PREPARATION METHOD, PRODUCT AND APPLICATION OF NON-FREE RADICAL PHOTO-CROSSLINKED HYDROGEL MATERIAL

Abstract

A method for preparing non-free radical photo-crosslinked hydrogels includes: dissolving component A that is a polymer derivative modified with o-nitrobenzyl phototrigger in a biocompatible medium to obtain solution A; dissolving component B that is a polymer derivative containing hydrazide, hydroxylamine or primary amine in a biocompatible medium to obtain solution B; mixing solution A and solution B to obtain a precursor solution of hydrogel; under light irradiation, crosslinking aldehyde generated from the o-nitrobenzyl with the hydrazine, hydroxylamine or primary amine to obtain a hydrogel by forming hydrazone, oxime or schiff base, respectively. A kit for preparation and application of the hydrogel in tissue repair, beauty therapy, and cells, proteins or drugs carriers is also described. The method or kit can achieve in situ photo-gelling on tissue surface or in situ forming thin gel on wounds in clinical treatment of wounds.

Claims

1. A preparation method of hydrogel by non-free radical photo-crosslinking reaction, comprising: obtaining solution A by dissolving component A in biocompatible medium, and obtaining solution B by dissolving component B that is a polymer derivative containing hydrazide, hydroxylamine or primary amine in biocompatible medium; obtaining a precursor solution of hydrogel by mixing solution A and solution B homogeneously; crosslinking the precursor solution to form a hydrogel under light irradiation via reacting aldehyde groups generated from o-nitrobenzyl of the component A after photo-activation with hydrazine, hydroxylamine, or primary amine groups of the component B, the reaction between the aldehyde groups and the hydrozin, hydroxylamine, or primary amine groups forming hydrazone, oxime and schiff base, respectively, wherein the component A is a derivative of a polymer modified with o-nitrobenzyl phototrigger as shown in structural formula I: ##STR00014##  where R.sub.1 is —H, ester bond selected from —CO(CH.sub.2).sub.xCH.sub.3, —CO(CH.sub.2CH.sub.2O).sub.xCH.sub.3, —CO(CH.sub.2).sub.x(CH.sub.2CH.sub.2O).sub.yCH.sub.3, ether bond selected from —(CH.sub.2).sub.xCH.sub.3, —(CH.sub.2CH.sub.2O).sub.xCH.sub.3, —(CH.sub.2).sub.x(CH.sub.2CH.sub.2O).sub.yCH.sub.3, ##STR00015##  carbonic ester bond selected from —COO(CH.sub.2).sub.xCH.sub.3, —COO(CH.sub.2CH.sub.2O).sub.xCH.sub.3, —COO(CH.sub.2).sub.x(CH.sub.2CH.sub.2O).sub.yCH.sub.3, or carbamate bond selected from —CONH(CH.sub.2).sub.xCH.sub.3, —CONH(CH.sub.2CH.sub.2O).sub.xCH.sub.3, —CONH(CH.sub.2).sub.x(CH.sub.2CH.sub.2O).sub.yCH.sub.3, where the x and y≧0 and are integer; R.sub.2 is —H or substituent group selected from —O(CH.sub.2).sub.xCH.sub.3, —O(CH.sub.2CH.sub.2O).sub.xCH.sub.3, —O(CH.sub.2).sub.x(CH.sub.2CH.sub.2O).sub.yCH.sub.3, where the x and y≧0 and are integer; R.sub.3 is selected from amino linkage bond —O(CH.sub.2).sub.xCONH(CH.sub.2).sub.yNH—, halogenated linkage bond —O(CH.sub.2).sub.x— and carboxyl linkage bond —O(CH.sub.2).sub.xCO—, wherein the ether bond end of R.sub.3 being connected with the benzene ring of the structural formula I and the other end of R.sub.3 being connected with P.sub.1, x and y≧1 and being integer; R.sub.4 is —H or —CONH(CH.sub.2).sub.xCH.sub.3, where the x≧0 and being integer; P.sub.1 is hydrophilic or water-soluble natural polysaccharide, protein or polypeptide, or hydrophilic or water-soluble synthetic polymer, wherein the component B is a polymer derivative containing hydrazide, hydroxylamine, or primary amine, as shown in structural formula IIA, IIB, IIC, respectively: ##STR00016##  wherein n≧2, P.sub.2, P.sub.3, P.sub.4 each independently are hydrophilic or water-soluble natural polysaccharide, protein, polypeptide, or hydrophilic or water-soluble synthetic polymer.

2. The preparation method of claim 1, wherein the natural polysaccharide is selected from hyaluronic acid, alginate, heparin, dextran, carboxymethyl cellulose, glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan, chitosan quaternary ammonium salt, and a modified derivative or degradation product thereof; the protein or polypeptide is selected from all kinds of hydrophilic or water-soluble plant or animal protein, collagen, serum protein, gelatin, and a modified derivative or degradation peptide thereof; the hydrophilic or water-soluble synthetic polymer is selected from two or multi-arms polyethylene glycol, polyethyleneimine, dendrimer, synthetic peptide, polylysine, (methyl) acrylate or (methyl) acrylamide polymer, and a modified derivative thereof.

3. The preparation method of claim 1, wherein the biocompatible medium is at least one medium selected from the group consisting of distilled water, saline, buffer, and cell culture medium solution.

4. The preparation method of claim 1, wherein, in the precursor solution of hydrogel, a molar ratio of the o-nitrobenzyl group to the hydrazine or hydroxylamine or primary amine group is from 1:0.02 to 1:50, preferably from 1:0.1 to 1:10; a total concentration of polymer is from 0.1 wt % to 60 wt %, preferably from 1 wt % to 10 wt %.

5. The preparation method of claim 1, wherein the illumination wavelength of the light source is 250 nm to 500 nm, preferably 300 nm to 400 nm, more preferably 365 nm.

6. A non-free radical photo-crosslinking hydrogel prepared according to the preparation method of claim 1.

7. A kit for preparing hydrogel through non-free radical photo-crosslinking using the method of claim 1, comprising: component A that is a derivative of a polymer modified with o-nitrobenzyl phototrigger shown as formula I; component B that is a polymer derivative containing hydrazide, hydroxylamine or primary amine as shown as formula IIA, IIB, or IIC; and instructions about the preparation and application of hydrogel.

8. The kit of claim 7, wherein the kit further includes a biocompatible medium including buffer and cell culture medium.

9. The kit of claim 7, wherein the instructions further include description of applications of hydrogel in tissue repair, beauty therapy, and cells, proteins or drugs carriers.

Description

DESCRIPTION OF FIGURES

[0054] FIG. 1 is the dynamic rheological curve of 10% wt hydrogel precursor solution (copolymer-NB+PEG-4ONH.sub.2) under light irradiation.

[0055] FIG. 2 shows the relationship between the swelling ratio and the polymer content of hydrogel precursor solution (HA-NB+HA-CDH).

[0056] FIG. 3 shows the scanning electron microscope image of the patterned hydrogel (10% wt, copolymer-NB+PEG-4ONH.sub.2) obtained from the non-free radical photo-crosslinking methods.

[0057] FIG. 4 shows the final healing results of the wounds treated with the non-free radical photo-crosslinking hydrogel on rat skin.

[0058] FIG. 5 shows the anti-adhesion effect of the non-free radical photo-crosslinking hydrogel on the rabbit abdominal defect model.

EXAMPLES

[0059] The following exemplary embodiments will provide more detailed description to this invention. The invention will be further described by combining with appended figures and exemplary embodiments. These examples are just for illustrating embodiments of the invention, but are not intended to limit the scope of this invention. Any other variations and modifications made by inventors abiding the spirit of invention and protection are still within the protection of this invention.

Exemplary Embodiment 1: Synthesis of o-Nitrobenzyl (HA-NB) Modified Hyaluronic Acid Derivative

[0060] ##STR00004##

[0061] (1) Synthesis of compound 1: compound 1 was synthesized according to a method reported in Pauloehrl, T.; Delaittre, G.; Bruns, M.; Meiβler, M.; Börner, H. G.; Bastmeyer, M.; Barner-Kowollik, C. Angew. Chem. Int. Ed. 2012, 51, 9181.

[0062] (2) Synthesis of compound 2: compound 1 (1 g, 3.3 mmol) and ethylenediamine (1.1 mL) were dissolved in methanol (50 mL). The mixture was refluxed overnight. After that, the solvent was evaporated in vacuum to obtain crude product. The crude product was dissolved in methanol and precipitated in ethyl acetate. After several times of dissolution-precipitation, filtration, vacuum drying, the pure compound 2 (0.93 g, yield 85%) was obtained.

[0063] (3) Synthesis of HA-NB: hyaluronic acid HA (400 mg) was dissolved in 50 mL distilled water and hydroxyl benzotriazole (HOBt, 153 mg) was added. Then the methanol solution of compound 2 (224 mg, 0.69 mmol) and 1-ethyl-(3-dimethyl amino propyl) carbodiimide hydrochloride (EDC-HCl, 200 mg) was added into the above solution. After stirring for 48 h at room temperature, the solution was firstly dialyzed against diluted HCl (pH=3.5) containing NaCl for 1 d, then dialyzed against purified water for 1 d. The solution was lyophilized to obtain HA-NB (410 mg) solid. The substitution degree of o-nitrobenzyl group was calculated according to the result of .sup.1H NMR as 7%.

Exemplary Embodiment 2: Synthesis of o-Nitrobenzyl Modified Dextran Derivative (Dextran-NB)

[0064] ##STR00005##

[0065] (1) Synthesis of compound 3: it was synthesized according to the public method in the reference Pauloehrl, T.; Delaittre, G.; Bruns, M.; Meiβler, M.; Börner, H. G.; Bastmeyer, M.; Barner-Kowollik, C. Angew. Chem. Int. Ed. 2012, 51, 9181.

[0066] (2) Synthesis of Dextran-NB: 1 g dried dextran was dissolved in dry DMSO. Compound 3 (0.23 g, 0.62 mmol), EDC-HCl (0.76 g, 3.96 mmol) and DPTS (0.12 g) were added sequentially to the above dextran solution followed by stirring for 48 h at room temperature. After completed reaction, the solution was poured into cold ethanol for precipitation and purified by three times of dissolution-precipitation and dried in vacuum to get pure dextran-NB (0.8 g). The substitution degree of o-nitrobenzyl group was calculated according to the result of .sup.1H NMR as approximate 10%.

Exemplary Embodiment 3: Synthesis of o-Nitrobenzyl Modified Chitosan Derivative (Chitosan-NB)

[0067] ##STR00006##

[0068] (1) Synthesis of bromated o-nitrobenzyl molecule:

##STR00007##

[0069] (2) Synthesis of compound 4: Vanillin 3-methoxy-4-hydroxybenzaldehyde (0.76 g, 4.9 mmol), K.sub.2CO.sub.3 (1.37 g, 9.9 mmol) and dibromoethane (1.28 g, 6.9 mmol) were dissolved in dry DMF solution together followed by stirring for about 1 h at 80° C. After completed reaction, the reaction solution was poured into ice water followed by filtering and washing to obtain crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=8:2) to get pure compound 4 (1.1 g, 80%).

[0070] (3) Synthesis of compound 5: compound 4 (2.00 g, 7.32 mmol) was completely dissolved in 15 mL ice concentrated H.sub.2SO.sub.4. The guanidine nitrate (0.9 g, 7.37 mmol) was slowly added to the above solution and reacted at room temperature for approximate 30 min. After completed reaction, the reaction solution was poured into ice water followed by filtering and washing several times. Then the crude product was recrystallized in ethanol to get pure compound 5 (1.5 g, 70%).

[0071] (4) Synthesis of compound 6: compound 5 (2.58 g, 8.5 mmol) was dissolved in methanol and NaBH.sub.4 (0.48 g, 12.75 mmol) was slowly added. After reacting at room temperature for 2 h, the solvent was evaporated in vacuum and purified by column chromatography (EA:PE=5:1) to get pure compound 6 (2.28 g, 88%).

[0072] (5) Synthesis of compound 7: compound 6 (0.88 g, 2.9 mmol) was dissolved in dichloromethane. 3,4-dihydro-2H-pyran (0.365 g, 4.3 mmol) and pyridine hydrochloride (72 mg, 0.6 mmol) were added sequentially. After reacting at room temperature for approximate 2 h, the solvent was evaporated in vacuum and recrystallized in diethyl ether to get pure compound 7 (0.73 g, 65%).

[0073] (6) Synthesis of Chitosan-NB: 10 g chitosan was added to 75 mL isopropanol to form a suspension. 25 mL NaOH solution (10 mol/L) was slowly added to the above suspension of chitosan in five times and the mixture was stirred for 0.5 h. Then compound 7 (20 g) was added to the above solution. After reacting at 60° C. for 3 h, the mixture solution was filtered and the filtrate was dialyzed three times with mixed solvent of methanol/water, and dialyzed twice in methanol, then lyophilized to get compound 7 modified chitosan (9.1 g). The compound 7 modified chitosan was dissolved in DMSO followed by adding p-toluenesulfonic acid to deprotect dihydropyran to get Chitosan-NB. The substitution degree of o-nitrobenzyl group was calculated according to the result of .sup.1H NMR as approximate 9%.

Exemplary Embodiment 4: Synthesis of o-Nitrobenzyl Modified Polyethylene Glycol Derivative (PEG-4NB)

[0074] ##STR00008##

[0075] PEG-4OH (1 g, 0.05 mmol) was dissolved in anhydrous acetonitrile followed by adding K.sub.2CO.sub.3 (55.3 mg, 0.4 mmol). The solution was stirred for 30 min. Then, compound 7 (0.16 g, 0.4 mmol) was added to react for 24 h at room temperature. After completed reaction, most of the solvent was removed and the residual solution was precipitated in the ether. The precipitates were filtrated, washed and dried to get PEG-4NB (1.1 g). The substitution degree of o-nitrobenzyl group was calculated according to the result of .sup.1H NMR as approximate 95%.

Exemplary Embodiment 5: Synthesis of o-Nitrobenzyl Modified Synthetic Copolymer (Copolymer-NB)

[0076] ##STR00009## ##STR00010##

[0077] (1) Synthesis of o-nitrobenzyl methyl acrylate monomer:

##STR00011##

[0078] (2) Synthesis of compound 8: compound 7 (0.5 g, 1.29 mmol) and ethylene glycol (0.24 g, 3.87 mmol) were dissolved in anhydrous acetonitrile and K.sub.2CO.sub.3 (0.5 g, 3.87 mmol) was added as alkali. Then the mixture was refluxed overnight. After completed reaction, the solvent was evaporated in vacuum and purified by column chromatography to get pure compound 8 (0.34 g, 72%).

[0079] (3) Synthesis of compound 9: compound 8 (0.64 g, 1.72 mmol) and triethylamine (0.34 g, 3.44 mmol) were dissolved in dry dichloromethane and methyl acryloyl chloride (0.27 g, 2.58 mmol) was slowly dropped to the above solution incubated in ice bath. After reacting overnight at room temperature, the solvent was evaporated in vacuum and the crude product was purified by column chromatography to get pure compound 9 (0.49 g, 65%).

[0080] (4) Synthesis of copolymer-NB: compound 9 (0.28 g, 0.63 mmol), comonomer PEG-MA (0.882 g, 2.52 mmol) and initiator azodiisobutyronitrile (11 mg) were added into Shrek tube and anhydrous THF was added to dissolve them. After several times of frozen-vacuum cyclic operation, the system reacted under the condition of 75° C. for 24 h., Then the reaction solution was poured into cold ether and washed with cold ether for three times. The collected precipitation was dried and dissolved in anhydrous DMSO, and p-toluene sulfonic acid was added to deprotect dihydrogen pyran group to get copolymer-NB (0.8 g). The graft ratio of o-nitrobenzyl group in copolymer was calculated according to the result of .sup.1H NMR as approximate 17%.

Exemplary Embodiment 6: Synthesis of Carbodihydrazide Modified Hyaluronic Acid Derivatives (HA-CONHNH.SUB.2., HA-CDH)

[0081] ##STR00012##

[0082] Hyaluronic acid HA (400 mg) was completely dissolved in 50 mL distilled water. Hydroxyl benzotriazole (HOBt, 153 mg), carbodihydrazide (CDH, 90 mg), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, 90 mg) were added to the above solution. After reacting at room temperature for 48 h, the solution was dialyzed against diluted HCl solution (pH=3.5) containing NaCl for 1 d, and then dialyzed against purified water for 1 d. The solution was lyophilized to obtain HA-CDH (410 mg). The product was characterized using TNBS assay as reporting in the following reference. [Oommen, O. P.; Wang, S.; Kisiel, M.; Sloff, M.; Hilborn, J.; Varghese, O. P. Adv. Funct. Mater. 2013, 23, 1273]. The modified degree of carbodihydrazide in the final product is approximate 6%.

Exemplary Embodiment 7: Synthesis of Hyaluronic Acid Derivatives Modified with Oxalic Acid Dihydrazide (HA-ODH)

[0083] According to the method in Exemplary Embodiment 6, HA-ODH was synthesized using the raw materials of hyaluronic acid, hydroxyl benzotriazole, oxalic acid dihydrazide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride. The grafting degree of HA-ODH is approximate 8% according to TNBS assay.

Exemplary Embodiment 8: Synthesis of Adipic Acid Dihydrazide Modified Hyaluronic Acid Derivative (HA-ADH)

[0084] According to the method in Exemplary Embodiment 6, HA-ADH was synthesized using hyaluronic acid, hydroxyl benzotriazole, adipic acid dihydrazide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride. The grafting degree of HA-ADH is approximate 6% according to the result of TNBS assay.

Exemplary Embodiment 9: Synthesis of Hydroxylamine Modified Four-Arm Polyethylene Glycol Derivative

[0085] ##STR00013##

[0086] The synthetic procedure of hydroxylamine modified four-arm polyethylene glycol derivative was based on the public method in the following reference [Grover, G. N.; Braden, R. L.; Christman, K. L. Adv. Mater. 2013, 25, 2937.]. Briefly, four arm polyethylene glycol (PEG-4OH, 2 g, 97.3 μmol) and N-hydroxyl phthalimide (634.6 mg, 3.89 mmol) were dissolved in dry dichloromethane. Then, triphenylphosphine (1.02 g, 3.89 mmol) was added slowly to the above solution incubated in ice bath followed by further reacting for approximately 30 min. Then, diisopropyl azodicarboxylate (765.9 μL, 3.89 mmol) was dissolved in dry dichloromethane and dropped slowly into the above solution. The solution reacted at temperature for 1 d, after which the solution was poured into ether and the participated solid was filtered. The product (0.25 g, 11.8 μmol) was redissolved in acetonitrile again and hydrazine monohydrate (22.9 μL, 473 μmol) was added. After stirring for 2 h. dichloromethane was added to the mixture solution followed by filtering through diatomite. The obtained liquid was evaporated in vacuum to get hydroxylamine modified four-arm polyethylene glycol (PEG-4ONH.sub.2).

Exemplary Embodiment 10: Synthesis of Hydroxylamine Modified Dextran Derivatives

[0087] According to the method in Exemplary Embodiment 9, the dextran modified with hydroxylamine was synthesized using dextran, N-hydroxyl phthalimide, triphenylphosphine, diisopropyl azodicarboxylate, hydrazine monohydrate.

Exemplary Embodiment 11: Hydrogel Preparation by the Non-Free Radical Photo-Crosslinking Gelation

[0088] According to the method of the present invention, different hydrogel precursor solutions were prepared at 37° C. with the composition of A:B=1:1 (w/w) as shown in Table 1.

TABLE-US-00001 TABLE 1 content B Dextran- Glycol- A HA-CDH HA-ODH HA-ADH PEG-4ONH.sub.2 ONH.sub.2 Chitosan Gelatin HA-NB 0.5-3 wt % 0.5-3 wt % 0.5-3 wt % 1-6 wt % 0.5-3 wt % 0.5-3 wt % 0.5-5 wt % Dextran-NB 0.5-3 wt % 0.5-3 wt % 0.5-3 wt % 1-6 wt % 0.5-3 wt % 0.5-3 wt % 0.5-5 wt % Chitosan-NB 0.5-3 wt % 0.5-3 wt % 0.5-3 wt % 1-6 wt % 0.5-3 wt % 0.5-3 wt % 0.5-5 wt % PEG-4NB   1-6 wt %   1-6 wt %   1-6 wt % 2-30 wt %    1-6 wt %   1-6 wt %  1-10 wt % Copolymer-   1-6 wt %   1-6 wt %   1-6 wt % 2-30 wt %    1-6 wt %   1-6 wt %  1-10 wt % NB

[0089] The hydrogels with different chemical compositions can be obtained by irradiating the precursor solutions at 365 nm (10 mW cm.sup.2) for 30 s-2 min. The hydrogel with different polymer component will exhibit different biological effects. Therefore, polymer components of the non-free radical photo-crosslinking hydrogel can be optimized according to the predesigned intention.

Exemplary Embodiment 12: Rheological Analysis of the Non-Free Radical Photocrosslinking Hydrogel

[0090] Rheological analysis was conducted by rheometer AR2000ex (TA). The rheological test was carried out at 37° C. testing platform (φ=20 mm). In this exemplary embodiment, the relationship between the storage modulus of the hydrogels and irradiation time or polymer concentration was investigated. FIG. 1 shows the gelation curve of the precursor solution containing 10% wt copolymer-NB (Exemplary Embodiment 5) and PEG-4ONH.sub.2 with equal mass ratio (Exemplary Embodiment 7) under irradiation FIG. 1 showed the hydrogel precursor solution gelled at about 30 s after light irradiation and approached the final modulus of 10.sup.4 Pa at about 100 s after light irradiation. In addition, the gel strength is proportional to polymer concentration, in which the hydrogel with higher polymer concentration exhibits lager gel strength. The gelation point and gel strength of the hydrogels composed with other polymers are shown in Table 2.

TABLE-US-00002 TABLE 2 Composition of hydrogel materials (A/B) Gel point (s) Gel strength (Pa) HA—CDH/HA—NB (2% wt) 20 1250 HA—CDH/Chitosan-NB (2% wt) 25 1506 HA—CDH/Copolymer-NB (5% wt) 23 5030 PEG—ONH.sub.2/HA—NB (5% wt) 28 3509 PEG—ONH.sub.2/Chitosan-NB (5% wt) 30 4020 PEG—ONH.sub.2/Copolymer-NB(10% wt) 30 10065 Gelatin/HA—NB (5% wt) 28 3080 Gelatin/Chitosan-NB (5% wt) 32 4027 Gelatin/Copolymer-NB (5% wt) 26 5490

Exemplary Embodiment 13: The Relation Between the Swelling Ratio and the Polymer Concentration of the Hydrogel Prepared by the Non-Free Radical Photocrosslinking Gelation

[0091] The hydrogels composed of HA-NB (Exemplary Embodiment 1) and HA-CDH (Exemplary Embodiment 2) with polymer concentration of 1% and 2% were exploited to investigate the relation between hydrogel swelling ration and polymer concentration. The swelling ratio of the hydrogels was tested after fully swelling in water for about 24 h. FIG. 2 showed that the hydrogels with lower polymer concentration exhibited higher swelling ratio. On the contrary, hydrogels with higher polymer concentration exhibited lower swelling ratio. It is mainly because that the precursor solution with higher polymer concentration forms a hydrogel with higher crosslinking density, thus resulting in the decrease of its water absorbing capacity.

Swelling ratio (%)=(W.sub.Swelling−W.sub.Dry)/W.sub.Dry×100%

[0092] In this experiment, the mass of the samples reached equilibrium state after 24 hours immersing in the medium. W.sub.Swelling is the gel weight after immersing in the medium for 24 h; W.sub.Dry is the gel weight after freeze-drying.

Exemplary Embodiment 14: The Non-Free Radical Photo-Crosslinking Gelation for Hydrogel Pattern

[0093] Copolymer-NB (Exemplary Embodiment 5) and PEG-ONH.sub.2 (Exemplary Embodiment 7) with equal mass were dissolved in distilled water to obtain the hydrogel precursor solution with polymer concentration of 10 wt %. The solution was then tiled onto a glass slide that was pretreated with piranha solution. After that, a chromeplate photomask was placed over the solution in glass slide and the distance between the mask and the glass slide was fixed at about 2 mm. After irradiating with 365 nm 365 nm light (10 mW cm.sup.2) for about 30 s-1 min and rinsing with secondary water slowly, the samples was observed by scanning electron microscopy (FIG. 3). The clear pattern in FIG. 3 demonstrates that the non-free radical photocrosslinking gelation has excellent spatiotemporal controllability.

Exemplary Embodiment 15: The Non-Free Radical Photocrosslinking Gelation for the Repair of Skin Wound in SD Rats

[0094] 4 complete skin defects with 1.8 cm diameter were made on the back of each rats. In order to ensure the faithfulness of the results, four materials (non-free radical photo-crosslinking hydrogels composed of 1% wt (a) or 2% (b) wt HA-NB and HA-CDH hydrogel; (c) commercialized 3M artificial skin; (d) no material treating) were respectively added to cover the four defects in one rat. The healing results of the defects were evaluated by microscopic observation and histological analysis. As shown in FIG. 4, the healing results of the defects treated by the non-free radical photo-crosslinking hydrogels (a, b) were much better than that of 3M artificial skin treated defects and sham control defect, suggesting the promotion effect of the non-free radical photo-crosslinking materials on wound repair. Furthermore, histological analysis results indicated that there was no obvious inflammation cells, demonstrating the biocompatibility of the hydrogel.

Exemplary Embodiment 16: The Non-Free Radical Photocrosslinking Gelation for Postsurgical Anti-Adhesion

[0095] Defects were made in the abdominal wall and cecum of the New Zealand rabbits (n=30). Then they were averagely and randomly divided into three groups based on the treatment of the defects: a. in situ formed HA-NB and HA-CDH hydrogel (2% wt); b. clinical used anti-adhesion film group; c. rising with saline. 14 days after surgery, all the rabbits were sacrificed by venous air embolism and the abdominal tissue adhesion result of each rabbit was evaluated. As shown in FIG. 5, serious tissue adhesion was observed in the rabbits from the sham control group (c). While, nine out of ten rabbits in the experiment group (a) developed no tissue adhesion at all and very slight adhesion was observed in the other rabbit of this group. In addition, no inflammation cells were observed in the histological staining image of the experiment group. These results demonstrate that hydrogel formed by the non-free radical photo-crosslinking method exhibits excellent biocompatibility and anti-adhesion ability.

Exemplary Embodiment 17: Non-Free Radical Photo-Crosslinking Gelation for Cell, Protein and Drug Delivery

[0096] Due to the unique properties of the non-free radical photocrosslinking gelation, it can achieve the in situ encapsulation of cell, protein and drug in hydrogel. Thus, the hydrogels prepared by this methods can be used as cell biology platform to mimic the natural extracellular matrix to manipulate cell fates by controlling the gel strength such as the differentiation of stem cells; it can also be used as in vivo controlled delivery systems for proteins and drugs. Next, we will introduce the application of the hydrogel prepared by the non-free radical photo-crosslinking gelation as a 3D scaffold material for mimicking the natural ECM. Equal weight of HA-NB and glycol chitosan was dissolved in α-MEM to prepare the hydrogel precursor solution. Mesenchymal stem cells were digested from plates by trypsin and centrifuged, and then added to the hydrogel precursor solution to get uniform cell suspension with a density 4×10.sup.6 cells/mL. After that, 25 μL above cell suspension was added into a 24-well plate and the suspension was further irradiated by 365-nm light of different time to form cell-encapsulated hydrogel with various gel strength. Next, the culture medium, antibiotics and culture was added to each well and the cells were cultured under the condition of 37° C., 5% CO.sub.2. Finally, the impacts of hydrogel strength on the biochemical properties of MSCs was evaluated according to the reported methods in the following reference: C. Yang, M. W. Tibbitt, L. Basta, K. S. Anseth, Nat. Mater. 2014, 13, 645-652; O. Jeon, D. S. Alt, S. W. Linderman, E. Alsberg, Adv. Mater. 2013, 25, 6366-6372.

Exemplary Embodiment 18: Application of the Non-Free Radical Photo-Crosslinking for the Moisturizing, Whitening and Anti-Wrinkle of Skin

[0097] Hyaluronic acid can make skin soft and smooth, increasing the elasticity and preventing the aging of skin largely exists in skin. Gelatin is the degradation product of collagen and has been widely used in cosmetology. Considering the easy operation and in situ controllability of the non-free radical photo-crosslinking gelation method, it can be used to prepare hydrogel mask containing hyaluronic acid and gelatin. For example, HA-NB (Exemplary Embodiment 11) can be used in combination with gelatin to produce hydrogel mask under light irradiation. This hydrogel mask can be formed directly on a face under light irradiation, adapting to profile of the face. Furthermore, essence factors can also be added into the HA-NB/gelatin hydrogel mask during its photo gelation process. Consequently, this hydrogel mask can perfectly attach the skin to preserve moisture, while the essence encapsulated in hydrogel mask can also be released to achieve better cosmetic effect.