METHOD OF CROSSLINKING GLYCOSAMINOGLYCANS

Abstract

A new hydrogel made of double crosslinked glycosaminoglycans, particularly crosslinked hyaluronic acid, chondroitin or chondroitin sulfate, having reversible linkages using boronic acid or boroxole derivatives leading to new benefits. Double crosslinked glycosaminoglycans, one linkage via two ether bonds with a hydroxyl group of each of two glycosaminoglycans and another linkage via an alkoxyboronate ester anion formed between a boronate hemiester grafted to one of the glycosaminoglycans and a diol function of to the other glycosaminoglycan. The diol function may be a backbone diol function or a diol portion of a diol functional moiety grafted the other glycosaminoglycan.

Claims

1-51. (canceled)

52. Glycosaminoglycans crosslinked by a first and a second linkage, wherein a) said first linkage comprises two ether bonds, one bond formed with a hydroxyl group of each of a first glycosaminoglycan and a second glycosaminoglycan; and b) said second linkage is via an alkoxyboronate ester anion formed between a boronate hemiester grafted to the first glycosaminoglycan and a diol function of the second glycosaminoglycan, wherein said diol function may be a backbone diol function or a diol portion of a diol functional moiety grafted to said second glycosaminoglycan.

53. Crosslinked glycosaminoglycans according to claim 52, wherein said second linkage is defined in Formula (I) ##STR00037## 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 first 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 wherein one of R.sup.2, R.sup.3 and R.sup.4 is a linker.

54. Crosslinked glycosaminoglycans according to claim 52, wherein said glycosaminoglycans are hyaluronic acid.

55. Crosslinked glycosaminoglycans according to claim 53, wherein R.sup.2 is a linker.

56. Crosslinked glycosaminoglycans according to claim 53, wherein said linker is NR.sup.9Y and forms an amide bond with said first glycosaminoglycans, 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.

57. Crosslinked glycosaminoglycans according to claim 53, 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 first 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.

58. Crosslinked glycosaminoglycans according to claim 52, wherein said boronate hemiester is selected from ##STR00038## wherein the boronate hemiester is grafted to said first glycosaminoglycan by that the NH.sub.2 group of the boronate hemiester forms an amide with a backbone carboxylate group of said first glycosaminoglycan.

59. Crosslinked glycosaminoglycans according to claim 52, said second linkage having a structure of Formula (II) ##STR00039##

60. Crosslinked glycosaminoglycans according to claim 52, wherein said diol function is a backbone diol function.

61. Crosslinked glycosaminoglycans according to claim 52, wherein said diol function is a diol portion of a diol functional moiety grafted to said second glycosaminoglycan, wherein said diol portion is selected from a monosaccharide, a disaccharide and an alditol or a derivative thereof, or wherein said diol portion is selected from maltose, fructose, lactose and sorbitol or a derivative thereof.

62. A method of crosslinking glycosaminoglycans, comprising the steps of: forming a linkage comprising two ether bonds, one bond formed with a hydroxyl group of each a first and a second glycosaminoglycan; grafting said first glycosaminoglycan with a boronate hemiester and crosslinking said first glycosaminoglycan with said second glycosaminoglycan by forming an alkoxyboronate ester anion linkage between the boronate hemiester of said first glycosaminoglycan and a diol function of said second glycosaminoglycan, wherein said diol function may be a backbone diol function or a diol portion of a diol functional moiety grafted to said second glycosaminoglycan.

63. A method according to claim 62, wherein said boronate hemiester is a compound of Formula (III), ##STR00040## 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 binding covalently to said first 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.

64. A method according to claim 62, wherein said first and said second glycosaminoglycans are hyaluronic acid.

65. A method according to claim 63, wherein R.sup.2 is a linker.

66. A method according to claim 63, wherein said linker is HR.sup.9NY and forms an amide bond with said first 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.

67. A method according to claim 63, 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 first 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.

68. A method according to claim 62, wherein said boronate hemiester is selected from ##STR00041## wherein the boronate hemiester is grafted to said first glycosaminoglycan by that the NH.sub.2 group of the boronate hemiester forms an amide with a backbone carboxylate group of said first glycosaminoglycan.

69. A method according to claim 62, wherein said diol function is a backbone diol function.

70. A method according to claim 62, wherein said diol function is a diol portion of a diol functional moiety grafted to said second glycosaminoglycan, wherein said diol portion is selected from a monosaccharide, a disaccharide and an alditol or a derivative thereof, or selected from maltose, fructose, lactose and sorbitol or a derivative thereof.

71. Polymer composition comprising crosslinked glycosaminoglycans according to claims 52 and an aqueous buffer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0159] FIG. 1: Gel obtained with HA-BOR.

[0160] FIG. 2: 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).

[0161] FIG. 3: 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).

[0162] FIG. 4: Rheological analysis of HA-BOR/HA-fructose mixtures in 0.01M HEPES buffer containing 0.15M NaCl at different pH (from 4 to 8).

[0163] FIG. 5: Schematic structure of double crosslinked glycosaminoglycans.

[0164] FIG. 6: 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.

EXAMPLES

[0165] The following terms and characteristics will be used in the examples and results shown. The definitions are the one hereafter:

[0166] MwMolecular Weight: The mass average molecular mass

[0167] 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

[0168] [PS]The polysaccharide concentration (g/L)

[0169] G: storage (elastic) modulus (in Pa)

[0170] G: loss (viscous) modulus (in Pa)

[0171] G 1 Hz: storage modulus (in Pa) measured at a frequency of 1 Hz

[0172] G 1 Hz: loss modulus (in Pa) measured at a frequency of 1 Hz

[0173] Gel-like behavior: G>G within the whole range of frequency covered (0.01-10 Hz)

[0174] Viscoelastic behavior: viscous (G<G) and elastic (G>G) behavior observed within the range of frequency covered (0.01-10 Hz).

[0175] ABOR: 5-Amino-2-methylphenylboronic acid

[0176] AMBOR: 6-(Aminomethyl)benzo[c][1,2]oxaborol-1(3H)-ol

[0177] APBA: 3-Aminophenylboronic acid

[0178] BDDE: 1,4-Butanediol diglycidyl ether

[0179] BDPE: 1,4-butanediol di-(propan-2,3-diolyl)ether

[0180] DMABOR: 6-Amino-3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol

[0181] DMF: Dimethylformamide

[0182] DMTMM: 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride

[0183] HEPES: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

[0184] PBS: Phosphate buffered saline

[0185] TNBS: 2,4,6-Trinitrobenzenesulfonic acid

[0186] 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

[0187] ##STR00030##

[0188] 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). A concentration of HA in the reaction medium of 3 g/L was used for HA samples of 75 and 100 kg/mol, whereas 2 g/L was used for HA with 600 kg/mol. Then, 5-amino-2-hydroxymethylphenylboronic acid hydrochloride (1-hydroxy-3H-2,1-benzoxaborol-amine, ABOR) 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) of HA-BOR was determined by .sup.1H NMR (DS.sub.NMR), and were 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. Table 1 summarizes the DMTMM/HA and BOR/HA molar ratios used for the syntheses with different M.sub.W HA, as well as the DS and the yields of HA-BOR conjugates.

[0189] HA-BOR: .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 : Synthesis of HA-PBA (Comparative Example)

[0190] ##STR00031##

[0191] Grafting of phenylboronic acid was done according to Example 1, but using 3-aminophenylboronic acid hemisulfate salt (APBA) instead of 5-amino-2-hydroxymethylphenylboronic acid hydrochloride (ABOR). The degree of substitution (DS) of HA-PBA was determined by .sup.1H NMR (DS.sub.NMR), and were 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. Table 1 summarizes the DMTMM/HA and PBA/HA molar ratios used for the syntheses with different M.sub.W HA, as well as the DS and the yields of HA-PBA conjugates.

[0192] 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).

TABLE-US-00001 TABLE 1 Syntheses of HA-BOR and HA-PBA. HA-boronic M.sub.w HA DMTMM/HA BOR or PBA/HA Yield acid derivative (Kg/mol) molar ratio molar ratio DS.sub.NMR.sup.a DS.sub.TNBS (%).sup.b HA-BOR 75 1 0.16 0.16 0.16 75 HA-BOR 100 1 0.16 0.12 0.14 85 HA-BOR 600 1 0.14 0.11 0.13 75 HA-PBA 75 1 0.16 0.16 0.16 75 HA-PBA 100 1 0.16 0.16 0.16 77 HA-PBA 600 1 0.14 0.14 0.14 78 .sup.aDS by .sup.1H NMR: 10% of accuracy. .sup.bHA-BOR or HA-PBA yield: calculation considering the DS.sub.NMR.

Example 3: Synthesis of HA-BOR Gels

[0193] HA-BOR gels were prepared by solubilizing the HA-BOR derivative in 0.01 M HEPES buffer with 0.15 M NaCl at physiological pH. The characteristics of the obtained gels are shown in Table 2.

TABLE-US-00002 TABLE 2 Characteristics of HA-BOR hydrooel ([PS] = 15 g/L). DS G G HA-boronic acid HA-boronic Mw HA 1 Hz 1 Hz Rheological derivative acid derivative (kg/mol) (Pa) (Pa) behavior HA-benzoboroxole 0.1 600 470 145 Gel HA-benzoboroxole 0.1 1000 56 36 Viscoelastic Native HA 600 2 8 Viscous Native HA 1000 27 33 Viscoelastic

[0194] Boronate ester bonds are formed between benzoboroxole and diol groups HA. Gels behavior has been demonstrated by rheological analysis.

[0195] Surprinsingly, when coupling HA chains with benzoboroxole only, obtained hydrogels present good gel behaviour (FIG. 1).

Example 4: Comparison of HA-BOR Gel to HA-PBA Gel and Native HA Gel

[0196] HA-BOR gel preparation:

[0197] 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.

[0198] HA-PBA and native HA samples preparation:

[0199] 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.

[0200] Results:

[0201] 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 mixtures are shown in Table 3 and in FIG. 2. Self-healing properties of a dynamic gel of HA-BOR (C.sub.HA=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, (FIG. 3). The characteristics of the resulting samples are shown in Table 3. The results show that HA-PBA gives a viscoelastic behaviour, whereas HA-BOR gives a gel with a number of different molecular weights.

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 5: Synthesis of Pentenoate-Modified HA

[0202] ##STR00032##

[0203] HA (1 g, 2.5 mmol, M.sub.W=100 kg/mol) was dissolved in ultrapure water (50 mL) under continuos stirring overnight at 4 C. DMF (33 mL) was then added dropwise in order to have a water/DMF ratio of (3/2, v/v). 4-pentenoic anhydride (0.454 g, 2.5 mmol) was added while maintaining the pH between 8 and 9 by adding 1 M NaOH for at least 4 h. The reaction was kept at 4 C. under stirring for one night. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-pentenoate was found to be 0.180.01 by .sup.1H NMR. A yield of 49% was calculated considering its DS.

[0204] .sup.1H NMR (400 MHz, D.sub.2O) .sub.H (ppm) 4.71 (H-1 from N-acetylglucosamine unit), 4.53 (H-1 from glucuronic acid), 4.13-3.2 (H-2, H-3, H-4, H-5, H-6 protons of HA), 2.1 (CH.sub.3CO from HA), 6.0 (m, 1H, CHCH2), 5.18 (m, 2H, CHCH.sub.2), 2.62 (m, 2H, CH.sub.2CO), 2.45 (m, 2H, OCCH.sub.2CH.sub.2).

Example 6: Synthesis of HA-Maltose

[0205] ##STR00033##

a. Maltose-Disulfide

[0206] To an aqueous solution of maltose (0.25 g, 0.694 mmol) in 25 mL of ultrapure water at room temperature, O-(carboxymethyl)hydroxylamine hemihydrochloride (0.0768 g, 0.694 mmol) was added. The pH was adjusted to 4.8 using 0.5 M NaOH. The reaction mixture was stirred for 24 hours at room temperature and then, was neutralized to pH 7 by addition of 0.5 M NaOH. The maltose-COOH derivative was then recovered by freeze-drying without further purification as a white powder (46 mol % of maltose-COOH/maltose). To a solution of maltose-COOH (0.25 g, 0.622 mmol) in dry DMF (50 mL), hydroxybenzotriazole (HOBt) (0.1875 g, 1.39 mmol), diisopropylcarbodiimide (DIC) (0.3483 g, 2.8 mmol) and cystamine dihydrochloride (0.094 g, 0.42 mmol) were successively added. The resulting mixture was stirred overnight at room temperature under nitrogen. After evaporation of most of the solvent, the residual syrup was poured dropwise into acetone (500 mL) under stirring. The white precipitate was collected by filtration, washed three times with acetone and dried to give the desired maltose-disulfide in 60% yield (0.295 g).

[0207] .sup.1H NMR (400 MHz, D.sub.2O) .sub.H (ppm) 7.75 (1H, anomeric H from linked glucose unit, NCH.sub.), 7.13 (1H, anomeric H from linked glucose unit, NCH.sub.), 5.4 (1H, anomeric H from pendant glucose unit of maltose), 5.19 (1H, anomeric H from linked glucose unit), 5.14 (1H, anomeric H from pendant glucose unit of maltose-disulfide), 4.7 (1H, anomeric H from pendant glucose unit), 4.66 (2H, NOCH.sub.2), 4.6 (1H, NCH.sub.,CH(OH) from linked glucose group), 3.4-4.2 (8H, H-3, H-4, H-5, H-6 from linked and pendant glucose groups), 2.95 (4H, NHCH.sub.2CH.sub.2).

b. HA-Maltose

[0208] The first step consisted in reducing the disulfide bond of maltose-disulfide. Thus, to an aqueous solution of this derivative (0.2 g, 0.211 mmol) in 4 mL of degassed phosphate buffered saline (PBS) pH 7.4 at room temperature, a solution of TCEP (91 mg, 0.317 mmol) in 1 mL of degassed PBS was added and the pH was adjusted to 5-5.5. The mixture was stirred for 15 min under nitrogen at room temperature to give maltose-SH. The pH was adjusted to 7.4 using 0.5 M NaOH and the mixture was added to HA-pentenoate solubilized in PBS in the presence of Irgacure 2959 (0.1%, w/v) as a photoinitiator. The grafting of maltose-SH moieties was performed under UV radiation (=365 nm, at 20 mW/cm.sup.2 for 15 min). The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying (80%). The degree of substitution (DS) of HA-maltose was found to be 0.10.01 by .sup.1H NMR.

[0209] .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), 1.85 (CH.sub.3CO from HA), 1.52 (m,2H,CH.sub.2CH.sub.2CH.sub.2S), 1.62 (m,2H,CH.sub.2CH.sub.2CH.sub.2S), 2.35 (m, 2H, OCCH.sub.2) 2.63 (m,2H, CH.sub.2CH.sub.2CH.sub.2S), 2.82 (m,2H, SCH.sub.2CH.sub.2NH), 7.63 (m, 1H, H anomer of maltose).

Example 7: Synthesis of HA-Lactobionic

[0210] ##STR00034##

a. Lactobionic-Disulfide

[0211] To a solution of lactobionic acid (0.5023 g, 1.39 mmol) in dry DMF (50 mL), hydroxybenzotriazole (HOBt) (0.3768 g, 2.79 mmol), diisopropylcarbodiimide (DIC) (0.705 g, 5.56 mmol) and cystamine dihydrochloride (0.141 g, 0.63 mmol) were successively added. The resulting mixture was stirred overnight at room temperature under nitrogen. After evaporation of most of the solvent, the residual syrup was poured dropwise into acetone (500 mL) under stirring. The white precipitate was collected by filtration, washed three times with acetone and dried to give the desired lactobionic-disulfide in 29% yield (0.2362 g).

b. HA-Lactobionic

[0212] A first step of reduction of the disulfide bond of the lactobionic-disulfide derivative (0.2 g, 0.211 mmol) dissolved in 1 mL of degassed PBS was performed by adding TCEP (91 mg, 0.317 mmol) in 1 mL of degassed PBS, with pH adjusted to 5-5.5. The mixture was stirred for 15 min under nitrogen at room temperature to give lactobionic-SH. The pH was adjusted to 7.4 using 0.5 M NaOH and the mixture was added to HA-pentenoate solubilized in PBS in the presence of Irgacure 2959 (0.1%, w/v) as a photoinitiator. The grafting of lactobionic-SH moieties was performed under UV radiation (=365 nm, at 20 mW/cm.sup.2 for 15 min). The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying (60%). The degree of substitution (DS) of HA-lactobionic was found to be 0.20.01 by .sup.1H NMR.

Example 8: Synthesis of HA-Fructose

[0213] ##STR00035##

[0214] 1-amino-1-deoxy-D-fructose hydrochloride (0.0121 g, 0.056 mmol) dissolved in 1 mL of ultrapure water was added to a solution of native HA (0.15 g, 0.374 mmol) 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) (0.1035 g, 0.374 mmol) as an amine-acid coupling agent. 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-fructose was determined by .sup.13C NMR (DS.sub.NMR=0.150.01), and was also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS=0.14). A yield of 84% was determined for HA-fructose (considering its DS.sub.NMR).

[0215] .sup.1H NMR (400 MHz, D.sub.2O) .sub.H (ppm) 4.62 (H-1 from N-acetylglucosamine unit), 4.46 (H-1 from glucuronic acid), 4.05-3.2 (18H, H-2, H-3, H-4, H-5, H-6 protons of HA and of fructose moieties), 2.02 (CH.sub.3CO from HA).

Example 9: Synthesis of HA-Sorbitol

[0216] ##STR00036##

[0217] 1-amino-1-deoxy-D-sorbitol hydrochloride (D-glucamine) (0.0088 g, 0.05 mmol) dissolved in 1 mL of ultrapure water was added to a solution of native HA (0.1305 g, 0.325 mmol) in ultrapure water in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (0.09 g, 0.325 mmol) as an amine-acid coupling agent. 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 164 h. The product was purified by diafiltration with ultrapure water and was recovered by freeze-drying. The degree of substitution (DS) of HA-sorbitol was determined by .sup.13C NMR (DS.sub.NMR=0.150.1), and was also estimated from the reaction kinetics performed using 2,4,6-Trinitrobenzene Sulfonic Acid (DS.sub.TNBS=0.1). A yield of 76% was determined for HA-sorbitol (considering its DS.sub.NMR).

[0218] .sup.1H NMR (400 MHz, D.sub.2O) .sub.H (ppm) 4.68 (H-1 from N-acetylglucosamine unit), 4.51 (H-1 from glucuronic acid), 4.1-3.3 (19H, H-2, H-3, H-4, H-5, H-6 protons of HA and of sorbitol moieties), 2.07 (CH.sub.3CO from HA).

Example 10: Preparation of HA-BOR/HA-Polyol Gel

[0219] Solutions of HA-BOR and of the HA-polyol derivatives (HA-fructose or HA-sorbitol) were prepared at 15 g/L in 0.01 M HEPES buffer containing 0.15 M NaCl pH 7.4, and were kept under stirring overnight at 4 C. Combinations of HA-BOR/HA-polyol derivative, were prepared by mixing a solution containing HA-BOR with a solution containing a HA-polyol derivative at physiological pH, at a total polymer concentration of 15 g/L and with BOR/polyol molar ratio of 1/1.

[0220] Results:

[0221] When gels were formed quasi-instantaneously upon mixing HA-BOR solution with a solution of a HA-polyol derivative. Characteristics of the resulting HA-BOR/HA-polyol mixtures are summarized in Table 4. A rheological analysis of HA-BOR/HA-fructose is shown in FIG. 4.

TABLE-US-00004 TABLE 4 Characteristics of HA-BOR/HA-Polyol hydrogel ([PS] = 15 g/L). DS HA- DS HA- G G BOR HA-polyol polyol Mw HA 1 Hz 1 Hz Rheological derivative derivative derivative (kg/mol) (Pa) (Pa) behavior 0.16 HA-maltose 0.12 75 34 20 Viscoelastic 0.16 HA-fructose 0.15 75 500 17 Gel 0.16 HA-sorbitol 0.15 75 250 125 Viscoelastic 0.12 HA-fructose 0.15 100 490 7 Gel 0.11 HA-fructose 0.08 600 250 80 Gel

Example 11: Doubly CL HA Gels

[0222] Two methods were employed to synthesize doubly crosslinked hyaluronic acid gels: i) cross-linking of a HA1000-BOR derivative and of a HA1000-fructose/HA1000-PBA mixture by reaction of HA hydroxyl groups with BDDE (method no. 1); ii) grafting of BOR or PBA or fructose moieties on HA-BDPE gel particles by a peptide-like coupling reaction (method no. 2). The products synthesized by the method no. 2 were purified by diafiltration (UF) with ultrapure water and were recovered by freeze-drying.

[0223] Results:

[0224] Table 5 summarizes the syntheses of doubly crosslinked gels by method no. 2.

TABLE-US-00005 TABLE 5 Summary of the syntheses of doubly crosslinked gels by method no. 2. Functional DMTMM/HA molecule/HA Membrane UF Yield Derivative molar ratio molar ratio MWCO (kDa) DS.sub.NMR DS.sub.TNBS (%).sup.d HA-BDPE/BOR 1 0.16 30 0.12.sup.b 0.14 100.sup.e HA- 1 0.15 30 0.1.sup.c 0.11 100.sup.e BDPE/fructose HA-BDPE/BOR 1 0.15 3 0.08.sup.b 0.12 100.sup.e alkaline treatment.sup.a HA-BDPE/PBA 1 0.15 3 0.11.sup.b 0.15 100.sup.e alkaline treatment.sup.a HA-BDPE 1 30 control .sup.aAlkaline treatment sequential to peptide coupling: 0.25 M NaOH (pH 13) at RT for 1 h. .sup.bDS by .sup.1H NMR after enzymatic degradation: 10% of accuracy. .sup.cDS by .sup.13C NMR after enzymatic degradation: 20% of accuracy. .sup.dYield calculated considering the DS.sub.NMR of the HA derivative. .sup.eImprecision of values probably related to variations of the concentration of HA in the initial syringes of HA-BDPE gel particles.

[0225] For clarity, the samples prepared following method no. 1, were named J1-3, whereas the ones obtained from method no. 2 were named T1-5. Scheme 1 illustrates the preparation of samples T1-5, by simply solubilizing modified HA-BDPE gel particles as a powder in a 1 mM phosphate/0.9% NaCl buffer pH 7.4 at a polymer concentration of 20 g/L. J1-3 samples were analyzed under the same conditions, and were recovered as hydrogels at the end of the cross-linking reaction of HA1000 derivatives using BDDE. Table 6 summarizes the rheological properties of these samples, measured by experiments of dependence on frequency of the rheological moduli. The results show that the HA-BDPE/BOR gel has the highest G and that it has improved properties after alkaline treatment than does HA-BDPE/PBA.

TABLE-US-00006 TABLE 6 Doubly crosslinked gels prepared by method no. 1 and 2 and their characterization by rheology. UF Rheo- MWCO logical G 1 Hz G 1 Hz Ref. Sample (kDa) DS.sub.NMR.sup.a behavior (Pa) (Pa) J1 HA/BDPE control Gel 909 132 J2 HA-PBA/HA- 0.14/0.1 Gel 32.5 4.6 fructose/BDPE J3 HA-BOR/BDPE 0.1 Gel 680 170 T1 HA-BDPE control 30 Gel 225 63.4 T2 HA-BDPE/BOR 30 0.12 Gel 1930 340 T3 HA-BDPE/BOR + 30 0.12/0.1 Gel 516 103 HA-BDPE/fructose T4 HA-BDPE/BOR 3 0.08 Gel 1320 250 alkaline treatment.sup.b T5 HA-BDPE/PBA 3 0.11 Gel 403 76 alkaline treatment.sup.b .sup.aDS of BOR- or PBA- or fructose-modified HA. .sup.bAlkaline treatment sequential to peptide coupling: 0.25 M NaOH (pH 13) at RT for 1 h.

Example 12: Self Healing Properties of Obtained Gels

[0226] The variation of G and G as a function of time immediately after injection through a 27 gauge needle of HA-BDPE/BOR and HA-BDPE control was investigated. Gels were prepared in 1 mM sodium sulphate/0.9% NaCl buffer pH 7.4, at a [PS]=20 g/L.

[0227] Results:

[0228] The hydrogel exhibited self-healing properties. Consequently, it 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. 6 shows the variation of G and G as a function of time immediately after injection of the HA-BDPE/BOR and HA-BDPE control gels through a 27 gauge needle. From this Figure, it can be seen that the three samples recovered into a solid gel quasi-instantaneously.