Method of crosslinking of polysaccharides using photoremovable protecting groups

Abstract

The invention discloses a method of preparation of crosslinked materials based on polysaccharides using electromagnetic radiation in an aqueous solution containing a polysaccharide with a bound carbamate photoremovable protecting group (PPG with group NHCOO) and a polysaccharide containing an aldehyde group CHO. The crosslinking process itself is carried out by means of a condensation reaction of the photochemically released amino group (NH.sub.2) with the aldehyde group (CHO) forming a bond of imine type (NCH). Both processes proceed simultaneously and they can be performed under physiological conditions. The advantage of the suggested solution is the temporal and spatial control of crosslinking that allows the preparation of advanced materials for tissue engineering where the crosslink density and thus the mechanical properties in the material structure can be tailored.

Claims

1. A method of preparation of crosslinked polysaccharide materials according to the general formula (I)
polysaccharide1-R.sup.1NCH-polysaccharide2(I) where polysaccharide1 and polysaccharide2 are identical or different polysaccharides and R.sup.1 is C.sub.1-C.sub.30 alkyl residue, C.sub.1-C.sub.30 alkylaryl residue or C.sub.1-C.sub.30 alkylheteroaryl residue, optionally containing one or more identical or different heteroatoms selected from the group comprising N, O, S, characterized in that an aqueous solution of aldehyde of polysaccharide 2 according to the general formula III
polysaccharide2-CHO(III), where the substitution degree of aldehyde in polysaccharide2 is within the range of 1 to 50%, is added to an aqueous solution of polysaccharide 1 substituted on the site of amino group by a photoremovable group, according to the general formula II
polysaccharide1-R.sup.1NHCOOCH.sub.2R.sup.2(II), where R.sup.1 is defined above; R.sup.2 is an aromatic system, and where the substitution degree of carbamate in polysaccharide 1 is within the range of 1 to 10%, and the formed mixture is subjected to electromagnetic radiation and deoxygenation simultaneously.

2. The method of preparation according to claim 1, characterized in that the substitution degree of carbamate in polysaccharide 1 is within the range of 3 to 10% and its molecular weight is 10 to 400 kDa.

3. The method of preparation according to claim 1, characterized in that the substitution degree of aldehyde in polysaccharide 2 is within the range of 3 to 25% and its molecular mass is 10 to 800 kDa.

4. The method of preparation according to claim 1, characterized in that polysaccharide 1 and polysaccharide 2 are selected from the group comprising hyaluronan, chondroitin sulfate, cellulose and pharmaceutically acceptable derivatives and/or salts thereof.

5. The method of preparation according to claim 1, characterized in that R.sup.1 is selected from the group comprising adipic acid dihydrazide and hexamethylene diamine.

6. The method of preparation according to claim 1, characterized in that R.sup.2 is selected from the group comprising pyrene, anthracene, phenanthrene, perylene, anthraquinone, coumarin and substitution derivatives thereof, that can contain atoms C, H, O, S, N in their structure and that exhibit absorption of electromagnetic radiation.

7. The method of preparation according to claim 1, characterized in that the weight ratio of polysaccharide 1 to polysaccharide 2 is within the range of 1:2 to 2:1.

8. The method of preparation according to claim 1, characterized in that the mixture is subjected to electromagnetic radiation for 0.25 to 2 hours at the temperature of 10 to 50 C.

9. The method of preparation according to claim 1, characterized in that the aqueous solutions of polysaccharides 1 and 2 further contain water soluble agents selected from the group comprising inorganic salts or buffers wherein pH of the solution is within the range of 6.5 to 7.5.

10. The method of preparation according to claim 1, characterized in that electromagnetic radiation of wavelength 320-400 nm is used.

11. The method of preparation according to claim 1, characterized in that the reaction is temporal controlled by means of a switch of the electromagnetic radiation source or by means of a pulse source of electromagnetic radiation or by means of shading of the reaction.

12. The method of preparation according to claim 1, characterized in that the reaction is spatially controlled by means of a photomask, focused electromagnetic radiation or a beam of electromagnetic radiation.

Description

DETAILED DESCRIPTION OF DRAWINGS

(1) FIG. 1 represents the use of upside-down method for determination of gelation in the reaction mixture. (a) Solution of two-component reaction mixture (Pmoc-DHA-HA and HA-aldehyde) before photolysis. (b) Hydrogel of the crosslinked product (HA-DHA-HA) after photolysis. (c) Hydrogel of crosslinked product (HA-DHA-HA) after 1 h in PBS (pH=7.4, c=0.9%, w/v).

(2) FIG. 2 illustrates the spatial control of the crosslinking reaction (Pmoc-DHA-HA and HA-aldehyde) using a photomask in the shape of a semi-circle on 50% of the surface of the reaction mixture. (a) the reaction mixture after photolysis, (b) the reaction mixture after 15 min in PBS (pH=7.4, c=0.9% w/v) and decanting of PBS solution, (c) the reaction mixture after 15 min in PBS (pH=7.4, c=0.9% w/v) and adding new portion of PBS.

(3) FIG. 3 shows microscopic photographs of freeze-dried samples. (a): the hydrogel surface (100), (b) the cross-section of the hydrogel (100), (c) the cross-section of the hydrogel after 1 h in PBS (pH=7.4, c=0.9% w/v).

EXAMPLES

(4) The term equivalent (eqv) used herein relates to a disaccharide of hyaluronic acid, disaccharide of chondroitin sulfate or monosaccharide of sodium carboxymethyl cellulose, if not stated otherwise. Percentage is used as mass percentage, if not stated otherwise.

(5) The molecular weight of the initial hyaluronic acid (source: Contipro Pharma a.s., Doln Dobrou, CZ) is the average molecular weight within the range of 10.sup.4 to 10.sup.6 g.Math.mol.sup.1 and was determined by SEC-MALLS.

(6) The molecular weight of the initial chondroitin sulfate (source: Sigma-Aldrich s.r.o., Prague, CZ) is the average molecular weight within the range of 410.sup.4 to 510.sup.4 Da or g.Math.mol.sup.1 and was determined with the method SEC-MALLS. The ratio of chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate (C6S) was 2:3. The material was isolated from an animal material.

(7) The molecular weight of the initial sodium carboxymethyl cellulose (source: Sigma-Aldrich s.r.o., Prague, CZ) is the average molecular weight within the range of 2210.sup.4 to 2510.sup.4 g.Math.mol.sup.1 and was determined with SEC-MALLS. The degree of alkylation with the carboxymethyl group was 70%.

(8) The degree of substitution or modification in the structure of glycosaminoglycans was determined by means of the following calculation:
DS=substitution degree=100%*(the molar amount of the bound substituent or modified disaccharide)/(the molar amount of all disaccharides)

(9) The degree of modification in the structure of sodium carboxymethyl cellulose was determined by means of the following calculation:
DS=substitution degree=100%*(the molar amount of the bound substituent or modified monosaccharide)/(the molar amount of all monosaccharides)

(10) PPG=photoremovable protection group

(11) DHA=dihydrazide adipate

(12) HMD=1,6-hexamethylenediamine

(13) Pmoc=pyren-1-ylmethoxycarbonyl

(14) UVA=near ultraviolet radiation within the range of wavelengths 320-400 nm, emitted by longwave ultraviolet source Black-Ray mercury spot lamp, model B-100A (UVP) with declared max=365 nm.

(15) The surface morphology of the freeze-dried gels was analyzed with scanning electron microscope Zeiss Ultra Plus.

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

(17) Oxidation of polysaccharides was performed according to Buffa R, et al.: WO2011069474 and WO2011069475.

Example 1. Preparation of Pmoc-Dihydrazide Adipate Hyaluronan (Pmoc-DHA-HA)

(18) HA aldehyde (100 mg, 0.265 mmol, DS=43%, Mw=1.3510.sup.5 g/mol) was dissolved in 5 mL of distilled water (solution I). Pmoc-DHA (54 mg, 0.126 mmol) was dissolved in 5 mL of DMSO (solution II). Both solutions were mixed and reacted for 24 h at room temperature. In the second step PicBH.sub.3 (81 mg, 0.754 mmol) was added. The reaction mixture was stirred for 48 h at room temperature. The product was precipitated with IPA.

(19) ##STR00004##

(20) DS=10%, Mw=0.3410.sup.5 g/mol, isolated yield 85%

(21) TABLE-US-00001 .sup.1H NMR (D.sub.2O) 1.60 (bs, 4H); 2.21 (bs, 2H); 2.25 (bs, 2H); 2.98 (bs, 1H, polymer-N.sub.6a); 3.26 (bs, 1H, polymer-N.sub.6b); 5.89 (s, 2H, CH.sub.2-pyr); 7.98-8.41 (m, 9H.sub.Ar) ppm H-H COSY (D.sub.2O) cross-peak 1.60-2.21; 1.60-2.25; 2.98-3.26 ppm HSQC (D.sub.2O) cross-peak 1.60 (.sup.1H)-24.6 (.sup.13C); 2.21 (.sup.1H)-33.0 (.sup.13C); 2.25 (.sup.1H)-33.1 (.sup.13C); 2.98 (.sup.1H)-50.0 (.sup.13C); 3.26 (.sup.1H)-50.0 (.sup.13C); 5.89 (.sup.1H)-64.3 (.sup.13C); 7.98 (.sup.1H)-124.2 (.sup.13C); 8.05 (.sup.1H)-125.3 (.sup.13C); 8.30 (.sup.1H)-129.6 (.sup.13C); 8.41 (.sup.1H)-131.2 (.sup.13C) ppm DOSY NMR (D.sub.2O) log D (1.60 ppm, 2x-CH.sub.2-linker)~10.70 m.sup.2/s log D (2.03 ppm, MeCONH-polymer)~10.70 m.sup.2/s log D (2.21 ppm, CH.sub.2CONHNH.sub.2)~10.70 m.sup.2/s log D (2.25 ppm, CH.sub.2CONHNH-polymer)~10.70 m.sup.2/s log D (2.98 ppm, polymer-N.sub.6a)~10.70 m.sup.2/s log D (3.26 ppm, polymer-N.sub.6b)~10.70 m.sup.2/s log D (5.89 ppm, CH.sub.2-pyr)~10.70 m.sup.2/s log D (7.98-8.41 ppm, CH.sub.2-pyr)~10.70 m.sup.2/s log D (4.72 ppm, H.sub.2O)~8.6 m.sup.2/s UV/Vis (0.01%, H.sub.2O) .sub.max1,2 = 350, 329 nm

Example 2. Preparation of Pmoc-Hexamethylene Diamine Hyaluronan (Pmoc-HMD-HA)

(22) HA aldehyde (100 mg, 0.265 mmol, DS=10%, Mw=1.9210.sup.5 g/mol) was dissolved in 5 mL of distilled water (solution I). Pmoc-HMD (19 mg, 0.05 mmol) was dissolved in 5 mL of DMSO (solution II). Both solutions were mixed and reacted for 24 h at room temperature. In the second step PicBH.sub.3 (81 mg, 0.754 mmol) was added. The reaction mixture was stirred for 48 hours at room temperature. The product was obtained by precipitation with IPA.

(23) ##STR00005##

(24) DS=7%, Mw=1.9210.sup.5 g/mol, isolated yield 71%.

(25) TABLE-US-00002 .sup.1H NMR (D.sub.2O) 1.34 (bs, 4H); 1.45 (bs, 2H); 1.66 (bs; 2H; 2H); 3.05 (bs; 2H; CH.sub.2NHCO-pyr); 3.15 (bs; 2H; CH.sub.2NH- polymer); 3.26 (bs; 1H; polymer-N.sub.6a); 3.48 (bs; 1H; polymer-N.sub.6b); 5.83 (bs, 2H, CH.sub.2-pyr), 8.00-8.45 (m, 9H.sub.Ar) ppm H-H COSY (D.sub.2O) cross-peak 1.34-1.45; 1.34-1.66; 1.66-3.05; 1.45-3.15; 3.26-3.48 ppm HSQC (D.sub.2O) cross-peak 1.34 (.sup.1H)-26.3 (.sup.13C); 1.45 (.sup.1H)-28.7 (.sup.13C); 1.66 (.sup.1H)-26.1 (.sup.13C); 3.05 (.sup.1H)-48.2 (.sup.13C); 3.15 (.sup.1H)-41.3 (.sup.13C); 3.26 (.sup.1H)-48.5 (.sup.13C); 3.48 (.sup.1H)-48.5 (.sup.13C); 5.83 (.sup.1H)-64.3 (.sup.13C); 8.00 (.sup.1H)-124.2 (.sup.13C); 8.09 (.sup.1H)-125.7 (.sup.13C); 8.26 (.sup.1H)-130.1 (.sup.13C); 8.45 (.sup.1H)-131.7 (.sup.13C) ppm DOSY NMR (D.sub.2O) log D (1.34 ppm, 2x-CH.sub.2-linker)~10.60 m.sup.2/s log D (1.45 ppm, CH.sub.2-linker)~10.60 m.sup.2/s log D (1.66 ppm, CH.sub.2-linker)~10.60 m.sup.2/s log D (2.03 ppm, MeCONH-polymer)~10.60 m.sup.2/s log D (3.05 ppm, CH.sub.2NHCO)~10.60 m.sup.2/s log D (3.15 ppm, CH.sub.2NH-polymer)~10.60 m.sup.2/s log D (3.26 ppm, polymer-N.sub.6a)~10.60 m.sup.2/s log D (3.48 ppm, polymer-N.sub.6b)~10.60 m.sup.2/s log D (5.83 ppm, CH.sub.2-pyr)~10.60 m.sup.2/s log D (8.00-8.45 ppm, H.sub.Ar)~10.60 m.sup.2/s log D (4.72 ppm, H.sub.2O)~8.6 m.sup.2/s UV/Vis (0.01%, H.sub.2O) .sub.max1,2 = 348, 330 nm

Example 3. Preparation of Pmoc-Dihydrazide Adipate Chondroitin Sulfate (Pmoc-DHA-CS)

(26) CS aldehyde (50 mg, 0.10 mmol, DS=14%, Mw=3.0-4.010.sup.5 g/mol) was dissolved in 2.5 mL of distilled water (solution I). Pmoc-DHA (8.7 mg, 0.02 mmol, 0.2 eqv.) was dissolved in 2.5 mL of DMSO (solution II). Both solutions were mixed and reacted for 24 h at room temperature. In the second step PicBH.sub.3 (32 mg, 0.3 mmol, 3 eqv.) was added. The reaction mixture was stirred for 48 h at room temperature. The product was obtained with precipitation with IPA.

(27) ##STR00006##

(28) DS=5-6%, Mw=3.0-4.010.sup.5 g/mol, isolated yield 84%

(29) TABLE-US-00003 .sup.1H NMR (D.sub.2O) 1.66 (bs, 4H); 2.25-2.32 (m, 4H); 3.00 (bs, 1H, polymer-N.sub.6a); 3.25 (bs, 1H, polymer-N.sub.6b); 5.89 (bs, 2H, CH.sub.2- pyr); 8.15-8.38 (m, 9H.sub.Ar) ppm H-H COSY (D.sub.2O) cross-peak 1.66-2.25; 1.66-2.32; 3.00-3.25 ppm HSQC (D.sub.2O) cross-peak 1.66 (.sup.1H)-25.0 (.sup.13C); 2.25 (.sup.1H)-31.2 (.sup.13C); 2.32 (.sup.1H)-32.8 (.sup.13C); 3.00 (.sup.1H)-50.6 (.sup.13C); 3.25 (.sup.1H)-50.6 (.sup.13C); 5.89 (.sup.1H)-64.6 (.sup.13C); 8.16 (.sup.1H)-124.8 (.sup.13C); 8.38 (.sup.1H)-125.6 (.sup.13C); 8.30 (.sup.1H)-129.6 (.sup.13C) ppm DOSY NMR (D.sub.2O) log D (1.66 ppm, 2x-CH.sub.2-linker)~10.50 m.sup.2/s log D (2.04 ppm, MeCONH-polymer)~10.50 m.sup.2/s log D (2.25-2.32 ppm, CH.sub.2CONHNH2, CH.sub.2CONHNHpolymer)~ 10.50 m.sup.2/s log D (3.00 ppm, polymer-N.sub.6a)~10.50 m.sup.2/s log D (3.25 ppm, polymer-N.sub.6b)~10.50 m.sup.2/s log D (5.89 ppm, CH.sub.2-pyr)~10.50 m.sup.2/s log D (8.15-8.38 ppm, CH2-pyr)~10.50 m.sup.2/s log D (4.72 ppm, H.sub.2O)~8.6 m.sup.2/s UV/Vis (0.01%, H.sub.2O) .sub.max1,2 = 343, 328 nm

Example 4. Preparation of Pmoc-Dihydrazide Adipate of Sodium Carboxymethyl Cellulose (Pmoc-DHA-CMCNa)

(30) CMCNa aldehyde (100 mg, 0.45 mmol, DS=4-5%, Mw=8.210.sup.5 g/mol) was dissolved in 5 mL of distilled water (solution I). Pmoc-DHA (19.4 mg, 0.045 mmol, 0.1 eqv.) was dissolved in 5 mL of DMSO (solution II). Both solutions were mixed and reacted for 24 h at room temperature. In the second step PicBH.sub.3 (144 mg, 1.345 mmol, 3 eqv.) was added. The reaction mixture was stirred for 48 h at room temperature. The product was obtained by precipitation with IPA.

(31) ##STR00007##

(32) DS=2%, Mw=0.8010.sup.5 g/mol, isolated yield 88%

(33) TABLE-US-00004 .sup.1H NMR (D.sub.2O) 1.60-1.65 (bs, 4H); 2.22 (bs, 2H); 2.38 (bs, 2H); 3.00 (bs, 1H, polymer-H.sub.6a); 3.37 (bs, 1H, polymer-H.sub.6b); 5.84-5.87 (bs, 2H, CH.sub.2-pyr); 8.05-8.33 (m, 9H.sub.Ar) ppm H-H COSY (D.sub.2O) cross-peak 1.60-2.22; 1.65-2.38; 3.00-3.37 ppm HSQC (D.sub.2O) cross-peak 1.60-1.65 (.sup.1H)-25.3 (.sup.13C); 2.22 (.sup.1H)-33.6 (.sup.13C); 2.38 (.sup.1H)-32.7 (.sup.13C); 3.00 (.sup.1H)-50.1 (.sup.13C); 3.37 (.sup.1H)-51.3 (.sup.13C); 5.84-5.87 (.sup.1H)-64.2 (.sup.13C); 8.05 (.sup.1H)-123.5 (.sup.13C); 8.30 (.sup.1H)-125.1 (.sup.13C); 8.33 (.sup.1H)-129.4 (.sup.13C) ppm DOSY NMR (D.sub.2O) log D (1.60-1.65 ppm, 2x-CH.sub.2-linker)~10.60 m.sup.2/s log D (2.22-2.38 ppm, CH.sub.2CONHNH.sub.2, CH.sub.2CONHNHpolymer)~ 10.60 m.sup.2/s log D (3.00 ppm, polymer-N.sub.6a)~10.60 m.sup.2/s log D (3.37 ppm, polymer-N.sub.6b)~10.60 m.sup.2/s log D (4.55-4.61 ppm, H1aH1-polymer)~10.60 m2/s log D (5.84-5.87 ppm, CH.sub.2-pyr)~10.60 m.sup.2/s log D (8.05-8.33 ppm, CH.sub.2-pyr)~10.60 m.sup.2/s log D (4.72 ppm, H.sub.2O)~8.6 m.sup.2/s FT-IR (KBr) CO st 1750-1680 cm.sup.1 (carbamate) NCOO st as 1270-1210 cm.sup.1 (carbamate) st sy 1050-850 cm.sup.1 (carbamate) UV/Vis (0.01%, H.sub.2O) .sub.max1,2 = 344, 329 nm

Example 5. Preparation of Pmoc-HMD-HA

(34) Pmoc-1-H-imidazole carboxylate (326 mg, 1 mmol) dissolved in 20 mL THF was added to 20 mL of an aqueous solution of HMD-HA (200 mg, 0.5 mmol, DS=36%) and the reaction mixture was stirred for 24 h at room temperature. The product (DS=8%, Y=40%) was obtained by precipitation with IPA.

(35) ##STR00008##

(36) The structural analysis of the product is shown in Example 2.

Example 6. Preparation of Pmoc-DHA-HA

(37) Pmoc-1-H-imidazole carboxylate (326 mg, 1 mmol) dissolved in 20 mL THF was added to 20 mL of an aqueous solution of DHA-HA (200 mg, 0.5 mmol, DS=25%) and the reaction mixture was stirred for 24 h at room temperature. The product (DS=6%, Y=45%) was obtained by precipitation with IPA.

(38) ##STR00009##

(39) The structural analysis of the product is shown in Example 1.

Example 7. Preparation of Pmoc-Deacetylated Hyaluronan (Pmoc-DEA-HA)

(40) Pmoc-1-H-imidazole carboxylate (326 mg, 1 mmol) dissolved in 20 mL of THF was added to 20 mL of an aqueous solution DEA-HA (200 mg, 0.5 mmol, DS=32%, Mw=0.3710.sup.5 g/mol) and the reaction mixture was stirred for 24 h at 40 C. The product was obtained by precipitation with IPA.

(41) ##STR00010##

(42) DS=7%, isolated yield 35%

(43) TABLE-US-00005 .sup.1H NMR (D.sub.2O) 3.70 (bs, 1H, N2); 5.86 (bs, 2H, CH.sub.2-pyr); 8.10-8.35 (m, 9H, pyr) ppm HSQC (D2O) cross-peak 3.70 (.sup.1H)-56.3 (.sup.13C); 5.86 (.sup.1H)-63.90 (.sup.13C); 8.10 (.sup.1H)-124.0 (.sup.13C); 8.20 (.sup.1H)-125.4 (.sup.13C); 8.30 (.sup.1H)-129.0 (.sup.13C); 8.35 (.sup.1H)-131.8 (.sup.13C) ppm UV/Vis (0.01%, H.sub.2O) .sub.max1,2 = 348, 329 nm

Example 8. Photolysis of Pmoc-DHA-HA in the Presence of HA-Aldehyde and Crosslinking

(44) Method 1: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of D.sub.2O in a quartz flask. HA-aldehyde (10 mg, 0.025 mmol, DS=11%, Mw=0.6610.sup.5 g/mol) was added. The sample was deoxygenated with a stream of nitrogen and was irradiated for 1 h in UVA under N.sub.2 at 25 C., pH=7 while stirred, while samples were taken in 15 minute intervals for .sup.1H NMR analysis. The increase of the crosslink density (=7.49 ppm, HA-CHN-HA) was monitored at particular time intervals (15/30/45/60 min) at the level (18/31/66/85%), respectively.

Example 9. Photolysis of Pmoc-DHA-HA in the Presence of ,-Unsaturated HA-Aldehyde and Crosslinking

(45) Method 1: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of D.sub.2O in a quartz flask. ,-Unsaturated HA-aldehyde (10 mg, 0.025 mmol, DS=5%, Mw=0.6810.sup.5 g/mol) was added. The sample was deoxygenated in the stream of N.sub.2 and irradiated for 1 h in UVA under N.sub.2 at 25 C., pH=7 while stirred, while samples for .sup.1H NMR analysis were taken in 15 minutes intervals. The increase of the crosslink density (=7.58 ppm (H6) and 5.60 ppm (H4), HA-CHN-HA) was monitored at particular time intervals (15/30/45/60 min) at the level (20/32/48/75%), respectively.

Example 10. Photolysis of Pmoc-DHA-HA in the Presence of Saturated HA-Aldehyde and Crosslinking

(46) Method 1: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL D.sub.2O in quartz flask. HA-aldehyde (10 mg, 0.025 mmol, DS=11%, Mw=0.6610.sup.5 g/mol) was added. The sample was deoxygenated in the stream of N.sub.2 and was irradiated for 1 h in UVA under N.sub.2 at 25 C., pH=7 while stirred whereas aliquots for .sup.1H NMR analysis were withdrawn in 15 minute intervals. After 60 min UV exposition 85% of hydrazone was formed (=7.49 ppm, HA-CHN-HA).

(47) Method 2: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of D.sub.2O in a quartz flask. HA-aldehyde (10 mg, 0.025 mmol, DS=45%, Mw=0.3510.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and irradiated for 1 h in UVA under N.sub.2 at 25 C., pH=7 while stirred, wherein samples for .sup.1H HMR analysis were taken in 15 minute intervals. After 60 min of UV exposition, 95% of hydrazone was formed (=7.49 ppm, HA-CHN-HA).

(48) Method 3: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA-aldehyde (10 mg, 0.025 mmol, DS=11%, Mw=5.1010.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and irradiated for 1 h in UVA under N.sub.2 at 37 C., pH=7 while stirred. After 1 h of UVA exposition, the viscosity of the solution increased.

(49) Method 4: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA-aldehyde (10 mg, 0.025 mmol, DS=11%, Mw=5.110.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2 at 50 C., at 25 C., pH=7 while stirred. After 1 h of UVA exposition, the viscosity of the solution increased.

(50) Method 5: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 3 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA-aldehyde (20 mg, 0.050 mmol, DS=11%, Mw=5.1010.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 1 h of UVA exposition, a gel was formed.

(51) Method 6: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 3 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA-aldehyde (20 mg, 0.050 mmol, DS=11%, Mw=5.1010.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 0.25 h in UVA, under N.sub.2, at 25 C., pH=6.5 while stirred. After 1 h of UVA exposition, the viscosity of the solution increased.

(52) Method 7: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 3 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA-aldehyde (20 mg, 0.050 mmol, DS=11%, Mw=5.1010.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 2 h in UVA, under N.sub.2 at 25 C., pH=7.5 while stirred. After 2 h of UVA exposition, a gel was formed.

(53) ##STR00011##

(54) DS=3%, hydrazone group

(55) TABLE-US-00006 .sup.1H NMR (D.sub.2O) 7.49 (bs, 1H, NCH) ppm HSQC (D.sub.2O) 7.49 (.sup.1H)-146.6 (.sup.13C) ppm cross-peak DOSY NMR (D.sub.2O) log D (2.04 ppm, AcNH-polymer)~11.5 m.sup.2/s log D (7.49 ppm, NCH)~11.5 m.sup.2/s log D (4.75 ppm, H.sub.2O)~8.6 m.sup.2/s

Example 11. Photolysis of Pmoc-DHA-HA in the Presence of ,-Unsaturated HA-Aldehyde and Crosslinking

(56) Method 1: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of D.sub.2O in a quartz flask. ,-Unsaturated HA-aldehyde (10 mg, 0.025 mmol, DS=5%, Mw=0.6810.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred, wherein samples for .sup.1H NMR analysis were taken in 15 minute intervals. After 60 min of UVA exposition, 75% of hydrazone (=7.58 ppm (H6) and 5.60 ppm (H4), HA-CHN-HA) was formed.

(57) Method 2: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 2 mL of PBS (0.9%, pH=7.4) in a quartz flask. ,-unsaturated HA-aldehyde (10 mg, 0.025 mmol, DS=4%, Mw=2.0510.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 1 h exposition, the viscosity of the solution increased.

(58) Method 3: Pmoc-DHA-HA (10 mg, 0.025 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 3 mL of PBS (0.9%, pH=7.4) in a quartz flask. ,-unsaturated HA-aldehyde (20 mg, 0.05 mmol, DS=4%, Mw=2.0510.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 1 h of UVA, the viscosity of the solution increased.

(59) Method 4: Pmoc-DHA-HA (20 mg, 0.05 mmol, DS=10%, Mw=2.6410.sup.5 g/mol) was dissolved in 3 mL of PBS (0.9%, pH=7.4) in a quartz flask, ,-unsaturated HA-aldehyde (10 mg, 0.025 mmol, DS=4%, Mw=2.0510.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 1 h of UVA, the viscosity of the solution increased.

(60) ##STR00012##

(61) DS=3%, hydrazone

(62) TABLE-US-00007 .sup.1H NMR (D.sub.2O) 7.58 (bs, 1H, NCH); 5.60 (bs, 1H, CHC) ppm HSQC (D.sub.2O) 7.58 (.sup.1H)-147.3 (.sup.13C); 5.60 (.sup.1H)-110.30 (.sup.13C) cross-peak ppm DOSY NMR (D.sub.2O) log D (2.04 ppm, AcNH-polymer)~11.2 m.sup.2/s log D (5.60 ppm, CHC)~11.2 m.sup.2/s log D (7.58 ppm, NCH)~11.2 m.sup.2/s log D (4.75 ppm, H.sub.2O)~8.6 m.sup.2/s

Example 12. Photolysis of Pmoc-DHA-CS in the Presence of Saturated HA-Aldehyde and Crosslinking

(63) Method 1: Pmoc-DHA-CS (10 mg, 0.020 mmol, DS=5%, Mw=2-410.sup.4 g/mol) was dissolved in 1 mL of D.sub.2O in a quartz flask. HA-aldehyde (8 mg, 0.020 mmol, DS=33%, Mw=0.4010.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 60 min of UVA exposition, 100% of hydrazone (=7.60 ppm, HA-CHN-DHA-CS) was formed.

(64) Method 2: Pmoc-DHA-CS (10 mg, 0.020 mmol, DS=5%, Mw=2-410.sup.4 g/mol) was dissolved in 1 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA aldehyde (8 mg, 0.025 mmol, DS=33%, Mw=0.4010.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 1 h of UVA exposition, 70% of hydrazone was formed.

(65) ##STR00013##

(66) DS=5%, hydrazone group

(67) TABLE-US-00008 .sup.1H NMR (D.sub.2O) 7.60 (bs, 1H, NCH) ppm HSQC (D.sub.2O) 7.60 (.sup.1H)-145.0 (.sup.13C) ppm cross-peak DOSY NMR (D.sub.2O) log D (2.04 ppm, AcNH-polymer)~11.2 m.sup.2/s log D (7.60 ppm, NCH)~11.2 m.sup.2/s log D (4.75 ppm, H.sub.2O)~8.6 m.sup.2/s

Example 13. Photolysis of Pmoc-DHA-CMCNa in the Presence of Saturated HA Aldehyde and Crosslinking

(68) Method 1: Pmoc-DHA-CMCNa (10 mg, 0.038 mmol, DS=3-4%, Mw=6-810.sup.4 g/mol) was dissolved in 1 mL of D.sub.2O in a quartz flask. HA-aldehyde (15 mg, 0.038 mmol, DS=33%, Mw=0.4010.sup.5 g/mol). The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7. After 60 min of UVA exposition, 100% of hydrazone (=7.55 and 7.60 ppm, HA-CHN-DHA-CMC) was formed.

(69) Method 2: Pmoc-DHA-CMCNa (10 mg, 0.038 mmol, DS=3-4%, Mw=6-810.sup.4 g/mol) was dissolved in 1 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. HA-aldehyde (15 mg, 0.038 mmol, DS=33%, Mw=0.4010.sup.3 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 1 h of UVA exposition, 90% of hydrazone was formed.

(70) ##STR00014##

(71) DS=4%, hydrazone group

(72) TABLE-US-00009 .sup.1H NMR (D.sub.2O) 7.55 and 7.60 (bs, 1H, NCH) ppm HSQC (D.sub.2O) 7.55 (.sup.1H)-148.2 (.sup.13C); 7.60 (.sup.1H)-148.2 (.sup.13C); ppm cross-peak DOSY NMR log D (2.04 ppm, AcNH-HA)~11.4 m.sup.2/s (D.sub.2O) log D (4.55-4.60 ppm, H1aH1-CMCNa)~11.4 m.sup.2/s log D (7.55 and 7.60 ppm, NCH)~11.4 m.sup.2/s log D (4.75 ppm, H.sub.2O)~8.6 m.sup.2/s

Example 14 Photolysis of Pmoc-DHA-CS in the Presence of Saturated CS-Aldehyde and Crosslinking

(73) Pmoc-DHA-CS (10 mg, 0.020 mmol, DS=5%, Mw=2-410.sup.4 g/mol) was dissolved in 1 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. CS (10 mg, 0.02 mmol, DS=5%) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2 at 25 C., pH=7, while stirred. After 60 min of UVA exposition, the viscosity increased.

(74) ##STR00015##

(75) .sup.1H NMR (D.sub.2O) 7.55-7.60 (bs, 1H, NCH) ppm

Example 15. Photolysis of Pmoc-DHA-CMCNa in the Presence of CMCNa-Aldehyde and Crosslinking

(76) Pmoc-DHA-CMCNa (10 mg, 0.038 mmol, DS=3-4%, Mw=0.60-0.8010.sup.5 g/mol) was dissolved in 1 mL of PBS (c=0.9%, pH=7.4) in a quartz flask. CMCNa-aldehyde (9 mg, 0.038 mmol, DS=3-4%, Mw=0.610.sup.5 g/mol) was added. The sample was deoxygenated in the stream of nitrogen and was irradiated for 1 h in UVA, under N.sub.2, at 25 C., pH=7 while stirred. After 60 min of UVA exposition, the viscosity increased.

(77) ##STR00016##

(78) .sup.1H NMR (D.sub.2O) 7.55-7.60 (bs, 1H, NCH) ppm