group of said second glycosaminoglycan.

18. A method according to claim 10, said boronate hemiester being ##STR00041##

19-27. (canceled)

28. Polymer composition comprising crosslinked glycosaminoglycans according to claim 1 and an aqueous buffer.

29. Crosslinked glycosaminoglycans produced according to the method according to claim 10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1: Rheological analysis: measurement of G and G for HA-BOR (15 g/I in 0.01 M HEPES buffer with 0.15 M NaCl, pH 7.4).

[0130] FIG. 2: Rheological analysis: measurement of G and G for HA-BOR with HA M.sub.w 500 kg/mol (HA500), [PS] of 15 g/L (HA-BOR derivative solubilized in ultrapure water at 30 g/L, followed by addition of 0.02 M HEPES buffer containing 0.3 M NaCl pH 7.4)

[0131] FIG. 3: Rheological analysis: measurement of G and G for HA-BOR with HA M.sub.w 600 kg/mol (HA600), [PS] of 15 g/L (HA-BOR derivative solubilized in ultrapure water at 30 g/L, followed by addition of 0.02 M HEPES buffer containing 0.3 M NaCl pH 7.4)

[0132] FIG. 4: Rheological analysis: measurement of G and G for HA-BOR with HA M.sub.w 1000 kg/mol (HA1000), [PS] of 15 g/L (HA-BOR derivative solubilized in ultrapure water at 30 g/L, followed by addition of 0.02 M HEPES buffer containing 0.3 M NaCl pH 7.4).

[0133] FIG. 5. Self-healing behavior of a HA-BOR hydrogel: application of gradually increasing stress values from 1800 to 2100 Pa for 2 min, intercalated with periods of application of a strain fixed at 5% for 3 min (frequency fixed at 1 Hz).

[0134] FIG. 6: Gel obtained with HA-BOR.

[0135] FIG. 7: Rheological analysis: measurement of G and G for HA-BOR and HA-DMABOR gels with HA M.sub.w 600 kg/mol (HA600), [PS] of 15 g/L (HA derivatives solubilized in ultrapure water at 30 g/L, followed by addition of 0.02 M HEPES buffer containing 0.3 M NaCl pH 7.4).

[0136] FIG. 8: Recovery of G and G as a function of time post-extrusion of a HA-DMABOR gel (M.sub.w=600 kg/mol) through a 27 gauge needle.

[0137] The following terms and characteristics will be used in the examples and results shown. The definitions are the one hereafter:

[0138] MwMolecular Weight: The mass average molecular mass

[0139] DSDegree of Substitution The term degree of substitution (DS) as used herein in connection with various polymers, e.g. polysaccharides, refers to the average number of substituting group per repeating disaccharide unit.

[0140] [PS]The polysaccharide concentration (g/l).

[0141] G: storage (elastic) modulus (in Pa)

[0142] G: loss (viscous) modulus (in Pa)

[0143] G 1 Hz: storage modulus (in Pa) measured at a frequency of 1 Hz

[0144] G 1 Hz: loss modulus (in Pa) measured at a frequency of 1 Hz

[0145] Gel-like behavior: G>G within the whole range of frequency covered (0.01-10 Hz)

[0146] Viscoelastic behavior: viscous (G<G) and elastic (G>G) behavior observed within the range of frequency covered (0.01-10 Hz).

[0147] The IUPAC names of the benzoboroxol derivatives in example 4-11 are generated using Biovia DRAW 4.2.

EXAMPLES

[0148] Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples.

Example 1: Synthesis of HA-BOR

[0149] ##STR00027##

[0150] The amine-acid coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was dissolved in 1 mL of water and was added to a solution of native HA in a mixture of water/DMF (3/2, v/v). The concentration of HA in the reaction medium depends on its molar mass (Table 1, M.sub.w HA). Then, 5-amino-2-hydroxymethylphenylboronic acid hydrochloride (i.e. 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride or BOR) solubilized in 1 mL of water was added to the reaction medium. The pH was adjusted to 6.5 using 0.5 M HCl or NaOH and the reaction was kept under stirring at room temperature for 24 h. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) was determined by .sup.1H NMR (DS.sub.NMR), and was also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS). This method consisted in quantifying the free primary amines in the reaction medium as a function of time.

[0151] Results:

[0152] Table 1 summarizes the concentration of HA (C.sub.HA), the DMTMM/HA and BOR/HA molar ratios used for the syntheses with different M, HA, as well as the DS and the yields of HA-BOR conjugates.

TABLE-US-00001 TABLE 1 Syntheses of HA-BOR. M.sub.w HA C.sub.HA DMTMM/HA BOR/HA molar (Kg/mol) (g/L) molar ratio ratio DS.sub.NMR.sup.a DS.sub.TNBS Yield (%).sup.b 100 3 1 0.16 0.12 0.14 85 500 2 1 0.14 (or 0.25) 0.1 (or 0.2) 0.12 (or 0.22) 91 (or 77) 600 2 1 0.14 (or 0.25) 0.11 (or 0.2) 0.13 (or 0.2) 75 (or 72) 1000 1 1 0.14 (or 0.26) 0.1 (or 0.2) 0.14 (or 0.23) 84 (or 60) .sup.aDS by .sup.1H NMR: 10% of accuracy. .sup.bHA-BOR yield: calculation considering the DS.sub.NMR.

[0153] HA-BOR (HA-1-hydroxy-3H-2,1-benzoxaborol-amine): .sup.1H NMR (400 MHz, D.sub.2O) .sub.H (ppm) 4.55 (H-1 from N-acetylglucosamine unit), 4.25 (H-1 from glucuronic acid), 3.9-3.1 (H-2, H-3, H-4, H-5, H-6 protons of HA), 2.08 (CH.sub.3CO from HA), 7.95 (s, 1H, NHCCHCB from Ph), 7.72 (m, 1H, CCHCHCCB from Ph), 7.55 (m, 1H, CCHCHCCB from Ph), 5.13 (s, 2H, CH.sub.2OB).

Example 2: Preparation of HA-Benzoboroxole (HA-BOR) Gel

[0154] HA-BOR gels were prepared by solubilizing 1-hydroxy-3H-2,1-benzoxaborol-amine (the HA-BOR derivative) in 0.01 M HEPES buffer with 0.15 M NaCl at physiological pH ([PS]=15 g/L).

[0155] Results:

[0156] Characteristics of the obtained HA-BOR hydrogels are summarized in Table 1. Boronate ester bonds are formed between benzoboroxole and HA hydroxy groups. Gels behavior has been demonstrated by rheological analysis (FIG. 1). Surprinsingly, when coupling HA chains with benzoboroxole only, obtained hydrogels present good gel behaviour (FIG. 6).

TABLE-US-00002 TABLE 2 Characteristics of HA-BOR hydrogel ([PS] = 15 g/L). DS HA- HA-boronic boronic acid acid Mw HA G 1 Hz G 1 Hz Rheological derivative derivative (kg/mol) (Pa) (Pa) behavior HA-BOR 0.11 600 470 145 Gel-like behavior HA-BOR 0.2 600 420 130 Gel-like behavior HA-BOR 0.1 1000 56 36 Viscolelastic behavior

Example 3: Comparison of HA-BOR Gel to HA-PBA Gel and Native HA Gel

[0157] HA-BOR Gel Preparation:

[0158] HA-1-hydroxy-3H-2,1-benzoxaborol-amine (HA-BOR derivative) was solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4 (final [PS]=15 g/L).

[0159] Results:

[0160] Within 8 h of stirring at 4 C., a final gel was obtained with a polymer concentration of 15 g/L and pH 7. Gels prepared using HA-BOR with M.sub.w of 1000 kg/mol may require a longer time of solubilization (24 to 48 h). Characteristics of the resulting gels or viscous solutions are are shown in Table 3. Plotted measurements of G and G for HA-BOR gels with different molecular weights and degrees of substitution, compared to native HA are shown in FIGS. 2, 3 and 4.

[0161] Self-healing properties of a dynamic gel of HA-BOR (CHA=15 g/L) at 25 C. were investigated by, while measuring G and G, applying successive stress values from 1800 to 2100 Pa for 2 min. These were intercalated with short time periods in which low stress values (corresponding to 5% strain) were applied for 3 min. This experiment demonstrated the stress recovery of the HA-BOR gel after 4 cycles of stress-induced breakdowns. Large stress (from 1800 to 2100 Pa) inverted the values of G (filled circles) and G (empty circles), indicating breakage of crosslinks and conversion to solution state. G was recovered under a small strain (5%) within few seconds. The obtained HA-BOR showed self-healing properties, see FIG. 5.

[0162] Synthesis of HA-PBA:

##STR00028##

[0163] 3-aminophenylboronic acid hemisulfate salt (APBA) dissolved in 1 mL of water was added to a solution of native HA in a mixture of water/DMF (3/2, v/v), in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as an amine-acid coupling agent. A concentration of 2 g/L was used for the sample HA M.sub.w 600 kg/mol. The pH was adjusted to 6.5 using 0.5 M HCl or NaOH and the reaction was kept under stirring at room temperature for 24 h. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-PBA was determined by .sup.1H NMR (0.140.01). HA-PBA was obtained with a yield of 78% (calculated considering its DS).

[0164] HA-PBA: .sup.1H NMR (400 MHz, D.sub.2O) .sub.H (ppm) 4.55 (H-1 from N-acetylglucosamine unit), 4.25 (H-1 from glucuronic acid), 3.9-3.1 (H-2, H-3, H-4, H-5, H-6 protons of HA), 2.08 (CH.sub.3CO from HA), 7.93 (s, 1H, NHCCHCB from Ph), 7.7 (m, 2H, CCHCHCHCB from Ph), 7.55 (m, 1H, CCHCHCHCB from Ph).

[0165] HA-PBA and Native HA Samples Preparation:

[0166] HA-PBA or native HA was solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4. The solutions were stirred during 8 h at 4 C.

[0167] Result: The characteristics of the resulting samples are shown in Table 3.

TABLE-US-00003 TABLE 3 Characteristics of obtained samples ([PS] = 15 g/L). HA DS HA Mw HA G 1 Hz G 1 Hz Rheological derivative derivative (kg/mol) (Pa) (Pa) behavior HA-BOR 0.1 100 0.043 0.44 Viscous HA-BOR 0.1 500 160 38 Gel HA-BOR 0.2 500 204 63 Gel HA-BOR 0.1 600 330 108 Gel HA-BOR 0.2 600 800 210 Gel HA-BOR 0.1 1000 45 29 Viscoelastic HA-BOR 0.2 1000 198 78 Gel HA-PBA 0.15 600 5.65 5.89 Viscoelastic Native HA 500 0.05 1.3 Viscous Native HA 500 0.1 1.96 Viscous Native HA 600 2 8 Viscous Native HA 1000 27 33 Viscoelastic

Example 4: Synthesis of HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine

[0168] ##STR00029##

[0169] Example 4 is performed according to Example 1, but using 1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine hydrochloride as the BOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 5: HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine Gel Preparation

[0170] HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine is solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4.

[0171] Another set of gels are prepared according to example 2, but using HA-1-hydroxy-7-methoxy-3H-2,1-benzoxaborol-6-amine instead of HA-BOR.

Example 6: Synthesis of HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine

[0172] ##STR00030##

[0173] Example 6 is performed according to Example 2, but using 7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine hydrochloride as the BOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 7: HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine Gel Preparation

[0174] HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine is solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4.

[0175] Another set of gels are prepared according to example 2, but using HA-7-fluoro-1-hydroxy-3H-2,1-benzoxaborol-6-amine instead of HA-BOR.

Example 8: Synthesis of HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine

[0176] ##STR00031##

[0177] Example 8 is performed according to Example 1, but using (1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine hydrochloride as the BOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 9: HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine Gel Preparation

[0178] HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine is solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4.

[0179] Another set of gels are prepared according to example 2, but using HA-(1-hydroxy-3H-2,1-benzoxaborol-6-yl)methanamine instead of HA-BOR.

Example 10: Synthesis of HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine

[0180] ##STR00032##

[0181] Example 10 is performed according to Example 1, but using 1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine hydrochloride as the BOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 11: HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine Gel Preparation

[0182] HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine is solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4.

[0183] Another set of gels are prepared according to example 2, but using HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine instead of HA-BOR.

Example 12: Synthesis of HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine

[0184] ##STR00033##

[0185] Example 10 is performed according to Example 1, but using 1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine hydrochloride as the BOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride.

Example 13: HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine Gel Preparation

[0186] HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine is solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4.

[0187] Another set of gels are prepared according to example 2, but using HA-1-hydroxy-3,4-dihydro-2,1-benzoxaborinin-7-amine instead of HA-BOR.

Example 14: Synthesis of HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine

[0188] ##STR00034##

[0189] Example 14 was performed according to Example 1, but using 1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine hydrochloride (DMABOR) as the BOR derivative instead of 1-hydroxy-3H-2,1-benzoxaborol-amine hydrochloride. The molecular weight of the Hyaluronic acid was 600 kg/mol.

Example 15: HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine Gel Preparation

[0190] HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine (HA-DMABOR) was solubilized in ultrapure water (pH 5-6) at 30 g/L for 24 h under continuous stirring at 4 C., followed by addition of 0.02M HEPES buffer containing 0.3M NaCl pH 7.4 (final [PS]=15 g/L).

[0191] Gels were prepared according to example 2, but using HA-1-hydroxy-3,3-dimethyl-2,1-benzoxaborol-6-amine (HA-DMABOR) instead of HA-BOR.

[0192] Result: Characteristics of the obtained HA-DMABOR hydrogel are summarized in Table 4, in comparison with HA-BOR gel. Boronate ester bonds are formed between DMABOR and HA hydroxyl groups. Gels behavior has been demonstrated by rheological analysis (FIG. 7). These hydrogel further exhibited self-healing properties. Consequently, they can be injected as preformed solids, because the solid gel can manage external damages and repair itself under a proper shear stress. Due to fast gelation kinetics after extrusion/injection, they recover their solid form immediately. As an example, FIG. 8 shows the variation of G and G as a function of time immediately after injection of a HA-DMABOR gel (in 0.01M HEPES/0.15M NaCl buffer pH 7.5, at a [PS]=15 g/L) through a 27 gauge needle. From this Figure, it can be seen that the sample recovered into a solid gel quasi-instantaneously.

TABLE-US-00004 TABLE 4 Characteristics of obtained gels ([PS] = 15 g/L). DS HA Mw HA G 1 Hz G 1 Rheological HA derivative derivative (kg/mol) (Pa) Hz (Pa) behavior HA-BOR 0.1 600 330 108 Gel HA-DMABOR 0.1 600 270 27 Gel