Derivatives of sulfated polysaccharides, method of preparation, modification and use thereof

Abstract

The invention relates to the preparation and the use of ,-unsaturated aldehydes in the structure of sulfated polysaccharides. It concerns the derivatives with a conjugated double bond in the 4th and 5th positions of the galactopyranose part situated in the 6th position with respect to the aldehyde, according to the general structural formula (I) or its hydrated form according to the general structural formula (II). The preparation of these derivatives derives from sulfated polysaccharides containing a galactopyranose ring sulfated in the 4th position that is bound in the polymer chain via (1.fwdarw.3) or (1.fwdarw.3) O-glycosidic bond. In the described solution, the sulfated polysaccharides undergo a regio- and chemoselective oxidation to form C6-saturated aldehyde, which, via a direct elimination of the sulfate group, provides the ,-unsaturated derivative according to the general formula (I) or (II). The described solution is technically advantageous, because it leads directly to ,-unsaturated aldehydes, without any elimination agents, higher temperature, or isolation of intermediates during the synthesis. The conjugation in the structure of ,-unsaturated aldehyde allows, under physiological conditions, to bind a wide variety of biocompatible amines in the structure of the sulfated polysaccharides. The proposed method allows to prepare materials suitable for pH-responsive drug delivery systems, or for the preparation of scaffolds in tissue engineering or regenerative medicine. Formulae for the abstract (I), (II) above, where R is OH, OSO.sub.2OH, OSO.sub.2ONa, or NHAc. ##STR00001##

Claims

1. A derivative of a sulfated polysaccharide having as a part of its polymer chain at least one galactopyranose ring modified according to the general formula I or II, said ring containing a double bond in the 4.sup.th and 5.sup.th position in a conjugation with an aldehyde in the 6.sup.th position according to the general formula I or next to its hydrated form according to the general formula II ##STR00011## where R is OH, OSO.sub.2OH, OSO.sub.2ONa or NHAc.

2. The derivative of a sulfated polysaccharide according to claim 1, characterized in that the polysaccharide is selected from the group comprising chondroitin sulfate, dermatan sulfate, carrageenan, and their pharmaceutically acceptable derivatives and/or salts.

3. The derivative of a sulfated polysaccharide according to claim 1, characterized in that the polysaccharide has the molecular weight within the range of 110.sup.3 to 510.sup.4 g.Math.mol.sup.1 and that the degree of modification of the polysaccharide to an alfa, beta-unsaturated aldehyde is within the range of 1 to 40%.

4. A method of preparation of the derivatives of sulfated polysaccharides as defined in claim 1, characterized in that the sulfated polysaccharide, that is soluble in water in its native form and that contains at least one galactopyranose unit sulfated in the 4.sup.th position which is bound via (1.fwdarw.3) or (1.fwdarw.3) O-glycosidic bond in the polymer chain, according to the general structural formula (III) ##STR00012## where R is OH, OSO.sub.2OH, OSO.sub.2ONa or NHC(O)CH.sub.3, R.sup.1 is H or Na, is oxidized to an aldehyde in the 6.sup.th position by means of the system 4-N-acetyl-TEMPO/NaClO in water or in an aqueous solution of inorganic salts, at 5 to 25 C., and immediately after the oxidation the reaction mixture is subjected to elimination in the same reaction mixture to form the derivative of the sulfated polysaccharide defined in claim 1.

5. The method of preparation according to claim 4, characterized in that a sulfated polysaccharide of the molecular weight within the range of 110.sup.4 to 510.sup.6 g.Math.mol.sup.1 is used.

6. The method of preparation according to claim 4, characterized in that a sulfated polysaccharide selected from the group comprising chondroitin sulfate, dermatan sulfate, carrageenan, and their pharmaceutically acceptable derivatives and/or salts is used.

7. The method of preparation according to claim 4, characterized in that the molar amount of 4-N-acetyl-TEMPO is 0.01 to 0.2 equivalent and the molar amount of NaClO is within the range of 0.1 to 2.0 equivalents with respect to a dimer of the sulfated polysaccharide.

8. The method of preparation according to claim 4, characterized in that the aqueous solution of inorganic salts is an aqueous solution containing an alkaline metal salt and/or a buffer.

9. The method of preparation according to claim 4, characterized in that the elimination proceeds at 5 to 25 C., and without the isolation of the saturated C6-aldehyde in the galactopyranose subunit sulfated in the 4.sup.th position.

10. A method of modification of the derivative defined in claim 1, characterized in that the derivative is reacted with an amine according to the general formula R.sup.2NH.sub.2, where R.sup.2 is an alkyl, aryl, hetaryl, linear, or branched C.sub.1 to C.sub.30 chain, optionally containing N, S, or O atoms.

11. The method of modification according to claim 10, characterized in that the amine is an amino acid or a peptide.

12. The method of modification of the derivative according to claim 10, characterized in that the derivative reacts with an amine which is a biologically acceptable polymer with a free amino group.

13. The method of modification according to claim 12, characterized in that the amino group is a direct part of the polymer selected from the group comprising gelatine, chitosan, deacetylated hyaluronic acid, and deacetylated chondroitin sulfate.

14. The method of modification according to claim 12, characterized in that the amino group is bound to the polymer via a linker containing an amino, hydrazine, hydrazide, aminoalkoxy, hydroxyl, carboxyl, thiol group, or any combination thereof.

15. The method of modification according to claim 10, characterized in that the amount of the amine is within the range of 0.05 to 3.0 equivalents with respect to a dimer of the sulfated polysaccharide.

16. The method of modification according to claim 10, characterized in that the reaction with the amine proceeds in water, phosphate buffer, or water-organic solvent system, at the temperature within the range of 20 to 60 C., for 10 min to 150 h.

17. The method of modification according to claim 16, characterized in that the organic solvent is selected from the group comprising water miscible alcohols, in particular isopropanol or ethanol, and water miscible polar aprotic solvents, in particular dimethyl sulfoxide, wherein the water content in the mixture is at least 50% vol.

18. The method of modification of the derivative according to claim 1, characterized in that the derivative is reacted with a water soluble biocompatible bi- and polyfunctional N-nucleophile selected from the group comprising alkyl amines, aryl amines, heteroalkyl amines, hetaryl amines, amino acids, peptides, polymers with a free amino group, hydrazines, hydroxylamines, hydrazides, semicarbazides, or thiosemicarbazides, wherein crosslinking of the derivative occurs.

19. The method of modification according to claim 18, characterized in that the N-nucleophile is hydrazide, deacetylated polysaccharide, or alkoxyamine, and in that the reaction proceeds in phosphate buffer.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates the formation of a hydrogel based on oxidized chondroitin sulfate with adipic dihydrazide (Example 21): (a) solution of ,-unsaturated aldehyde of chondroitin sulfate in PBS, (b) gelation of the solution after the addition of adipic dihydrazide solution in PBS, (c) the hydrogel after 1 h in PBS (pH=7.4, c=0.9% w/v), (d) the use of an illustrative form for the preparation of the hydrogel, (e) the use of a defined form for the preparation of the hydrogel, (f) a detailed view of a segment of the hydrogel.

(2) FIG. 2 Photographs of the lyophilized hydrogel based on the oxidized chondroitin sulfate with adipic dihydrazide taken by the scanning electron microscope: (a) a cross section, magnification 200, detection of secondary electrons, (b) an enlarged section of the porous structure of the hydrogel with the measured diameter.

(3) FIG. 3 illustrates the results of the cell viability tests of 3T3 fibroblasts in ,-unsaturated aldehyde of chondroitin sulfate (Mw=410.sup.4 g/mol, DS=20%). The activation vs inhibition curve, expressed in %, with respect to the control in time T=0 h (100%). Evaluation by means of MTT method, six repetitions.

(4) FIG. 4 Kinetics of the gelation in Example 30, with the determination of the gel point (Tg 97 s, T=45 C.). A graphical representation of the dependence of elastic (G) and viscous (G) modulus in time.

EXAMPLES

(5) The term equivalent (eq), as used herein, relates to the dimer unit of the sulfated polysaccharide, if not stated otherwise. The percentage is expressed as weight percentage, if not stated otherwise. The molecular weight of the initial chondroitin sulfate (the source: Sigma Aldrich, Ltd., Prague, CZ) is the weight-average molecular weight within the range of 410.sup.4 to 510.sup.4 g.Math.mol.sup.1.

(6) The ratio of chondroitin-4-sulfate (type A) and chondroitin-6-sulfate (type C) was 3:2. The material was isolated from an animal material.

(7) The sodium salt of dermatan sulfate (chondroitin sulfate B sodium salt) of the solubility of 5 mg/ml in water was purchased from Sigma Aldrich. The material was isolated from an animal material.

(8) Lambda carrageenan of the solubility of 10 mg/ml in water was purchased from Sigma Aldrich and was isolated from sea algae without gelation properties in the native form.

(9) The degree of substitution of ,-unsaturated aldehyde in the structure of the sulfated polysaccharide was determined according to the following calculation:
DS=the degree of substitution of ,-unsaturated aldehyde=100%*(the molar amount of the modified dimer of the sulfated polysaccharide/(the molar amount of all dimers of the sulfated polysaccharide)

(10) The degree of substitution of the amination reaction in the structure of the sulfated polysaccharide was determined according to the following calculation:
DS=the degree of substitution for amination=100%*(the molar amount of the modified dimer of the sulfated polysaccharide/(the molar amount of all dimers of the sulfated polysaccharide)

(11) FT-IR spectra were measured within the range of 4000-400 cm.sup.1 in KBr, by means of the spectrometer Nicolet 6700 FTIR. UV-VIS spectra were measured by means of the apparatus Shimadzu UV-2401PC within the range of 200-600 nm and processed by means of the UV Probe software, version 2.00.

(12) The gelation kinetics was determined by means of the apparatus AR-G2 and TA Analysis was used as the evaluation software. The gel point (Tg) was determined from the time dependence of the elastic and viscous modulus.

(13) The mechanical properties of the selected gels were measured by the compression test by means of the apparatus Instron 3433 and evaluated by means of Bluehill software. The determined parameters for each sample were as follows: Young's modulus for compression, compressive strength, deformation at ultimate strength, and tenacity.

(14) The surface morphology of the lyophilized materials was analysed by means of the electron microscope Zeiss Ultra Plus.

(15) Deacetylated hyaluronic acid was prepared by deacetylation with hydrazine according to Buffa R., et al CZ304512.

(16) Aminopropoxyle and hydrazine derivative of hyaluronic acid were prepared by the reductive amination according to Buffa R., et al.: WO2011069474.

LIST OF ABBREVIATIONS

(17) TEMPO2,2,6,6-tetramethyl-1-piperidinyloxy radical 4-AcNH-TEMPOTEMPO with an acetamide group in the 4.sup.th position PBSphosphate buffered saline RGD peptidepeptide of the sequence of aminohexane acid-Gly-Arg-Gly-Asp-NH.sub.2 AMKamino acid IPAisopropylalcohol DMSOdimethylsulfoxide

Example 1

(18) Preparation of ,-Unsaturated Aldehyde of Chondroitin Sulfate

(19) Method 1: An aqueous solution of sodium hypochlorite (0.8 eq, 11% of active chlorine) was gradually added into a 2% aqueous solution of chondroitin sulfate (200 mg, Mw=4.510.sup.4 g.Math.mol.sup.1) cooled to 5 C., containing disodium phosphate dodecahydrate (2.2 eq), sodium bromide (0.8 eq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 h at 5 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature.

(20) The product was isolated by precipitation with IPA and analysed by NMR.

(21) DS=23% (determined by NMR), Mw=2.110.sup.4 g.Math.mol.sup.1 (determined by SEC MALLS)

(22) Method 2: An aqueous solution of sodium hypochlorite (0.8 eq, 11% of active chlorine) was gradually added into a 2% aqueous solution of chondroitin sulfate (200 mg, Mw=4.510.sup.4 g.Math.mol.sup.1) cooled to 5 C., containing sodium bromide (0.8 wq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 h at 5 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature. The product was isolated by precipitation with IPA and analysed by NMR.

(23) DS=20% (determined by NMR)

(24) Spectral analysis of ,-unsaturated aldehyde of chondroitin sulfate: NMR .sup.1H (500 MHz, D.sub.2O, ppm): 2.02 (3H, AcNH, bs), 4.31 (1H, H2, bs), 4.49 (1H, H3, bs), 5.20 (1H, H1, bs), 6.34 (1H, H4, bs), 9.21 (1H, H6, bs);

(25) NMR .sup.1H-.sup.1H COSY (D.sub.2O), crosspeaks, ppm: 4.31-4.49, 4.31-5.20, 4.49-6.34; NMR .sup.1H-.sup.13C HSQC

(26) (D.sub.2O), crosspeaks, ppm: 2.02-25.1, 4.31-51.0, 4.49-73.1, 5.20-98.6, 6.34-122.0, 9.21-189.0;

(27) NMR DOSY (D.sub.2O), log D ((2.02, AcNH), (4.31, H2), (4.49, H3), (5.20, H1), (6.34, H4), (9.21; H6))10.3 m.sup.2 s.sup.1, log D (4.72, H.sub.2O)8.6 m.sup.2 s.sup.1;

(28) IR (KBr, cm.sup.1): 1725, 1650 (v CO st), 1615, 1663 (v CC st);

(29) UV/Vis (0.1%, H.sub.2O); .sub.max1,2 (C.sub.C.sub.CO)=254 nm (.fwdarw.*), 300-350 (.fwdarw.*).

Example 2

(30) Preparation of ,-Unsaturated Aldehyde of Chondroitin Sulfate

(31) An aqueous solution of sodium hypochlorite (0.4 eq, 11% of active chlorine) was gradually added into a 2% aqueous solution of chondroitin sulfate (200 mg, Mw=4.510.sup.4 g.Math.mol.sup.1) cooled to 5 C., containing disodium phosphate dodecahydrate (2.2 eq), sodium bromide (0.4 eq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 hours at 5 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature. The product was isolated by precipitation with IPA and analysed by NMR.

(32) DS=2% (determined by NMR), Mw=2.810.sup.4 g.Math.mol.sup.1 (determined by SEC MALLS). The structural analysis of the product is presented in Example 1.

Example 3

(33) Preparation of ,-Unsaturated Aldehyde of Chondroitin Sulfate

(34) An aqueous solution of sodium hypochlorite (1 eq, 11% of active chlorine) was gradually added into a 2% aqueous solution of chondroitin sulfate (200 mg, Mw=4.510.sup.4 g.Math.mol.sup.1) cooled to 5 C., containing disodium phosphate dodecahydrate (2.2 eq), sodium bromide (1 eq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 hours at 5 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature. The product was isolated by precipitation with IPA and analysed by NMR.

(35) DS=21% (determined by NMR), Mw=2.010.sup.4 g.Math.mol.sup.1 (determined by SEC MALLS). The structural analysis of the product is presented in Example 1.

Example 4

(36) Preparation of ,-Unsaturated Aldehyde of Chondroitin Sulfate

(37) An aqueous solution of sodium hypochlorite (2 eq, 11% of active chlorine) was gradually added into a 2% aqueous solution of chondroitin sulfate (200 mg, Mw=4.510.sup.4 g.Math.mol.sup.1) cooled to 5 C., containing disodium phosphate dodecahydrate (2.2 eq), sodium bromide (2 eq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 hours at 5 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature. The product was isolated by precipitation with IPA and analysed by NMR.

(38) DS=21% (determined by NMR), Mw=1.810.sup.4 g.Math.mol.sup.1 (determined by SEC MALLS). The structural analysis of the product is presented in Example 1.

Example 5

(39) Preparation of ,-Unsaturated Aldehyde of Dermatan Sulfate

(40) An aqueous solution of sodium hypochlorite (0.8 eq, 11% of active chlorine) was gradually added into a 2% aqueous solution of dermatan sulfate (200 mg, 0.42 mol) cooled to 5 C., containing disodium phosphate dodecahydrate (2.2 eq), sodium bromide (0.8 eq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 hours at 5 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature. The product was isolated by precipitation with IPA and analysed by NMR.

(41) DS=20% (determined by NMR)

(42) Spectral analysis of ,-unsaturated aldehyde of dermatan sulfate: NMR .sup.1H (500 MHz, D.sub.2O, ppm): 2.01 (3H, AcNH, bs), 6.30 (1H, H4, bs), 9.20 (1H, H6, bs).

Example 6

(43) Preparation of ,-Unsaturated Aldehyde of Carrageenan

(44) An aqueous solution of sodium hypochlorite (0.8 eq, 11% of active chlorine) was gradually added into a 1% aqueous solution of carrageenan (200 mg, 0.31 mol) cooled to 10 C., containing disodium phosphate dodecahydrate (2.2 eq), sodium bromide (0.8 eq) and 4-AcNH-TEMPO (0.01%). The mixture was stirred for 2 hours at 10 C. Then ethanol (10 eq) was added to the reaction which was stirred for an additional hour at the room temperature. The product was isolated by precipitation with IPA and analysed by NMR.

(45) DS=10% (determined by NMR)

(46) Spectral analysis of ,-unsaturated aldehyde of carrageenan: NMR .sup.1H (500 MHz, D.sub.2O, ppm): 6.30 (1H, H4, bs), 9.20 (1H, H6, bs).

Example 7

(47) Binding of Hydrazine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(48) Hydrazine hydrate (2 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. The reaction was stirred for 24 h at the room temperature. The product was analysed in the form of a crude reaction mixture.

(49) DS=20% (determined by NMR)

(50) NMR .sup.1H (500 MHz, D.sub.2O, ppm): 5.40 (1H, CHCCHN, bs), 7.38 (1H, CHCCHN, bs)

Example 8

(51) Binding of Butylamine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(52) Method 1: Butylamine (0.2 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. The reaction was stirred for 24 h at the room temperature and pH=11.20. The product was analysed in the form of a crude reaction mixture.

(53) DS=20% (determined by NMR)

(54) NMR .sup.1H (500 MHz, D.sub.2O, ppm): 5.67 (1H, CHCCHN, bs), 7.74 (1H, CHCCHN, bs)

(55) Method 2: Deuterated acetic acid (14.5 L) was added to a NMR sample of method 1. The measured pH was 4.10 and the sample was then analysed by NMR.

(56) DS=0% (determined by NMR)

Example 9

(57) Binding of Butylamine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(58) Butylamine (0.2 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in deuterated PBS. The reaction was stirred for 24 h at the room temperature and pH=7.30. The product was analysed in the form of a crude reaction mixture.

(59) DS=0% (determined by NMR)

Example 10

(60) Binding of Hexane-1,6-Diamine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(61) Hexane-1,6-diamine (0.5 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. The reaction was stirred for 24 h at the room temperature and pH=11.60. The product was analysed in the form of a crude reaction mixture.

(62) DS=20% (determined by NMR)

(63) NMR .sup.1H (500 MHz, D.sub.2O, ppm): 5.68 (1H, CHCCHN, bs), 7.74 (1H, CHCCHN, bs)

Example 11

(64) Binding of Propoxyamine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(65) Propoxyamine hydrochloride (0.5 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. The reaction was stirred for 24 h at the room temperature and pH=3.90. The product was analysed in the form of a crude reaction mixture.

(66) DS=20% (determined by NMR)

(67) NMR .sup.1H (500 MHz, D.sub.2O, ppm): 5.57 and 6.88 (1H, CHCCHN, bs), 7.52 and 7.70 (1H, CHCCHN, bs)

Example 12

(68) Binding of Lysine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(69) Lysine hydrochloride (0.5 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in deuterated PBS. The reaction was stirred for 24 h at the room temperature and pH=7.46. The product was analysed in the form of a crude reaction mixture.

(70) DS=4% (determined by NMR)

(71) NMR .sup.1H (500 MHz, D.sub.2O, ppm): 5.69-5.75 (1H, CHCCHN, bs), 7.70-7.75 (1H, CHCCHN, bs)

Example 13

(72) Binding of Lysine to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(73) Lysine hydrochloride (0.5 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. pH of the reaction was adjusted to 8.40 by adding sodium bicarbonate (2 eq). The reaction was stirred for 24 h at the room temperature. The product was analysed in the form of a crude reaction mixture.

(74) DS=7% (determined by NMR)

(75) The structural analysis is presented in Example 12.

Example 14

(76) Binding of RGD Peptide to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(77) RGD peptide (0.2 eq, sequence Ahx-Gly-Arg-GlyAsp-NH.sub.2) was added to 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. The reaction was stirred for 24 h at the room temperature and pH=4.22. The product was analysed in the form of a crude reaction mixture.

(78) DS=0% (determined by NMR)

Example 15

(79) Binding of RGD Peptide to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(80) RGD peptide (0.2 eq, sequence Ahx-Gly-Arg-GlyAsp-NH.sub.2) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in deuterated PBS. The reaction was stirred for 24 h at the room temperature and pH=7.22. The product was analysed in the form of a crude reaction mixture.

(81) DS=4% (determined by NMR)

(82) NMR .sup.1H (500 MHz, D.sub.2O, ppm): 5.68 (1H, CHCCHN, bs), 7.74 (1H, CHCCHN, bs)

Example 16

(83) Binding of RGD Peptide to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(84) RGD peptide (0.2 eq, sequence Ahx-Gly-Arg-GlyAsp-NH.sub.2) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. pH of the reaction was adjusted to 10.49 by adding sodium bicarbonate (2 eq). The reaction was stirred for 24 h at the room temperature. The product was analysed in the form of a crude reaction mixture.

(85) DS=20% (determined by NMR)

(86) The structural analysis is presented in the Example 15.

Example 17

(87) Binding of Aniline to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(88) Aniline (0.3 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. pH of the reaction was adjusted to 4.22 by adding deuterated acetic acid (8.8 L). The reaction was stirred for 24 h at the room temperature. The product was analysed in the form of a crude reaction mixture.

(89) DS=0% (determined by NMR)

Example 18

(90) Binding of Aniline to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(91) Aniline (0.3 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in deuterated PBS. The reaction was stirred for 24 h at the room temperature and pH=7.42. The product was analysed in the form of a crude reaction mixture.

(92) DS=5% (determined by NMR)

(93) NMR .sup.1H (500 MHz, D.sub.2O, S ppm): 5.93 (1H, CHCCHN, bs), 8.03 (1H, CHCCHN, bs)

Example 19

(94) Binding of Aniline to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(95) Aniline (0.3 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. pH of the reaction was adjusted to 10.73 by adding sodium carbonate. The reaction was stirred for 24 h at the room temperature. The product was analysed in the form of a crude reaction mixture.

(96) DS=25% (determined by NMR)

(97) The structural analysis is presented in the Example 18.

Example 20

(98) Binding of Dihydrazide Adipate to ,-Unsaturated Aldehyde of Chondroitin Sulfate

(99) Dihydrazide adipate (3 eq) was added to a 2% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in D.sub.2O. The reaction was stirred for 24 h at the room temperature and pH=7.50. The product was analysed in the form of a crude reaction mixture.

(100) DS=20% (determined by NMR)

(101) NMR .sup.1H (500 MHz, D.sub.2O, S ppm): 1.64 (4H, DHA.sub.2,3, bs), 2.04 (3H, AcNH, bs), 2.34 (4H, DHA.sub.1,4, bs), 4.28 (1H, H2, bs), 4.36 (1H, H3, bs), 5.20 (1H, H1, bs), 5.62 (1H, H4 cis, bs), 5.68 (1H, H4 trans, bs), 7.52-7.48 (1H, H6 cis, bs), 7.61 (1H, H6 trans, bs);

(102) NMR .sup.1H-.sup.1H COSY (D.sub.2O), crosspeaks, ppm: 1.64-2.34, 4.28-5.20, 4.36-5.68; NMR .sup.1H-.sup.13C HSQC

(103) (D.sub.2O), crosspeaks, ppm: 1.64-24.9, 2.34-34.1, 4.28-51.0, 4.36-73.6, 5.20-98.8, 5.68-111.3, 7,61148.5;

(104) NMR DOSY (D.sub.2O), log D ((2.04, AcNH), (4.28, H2), (4.36, H3), (5.20, H1), (5.62 and 5.68,

(105) H4 cis/trans), (7.52 and 7.68, H6 cis/trans))10.4 m.sup.2 s.sup.1, log D (4.72, H.sub.2O)8.6 m.sup.2 s.sup.1;

(106) IR (KBr, cm.sup.1): 1640-1650 (CN-st);

(107) UV/Vis (0.1%, H.sub.2O); .sub.max1,2 (CN)=280 nm (.fwdarw.*).

Example 21

(108) Crosslinking of ,-Unsaturated Aldehyde of Chondroitin Sulfate with Dihydrazide Adipate

(109) Dihydrazide adipate (0.12 eq, binding sites ratio 1:1) in PBS was added to an 8% solution of ,-unsaturated aldehyde of chondroitin sulfate (40 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in PBS (pH=7.40, c=0.9% w/v). After the addition of the solution of dihydrazide adipate, the gelation occurred in time (Tg=34 s). The elastic gel was photographed (FIG. 1), lyophilized, and analyzed by SEM (FIG. 2).

(110) Young's modulus of elasticity in compression=910.sup.3 Pa

(111) Ultimate strength in compression=6410.sup.3 Pa

(112) Deformation at ultimate strength=64%

(113) Tenacity=3668 J.Math.m.sup.3

Example 22

(114) Crosslinking of ,-Unsaturated Aldehyde of Dermatan Sulfate with Dihydrazide Adipate

(115) Dihydrazide adipate (0.1 eq, binding sites ratio 1:1) in PBS was added to an 8% solution of ,-unsaturated aldehyde of dermatan sulfate (40 mg, DS=20%, Mw40 kDa) in PBS (pH=7.40, c=0.9% w/v). After the addition of the solution of dihydrazide adipate, the gelation proceeded.

Example 23

(116) Crosslinking of ,-Unsaturated Aldehyde of Carrageenan with Dihydrazide Adipate

(117) 40 L of dihydrazide adipate (0.05 eq) in PBS was added to an 8% solution of ,-unsaturated aldehyde of carrageenan (40 mg, DS=10%, Mw50 kDa) in PBS (pH=7.40, c=0.9% w/v). After the addition of the solution of dihydrazide adipate, the viscosity increased.

Example 24

(118) Crosslinking of ,-Unsaturated Aldehyde of Chondroitin Sulfate with Hydrazide Derivative of Hyaluronic Acid

(119) A 4% solution of hydrazide derivative of hyaluronic acid (9.2 mg, DS=25%, Mw=13810.sup.3 g.Math.mol.sup.1) in PBS (pH=7.4, c=0.9% w/v) was added to a 4% solution of ,-unsaturated aldehyde of chondroitin sulfate (10 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in PBS. After mixing the solutions, the gelation occurred in time (Tg=109 s).

(120) Young's modulus of elasticity in compression=610.sup.3 Pa

(121) Ultimate strength in compression=84010.sup.3 Pa

(122) Deformation at ultimate strength=96%

(123) Tenacity=11978 J.Math.m.sup.3

Example 25

(124) Crosslinking of ,-Unsaturated Aldehyde of Chondroitin Sulfate with Deacetylated Hyaluronic Acid

(125) A 2% solution of deacetylated hyaluronic acid (2 eq, DS=11%, Mw=116 kDa, binding sites ratio 1:1) in PBS (pH=7.4, c=0.9% w/v) was added to a 4% solution of ,-unsaturated aldehyde of chondroitin sulfate (10 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in PBS (pH=7.40, c=0.9% w/v). The reaction mixture was stirred at the room temperature, wherein an increase in viscosity was observed after 0.5 h, and an elastic gel was formed after 1 h.

(126) Young's modulus of elasticity in compression=310.sup.3 Pa

(127) Ultimate strength in compression=39510.sup.3 Pa

(128) Deformation at ultimate strength=95%

(129) Tenacity=14670 J.Math.m.sup.3

Example 26

(130) Preparation of the Acid Form of Chondroitin Sulfate

(131) A 1% solution of chondroitin sulfate (500 mg, 1.1 mmol) in distilled water was prepared. The solution was cooled to 5 C. and 1.2 ml of Amberlite IR 120 Na(H.sup.+) catex was added. The reaction mixture was stirred for 24 h at 5 C. The catex was filtered off, the product was frozen and lyophilized. Its solubility in DSMO was tested and found satisfactory.

Example 27

(132) Preparation of Deacetylated Chondroitin Sulfate

(133) A 1% solution of acid form of chondroitin sulfate was prepared (200 mg, 0.44 mmol, Mw40 kDa) in DMSO. The solution was degassed by a stream of nitrogen. 10.6 mL of hydrazine hydrate and 3 eqs of hydrazine sulfate were added. The reaction mixture was stirred for 24 h at 60 C. under nitrogen. Then NaHCO.sub.3 was added to the reaction mixture. The product was isolated by precipitation with IPA.

(134) DS=10% (determined by NMR), Mw=1.810.sup.4 g.Math.mol.sup.1 (determined by SECMALLS)

(135) NMR .sup.1H (500 MHz, 1% NaOD v D.sub.2O, ppm): 3.01 (1H, CHCCHN, bs) HSQC (500 MHz, D.sub.2O, ppm): crosspeak: 3.42-52.2 ppm

Example 28

(136) Crosslinking of ,-Unsaturated Aldehyde of Chondroitin Sulfate with Deacetylated Chondroitin Sulfate

(137) An 8% solution of deacetylated chondroitin sulfate (2 eq, DS=10%, Mw=1.810.sup.4 g.Math.mol.sup.1, binding sites ratio=1/0.85) in PBS (pH=7.4, c=0.9% w/v) was added to an 8% solution of ,-unsaturated aldehyde of chondroitin sulfate (20 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in PBS (pH=7.40, c=0.9% w/v). The reaction mixture was stirred at the room temperature, wherein an increase in viscosity was observed after 0.5 h, and an elastic gel was formed after 3 h.

(138) Young's modulus of elasticity in compression=310.sup.3 Pa

(139) Ultimate strength in compression=77410.sup.3 Pa

(140) Deformation at ultimate strength=95%

(141) Tenacity=16489 J.Math.m.sup.3

Example 29

(142) Crosslinking of ,-Unsaturated Aldehyde of Chondroitin Sulfate with Propoxyamine

(143) A PBS solution of propoxyamine hydrochloride (0.12 eq, binding sites ratio=1/1) was added to a 10% solution of ,-unsaturated aldehyde of chondroitin sulfate (50 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in PBS (pH=7.40, c=0.9% w/v), wherein a gel was formed in time (Tg=110 s).

(144) Young's modulus of elasticity in compression=810.sup.3 Pa

(145) Ultimate strength in compression=7410.sup.3 Pa

(146) Deformation at ultimate strength=65%

(147) Tenacity=3768 J.Math.m.sup.3

Example 30

(148) Measurement of Gelation Kinetics of Crosslinking Reaction of ,-Unsaturated Aldehyde of Chondroitin Sulfate with Aminopropoxyl Derivative of Hyaluronic Acid

(149) The measurement of gelation kinetics was performed by means of a 4% sample of ,-unsaturated aldehyde of chondroitin sulfate (10 mg, DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1) in PBS (pH=7.40, c=0.9% w/v) with a 4% solution of aminopropoxyl derivative of hyaluronic acid (1 eq, DS=25%, Mw=66 kDa) in PBS (pH=7.4, c=0.9% w/v). The gelation time, i.e. the stage, when the first macroscopic gel network was formed, was determined to be (Tg=97 s, FIG. 4).

Example 31

(150) Comparison of Mechanical Properties of Hydrogels Based on the Crosslinked ,-Unsaturated Aldehyde of Chondroitin Sulfate and ,-Unsaturated Aldehyde of Hyaluronan

(151) Solution 1: 4% solution of ,-unsaturated aldehyde of chondroitin sulfate (DS=23%, Mw=2.110.sup.4 g.Math.mol.sup.1, Example 1) in PBS (pH=7.40, c=0.9% w/v).

(152) Solution 2: 4% solution of ,-unsaturated aldehyde of hyaluronic acid (DS=7%, Mw=2.510.sup.4 g.Math.mol.sup.1) in PBS (pH=7.40, c=0.9% w/v).

(153) Solution 3: 4% solution of aminopropoxyl derivative of hyaluronic acid (DS=25%, Mw=66 kDa) in PBS (pH=7.40, c=0.9% w/v).

(154) Hydrogels were prepared from the said solutions by mixing their equivalent volume ratios in the following combinations: solution 1+solution 3 (sample A) and solution 2+solution 3 (sample B). Samples A and B were left to mature at the room temperature for 3 hours. Then the mechanical properties of the materials were measured, namely Young's modulus of elasticity in compression, ultimate strength in compression, and deformation at ultimate strength (Table 2).

(155) TABLE-US-00003 TABLE 2 Comparison of the mechanical properties of the hydrogels based on crosslinked ,-unsaturated aldehydes derived from chondroitin sulfate and hyaluronic acid Young's modulus of ultimate strength Elongation in elasticity in compression in compression ultimate strength Sample (kPa) (kPa) (%) A 11 30 52 B 7 58 76

(156) The measured data indicate the advantages of the use of the material of a higher degree of substitution in the chondroitin sulfate derivative (sample A), because the hydrogels prepared from this derivative have a higher rigidity and show a lower deformation rate in comparison with the hyaluronic acid derivative (sample B). As the samples were of the same molecular weight and were analysed under the same conditions, this fact seems to be the consequence of a higher crosslink density and it directly correlates, while maintaining the same molecular weight, with the higher degree of substitution of the ,-unsaturated aldehyde in the structure of the modified polysaccharide.

Example 32

(157) Viability Tests of 3T3 Fibroblasts in the Presence of ,-Unsaturated Aldehyde of Chondroitin Sulfate

(158) The tested substance, ,-unsaturated aldehyde of chondroitin sulfate (DS=20%, Mw=40 kDa), was dissolved in a complete 3T3 medium. The solution was filtered via a 0.22 m filter. The final test concentrations of the tested solution were 10, 100, 500, and 1000 g.Math.mL.sup.1. 3T3 cells of the density of 3000 cells per a well were inoculated into 96-well plates. Before the treatment, the cells were cultured for 24 hours in a complete medium. The cell viability was evaluated by spectrophotometry by means of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide (MTT method) in the intervals of 0, 24, 48, 72 hours. The whole experiment was supplemented with a set of non-treated controls and blank samples. The measured optical density data were converted into percentage formulation related to the control in the time T0 hours (the ratio of the optical density of the treated sample with respect to the optical density of the non-treated control T0, multiplied by 100) and the standard error of the mean (SEM) was calculated. The results of the test are graphically presented at FIG. 3.